Plasmonic enhancement of Gold Nanoparticles in a microuidicbiochip

Ana Rita Trindade Antunes

Thesis to obtain the Master of Science Degree in

Biomedical Engineering

Supervisor(s): Doctor João Pedro Estrela Rodrigues Conde

Doctor João Garcia da Fonseca

Examination Committee

Chairperson: Prof. Luís Humberto Viseu Melo

Supervisor: Prof. João Pedro Estrela Rodrigues Conde

Member of the Committee:

Dr.Pedro Miguel Neves Ribeiro Paulo

Eng.Sandro Miguel Pinto Bordeira

March 2016

ii

The known is finite, the unknown infinite; intelectually we stand on as islet in the midst of an

illimitable ocean of inexplicability. Our business in every generation is to reclaim a little more land.

T. H. Huxley, 1887, from Cosmos

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Agradecimentos

Finda esta longa jornada, cumprida com esforco, dedicacao e acima de tudo perseveranca, olho para tras

e surpreende-me ja terem decorrido sete anos. Foram sete anos de aprendizagem nao somente academica,

mas de enriquecimento pessoal: todos os que conheci ensinaram algo sobre mim mesma e sobre que pessoa

quererei ser.

Sendo a dissertacao o apogeu final do curso, nao poderei esquecer nem deixar de reconhecer a ajuda

inqualificavel de todos os intervenientes. Como tal, um primeiro profundo agradecimento ao meu orienta-

dor Professor Joao Pedro Conde, por melhorar o meu espırito crıtico sob as adversidades encontradas ao

longo das experiencias, bem como no incentivo de que qualquer trabalho pode ser sempre aperfeicoado.

Um genuıno obrigado ao meu co-orientador Dr. Joao Fonseca, pelo seu contributo nas varias sessoes na

Biosurfit. Nao poderei esquecer o Dr. Denis Roda dos Santos, Ruben Soares e Rui Pinto pela disponi-

bilidade que mostraram nas varias duvidas que os presenteei. Os meus sinceros agradecimentos ao Dr.

Narayanan Srinivasan por me ter dado nao so o seu tempo a discutir resultados e problemas encontrados,

mas tambem a sua amizade. Por fim, agradeco a todos os restantes colegas do INESC-MN, em particu-

lar a Giulia Petrucci, Joana Chim e Catarina Caneira pela amizade que criaram comigo ao longo deste

percurso, aliviando assim momentos menos bons.

Muitas pedras no caminho foram encontradas ao longo deste ano, guardei-as e vou construındo um

castelo. Esse castelo nao seria possıvel sem o apoio incondicional do meu Pai, que possibilitou toda

esta aventura e que me deu forca e motivacao nos momentos mais difıceis. Nao teria conseguido todo

este processo sem a incomparavel amizade e ajuda do Ruben Antonio, por acreditar em mim em alturas

que nem eu acreditava; nao teria conseguido sem a imensa amizade, dedicacao e preocupacao da Monica

Loureiro. Terei que reconhecer tambem a Monica Araujo, Andreia Oliveira e Susana Barroso pelo carinho,

ajuda e preciosas amizades. Todas me marcaram com excelentes memorias do curso. Sem elas, os

incontaveis almocos de boa disposicao que permitiram ver o bright side of your life em dias tristes nao

seriam possıveis.

Por fim, quero dedicar esta tese ao Dr.Carl Sagan, que atraves do seu livro Cosmos me relembrou o

quao pequenos somos, a paixao pela Ciencia e o deslumbre para com o desconhecido que nos rodeia.

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Resumo

As Nanopartıculas de Ouro exibem propriedades extraordinarias, diferentes do material comum, nas

quais propriedades opticas como o Localized Surface Plasmon Resonance (LSPR) sao dependentes da

sua dimensao e forma. Este trabalho apresenta a adsorcao de nanopartıculas esfericas estabilizadas

em citrato, de diametro 20 nm, num biochip microfluıdico, em que a aquisicao do LSPR foi realizada

atraves de fotodıodos e fotoconductores. Para a adsorcao de partıculas ocorrer nas superficies dos canais

microfluıdicos foi necessario a sua funcionalizacao com APTES durante 10 minutos, onde a interaccao

electrostatica entre as partıculas e o silano resultou num canal microfluıdico com total coloracao rosea.

A imobilizacao das nanopartıculas foi bem-sucedida utilizando um fluxo ininterrupto de 1 µL/min em

experiencias de 10, 20, 30 e 75 minutos de duracao, onde a funcionalizacao foi tambem realizada com

sucesso. O pico LSPR das nanopartıculas esfericas coloidais foi confirmado por Espectroscopia UV-Visıvel,

com 0.29 de absorvancia maxima registada a 520 nm. No sentido de detectar e avaliar o pico LSPR em

cada canal microfluıdico, utilizando fotodetectores, foi necessario acoplar no topo destes dispositivos,

barreiras de luz dispersa alinhadas com o biochip. As fotocorrentes obtidas dos dispositivos permitiram

a aquisicao do espectros, a fim de medir o pico LSPR, como tambem as fotocorrentes em funcao do

tempo. A diminuicao das fotocorrentes assinalada a 520 nm, em relacao aos valores obtidos com a

solucao de APTES no canal, apos 10 minutos da introducao das nanopartıculas sugere que a imobilizacao

nas superfıcies do canal tera sido profıcua. A acquisicao dos espectros foi realizada apos introduzir as

nanopartıculas no canal, com o objectivo de calcular os valores de absorvancia a cada comprimento

de onda. A 520 nm, o pico de absorvancia maximo foi obtido a 20, 30 e 75 minutos nas experiencias

de imobilizacao em fotodıodos e fotoconductores. No sentido de desenvolver um setup, acessıvel para

biodeteccao em sistemas Lab-on-a-Chip, os resultados aqui identificados asseguram futuras possibilidades

na monitorizacao em tempo real da interaccao entre nanopartıculas e moleculas biologicas, num robusto,

economico e reprodutıvel chip microfluıdico.

Palavras-chave: Nanopartıculas esfericas, LSPR, interaccao electrostatica, microfluıdica, fotodetec-

tores.

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Abstract

Gold Nanoparticles exhibit extraordinary properties which are quite unlike those of bulk material, since

the optical properties, such as localized surface plasmon resonance (LSPR), are dependent on the dis-

played size and shape. This work presents the adsorption of citrate stabilized spherical gold nanoparticles

of 20 nm size in a microfluidic biochip, in which the LSPR acquisition was made using photodiodes and

photoconductors. For particle adsorption on channel surfaces, functionalization was successfully accom-

plished by flowing APTES inside the channel for 10 min, in which the electrostatic interaction between

the gold nanoparticless and the silane resulted in full-coloured red microfluidic channels. The immobiliza-

tion of the nanoparticles was successful flowing uninterruptedly at 1 µL/min for 20, 30 and 75 minutes,

in all experiments in which the surface silanization was also well accomplished. The LSPR peak of these

colloidal gold nanoparticles was confirmed by UV-Vis Spectroscopy, having maximum absorbance of 0.29

at 520 nm wavelength. To detect and evaluate the LSPR peak in each microchannel using photodetectors,

it was necessary to couple the photodetectors with light scattering barriers aligned below the microflu-

idic chip. The obtained photocurrents from both devices allowed the acquisition of current spectra, in

order to measure the LSPR peak, and the photocurrent measurement over time. The photocurrents

measurement at 520 nm decreased from initial value measured with APTES, after 10 min of flowing the

gold nanoparticles, suggesting that were successfully immobilized on the channel surfaces. The spectrum

acquisitions were performed after flowing the gold nanoparticles, in order to calculate absorbances values

at each wavelength. The absorbance value registed a peak at plasmonic wavelength of 520 nm, in 20, 30

and 75 min immobilization experiments, which was successfully calculated using photodiodes and pho-

toconductors. Towards the understanding and development of simple setup for biosensing purposes in a

Lab-on-a-Chip system, these findings show the possibilites in monitorizing in real-time gold nanoparticle

interaction with biological molecules, in a robust, low cost and easily fabricated microfluidic biochip.

Keywords: Spherical Gold Nanoparticles, LSPR, electrostatic interaction, microfluidics, photode-

tectors.

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Contents

Agradecimentos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Resumo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx

1 Introduction 1

1.1 Gold Nanoparticles: to plasmon or not to plasmon? . . . . . . . . . . . . . . . . . . . . . . 2

1.2 From Microfluidics to Biomicrofluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.3 Photodetectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.3.1 Theoretical concepts in semiconductors . . . . . . . . . . . . . . . . . . . . . . . . 8

1.3.2 Hydrogenated Amorphous Silicon p-i-n junction photodiodes . . . . . . . . . . . . 11

1.3.3 Intrinsic Hydrogenated Amorphous Silicon Photoconductors . . . . . . . . . . . . . 14

1.4 State-of-the-Art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.5 Problem Description and Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

1.6 Thesis Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2 Experimental Methods 19

2.1 Moulds Fabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

2.1.1 Surface Functionalization of PDMS Microchannels . . . . . . . . . . . . . . . . . . 21

2.2 Immobilization of Gold Nanoparticles (AuNPs) in a microfluidic channel . . . . . . . . . . 22

2.2.1 The role of Diffusion and Convection Phenomena in the immobilization step . . . . 22

2.3 Data Acquisition and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3 Results and Discussion 31

3.1 Gold Nanoparticles: making their way into channels . . . . . . . . . . . . . . . . . . . . . 32

3.1.1 A PDMS/PDMS substrate experiment . . . . . . . . . . . . . . . . . . . . . . . . . 39

3.2 Scanning Electron Microscopy as a tool for insight . . . . . . . . . . . . . . . . . . . . . . 40

3.3 Localized Surface Plasmon Resonance Detection . . . . . . . . . . . . . . . . . . . . . . . 47

3.3.1 Localized Surface Plasmon Resonance (LSPR) detection in microfluidics using pho-

todiodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

xi

3.3.2 LSPR detection in microfluidics using photoconductors . . . . . . . . . . . . . . . 53

4 Conclusions and Future Challenges 61

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

4.1 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

A Appendix 73

A.1 Photodiodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

A.2 Photoconductors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

A.3 Photodetectors Runsheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

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List of Tables

2.1 Different flow rates Q assumed and derived calculations. . . . . . . . . . . . . . . . . . . . 24

3.1 Experimental time calculated for each flow rate used. . . . . . . . . . . . . . . . . . . . . . 33

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xiv

List of Figures

1.1 Gold Nanoparticles synthesized by the Turkevich method. . . . . . . . . . . . . . . . . . . 2

1.2 Effect of light interaction in a nanoparticle. . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.3 Different efficiencies corresponding to AuNPs sizes. . . . . . . . . . . . . . . . . . . . . . . 6

1.4 Silicon: energy levels splitting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.5 Band diagram and Fermi-Dirac distribution function. . . . . . . . . . . . . . . . . . . . . . 10

1.6 2D representation of a dopped Silicon (Si) lattice. . . . . . . . . . . . . . . . . . . . . . . . 11

1.7 Representation of a p-i-n photodiode and associated energy bands. . . . . . . . . . . . . . 12

1.8 Quantum efficiency of a Si photodiode. [18] . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.9 A p-i-n photodiode responsivity compared with several quantum efficiencies of semicon-

ductors. [21] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.10 Representation of photoconductor structure. . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.11 Comparison of gain and response times of distinctive photodetectors. . . . . . . . . . . . . 15

2.1 SU-8 mould used for PDMS channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

2.2 SU-8 mould resultant microfluidics channels and associated dimensions. . . . . . . . . . . 20

2.3 SU-8 mould on PMMA sheets used in photodetectors. . . . . . . . . . . . . . . . . . . . . 21

2.4 Shematic of PDMS channels fabrication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.5 Silanization of the channel surfaces. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.6 Parameters of a microfluidic channel for transport analysis. Adapted from [47]. . . . . . . 23

2.7 Example of an area inside a microfluidic channel obtained by ImageJ. . . . . . . . . . . . 25

2.8 PDMS channel aligned with the photodetector dye. . . . . . . . . . . . . . . . . . . . . . . 26

2.9 Optical setup for the photodetector experiments. . . . . . . . . . . . . . . . . . . . . . . . 26

2.10 Current-Voltage (I-V) characterization values of dark photocurrent measured in photodiode. 27

2.11 I(t) characterization values of dark photocurrent measured in photodiode. . . . . . . . . . 27

2.12 The I-V values measured in dark environment of a used photoconductor. . . . . . . . . . . 27

2.13 The measured I(t) values in a dark environment of the same photoconductor. . . . . . . . 27

2.14 Aluminum (Al) barrier to exclude the scattered light. . . . . . . . . . . . . . . . . . . . . 28

2.15 Aluminum barrier fabrication scheme. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

2.16 Fabrication step scheme of second generation barrier: TiW . . . . . . . . . . . . . . . . . 29

2.17 Image of Al barriers fabricated. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

xv

2.18 Titanium Tungsten (TiW) barriers fabricated. . . . . . . . . . . . . . . . . . . . . . . . . . 29

3.1 Absorbance of original solution obtained by Ultraviolet-visible (UV-Vis) spectroscopy. . . 32

3.2 The incubation experiment image acquisitions, at 0 and 75 minutes. . . . . . . . . . . . . 34

3.3 PDMS sealed channels on glass were immobilization of AuNPs occured: a roseate channel

is visible. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.4 Olympus Microscope acquisitions of AuNPs immobilization assay for 75 min in micofluidic

channel, at Q = 1µL/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.5 Olympus Microscope acquisitions of AuNPs immobilization assay in a microfluidic channel

at Q = 5µL/min, during 75min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

3.6 Transmittance values calculated through mean intensity values from ImageJ for two dif-

ferent flow rates Q. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

3.7 Image acquisitions comparing two channel of different used flow rates. . . . . . . . . . . . 37

3.8 Control experiment, a channel without (3-Aminopropyl)triethoxysilane (APTES) surface

modification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3.9 Phosphate Buffered Saline (PBS) washing experiment in a previous AuNPs immobilization

assay channel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.10 Comparison among three channels, where different flow rates were used: 0.05, 0.5 and 5

µL/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.11 Transmittance values comparison of different AuNPs immobilization repetition experi-

ments using 1 µL/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

3.12 Immobilization of gold nanoparticles only on PDMS surfaces. . . . . . . . . . . . . . . . . 39

3.13 Transmittance values of assays perfomed in PDMS channel sealed on glass and on Poly(dimethylsiloxane)

(PDMS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

3.14 Acquisition of a full channel with AuNPs immobilized. . . . . . . . . . . . . . . . . . . . . 40

3.15 Channel with AuNPs immobilized used as sample for Scanning Electron Microscopy (SEM). 41

3.16 Pealing the PDMS channel of the glass substrate for SEM analysis. . . . . . . . . . . . . . 41

3.17 Top view of de-sealed channel acquisition in SEM. . . . . . . . . . . . . . . . . . . . . . . 42

3.18 SEM acquisition image of area 1, scale of 200 nm. . . . . . . . . . . . . . . . . . . . . . . . 42

3.19 SEM acquisition image of area 2, scale of 200 nm. . . . . . . . . . . . . . . . . . . . . . . 43

3.20 SEM acquisition of area 3, scale of 20 nm. . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

3.21 SEM acquisitions of area 4, scale of 20 and 200 nm. . . . . . . . . . . . . . . . . . . . . . 44

3.22 SEM acquisitions of inlet and outlet zones. . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3.23 Comparative results: SEM acquisition of 0.5 and 1 nM AuNPs. [48] . . . . . . . . . . . . 45

3.24 Comparative results: AFM acquisitions of immobilized AuNPs of two concentrations of

APTES. [50] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

3.25 APTES solution stability dependence on pH and temperature . . . . . . . . . . . . . . . . 47

3.26 Comparison between plasmon peak obtained by UV-Vis Spectroscopy and photodiode

acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

xvi

3.27 Absorbances values calculated for a 30 min AuNPs immobilization. . . . . . . . . . . . . . 50

3.28 Channel on top of a photodiode dye using Al barrier. . . . . . . . . . . . . . . . . . . . . . 50

3.29 Absorbance spectrum acquired after 20 min of AuNPs immobilization. . . . . . . . . . . . 51

3.30 Absorbance spectrum acquired after 20 + 30 min of AuNPs immobilization. . . . . . . . . 51

3.31 Transmittance calculated over time acquired for 20 min of AuNPs immobilization. . . . . 52

3.32 Transmittance calculated over time acquired for 30 min of AuNPs immobilization. . . . . 52

3.33 Absorbance spectrum over 30 min of immobilization in photoconductor. . . . . . . . . . . 54

3.34 Calculated Transmittance over time of AuNPs immobilization in photoconductor. . . . . . 55

3.35 Absorbance spectrum of Bovine Serum Albumine (BSA) compared with AuNPs. . . . . . 56

3.36 Absorbance spectrum of 75 min immobilization acquired by photoconductor. . . . . . . . 56

3.37 Evolution of transmittance over 75 min of immobilization in photoconductor. . . . . . . . 57

3.38 TiW barrier aligned on top of photoconductor. . . . . . . . . . . . . . . . . . . . . . . . . 58

3.39 External Quantum Efficiency (EQE) dependency on bias voltage of a photoconductor. [57] 58

3.40 Responsivity of a-Si:H photoconductor and dependency on electrode spacing. [57] . . . . . 59

A.1 Figure of merit of photodiode using five different Neutral Density (ND) filters. . . . . . . 74

A.2 Photodiode photocurrent acquisition of black ink channel. . . . . . . . . . . . . . . . . . . 74

A.3 Typical photocurrents acquisition from 30 min immobilization of AuNPs. . . . . . . . . . 75

A.4 Spectra acquired of ink in two different microfluidic channels. . . . . . . . . . . . . . . . . 75

A.5 Hydrophobicity of the black ink inside a microfluidic channel. . . . . . . . . . . . . . . . . 76

A.6 Photocurrent spectrum acquisitions of the 20+30 min assay of AuNPs immobilization. . . 76

A.7 Comparison between photocurrents acquisition in photodiode, of each main step of the

20+30 min immobilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

A.8 Characterization: dark photocurrent of photoconductor. . . . . . . . . . . . . . . . . . . . 78

A.9 Characterization: ND3 yield photocurrent of photoconductor. . . . . . . . . . . . . . . . . 78

A.10 Characterization: ND3 yield photocurrent of black ink channel. . . . . . . . . . . . . . . . 78

A.11 Photocurrent spectrum acquisitions in photoconductor of 30 min immobilization assay. . . 79

A.12 Comparison between photocurrents acquisition in photoconductor, of each main step of 30

min immobilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

A.13 Photocurrents acquisitions of 75 min immobilization acquired by photoconductor. . . . . . 80

A.14 Comparison of photocurrents acquisitions of 75 min immobilization acquired by photocon-

ductor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

A.15 Photocurrent spectrum acquisition of 75 min of AuNPs immobilization in photoconductor

using TiW barrier. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

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xviii

Nomenclature

Al Aluminum

As Arsenium

AFM Atomic Force Microscopy

APTES (3-Aminopropyl)triethoxysilane

AuNPs Gold Nanoparticles

a-Si:H Hydrogenated Amorphous Silicon

B Boron

B2H6 Diborane

Bi Bismuth

BSA Bovine Serum Albumine

DI-water Deionized water

EQE External Quantum Efficiency

Ge Germanium

H Hydrogen

Hg(II) Mercury

In Indium

ITO Indium-Tin-Oxide

I-V Current-Voltage

LoC Lab-on-a-Chip

LSPR Localized Surface Plasmon Resonance

N2 Nitrogen gas

ND Neutral Density

xix

NIR Near Infrared Reagion

P Phosphorus

PBS Phosphate Buffered Saline

PDMS Poly(dimethylsiloxane)

PeH Peclet number

PeS Shear Peclet number

PH3 Phosphine

PMMA Poly(methyl 2-methylpropenoate)

PoC Point-of-care

Re Reynolds number

RF-PECVD Radio-Frequency Plasma Enhanced Chemical Vapor Deposition

SEM Scanning Electron Microscopy

SERS Surface-enhanced Raman Spectroscopy

Si Silicon

SiH4 Silane gas

SiNx Silicon Nitride

SPR Surface Plasmon Resonance

TiW Titanium Tungsten

UV-O Ultraviolet Ozone

UV-Vis Ultraviolet-visible

xx

1Introduction

Contents

1.1 Gold Nanoparticles: to plasmon or not to plasmon? . . . . . . . . . . . . . 2

1.2 From Microfluidics to Biomicrofluidics . . . . . . . . . . . . . . . . . . . . . . 6

1.3 Photodetectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.4 State-of-the-Art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.5 Problem Description and Motivation . . . . . . . . . . . . . . . . . . . . . . 17

1.6 Thesis Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

1

1.1 Gold Nanoparticles: to plasmon or not to plasmon?

Gold and iron at the present day,

as in ancient times, are the rulers

of the world.

William Whewell, 1851

Through ages, Man has mastered the art of extracting and crafting gold for its own purposes, from

sacred symbols, monarchists purposes, as well as decoration in ceramics or glass, to medicine. The use

of gold in therapeutics dated back to thousand of years ago in India and later in the Medieval Europe,

due to its intrinsic characteristics: non-toxic, non-irritating and resistant to chemical corrosion.

In the present chapter, the nanoscale properties of gold particles are discussed. The following charac-

terization attempts to demonstrate, to several readers, the physics and the optical advantages behind

this material, as well as the broad applicability, impossible to report in full extension. Gold Nanoparti-

cles (AuNPs) possess unique optical, physical and electronic properties that enables applications in diverse

fields. These properties are totally dependent on size and shape, these key parameters are related to light

scattering and surface chemical activity; therefore the production through different methods should be

systematic and controlled over morphology and composition. There are well-known liquid-phase syn-

thetic methods performed by the reduction of gold percursors introduced in organic or aqueous media,

adding surface stabilizers, depending on the size features required. Turkevich et al. [1] created the prime

reduction method, synthesizing colloidal gold, in aqueous media, using sodium citrate (C6H5Na3O7) as

reductant of HAuCl4 and also citrate as a surface stabilizer agent. Aggregation processes are needed

to construct these particles. Therefore, the decrease of citrate ions, through reduction of initial sodium

citrate, enables stabilization of the AuNPs, leading to the aggregation of small particles forming larger

ones. This method yields monodispersed, spherical of 10 nm to 20 nm range-size particles, depicted in

1.1 that are suspended in water.

Figure 1.1: Electron micrograph of AuNPs, using the Turkevich et al. with a magnification 50,000

diameters. [1]

2

As mentioned previously, size and shape are the main determinants for the physical occurances, by

which optical properties are influenced. AuNPs can be classified by size, and, in this work, 20 nm di-

ameter particles were used and categorized to a defined class range of 10-300 nm . [2] In this size scope,

it is designated by a plasmonic crystal whose more interesting properties are Localized Surface Plasmon

Resonance (LSPR) and Surface Plasmon Resonance (SPR), which will be described below.

The physics behind AuNPs can be described by analysing the interaction between an electromagnetic

wave and a metallic surface. In the visible range, an incident electromagnetic wave on a metal does not

penetrate further than the designated skin depth (or penetration depth), where for example a 500 nm

wavelength light beam would have a penetration depth of 20 nm. [2] Penetration depth is considered to

be a measure of electomagnetic wave decay inside a material, which can be described by Beer-Lambert

Law. Therefore, the penetration depth depends greatly on the incident wavelength. When a light beam

is incident on a surface of a metal, it creates a thin sheet of polarization with penetration depth thickness

at the surface, where the electrons in the conduction band of gold act as free polarized particles, with

certain detachment from the nucleus. This occurs in two specific cases, first in systems where the size

of metal structures have the same order magnitude of the penetration depth and second in the case of

a flat theoretically infinite surface. Concerning the two cases described above, it is needed to clarify the

difference between LSPR and SPR.

In respect to the first case, where the size of the AuNPs is inferior to the incident beam’s wavelength

and in many cases it is also smaller than the penetration depth, a particular phenomena occurs on the

oscillation of charges. The electric field from the incident electromagnetic wave attains the free electrons

from the valence band, polarizing the whole surface of the nanoparticle, since the particle size is the

same order of magnitude of the penetration depth, shown in 1.1 (c). The excitation of the incident wave,

e.g. an optical beam, induces a resonant oscillation of those electrons at a specific frequency, ωplasmon,

creating an electric dipole at the AuNPs surface. The given equation 1.1, seen in [3], describes the

dependence of the plasmon frequency from conduction electrons density Ne, the electron charge e and

the conduction electrons effective mass m′. This equation was obtained through the theoretical study

of AuNPs dependence on size and wavelength, using the mean free path correction in Mie’s theory, seen

in [3].

ωplasmon =4πNee

2

m′(1.1)

This dipole formation through the acumulation of the charges on the particles’ opposite ends, con-

senquently creates an electric field inside the AuNPs, opposing to that of the incident light. This field

will compel the polarized electrons to restore equilibrium positions, designated by restoring force. Fur-

thermore, the yield resonant oscillation is somewhat repressed through light scattering and heat creation

processes. For these reasons, the above mentioned optical characteristics of AuNPs describes the LSPR,

demonstrated in figure 1.2, which is responsible for the red colour of colloidal spherical AuNPs with a 20

3

nm diameter size.

Figure 1.2: Schematic of the light interaction with a nanoparticle, resulting in the creation of an electric

field (c). Through the collective oscillations, cross-sections of absorption and scattering are originated.

The LSPR at the plasmon wavelength for different size ranges are presented in (h), (i). Adapted image

from [4].

The light incident of nanoparticles depending on size can be absorbed or scattered. In order to analyze

the efficiency of such processes, it is necessary to characterize both in cross-sections, for absorption and

elastic scattering, so that the sum of the two processes causes light attenuation, characterized by the

extinction cross-section. According to the simplified scattering model, applied to small and homogeneous

spherical particles, where a first order in multipole expansion in Mie theory is used to calculate the

extinction cross section σext and scattering cross-section σscatt. To derive the light intensity of a wave

being absorbed, the σabs relates the three cross-sections described in equation 1.2, seen in [2] .

σabs = σext − σscatt (1.2)

AuNPs of 20 nm size, when isolated and in colloidal form, display a predominant absorption cross-

section, with plasmon resonance peak at 520 nm wavelength, causing the extinction of respective green

wavelengths, although transmission of red colours arise. When agglomerated, the plasmon resonance

shifts for longer wavelengths and the peak itself is broadened, therefore the red colour is consequently

absorbed.

According to [2], the fundamental properties of LSPR can be described:

• ωplasmon: When a light beam is incident on monodispersed AuNPs, it is partly absorbed at resonance

frequency ωplasmon, which appears to be in Near Infrared Reagion (NIR) or in the visible range, as

seen in figure 1.3.

• Absorption vs Scattering: Along with the absorption, AuNPs show a wide cross section of light

scattering. As also seen in figure 1.3, the 20 and 40 nm diameter particles a) and b), respectively,

show that the absorption efficiency prevails over the scattering efficiency. On the contrary, the

4

scattering efficiency is visible for the 80 nm diameter particle. Therefore, the increase in particle

size implies dominance of scattering over absorption.

• towards LSPR Biosensing: The possibilities of using AuNPs widens as biosensing plataforms, in

colorimetric assays using Lab-on-a-Chip (LoC) systems, since the position of LSPR peak, in ex-

tinction spectrum is deeply influenced by the surrounding medium refractive index. The LSPR

is extremely sensitive to the surrounding medium, whereas the conduction electrons frequency of

oscillation is most dependent on the external dielectric constant thereby related to the refractive

index. In focus, the resonance peak is shifted to longer wavelengths, as the refractive index (n)

increases, as illustrated in equation 1.3, from [5]. This equation is verified in cases where the change

in refractive index is due to the absorption of a certain layer in the surroundings of the nanopar-

ticles, specifically where the layer’s thickness is inferior to the electric field decay. The shift of

plasmon resonance peak is also directly dependent on the responsiveness m of the nanoparticles.

This finds purpose in biosensing for targeted molecules, in which if a target binds specifically to

the AuNPs, it leads to a higher average refractive index in the neighbouring medium, causing the

LSPR to red shift. The increase of absorption and scattering processes, utterly dependent upon

size, are of major importance in the biosensing and enhancement on sensitivity processes, thereby

a significant interest in developing a highly sensitive biological and chemical sensors is based on

these nanosystems.

4 (λmax) = m(nA − nE)[1− e−2dld ] (1.3)

Being nA the refractive index of the adsorbate layer, nE the refractive index of the particle envi-

ronment, d the thickness of the adsorbate layer and ld is the electric field decay length.

The efficiencies demonstrated in figure 1.3 were calculated from Mie theory for three particle sizes.

Moreover, in 1908, Gustav Mie [6] solved the Maxwell’s equations and calculated an analytical and exact

solution of the surface plasmon of spherical nanoparticles and the intensity of light absorption, using the

assumption that the particles would be distant enough, so that the electric field created among them

would not affect all individually. This theory postulates that when the size of the nanoparticle is smaller

that the incident light’s wavelength, the electric field of the nanoparticle is spatially constant, but has

phase variable which is dependent on time. This so called dipolar approximation theory is valid for

particles with size inferior to 60 nm.

5

Figure 1.3: The different efficiencies: green line represents exctintion, red dashed line represents absorp-

tion and black dots represent scattering. Where a) 20 nm, b) 40 nm and c) 80 nm diameters. [7]

The processes of absorption and scattering are of main importance to the present work. As formerly

pointed regarding the absorption efficiency, it theoretically describes the geometrical section of an ideal

opaque particle, which absorbs the equal number of photons as the one particle in study. Therefore, the

absorption efficiency is handed out by its absorption cross section. [8] Besides absorption, as light interacts

with AuNPs it can also be scattered, so a scattering cross section can be defined as the geometrical section

of an ideal scattering particle having the same efficiency as the particle in study. Finally, the extinction

cross section can be defined as the sum of the absorption and scattering cross section, representing the

ability of a particle to extract photons from incident light through both processes. [8]

As specified above, another interesting property is the SPR. When an electromagnetic wave is incident

between an infinite flat metal surface and an insulator, the two having different dielectric functions, a

surface wave is created and it is restrained close to the interface between them. The surface wave is

named polariton, a charge density with a longitudinal direction, opposed to the transversal direction of

the incident wave. Since these two wave couple, when the incident beam possess a given angle, capable

to excite, a surface plasmon wave is arised. Considering that SPR is only seen on flat planes of infinite

extension, this topic falls out of the scope in this work.

1.2 From Microfluidics to Biomicrofluidics

The Microfluidics field rose from two different science fields: analytical chemistry and microfabrication.

The urgency of solving both chemically and biochemically relevant problems led to the increased usage

and development of microdevices. Nowadays, the scope of microfluidics is seen in fields such as bio-

chemistry, biology and bioengineering, giving birth to the claimed Biomicrofluidics. This field hinges on

transport phenomena and flow physics in nano and micro length scale systems, operating and controlling

small volumes of gases or liquid, the latter from fento-litre to micro-litre. Hence offering different appli-

cations through varied geometric shapes of small channels. In this section, the fundamental aspects of

fluids flowing over microscopic scales, along with its mechanisms and implications, will be presented.

The Navier-Stokes equations are a common tool used to understand the behaviour of a fluid inside a

microfluidic set, using the fluid’s characteristics such as velocity, pressure, density and dynamic viscosity.

6

The terms of the expressions that constitute the equation represent the several forces in which the fluid

is exposed to: inertial, pressure, viscous and external forces. The underlined calculations is build upon

a continuum hypothesis, which states that there are enough molecules to establish statistical properties

when using small volumes. [9] Then, if the fluid is assigned to as a continuum, these equations are

accurately aplied to liquids in microsystems, concerning that the physical dimension of the channel(L)

is much wider than fluid’s molecules mean free path (λ), represented by the Knudsen number given in

equation 1.4. [10]

Kn =λ

L(1.4)

The mean free path is the average linear displacement between two moving molecules that collide

and thereby, change directions. These collisions are naturally associated with temperature, as can be

seen in gases and liquids. [9] Considering that the mean free path is smaller that the characteristic size

of a channel in liquids, the continuum hypothesis is still viable. The simplification of the Navier-Stokes

equations can be accomplished depending on flow’s regime, which can be defined by a non-dimensional

value, Reynolds number, according to equation 1.5. [10]

Re =ULρ0µ

(1.5)

Where U is the characteristic velocity, L is length of the flow, ρ0 is the constant fluid’s density and

µ is the constant kinematic viscosity of the fluid. It is to be noticed that the Reynolds number (Re) is

not a property of the fluid but it is a parameter that combines the fluid and geometric properties. The

equation 1.5 represents the ratio of the inertial forces over the viscous forces, measuring the turbulence

of the flow. If Re is low, Re ≤ 1, the interaction between the viscous forces amidst the wall and the fluid

is intensified, with no turbulences and vortices occuring, so the flow is laminar; if Re is higher, the flow

is turbulent. Commonly in microfluidics, specifically LoC, laminar flow is the most present regime (if

the application of interest does not require larger channels or higher speeds). By assumption, if U is less

than cm.s−1 and L in the range order of µm, then the Re ≤ 0.1. [11]

The molecules in a gas or liquid have a peculiar erratic movement, a Brownian motion behaviour,

which is parallel in macromolecules and microparticles, which behaviour can be described analogously.

The conception of Diffusion is built upon Brownian theory, where a initially confined group of particles

of a certain volume starts erratic movements over time and are continuously dispersed in a buffer liquid.

The presented equation 2.1 is based on the preceding continuum hypothesis. [10]

D =KBT

6πµRH(1.6)

Where KB is the Boltzmann Constant, T the absolute temperature in K, µ the viscosity of the

solvent fluid and RH the hydrodynamic radius of solute particles. The accepted Diffusion coefficient for

commonly tested biomolecule analytes is approximately 10−10 to 10−9 m2/s. [10] In the process of particle

spreading, a diffusive flux JD can be analysed. According to Flick’s Law in equation 1.7, the number of

7

particles crossing a unit surface, in time t, is in fact proportional to the gradient of concentration (with a

negative constant of proportion, since diffusion occurs reversed from gradient concentration) and to the

Diffusion Coefficient. [10]

JD = −D5c (1.7)

Inside a confinement, the particles of a fluid are performing diffusion, but are also creating the advec-

tion of a velocity field along the fluid. The competition between advection and diffusion is the mechanism

behind several mass and chemical transports. In order to evaluate the predominance of advection in re-

lation to diffusion, the Peclet, PeH , number was defined, as seen in equation 2.2. [11]

PeH =Ul

D(1.8)

Polymeric material, such as Poly(dimethylsiloxane) (PDMS), is commonly used for LoC purposes.

The usual microfabrication technique applied is Soft Lithography, being a low cost and a fast procedure.

There are different techniques that are included in Soft Lithography processes, sharing the general proto-

col of fabrication, although they differ in the way that a polymer stamp is used to reproduce its form. [12]

PDMS has been one of the leading choices to fabricate microchannels and has the potential to integrate

valves, mixers and pumps on-chip. These contributions have settled the foundations of micro total anal-

ysis systems (µTAS). As an elastomer, PDMS presents characteristics that are useful and advantageous

for biological assays, since it is chemically inert, has no swelling properties in humid environments, is

biocompatible and has a structural compliance when in contact with large area surfaces. Moreover, this

polymer possess good permeability to gases, is homogeneous, isotropic and is compatible with several

optical detection systems, since it is optically transparent from 300 nm to IR range. After the fabrication

process of PDMS conformations needed, surface modification developments take place. The hydrophobic

nature of this material it is prone to non-specific protein adsorption, thus it is necessary to perform

surface treatment in order to minimize these occurrences. Surface treatments are highly dependent of the

biological goals, for that purpose some of the most common techniques are Ultraviolet Ozone (UV-O),

Oxigen Plasma or Corona Discharge, as seen in [13].

1.3 Photodetectors

1.3.1 Theoretical concepts in semiconductors

Photodetectors are made of semiconductor materials that detect incident light by photon absorption

and originate a flow current, proportional to the initial light intensity. To address photodiodes operation

modes, it is essential to elucidate theoretical concepts on semiconductor materials and quantum mechanics

principles.

Silicon (Si) has four valence electrons that establish covalent bonds with surrounding silicon atoms

when creating a lattice. A pure silicon crystal behaves as an insulator, since the outer electrons are all

involved in covalent bonds, with no free electrons to conduct electric current. Although these atoms

8

bond covalently, the bonds are substantially weaker than carbon-carbon ones, present in insulators such

as glass, diamonds and polymers. The excitation energy that electrons need to surpass, to migrate from

the valence band to the conduction band, is called the forbidden gap (Eg), in which its values define

whether a material is an insulator, a semiconductor or a conductor. If the Eg is large, then the electron

energy from an applied electric field will not be sufficient for an electron in the valence band to enter

the conduction band. Thus, this electron would not be freed and no current would flow, as it is seen in

insulators.

In the case of semiconductors, although Eg is smaller, they are not electrically conductive in low

temperatures. Yet, through thermal excitation some electrons involved in covalent bonds would become

free and become conduction electrons carrying enough energy to overwhelm the forbidden gap. In oppo-

sition to semiconductors, the forbidden gap is inexistent in conductors, due to the overlap between the

valence and conduction bands. [14] As pointed out, thermal excitation leads to electric conductivity in

semiconductors. When temperature increases, Eg tends to decrease, since the interatomic distances are

greater, causing a reduction in the average potential. [15]

The formation of energy bands is intimately related with discrete electronic states and associated

energies, in which electrons are allowed to occupy. Energy bands in crystalline materials are individually

spaced energy levels of electrons, enclosing each atom. Accordingly to Pauli’s exclusion principle, when

two wave functions belonging to two neighbouring electrons overlap, they cannot share the same quantum

number. Hence, this discrete energy level has to be split in closely spaced levels, forming energy bands

separated by forbidden energies, such that electron fills a different quantum state, 1.4. The minimum

quantum states existent in a band are twice the number of atoms present on the material, considering

that any energy level is able to contain two electrons of opposite spins. [15]

Figure 1.4: Valence states (3s and 3p) splitting of Si, into forbidden and allowed energies, where r-axis

represents the interatomic distance. [16]

Considering that two electrons having opposite half-integer spins are Fermions in an energy state,

these particles have an associated occupancy probability distribution in a given system. The Fermi-Dirac

distribution function provides the probability of an energy state (E ), in thermal equilibrium with another

system, being occupied by a Fermion. As thermodinamics brings to light, for a given system to be in

9

thermal equilibrium, it must have the lowest energy configuration when subjected to thermal agitation.

For that reason, Fermions fill first the lower energy states and higher ones are filled next. When the system

is at absolute zero, 0 K, Fermions will fill the states to a maximum energy level called the Fermi energy

level (EF ), with no higher energy states filled. The Fermi energy level remains constant throughout the

system, as long as thermal equilibrium is present. [15]

The approach referred implies that each electron is defined as an undistinguishable particle with an

expected probability of being in an available energy level, in accordance with Pauli’s Exclusion Principle.

The figure below 1.5 illustrates that when E is increasing, becoming higher than E-EF , the probability

involved of an electron occupying that energy level f(E) decreases exponentially. At lower energy E, the

probability increases, which means that the low energy states favor to be fully occupied.

Figure 1.5: Schematic band diagram, associated density states and carriers. Fermi-Dirac Distribution

plot and mathematical expression(in box). Adapted image from [14].

Semiconductors can be defined in two types: intrisic and extrinsic. Two of the most common intrinsic

semiconductors are Si and Ge, with no impurities on their crystal structure. In this case, whenever an

electron is excited and migrates to the conduction band, it leaves a hole behind in the valence band, hence

a formation of carriers (holes and electrons). A hole can be defined as an absence of negative charge,

or can be defined as a void with a higher electric potential. At room temperature, the small density of

holes left in the valence band is equal to the density of electrons on the conduction band, where these

hole-electron pairs need approximately 1.1 eV to be formed. [14] In order to increase density of carriers in

Si, there is a need for a doping process, which is the addition of suitable impurity atoms to this specific

lattice. This process yields an extrinsic semiconductor material, with controlled electrical conductivity,

seen in 1.6. The doping process can be accomplished by using selected compounds that have higher or

lower valence electrons than Si. Mostly, group V elements, such as As, P or Bi are used as electron donors

when introduced into the Silicon lattice, along with no holes formation. Semiconductor materials that

have increased conductive behaviour due to moving electrons in the lattice are titled type N. In contrast,

a semiconductor doped with group III atoms, commonly B, Al or In are named type P. These dopant

elements are used as electron acceptors, leading to higher density of holes with positive charge. Hence,

in this case, the motion of holes as carriers is also responsible for current conduction. [17]

It is important to notice that Fermi-Dirac distribution function is a statistical analysis that hypothesize

the number of electrons/fermions and the system’s total energy are held constant. This approach is also

10

applied on impurities, present in semiconductors materials. Moreover, it is possible to define the Fermi

level in both types of doped semiconductors. The Fermi level on a n-doped semiconductor is localized

near the conduction band, as for the p-doped semiconductor is localized near the valence band.

Figure 1.6: Two-dimensional representation of a dopped lattice. Semiconductor doped with a) donor

(As) and b) acceptor (B). [14]

In doped semiconductors, Fermi-Dirac distribution function of impurities contrasts with one previously

described above, considering the possible and unknown quantum states that are implied for the donation

or acceptance of electrons. When a donor from an impurity element provides an electron, the resulting

form is ionized (positive charge), with an energy level that contains an electron that could be in two

possible quantum states (spin pointing upwards or downwards). This aspect contributes to the one-half

degeneration factor presented in the equation 1.9 [15]. As for the acceptor element, which receives an

electron, it will occupy a certain acceptor level in also two possible different quantum states. Furthermore,

it is usual that semiconductors show dual degenerate valence band, by which the sum of all aspects yields

a degeneration factor of four affecting the Fermi-Dirac function, demonstrated in equation 1.10 [15].

fdonor =1

1 + 12e

(Ed − EF )/kT(1.9)

facceptor =1

1 + 4e(Ea − EF )/kT(1.10)

Being Ed the donor energy level, Ea the acceptor energy level, EF Fermi energy level and kT the

thermal energy.

Along this topic, electron excitation in semiconductors has been reviewed. Particularly, in carriers

generation from light incident on semiconductors in two types of devices are on focus.

1.3.2 Hydrogenated Amorphous Silicon p-i-n junction photodiodes

A non-regular crystalline form of Si is seen in (a-Si:H). The addition of H atoms allows the dangling bonds

of amorphous Si to be passivated, so that the process of hydrogenation plays a key role on stabilizing

this amorphous structure. To acquire the desired optic and electronic features of a-Si:H structure, it

is mandatory to control and optimize the deposition process and growth conditions, such as substrate

temperature, gas composition, gas pressure and flow. It also depends on the power of RF-PECVD, which

is the usual method used to control the surface growth. The plasma decomposes gas SiH4 and the added

dopants, in this case, a layer of 100 A of n-doped Si (using PH3) was deposited, a 5000 A of intrinsic

11

Si layer and 100 A of p-doped Si, using B2H6 were also deposited. Hence, the intrisic layer is wider and

has less doping. In addition, a 2000 A layer of ITO for top contact deposition was performed also, as a

anti-reflection coating. As referred above, p-i-n photodiode is a structure of an intrinsic region between

two differently doped layers. In the figure below 1.7 there is a simple representation of a p-i-n diode,

lacking the top and bottom metal contacts, since the doped layers are not sufficiently conductive.

Figure 1.7: Structure of a p-i-n photodiode (left). [18] The correspondent schematic energy bands diagram,

showing the electron diffusion in p-doped layer and hole’s diffusion in n-doped layer (right). [19]

The detection process using these devices goes through a physical principle: internal photoelectric

effect, where electron-holes pairs are generated by the photon’s energy, being absorbed in the intrinsic

layer. As said previously, the energy of an incident photon has to be higher than Eg to break the covalent

bonds and excite the collided electron from the valence band to the conduction band. Thereupon, the

excitation depends on Eg, h is Planck’s constant and c the speed of light. If the used wavelengths are

afar of λc, the incident photon energy is absorbed by the semiconductor and carreirs are generated. This

defines the cut-off wavelength, λc, as shown in equation 1.11.

λc =hc

Eg(1.11)

The p-i-n diodes possess two different operation regimes, as photodetector (reverse bias) or as photo-

voltaic (forward bias). In the first case, the power is supplied through an external source, whereas in the

second case, it is by energy harvesting (solar cell). [18] A reverse bias operation mode is central in this

work, accomplished by reversing the polarization, such as applying a positive potential (Vr) source to the

n-type doped layer. Although the doped layers provide the built-in potential of the junction, they do not

contribute to light sensitivity. The life span of the minority carriers formed in those thin layers is not

significant, since the holes in the n layer and the electrons in the p layer recombine before crossing the

intrinsic layer. [18] The depletion region associated to the small size of each doped layer is formed and

it is smaller than the depletion region originated from the intrinsic layer in between. In constrast, the

intrinsic layer is responsible for light sensitivity, because it collects drifting electron-holes pairs efficiently,

due to its higher degree of crystalinity. A thicker depletion gives a larger capture area, advantageous for

a maximum absorbance of the photon flux. [18]

While using a reverse bias operation, a reverse bias dark current is generated. The dark current can

be defined as the current that flows throught the photodiode when no light is incident on the device,

12

resulting in a noise source in this operation mode, therefore this current is controlled by the internal

energy barrier. The resultant dark current, or leakage current, is a key parameter in a diode performance

evaluation. A highly efficient photodiode is related to the lowest dark current possible, which is only

accomplished by having thermal generation current in a full depleted diode. The bulk thermal current

is a result of electron excitation from the valence band to a defect state that is singly occupied, due to

thermal energy. This major contributor to the dark current can be reduced by decreasing the deffect’s

density and by increasing the Eg. In addition, the doped contact injection and edge leakage of the

component may be also contributing factors to the dark current. [20]

Besides, under reverse bias, a photocurrent Iph is created resulting from light exposure, throught

the electron-holes pairs being generated in the intrinsic layer and drifted across it by the electrical field.

The photocurrent is directly proportional to the quantum effiency, ηQE , given by the ratio between the

number of electron-hole carrier pairs generated per incident and absorbed photon with energy hv, shown

in the equation 1.12. [21]

ηQE =Iph/q

Pin/hv(1.12)

Where Iph is the photocurrent, q the elementary charge, Pin the optical power at wavelength λ, h

represents the Planck’s constant and v the frequency of the incident photon. In figure 1.8, incident λ

is decreasing to an energy inferior to Eg and consequently ηQE decreases to zero, since the majority of

photons are absorbed at the surface. Also, figure 1.10 shows the spectral response, in particular, of Si

photodiode, where the relation betweeen the amount of current produced with wavelength is presented,

presuming that the used wavelengths are at the same light level.

On a related matter, other important parameter to consider is the photodiode responsivity, R, typically

used to evaluate the sensitivity of a photodiode, demonstrated in figure 1.9. Commonly, it is defined by the

ratio of Iph, calculated in Amperes by the incident optical power measured in Watts. If the device is more

sensitive, a higher responsivity is acknowledged, therefore it can generate greater currents. Responsivity

is linearly proportional to ηQE and to the used λ, demonstrated in 1.13 [22]:

R =ληQE

hv(1.13)

13

Figure 1.8: Quantum efficiency. [18]

Figure 1.9: A p-i-n photodiode responsivity com-

pared with several quantum efficiencies of semicon-

ductors. [21]

1.3.3 Intrinsic Hydrogenated Amorphous Silicon Photoconductors

Photoconductors are another type of device reactive to light, embedded with different characteristics and

operation modes. Concerning the fabrication, RF-PECVD was used to deposite an intrinsic layer of 5000

A of a-Si:H and to deposite SiNx, a passivation layer deposition of 2000 A. The device was also set with

2000 A of Al as electrods pads.

These devices are the simplest conceivable photodetector, where an intrinsic semiconductor is nested

with electrical (Aluminum (Al)) contacts, operating under an applied external voltage 1.10. One of

the three essential absorption mechanisms, free carrier absorption, absorption with associated forbidden-

gap energy levels, only intrinsic (band-to-band), is in discussion. [23] When incident light is upon the

photoconductor, for every arriving and absorbed photon, an electron-hole pair is generated. Due to the

influence of an applied electric field between the two metal contacts on these carriers, they migrate to

the opposite poles: the electron to the positive and the hole to the negative.

Figure 1.10: Schematic of a usual structure of a intrinsic photoconductor and its working principle. [24]

Intrinsic band-to-band is the most common effect in photoconductors. Similarly, as described in the

14

prior topics, the hole-pair formation follows the same principle, as to intrinsic layer in photodiodes. An

incident photon must comprise sufficient energy to excite the electron from the valence band, therefore

the incident light should carry a certain wavelength to exceed the forbidden gap energy, seen in 1.11.

According to the literature, an intrinsic Si photoconductor has a forbidden gap energy of 1.12 eV, with

a typical operating range of 500 to 900 nm. [23]

As mentioned, the movement of the carriers to the opposite poles in response to an applied voltage

creates a photocurrent, which is proportional to the incident photon flux, φ. Thereby, the increase of

conductivity is a result of increased number of carriers. [25] If the number of carriers reaching the contact

pads are taken into consideration in terms of time (seconds), a parameter for evaluating the detector’s

perfomance is considered. For each carrier migrating between the poles per second and for each photon

absorbed also per second, which is the definition of gain, the figure 1.11 shows the typical gain values. [22]

Figure 1.11: Classic photoconductive gain and response times values in diverse photodetectors. [22]

Concluding this chapter, it is possible to say that an ideal photodetector should be highly sensitive

with no associated dark current. The main differences between photodiodes and photoconductors are the

structural distinctions, photoconductors possess electrical contacts (ohmic), in opposition to photodiodes

which has doped layers. The advantage in using photoconductors is that the photoconductive gain is

higher, although it costs greater response times, seen in 1.11 and higher dark currents. As for photodiodes,

the notorious low response time and low dark current are the more interesting characteristics for sensitive

optical detection.

1.4 State-of-the-Art

Over the past fifteen years, there has been a sort of Big Bang in the study of AuNPs properties and

applications, rediscovering all the advantageous characteristics of this material. AuNPs have endured a

long path to understanding, which is seen in biomedical applications, as well as personalized medicine,

making their way to the possibility of adjoining diagnostics and therapeutics. Due to SPR and LSPR

characteristics, the ability to manipulate the intensity and amplification of light at the nanoscale, along

with the possibility of synthesizing various sizes and shapes in aqueous media and taking advantage of

low tissue toxicity high chemical stability, making these nanoparticles the object of extensive study and

applications in many areas.

Biomedical analysis is the proeminent field in which AuNPs have been a successful tool for the

15

diversed applications. In behalf of some properties owing to the individual interaction at the same

scale, the size of small AuNPs can be in the order of magnitude of some biological entities such as

DNA chains, cells, bacteria and even viruses. Furthermore, this individual interaction is facilitated by

a straightforward functionalization step at the surface of the nanoparticle that can couple to organic

molecules, in a biocompatible approach. Efforts have been made to discover and understand, not only

the genetic but also physiological processes that contribute to several diseases. This information has

been tied to the discovery of biomarkers field, in order to develop targeted therapeutics, where a strategy

for selective interference with disease hallmarks is implemented. The creation of molecular targeted

therapeutics has been implemented in diseases such as cancer, where tumor cells are targeted for a

specific drug. This brings improvements not only in efficacy, but in decreased toxicity and with no

limitations in drug penetration on tumor, often seen in conventional therapies.

The usual drug delivery strategies are based on releasing a coated drug specifically on targeted (dis-

eased) cells. When the drug arrives to the targeted region, the coating structure is disaggregated, allowing

the interaction of the drug. This drug release must be a controlled and precise approach, in order to

promote effectiveness. Metallic nanoparticles, such as gold nanocages, as seen in [26], are one of the ex-

amples of the various synthesized structures obtained, with porous walls, a hollow core and characterized

by a photothermal effect. Through photolysis with a NIR laser, the bioactive compound is released in a

controlled way. The converted heat of the light absorption triggers the dissociation of the smart polymer,

which covers the nanocage surface. By turning off the laser, the chains of the smart polymer acquire initial

conformation, ceasing the drug release. Later, similar work is seen in [27], using spherical gold nanopar-

ticles of 40 nm diameter, with a double coating functionalization. This AuNP/PEG-INU/Doxorubicin

system was used to transport an increased mass of doxorubicin, an anticancer drug, to evaluate cyto-

toxicity for in vivo cells. Likewise, another nanoplatform based on doxorubicin was used in [28], which

showed that using coated nanoparticles induced tumour cell apoptosis successfully and efficiently, through

an improvement in cellular uptake with no cytotoxicity. To transform nano-theranostics from a concept

to a practical medical approach, there have been several studies about the toxicological repercussion of

AuNPs on in vivo tissues, as seen in [29]. Actually, the toxicity is deeply related to the size and shape

of the nanoparticles used, see [30], as well as the administration route admitted and the type of surface

coverage. Further studies have been done in the interaction between colloidal AuNPs and cells, as seen

in [31]. In addition, the surface charge of the particle plays a central role in the internal uptake by the

cell, through electrostatic interactions. [32]

Selective labelling using AuNPs for specific disease type has demonstrated great potential, in which

the excitation of the nanoparticles is used to intensify and cause other optical processes, such as Surface-

enhanced Raman Spectroscopy (SERS) and dye fluorescence. An illustration of this matter is the creation

of 30 and 60 nm gold nanostars as radiolabelling probes inside tumor cells, in order to explore and compare

the nanoparticle distribution and cellular uptake for each size. [33]. Comparable work in labeling for

localization is seen in [34], where AuNPs were coated with polyethylene glycol coupled with antibody

for breast cancer marker. Through this technique it was possible to microlocalize the gold, resulting in

tumour and non-tumour tissue densities identification.

16

In order to give the reader an insight of the astonishing and incommensurable applications of AuNPs,

some other interesting applications are seen in DNA nanotechnology [35], and energy harvesting, where

configuration of AuNPs are activated by light that collect sunlight and transfer this energy to highly

excited electrons. This innovation could increase efficiencies and reduce costs in converting solar to

electric energy. [36] Furthermore, chemical sensors such as an optical microfluidic system with AuNPs

with surface modification is used to detect if Mercury (Hg(II)) is present in water samples. The detection

principle is based on ion recognition which originates a change in SPR band. [37] In extension, another

chemical sensor used colorimetric detection of As in water samples, through LSPR signal. [38] As seen here,

the myriad of AuNPs applications is not fully portrayed, given the confirmation that these nanoparticles

are not only a proof of concept, but a pratical tool to solve some of today’s problems.

1.5 Problem Description and Motivation

Biosensing devices based on LSPR provides a sensitive, easily acessed and label-free detection in low-cost

fabricated systems. Hence, these biosensors are stated to be a resourceful tool in constrained environ-

ments, by which costs, detection time and transport mechanisms are crucial. [39] An LSPR sensing

plataform have been widely used for diagnostic in Point-of-care (PoC) applications, where the inter-

actions between AuNPs and biomolecules are developed comprehensively through the incorporation of

multiplexed and microfluidic devices. [40] [41] [42] So far, it has been reported the detection and char-

acterization of AuNPs by Ultraviolet-visible Spectroscopy [43] [44], using the refractive index sensing

capabilities of AuNPs in a near-surface environment as an attractive employment for protein detection.

The high surface area, stability, biological compatibility and controllable morphology are excellent fea-

tures for immunoassay bionsensing plataforms.

Here, this work finds the motivation in the detection of LSPR-based sensing system in a simple

microfluidic device by the use of photodetectors for real-time acquisition. As seen in previous work, pho-

todiodes have been used for PoC applications [45] [46], showing that the integration between microfluidic

systems and photodetectors are not only successful but also versatile. The goal was the acquistion of the

LSPR peak, when the AuNPs were adsorbed in a microfluidic channel using two different devices: photo-

diodes and photoconductors. The immobilization premise was based on electrostatic interaction between

the negative charged surfaces of citrate stabilized AuNPs and the positive charges on the channel surfaces.

To ensure this adsorption of the nanoparticles, the microfluidic channel surfaces were functionalized by

APTES previously and correspondent photocurrents were acquired. To achieve the absorbance spectrum

calculation, photocurrents acquisition were performed during the experiment. With the applied methods

it was possible to acquire the LSPR using both devices and to monitor the interaction with BSA protein.

17

1.6 Thesis Outline

This dissertation will be branched into four essential chapters:

1. Introduction

This chapter intends to highlight summarily the theoretical concepts that support the work presented.

The fundamental sections are a brief introduction to the physical concepts behind spherical AuNPs, fol-

lowed by the theoretical concepts of microfluidics and an elucidation of semiconductor photodetector

operation principles, namely photodiodes and photoconductors.

2. Experimental Methods

The techniques and procedures undertake towards the accomplishment of the motivation are described

in this chapter. General devices and methods of data acquisition and analysis will be described in fol-

lowing sections. The details on mould fabrication process to achieve a microfluidic device are given, as

well as the surface chemistry needed on the PDMS in order to execute nanoparticles assays. This chapter

ends with summary explanation on the acquisition steps using photodetectors.

3. Results and Discussion

The discussion on this chapter will focus on the immobilization of AuNPs in PDMS microfluidic

devices and its detection using an optical microscope. To clarify the obtained results from the immo-

bilization, a Scanning Electron Microscopy probing method is used. Furthermore, the detection of the

Localized Surface Plasmon Resonance through photodiodes, photoconductors and associated challenges

are detailed.

4. Conclusions and Future Work

The last chapter of this thesis includes the outcomes and perspectives taken on the performed work,

some of the possible improvements and suggestions to the forthcoming work.

18

2Experimental Methods

Contents

2.1 Moulds Fabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

2.2 Immobilization of AuNPs in a microfluidic channel . . . . . . . . . . . . . . 22

2.3 Data Acquisition and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 25

19

2.1 Moulds Fabrication

The approach used in this work was based on microfluidics, as a plataform for the experiments and

photodetectors, and as a tool to observe, acquire and analyse the output data. For this purpose, SU-8

moulds used were from INESC-MN, as seen figure 2.1, and developed previously to this work.

Figure 2.1: The first SU-8 mould used to fabricate PDMS channels, of INESC-MN. The SU-8 mould is

fixed to the Petri dish, with adhesive tape and with cured PDMS.

From figure below 2.2, the design was performed to be a simple straight channel with two peripheral

areas, the inlet and the outlet, being the entrance and exit of the fluids, respectively.

Figure 2.2: Two dimensional representation of a SU-8 mould channel dimensions, with a channel height

of 20 µm.

Another SU-8 mould was used, also from INESC-MN, with PMMA sheets that were specifically used

for the photodetector experiments, to assure that it would obtain the most possible smooth topography on

the the surface of PDMS substrate. A smooth surface would result in minimal scattered light from incident

beam. There were three Poly(methyl 2-methylpropenoate) (PMMA) sheets, with already fabricated holes,

as shown in 2.3, where the PDMS would be poured onto the SU-8, which was glued to the bottom PMMA

sheet. The yielded channel assume almost same dimensions of the first one mentioned: 200 µm of width,

20 µm in height and 10000 µm in length.

20

Figure 2.3: PMMA sheets that held the SU-mould in order to produce PDMS microfluidic channels.

In order to obtain several channels as a platform for the experiments, PDMS was prepared using

the base (SYLGARD 184 silicone elastomer by DOW CORNING) and a curing agent (SYLGARD 184

silicone elastomer by DOW CORNING) using a ratio 10:1 (w:w), respectively. After stirring both in

a plastic cup, it was degassed in a vacuum chamber for 1 hour and 30 min, so that air bubbles were

removed. This mixture was poured on top of each SU-8 mould and put in Memmert oven, at 70 C to be

baked for 1 hour and 15 min, showed schematically shown in figure 2.4. The PDMS structure was cut

and separated from the mould, then it was necessary to punch holes in the outlet and inlet spots to allow

the entrance of adapters and tubes, using a syring needle tip.

Figure 2.4: PDMS Microchannel fabrication scheme, with fundamental steps (not at real scale).

2.1.1 Surface Functionalization of PDMS Microchannels

The PDMS prepared was sealed on thin glass, Menzel-GlaserTM, 50× 24 mm. Each glass was washed in

Alconox for 30 min, then washed with acetone, isopropyl alcohol and water, finishing with N2 blow-dry

gun. The first surface derivatization process was performed with UV-O treatment (UVO cleaner 144AX,

Jelight Company Inc.TM), where a UV lamp would create reactive hydroxyl groups at the surfaces during

11 min (6 min to clean and 5 min to exhaust). Since, during this project, INESC-MN acquired the

Plasma equipment, all the following described experiments were initialized by using this prefered sealing

process. PDMS and glass surfaces were put in Plasma Cleaner (Harrick PlasmaTM), where an Oxygen

plasma would activate these surfaces for 1 min, creating reactive OH negative groups. Then, PDMS and

glass were assembled together, gently and manually pressed to seal together, and put over a hotplate at

130 C for 5 min.

21

2.2 Immobilization of AuNPs in a microfluidic channel

The testing plataform used was a PDMS structure with several channels sealed on a glass slide. To

perform the respective assays, syringe pumps (SyringPumpTM) were used to push solutions into the

channels. The first step in every experiment made was the silanization of the glass and PDMS surfaces,

which was achieved using APTES, a (1%) solution prepared on Deionized water (DI-water) (99%), from

ACROS ORGANICS TM. (3-Aminopropyl)triethoxysilane (APTES) reacts with hydroxyl groups on the

surfaces, resulting in siloxane covalent bonds, while its amine groups are spatially available to interact.

This functionalization process involved all the surfaces inside the channel, not only glass but also the

PDMS surfaces, thereby for simplicity it is shown in figure 2.5 only the glass silanization.

Figure 2.5: Covalent reaction between APTES and hydroxyl groups present after the Plasma Cleaner

treatment.

2.2.1 The role of Diffusion and Convection Phenomena in the immobilization

step

Every microfluidic system is unique, not only regarding the dimensions, design and fabrication imple-

mented, but also due to the surface chemistry applied which is thereby dependent on the application of

interest. In these systems, the analyte transport plays a critical a role, since it is dependent on the com-

peting physical processes occurring inside the channels, such as diffusion and convection. The following

matter is a theoretical study based on a previous work [47], in order to find which microfluidic condi-

tions would be optimal to capture a certain number of AuNPs in the microfluidic channel, which would

correspond visually to their presence. Squires, et al., [47] presented a description of these effects, using

finite-element computational tools to assist and to model the analyte target transport and interaction

with a sensing area, in different sizes of microfluidic biosensors. A considerable difference between the

following analysis and the work seen in Squires, et al. is the target area. In current work, the sensing

area is ideally a surface (area of WcL) that is functionalized with APTES, while in Squires,et al. this

area is a small targeted-functionalized region, compared with the channel size, in the bottom surface as

well.

Therefore, this theoretical comprehensive treatment will be partially used in this work to understand

which flow rates should be used in order to have a successful AuNPs immobilization over the surface

22

reaction area. This surface is theoretically of width Wc, where a solution of AuNPs with concentration

C0 and diffusivity D, flows with flow rate Q. A simple design of microfluidic channel can be represented

as seen in figure 2.6, having a height H, a channel width Wc and length L, subjected to a volumetric flow

rate Q.

Figure 2.6: Model of a microfluidic channel and its characteristics, adapted from [47].

Where L is 9000 µm (0.9 cm), Wc is 200 µm (0.02 cm) and H is 20 µm (0.002 cm). The Q values are

the evaluated parameter, in order to adress the immobilization process.

A concentrated solution of AuNPs was purchased from PlasmaChemTM, with particle average size

of 20±3 nm diameter, stabilized in citrate buffer, with an initial concentration of 0.05 mg/mL (1 OD)

and at ca. pH 8,0. An initial concentration of 0.05 mg/mL holds 6.8 × 1011 particles/cm3, flowing in a

channel volume of 3.6× 10−5 cm3, reaching a reaction surface area of 1.8× 10−2 cm2 (of width Wc).

As considered in the Biomicrofluidics chapter, the diffusion coefficient given in 2.1 is useful to char-

acterize the analyte displacement in a certain volume and applied to the system used in this work. The

below value yields the theoretical diffusivity of the AuNPs buffered in citrate at room temperature.

D =KBT

6πµRH=

(1.38× 10−23)× 298.15

6π × 0.001× (10× 10−9)≈ 2.18× 10−11cm2/s (2.1)

Where KB = 1.38× 10−23m2kgs−2K−1, µ = 0.001 kg/ms is the water buffer dynamic viscosity, T in K

and RH hydrodynamic diameter of the AuNPs.

In this theoretical approach, the PeH is calculated to understand if AuNPs are reaching the sensing

area by convection or diffusion. With the diffusion coefficient obtained above, PeH is given in equation

2.2. To calculate this dimensionless value, it is necessary to assign different values of flow rate Q shown

in Table 2.1 to analyze which would be the best for the current work, also the reaction surface area Wc.

PeH =Q

WcD(2.2)

Furthermore, in this approach [47], as the analyte molecules diffusion throught channels are collected

near the sensing area, a depletion zone is formed with a certain size. This depletion zone is given by

equation 2.3, being D the diffusivity of the analyte molecule and t the time concerned for a molecule to

arrive to the sensing region. A valid approach in an simple and ideal sensor, considering molecules that

bind promptly upon reaching the sensing area.

δ =√Dt (2.3)

23

To describe the number of analytes collected while the depletion zone grows thicker, it is necessary

to define flux (molecules/ time), since these analytes diffuse according to the initial concentration and

through the distance on which the concentration varies. In this current case, the depletion zone is

broadened through the channel so that the total flux through the cross-section area (Wc×H) is given by

equation 2.4. A dimensionless flux (Sherwood number for mass-transport systems) needed to calculate a

dimensional flux is defined by equation 2.5 for very fast flow fluxes. In this regime, particles are swept

downstream in the channel, hence the capture of the particles occurs in a thin layer above the sensing

region.

JD = FWcC0D (2.4)

Being C0 the initial concentration, D the diffusion coefficient, Wc the reaction area (sensing region)

and F the dimensionless flux represented below.

F ≈ 3√PeS (2.5)

A dimensionless parameter appears within these calculations, λ, being the sensor size, a ratio between

the length L and the height H of the channel given by the equation in 2.6. The second Peclet number,

PeS , is deeply related to shear rate and the length of the sensing region, represented in equation 2.7:

λ =L

H(2.6)

PeS = 6λ2PeH (2.7)

Both Peclet numbers define not only the competing effects of convection and diffusion, but also the

depletion region. As referred in [47], PeH associates depletion region with channel size, whereas PeS

relates the depletion zone to the size of the sensing area itself.

In the table below, there are values from performed calculations of the above dimensional and adi-

mensional parameters, involving each tested volumetric flow rate.

Table 2.1: Different flow rates Q assumed and derived calculations.

Q (µL/min) PeH PeS F JD (AuNPs/s)

0,05 1.91× 106 2.32× 1012 1.32× 104 3.93× 103

0,5 1.91× 107 2.32× 1013 2.85× 104 8.46× 103

1 3.82× 107 4.64× 1013 3.59× 104 1.07× 104

5 1.91× 108 2.32× 1014 6.15× 104 1.82× 104

24

2.3 Data Acquisition and Analysis

The immobilization of AuNPs in the microfluidic channel was visualized over time using inverted Olympus

Microscope. Then, it was possible to acquire selected areas of the channel at a given t. After the

experiment, the transmittance was calculated by selecting areas using ImageJ (NIH) software as a tool,

in order to measure the pixels intensity by mean values of gray scale, shown in figure 2.7. It is important

to notice that this calculation is made using a control, by which the initial intensity is given by an APTES

solution inside the channel at the beginning of the immobilization step (t=0). So the transmittance is

obtained using the expression 2.8. The values obtained by this equation characterize the LSPR effect

inside a microfluidic channel over time.

T =It

It=0

(2.8)

Figure 2.7: Selected area of the channel using ImageJ and correspondent mean intensity value, to provide

transmittance calculation.

The second component of this work focused on acquiring the spectrum of the AuNPs as function of

the absorbance, in order to obtain the LSPR peak. Additionally, the transmittance through time was

calculated at the end of the assays. As visualized in figure 2.8, a microfluidic PDMS structure, with

four channels was fixed on top of the dye, where the experiment channel was manually aligned to the

photodetector, using zoom stereo microscope (Nikon 75519) and temporarily fixed after positioning for

acquisition.

25

Figure 2.8: Top view of the PDMS channels above the photodetector dye.

A systematic approach in acquisition protocol was followed in these experiments, verifying the data

in each step. All the acquisitions were made at room temperature (24C), in a most possible dark

environment, using the apparatus shown in figure 2.9, with a 24 V Tungsten-Halogen Lamp, coupled to

an Oriel monochromator. Spectrums were acquired in the range of 400 nm to 650 nm, while photocurrent

vs time was otained exclusively using 520 nm wavelength of interest.

Figure 2.9: Optical setup used in photodetector measurements seen from different angles.

The acquisition setup involved photodiodes and photoconductors of 200× 200 sq. µm to acquire the

data of the AuNPs immobilization. When evaluating each type of photodetector response, several peaks

were obtained and used as representative of each step accomplished on the experiment using a lock-in

amplifier (EGG Princeton Applied Research, model 5209). The typical response obtained in photodiodes

and photoconductors is shown in the graphics below, where it is possible to observe that both have

different ways to operate, thus it is necessary to characterize separately and erstwhile each assay. The

first step was to acquire in a dark environment, the Current-Voltage (I-V) plot in range of [-1,1]V values

with a 0.1 V steps, using photodiodes, seen in figure 2.10, and the initial values of the dark photocurrent in

function of time, demonstrated as seen in figure 2.11. As for photoconductors, the I-V plot was acquired

with an applied voltage of [0,20] V represented by figure 2.12 and also the photocurrent in function of

26

time, illustrated by figure 2.13. Twain acquisitions were made using a picoammeter Keithley 237.

Figure 2.10: The I-V values measured in dark

environment of a used photodiode.

Figure 2.11: The measured I(t) values in a dark

environment of the same photodiode.

Figure 2.12: The I-V values measured in dark

environment of a used photoconductor.

Figure 2.13: The measured I(t) values in a dark

environment of the same photoconductor.

In this work there was a concern regarding scattered light of the environment or external sources, e.g.

the used lamp and all the other machinery involved in this acquisition. So, towards the understanding of

which amount of scattered light was introduced in acquisition and affected the measurements, a channel

was filled with black ink, purchased from PelikanTM. This procedure was defined by the background

photocurrent acquisition. It was based on the premise that this ideal opaque black ink channel would

yield the amount of light that would still reach to each photodetector. Consequently, the final step in each

experiment was to fill the channel with the cited black ink, covering the prior flown AuNPs. Furthermore,

two barriers were manufactured to make the acquisition process as clean as possible from this noise.

Hence, two glass masks, with different material deposition, were fabricated to cover the photodetectors’

surroundings, in order to prevent all the light that reaches the photodetectors from external sourcers and

due to probable misalignments, between the photodetector itself and the channel. An illustration of this

strategy is seen in the figure 2.14:

27

Figure 2.14: Lateral view of the prepared setup: the outcome of the manual alignments of the barrier on

top of the photodetector followed by the channel on top of the barrier.

Much importance was given to achieve a barrier as opaque as possible, so a 2000 A thickness of Al was

deposited on a glass slide. The fabrication process displayed in figure 2.15 yielded three glass barriers

with a row of squares without deposition, so that the incident light beam could pass through and be

retained in the outer remaining area.

Figure 2.15: Steps of Al barriers fabrication process, where the main procedures are included. Images

are scaled.

The second generation mask was made of an 1500 A Titanium Tungsten (TiW) thickness deposited

on glass, however another patterning was implemented since some alignment difficulties were encountered

with the first barrier. Thence, this barrier had the same row of squares and an auxiliar patterning to

align with the photodetector dye was implemented. The fabrication process is illustrated below in figure

2.16, in which three barriers were created: two of them with the deposition performed, including the rows

of squared holes differing in size, and the third without the complete deposition and with a small row of

deposited alloy squares.

28

Figure 2.16: Steps of TiW barriers fabrication process. Images are not representative of a real scale.

Figures 2.17 and 2.18 show the real images of the produced barriers, in which the red arrow indicates

the row of holes, where the light enters in two first barriers.

Figure 2.17: Al barriers, with a row of 100×100

µm2 holes. These holes were to be aligned with

the photodetector.

Figure 2.18: TiW barriers: the first from the

left was designed to bear a row of 100 × 100

µm2 holes (red arrow); the middle barrier, a

row of 70 × 70 µm2 holes and the last had the

negative patterning, with a row of 100×100 µm

deposited squares to block light.

These barriers, if well aligned with the photodetectors, should by principle, cut the light intensity

reaching the sensors. The incident photons are also controlled by the Neutral Density (ND) filters, which

reduce the photon flux by an estabilished factor. Therefore, by managing the number of photons it

is possible to control the consequent photocurrent, since the photon flux is related to the formation of

electron-hole pairs. The photon flux, demonstrated in equation 2.9, corresponds to the number of photons

that is absorbed in the surface of the photodetector, per unit of area and per unit of time, at a certain

used wavelength.

φ(λ) =I(λ)λ

SR(λ)ch(2.9)

29

Where I(λ) is the photocurrent, R(λ) responsivity of the photodetector, h Planck’s Constant, S the

surface area (cm2) and c the speed of light.

Transmittance can be calculated using the above mathematical relationship, since the incident light

on the channel I0 is partially absorbed and a fraction of it is acquired Isample. At the end of each

acquisition, it was necessary to analyze in detail every photocurrent obtained, in which a correction of

the formula seen in the equaton 2.10 was applied. As a matter of fact, this correction was made by

measuring the photocurrent yield from black ink channel. These values were subtracted to the sample

photocurrent ones. Finally, the optical depth was calculated, using equation 2.11 to identify the behaviour

of the AuNPs inside the channel and to evaluate its LSPR peak.

Tcorrected =Isample − IblackI0 − Iblack

(2.10)

A = −ln(Tcorrected) (2.11)

30

3Results and Discussion

Contents

3.1 Gold Nanoparticles: making their way into channels . . . . . . . . . . . . . 32

3.2 Scanning Electron Microscopy as a tool for insight . . . . . . . . . . . . . . 40

3.3 Localized Surface Plasmon Resonance Detection . . . . . . . . . . . . . . . 47

31

3.1 Gold Nanoparticles: making their way into channels

The plasmon peak was obtained in a spectrophotometer from the AuNPs, of 20 nm diameter, using

a circular cuvette with the original 0.05 mg/mL concentrated solution from a 10 mL flask, at room

temperature of 24 C. As stated previously in Introduction section, AuNPs in this size range, colloidal

and monodispersed is characterized by LSPR. This optical property causes light absorption in the

spectrum portion inferior to 450 nm wavelengths, while red light is reflected in higher wavelengths (≈

700 nm), hence causing the visible red colour. Therefore, these particles in colloidal solution show a red

colour with the plasmonic peak confirmed at 520 nm wavelength, correspondent to a retrieved absorbance

value of 0.29 seen in figure 3.1:

Figure 3.1: Spherical nanoparticles with direct retrieved absorbances values for each wavelength.

From Beer-Lambert law, it is possible to determine the molar absorptivity of the AuNPs solution,

given by the expression 3.1, where A is the absorbance of the solution measured, C the molar concentration

(1.14 × 10−9 M) and L the optic path length (0.6 cm). Therefore, the molar absorptivity, ε , for this

solution is 4.19× 108 M−1 cm−1.

ε =A

CL(3.1)

Theoretically, by creating a 2D surface of a cuvette with full coverage, entirely filled by AuNPs

disposed on the same plane, it is possible to define the molar absorptivity for each nanoparticle (ε′).

Let it be used the measured absorbance value of 0.29 with the respective 6.8× 1011 particles/cm3 (flask

concentration) and the used optical path to infer on the value of ε′. Hence, by equation 3.1, it would

yield ε′ = 6.99× 10−13 cm2/particle. The calculation from Mie’s theory, in which the extinction straight

section of the AuNPs was calculated and yielded ε′ = 4.18×10−12 cm2/particle (courtesy correction of Dr.

Pedro Paulo that will be used in the following calculations). Also, by assuming a uniform and standard

size spherical AuNPs, the area occupied by each particle in this surface would be 3.14 × 10−12 cm2.

Consequently, in a theoretical cuvette with optical path length of 1 cm, there would be σ = 3.18× 1011

32

particles/cm2. Then it is possible to define an absorbance of the full covered surface, given by the equation

3.2. This absorbance value corresponds theoretically to surface covered by a full monolayer of AuNPs,

aligned in the same plane of the cuvette.

A = ε′σ = 4.18× 10−12 × 3.18× 1011 ≈ 1.33 (3.2)

Since an absorvance of 0,02 is the minimum value to be detected, using the photodetectors, the number

of AuNPs needed per cm2 to be incubated is given by 3.3:

σ =A

ε′≈ 4.78× 1010particles/cm2 (3.3)

Applying this approximation to the used microfluidic channel with area Achannel= 1.8 × 10−2 cm2,

the number of particles needed to yield the minimum detectable absorbance value, is given by equation

3.4:

Nparticles = σ ×Achannel = 4.78× 1010 × 1.8× 10−2 = 8.61× 107AuNPs (3.4)

If this number of particles were by chance, planar monodispersed in one surface of this channel, for

a given flow rate and consequently, for a certain JD, what time should the AuNPs be flown to yield an

absorbance of 0.02 value? To answer this question, the time involved in each assay needs to be calculated,

in accordance with the designed flow rate. Table 3.1 clusters the speculated values of flow rates (seen

already in the Experimental Methods section) and the associated time of the experience, using equation

3.5, for Nparticles in a channel.

JD 4 t = 8.61× 107 (3.5)

Table 3.1: Experimental time calculated for each flow rate used.

Q (µL/min) JD (AuNPs/s) 4t (min)

0,05 3, 93× 103 366

0,5 8, 46× 103 170

1 1, 07× 104 135

5 1, 82× 104 79

The strength of the electrostatic interaction between the available amine groups of the APTES and

the citrate at the AuNPs’ surface was tested. Silanization of all the surfaces inside a microfluidic channel

was a crucial step for the immobilization, hence the APTES prepared solution was flowed in each new

and empty channel, for 10 minutes at 0.5 µL/min rate. These flow rate and time parameters were suited

for the initial functionalization of every channel, according to previous work at INESC-MN.

Before adressing which flow rates were used, an incubation experiment was made. After flowing

APTES, AuNPs were introduced onto the channel and remained for 75 minutes, with no flow rate

33

associated.

Figure 3.2: The incubation experiment, where the AuNPs were not continuosly flowed, but remained in

suspension inside the channel. The left image shows the channel with no AuNPs at 0 minutes and the

right image shows the channel with AuNPs inside after 75 minutes incubating.

By the result shown above, it is possible to infer that in order to immobilize AuNPs inside the mi-

crofluidic channel it is necessary to apply a flow rate, so that these particles may be diffused through

the channel and interact with surfaces. There was no change in colour, nor decrease in mean intensity,

since probably the introduced particles were inferior to the estimated JD. For that reason, these particles

would be insufficient to interact with amine groups and to form layers at the channel surfaces.

The next experiments were made by continuously flowing AuNPs within a given time window. As

seen in the Experimental Methods section, the calculation of four flow rates Q were implemented for

the immobilization of the AuNPs in a microfluidic channel. These Q values were chosen considering the

previous work at INESC-MN, by which Q values inferior to 0.05 would be considered ”too slow” flows

and higher than 5 would be ”too fast” flows, along with an increased expense of material. Therefore, the

four flow rates were tested in order to achieve an optimized immobilization process. The table 3.1 yields

the theoretical assay times involved in each Q, for a given absorbance. A Q value of 1 µL/min was the

preferred flow rate for the experiments, since it has a reasonable time window to reach the theoretical

absorbance value and the considerable volume of AuNPs spent in each assay. In figure 3.3, the used

PDMS channels for these experiments are showed. A naked-eye visible roseate colour appears in two of

the channels after flowing AuNPs. All the immobilizations are built upon a sucessful previous step of

activating the surface with APTES.

34

Figure 3.3: PDMS structure with microfluidic channels sealed on glass, the roseate channel shows the

AuNPs captured inside.

The following experiments were made to compare both flow rates of 1 and 5 µL/min (figures 3.4

and 3.5) acquired in time t, in order to see in loco which one yield the optimized immobilization. Both

flow rates allowed a fast immobilization of the AuNPs, noticing the formation of ”spots” along with an

appearance of the roseate colour in the center of the channel, which spread over the limits of the channel.

Figure 3.4: Nanoparticles immobilized in a microfluidic channel at Q = 1µL/min, during 75min. Ac-

quisitions were made at each 15 minutes and images were acquired in Olympus microscope using 20x

magnification and exposure time of 500 µs and 0× gain.

When a fluid is inside a microenvironment is subjected to significant viscous effects, causing the flow

to be reproducible, which is usually laminar and possible to be controlled. The most common way to

create these flows is to apply a gradient pressure between the entrances of a channel, where the flow

speed varies linearly with the applied pressure. In these microfluidic channels, if it is assumed that there

is no gravity influence, the viscous forces are predominant and a fluid flows in the laminar regime (low

Re). Since the velocity varies through the channel, with high speed near the center of the channel and

low speed at the boundaries, it is natural to consider that the residence time in the middle of the channel

as being minimized, whereas the resident time near the walls is high. This present pressure-driven flow,

according to Poiseuille flow, is defined by velocity varying parabolically with position, perpendicular to

the direction imposed by the flow. Hence, it also comes naturally that in throughout experiments, the

35

immobilization of AuNPs occurred primarily at the center of the channel where the resident time was

lower. As a result, the first roseate colour appeared in the center of the channel, seen in figure 3.4. This

immobilization spread to the boundaries of the channel, after flowing for a longer time, thus surpassing

the higher boundary residence time.

Figure 3.5: Nanoparticles immobilized in a microfluidic channel at Q = 5µL/min, during 75min. Ac-

quisitions were made at each 15 minutes and images were acquired in Olympus microscope using 20x

magnification and exposure time of 500 µs and 0× gain.

It is seen in both figures 3.4 and 3.5 that by flowing AuNPs through time, some features appear in the

channel (spots), which might be explained by the cluster formation of several AuNPs. It is also seen in

those figures that the immobilization, when well succeeded, provides an homogenous roseate colour in the

channel. In figure 3.6 the transmittance calculated from both experiments referred in figures 3.4 and 3.5

are plotted. These transmittances, calculated as explained in the Methods section, decrease through time

as expected. The initial value for both is 1, with no AuNPs inside the channel. Only APTES and after

45 minutes of flowing the AuNPs, a plateau formation in both flow rates is seen, suggesting a saturation

within the channel.

0 15 30 45 60 75

0,86

0,88

0,90

0,92

0,94

0,96

0,98

1,00

1 uL/min 5 uL/min

T

Time (min)

(75, 0,92)

(75, 0,89)

Figure 3.6: Transmittance values calculated through mean intensity values from ImageJ for flow rates Q

of 1 and 5 µL/min.

The two used flow rates are compared in another experiment, seen in figure 3.7. The 5µL/min

36

immobilization flow rate showed a similar behaviour to the one seen when 1µL/min was performed,

where AuNPs started to interact in the center of the channel. This fact is not seen in figure 3.5, where

at the same acquisition time (15 minutes) the channel was fully coloured. Same experiments show a

successful immobilization but differ slightly in homogeneity, possibly due to the silanization process that

took place in each experiment.

Figure 3.7: Comparison of both Q of 1 (first row) and 5 µL/min (second row).

To ensure that APTES is crucial to immobilize AuNPs on the surface of the channel, a control

experiment was made by eliminating the first condition above described. In this experiment, only the

nanoparticles were flowed for 15 minutes, not APTES. No colour on the channel, as expected, shown in

figure 3.8. The mean intensity of the channel at 0 minutes was 4253 a.u. and at 15 minutes was 4359

a.u., showing that the mean intensity did not decrease its value.

Figure 3.8: Microfluidic channel containing particles in suspension, with no surfaces functionalized with

APTES. Images acquired in Olympus microscope using 20x magnification and exposure time of 500 µs

and 0× gain.

Likewise, C.Jen et al., [48] described a silanization process using APTES in order to form self-assembly

monolayers of three different sizes of AuNPs as a pattern on a glass substrate. These AuNPs were used

to form a junction gap between microchannels in a microchip, for protein bonding tests. Moreover,

it was discovered in Zhang, F. et al., [49], un-modified AuNPs multilayer thin films could be grown

through layer-by-layer assembly, into aminosilane (poly(allylamine hydrochloride)) functionalized sub-

strates. The mechanisms of layer-by-layer construction are electrostatic interaction and coordination

chemistry between particles and aminosilane compound.

Since the negatively charged AuNPs interact electrostatically with the positive group of APTES, an

experiment with Phosphate Buffered Saline (PBS) (143 mM, pH of 7.4) was used to test once more this

interaction strength. After 75 minutes of immobilizying AuNPs, an ionic solution was flown at 5 µL/min

37

for 10 min to test if the particles would interact electrostatically with PBS over APTES. The output

showed no alteration in the colour presented initially and no alteration in mean intensity. The high

concentration of PBS, its salt effect and ionic charges did not disrupt the electron charge over the AuNPs

surface, therefore no change in color was visible, due to this ionic strength in buffer. The roseate-blue

colour shown in figure 3.9 can be explained by an higher agglomeration effect of the AuNPs on the channel

surfaces. As agglomerates’ size increase, the wavelength of LSPR absorption shifts to longer wavelengths,

where red ligh is absorbed and blue-purple light is reflected, hence the dark colour in the channel.

Figure 3.9: On the left side, a channel with immobilized AuNPs using 1 µL/min after 75 minutes is

shown, in which the given mean intensity is 2252 a.u. . In contrast, the right image shows the same

channel after washing with PBS and using Q = 5µL/min for 10 minutes, with mean intensy of 2270 a.u.

. Images acquired in Olympus microscope with 20x magnification and exposure time of 500 µs and 0×

gain.

The chosen time-window of 75 minutes was chosen after calculating the times (seen in table 3.1) as

a reasonable time window for the experiments, and also since the experiments revealed that at the end

of each experiment, the channel had full immobilization of AuNPs using this time-window. The tests

with Q=0.05 and Q=0.5 µL/min were rejected, since the high time-window for each experiment was not

reproducible. The Q = 0.05µL/min yielded no aparent immobilization and the Q = 0.5µL/min showed

a poor immobilization, demonstrated in figure 3.10, while using Q=5 µL/min showed a channel with

notably AuNPs immobilization, as expected.

Figure 3.10: Comparison among three different microfluidic channels with AuNPs flown at each designed

flow rate for 75 min. Images acquired in Leica microscope using 20x magnification and exposure time of

1 ms and 1× gain and were modified for 0.2% of enhanced contrast in ImageJ.

Here, some of the experiments individually performed in channels are presented, with chosen Q=1

µL/min during 75 min. Although the theoretical predictions of calculated assay time, aimed for these

flow rates, showed higher values than the ones seen experimentally, the transmittance value related to

an 0.02 absorbance is 0.98, which is accomplished within the first 10 minutes of assay. This fact implies

38

that the theoretical calculations made are far from the experimented results. Figure 3.11 shows five

experiments of flowing AuNPs demonstrating the decrease of transmittance over time, reaching a usual

20% of initial value and, in a specific case, decreases up to 50% of initial value. These repetitions are

calculated regarding the control experiment, where only APTES was flown in a single channel.

Figure 3.11: Comparison of calculated transmittance of different microfluidic channels with AuNPs flown

at 1 µL/min flow rate for 75min. Standard deviation calculated of the represented assays for each time.

3.1.1 A PDMS/PDMS substrate experiment

All the above cited experiments were on PDMS channels sealed on glass. As a proof of concept, AuNPs

were immobilized in PDMS channels sealed with a 500 µm sheet of PDMS interface. This time a channel

with 5000 µm length and same height and width was used. In the figure 3.12, the outcome of the

immobilization is seen, using flow rate of 1µL/min. This experiment was compared with ones performed

in the previous conditions, shown in figure 3.13, where the Control assay was done in a channel, flowing

APTES solution for 75 minutes.

Figure 3.12: Channel with AuNPs after 75 minutes of immobilization on PDMS surfaces. Images acquired

in Olympus microscope using 20x magnification and exposure time of 500 µs and 0× gain.

39

Figure 3.13: Three different PDMS channels: The transmittance values of Control (black) on PDMS/glass

channel, immobilization on PDMS/glass, Rep4(red) and immobilization on PDMS/PDMS (blue).

The immobilization of AuNPs channel sealed on PDMS showed a decrease of 33% in transmittance

at the end of 75 minutes flowing, whereas the Rep4 (red curve) seen previously in graphic of figure 3.11,

showed a decrease of 22% of initial value. Figure 3.14 illustrates the experiment Rep4 obtained from this

channel.

Figure 3.14: Full channel with AuNPs after 75 minutes of immobilization. The inlet region (left) bears

several blue spots in constrast with outlet (right). Images acquired in Olympus microscope using 10x

magnification and exposure time of 500 µs and 0× gain.

3.2 Scanning Electron Microscopy as a tool for insight

The Brownian movement inside the channel of AuNPs and its arrangement needed to be clarified.

Scanning Electron Microscopy (SEM) allowed an observation about how these particles were distributed

on the surface, since the microfluidic channel had visible roseate colour at the end of each experiment.

To perform SEM in RAITH 150 and unveil any pattern of immobilization inside the channel, a de-

sealing process was needed for the glass to be exposed to electron gun. If the PDMS remained, it would

not be possible to use this technique, due to the high noise associated with electron scattering at the

PDMS surface. For this reason, the surface modification accomplished in UV-O was a reduced exhaust

time, only 3 min against the usual 5 min. With less exposure time it was possible to seal less efficiently

40

the PDMS onto the glass, so that it could be pealed off afterwards. In fact, the assay took place only for

32 min, since a leakage ocurred and so the assay was stopped, shown in figure 3.15. Consequently, the

de-sealing of the PDMS was performed manually, which was a substantially abrupt and violent process.

It was a critical step, since several trials to de-seal resulted in a loss of the whole channel structure, where

PDMS remained fixed to the glass substrate. After this successfull de-sealing process, seen in figure 3.16,

was then subjected for study.

Figure 3.15: The used channel with

AuNPs immobilized at 32 min. Images ac-

quired in a Olympus microscope using 20x

magnification and exposure time of 500 µs

and 0× gain.

Figure 3.16: The glass substrate without

the PDMS channels on top, where it is

possible to seen a thin roseate line rounded

by red marker, indicating the remained

AuNPs.

The above shown glass substrate was used as a sample for the SEM. Since the resolution limit of

RAITH 150 was 20 nm, which is as the same order of magnitude as the used AuNPs, it was necessary

to deposit a 30 A layer of Tantalum on this sample. This deposition was crucial in order to increase the

secondary electrons conductivity, consequently enhancing contrast on image acquisiton. The following

figures show the E-beam acquisitions of the remained AuNPs channel. The figure 3.17 shows the channel

center area chosen for acquisitions, where the colour seen in figutr 3.16 was more intense. Through the

increase in conductivity of Tantalum secondary electrons, it was possible to visualise the conformation

of the AuNPs that remained after the de-sealing process. Although, this technique yielded information,

which was unknown so far, the manually de-sealing was abrupt and caused damage to the actual channel

structure. As seen in figure 3.17, four interest areas were selected to acquire with increased resolution.

These areas were deliberately chosen across the channel, since the immobilization occurs from the center

of the channel to the edges.

41

Figure 3.17: SEM image acquisition of the center zone of the remained channel, with four interest areas.

The green lines identify the channel boundaries selected in the SEM software, where it was acquired with

a 20 µm resolution, 240× magnification and EHT=10 kV.

In figure 3.17 it is possible to observe different densities in center of the channel, as well as on the

edges, seen as an accumulation of small bright areas. When focusing on area 1, the obtained acquisition

in figure 3.18 showed two regions, one corresponds to the channel boundary in which the aggregates of

AuNPs are easily spotted. On the other region, where the AuNPs were covered, it is possible to infer as

being the remains of un-pealed PDMS.

Figure 3.18: SEM image acquisition area 1. The image was acquired with magnification of 36.27 kX,

EHT=10 kV and analyzed in ImageJ with 0.2% enhanced contrast and FFT.

The area 2 is at the edge of the channel’s center region, where the acquisition in figure 3.19 showed

similar features similar features to the ones seen in figure 3.18. Both acquisitions display AuNPs constructs

that are apparently random, with no geometric arrangement. Instead, large clusters of AuNPs were

formed, specifically under PDMS residues.

42

Figure 3.19: SEM image acquisition area 2. The image was acquired with magnification of 30.00 kX,

EHT=10 kV and analyzed in ImageJ with 0.2% enhanced contrast and FFT.

The center of the channel was the most interesting region to investigate, since a higher number of

AuNPs was expected to be present. The figure below 3.20 corresponds to area 3, in which it was possible

to concieve several cluster aggregates of AuNPs, with notably less spacing between each other. This

suggests a higher concentration of these AuNPs in this area, compared with the boundary area.

Figure 3.20: SEM image acquisition area 3. The image was acquired with magnification of 146.00 kX,

EHT=10 kV and analyzed in ImageJ with 0.2% enhanced contrast and FFT.

Regarding area 4, in figure 3.21, shows the second boundary. This acquisition confirms what has been

seen in the previous figures, that AuNPs form different sized clusters of aggregates that are randomly

dispersed through the channel. At the edges of the channel there are aggregates with inferior size (3.21 a)

), compared to the ones in the center channel. In figure 3.21 b) it is possibe to see with limited resolution

one of the aggregates spotted on a).

43

Figure 3.21: SEM image acquisition area 4. The images was acquired with magnification of a) 86.00 kX,

b) 145.00 kX EHT=10 kV and analyzed in ImageJ with 0.2% enhanced contrast and FFT.

In figure 3.22 it is possible to recognize the inlet (a) and outlet (b) areas, where in (b) can be visualized

the leakage occured during the assay. In addition, these acquisitions demonstrate the disorder caused

by the de-sealing process, which influenced the obtained images. Still, in (c) it is possible to perceive

the higher density of AuNPs, where the brighter areas form considerable aggregates. The discernible

difference between the outlet and inlet figures did not come as a surprise, since the inlet of a channel

is usually filled with increased darker areas (blue spots) than the outlet, as seen in inverted Olympus

microscope experiences. The acquisition (d) confirms once more the agglomerates that are transversal to

the whole channel.

Figure 3.22: SEM image acquisitions of a) inlet with magnification of 182 kX, scale = 20 µm; b) outlet

with magnification of 155 kX, scale = 20 µm; c) center area near inlet with magnification 93.00 kX and d)

area near outlet with magnification of 86.00 kX. All the acquisitions shown were obtained with EHT=10

kV and analyzed in ImageJ with 0.2%enhanced contrast and FFT.

44

All the shown figures were obtained from the sealed channel, seen in figure 3.15, on which the coloration

presented at 32 min was consistent with a transmittance value of 0.85 (absorbance value of 0.16). Using

previous calculations presented in this chapter, the calculated absorbance is higher than the theoretic

limit value of 0.02, on which there would be needed JD particles to attack the surface. Thus, it is

possible to conclude that the obtained absorbance value implies a number of AuNPs higher than JD

value. Moreover, it is possible to infer that the use of APTES for silanization may not be uniform across

the channel and is responsible for the two-dimensional aggregation of AuNPs.

On a related subject, Jen, C. et al., [48], used different concentrations of colloidal AuNPs in their work,

with mean diameter of 13.7 nm, to form self-assembly disposition after glass silanization. The process of

silanization was performed using APTES (0.1 v/v solution on water) for 1 min. Different concentrations

of AuNPs yielded different arrangements on silanized glass, as seen in figure 3.23, where the authors used

SEM to discover which concentration would optimize the process for protein preconcentration.

Figure 3.23: Acquisitions using SEM of different AuNPs concentrations: 1.0 nM (left) and 2.0 nM (right).

Adapted image from [48].

In the presented image above, there are no aggregates of AuNPs and defined pattern of self-assembly

are seen. However, the silanized glass substrate suffered washing processes afterwards; also, the different

concentrations each of 30 µL of AuNPs were deposited on the silaned area for 1 hr and washing processes

were applied at the end of the immobilization. Therefore, no flow rates were applied, the silanization

process diverges and washing process are introduced. For this reason, the SEM acquisitions shown above

indicate that, by applying a flow rate on the immobilized particles, it has a direct impact on AuNPs

template at the channel surfaces. This template can also be influenced by the time-window of the

channel silanization and further washing processes, since the 10 min used in this work are according to

the previous protocol identified by INESC-MN but are not specifically used for these experiments.

Other work is found in literature, Trung, N. and co-workers, [50], in which colloidal citrate stabilized

AuNPs with 100 nm average size were immobilized, in a silanized PDMS surface. It was reported that

the used concentration of APTES influenced the distribution and the density of the AuNPs at the surface

of the PDMS. They used Atomic Force Microscopy (AFM) to visualize the immobilized particles with

different APTES concentrations, as seen in figure 3.24:

45

Figure 3.24: Acquisitions using AFM of immobilized AuNPs on PDMS with two different concentrations

of APTES: 1% (left) and 15% (right). Adapted image from [50].

In Trung, N. and co-workers, the APTES solutions were prepared with ethanol instead of water, with

different concentrations, and the PDMS surface silanization process endured for 15 min, in incubation,

followed by ethanol washing and annealing for 2 h at 130 C. Moreover, the immobilization of AuNPs

was performed in different incubation times, followed by washing with DI-water and annealed at 120C

for 30 min. As said, this work brought to light the importance of APTES concentration, which was

found as a key parameter for the morphology of APTES silanization layer and consequently on the

adsorption of AuNPs. The described process was performed in order to minimize the aggregation of

AuNPs, seen in figure 3.24 (left) where a decreased size in aggregation formation is shown when using

1% solution of APTES. In contrast with the present work, the silanization process differs in solution

preparation, incubation methods and modified surfaces. The channel images obtained by SEM reveal no

layer formation, instead several different size aggregates and scattered AuNPs are observed, which may

be validated by the concentration used of APTES used and by the flowing silanization process.

The microenvironment inside a microfluidic channel is difficult to control, where many competing

occurrences take place. One of the factors that could also cause the aggregation of AuNPs as investigated

in Wager, K. and co-workers, [51], is the influence of pH on the stability of these particles. The citrate

stabilizer prevents the aggregation, due to the negative charges over the particle’s surface, guarantee-

ing the electrostatic repulsion among these. Hence, the AuNPs remained in suspension. If the pH of

surrounding environment changes, aggregation processes take place. In Wager, K., several pH values

were tested to investigated AuNPs aggregation: at low pH the citrate protonation occurs, consequently

decreasing the number of negative charges and forming aggregates. For neutral pH (above pKa acitric

acid values) no aggregation took place, since citrate groups were available to stabilish repulsion. There-

fore, the authors chose neutral pH in order to use non-aggregated form of AuNPs for biological assays.

Here, several explanations were given, in order to find possible reasons for the AuNPs aggregation and

dispersion throughout the channel, as visualized in SEM. The silanization of the channel is a key point,

since if there is no positive charges spread in the surfaces, the AuNPs will not interact and thus will

not be adsorbed onto the surfaces. For this reason, another study involving the solution preparation of

APTES is presented. According to [52], the half-life of a prepared solution of APTES on water is deeply

dependent upon temperature and pH environment, as shown in figure 3.25.

46

Figure 3.25: The Half-life variation of APTES on water solution, depending on pH and temperature. [52]

The half-life characterizes the hydrolyzing process of the ethoxy groups compared to the Si-C bond.

Only the first are hydrolized yielding ethanol and trisilanols, while the bond with the amine groups

are regenerated. Since the used AuNPs had a ca. pH of 8.0 and the acquisitions were made at room

temperature ca. 25 C , the environment inside the channel when flowing particles is changed. The pH

inside the channel is not favorable to APTES’ stability, assuming a half-life between 0.15 (9 min) and 8.4

h (504 min), since the regeneration of aminopropyl-functional resins produce local electrostatic conditions

for the AuNPs. This may ultimately increase the number of aggregates present randomly in the channel.

3.3 Localized Surface Plasmon Resonance Detection

3.3.1 LSPR detection in microfluidics using photodiodes

Photodiodes were the first device used on which the detection of the plasmon peak was made. As

referred, the aim was to obtain the same peak by acquiring the photocurrents vs wavelength after the

immobilization occurred. In addition, it was possible to monitor transmittance’ behaviour, concerning

different time-windows of flowing AuNPs. The data obtained by the spectrophotometer states that

a plasmon peak is visible at 520 nm wavelength, which corresponds to an absorbance value of 0.29.

Hence, future experiments (absorbance vs time) had been done at same wavelength, using transmittance

and absorbance calculations to evaluate the success of the each assay. These calculations are made

possible due to the External Quantum Efficiency (EQE) of these devices, estimated at ca. 1,which

allowed the number of electron-hole pairs detected to be considered as an intensity. The sensibility of the

photodiode was first tested in the acquisition of the plasmon peak and compared to the data yielded by

the spectrophotometer. The goal was to recreate some the conditions performed in a spectrophotometer,

therefore the photocurrents vs wavelengths of an empty channel were acquired, as reference. Then, AuNPs

were flown for 75min at 1 µL/min in a PDMS microfluidic channel. The absorbances were calculated

using the empty channel photocurrents (I0) and the photocurrents from the AuNPs (Isample). Therefore,

to illustrate this result, figure 3.26 shows the detection of AuNPs’ plasmonic peak. These calculated

absorbance values were not corrected with the black ink, as referred in the Experiment Methods section

and were obtained with full intensity of the light beam.

47

Figure 3.26: Comparison of absorbance values between the peak obtained by spectrophotometer (black

curve) of colloidal AuNPs and the peak obtained by photodiode (orange curve), immobilized AuNPs

within the microfluidic channel.

The yield absorbance value from photodiode measurement was ≈ 0.28, near the obtained 0.29 from

the spectrophotometer for λplasmon = 520 nm. The obtained curves in both devices are in accordance

for colloidal AuNPs with 20 nm size, as seen elsewhere in literature for Ultraviolet-visible (UV-Vis)

Spectroscopy acquisitions. [53] [54] [55]

Throughout experiments, several issues arose, regarding the amount of light shone in to the pho-

todiode. In the optics apparatus given in introduction, wheels with integrated filters were part of the

optic system. These wheels carried different neutral density filters, ND, which reduced the number of

photons reaching the sensor by the rule 1 × 10x, being x the order of the filter used. For example, by

applying a ND 2 filter, the number of photons reaching the detector would be decreased by 100×. The

ND filters were tested directly with the potodiodes’ dye, by applying each filter in light crescent order

ND 5, ND 4, ND 3, ND 2 and ND 1. Due to the performed tests it was possible to define which filter

should be appropriate to photodiodes tests, without increasing the dark photocurrent of the device. The

dark photocurrent measurement after each acquisition was a key point to define the device status, hence

a good operation value for the dark photocurrent was from 10−13 to 10−14 A (the picoammeter detection

limit), as demonstrated previously in figure 2.11 (Experimental Methods section). Also, in the figure A.1

in Appendix, it is shown the photodiode figure of merit, where all photocurrent vs time acquisitions were

made using a 520 nm wavelength of interest for the different ND filters referred. A trade-off between the

ND filter, with associated dark photocurrent and the signal acquisition from the immobilization of the

AuNPs was considered. The filter should not decrease the beam light to the point where the assay yield

signal was not acquired. Thence, the ND 3 filter was chosen for process optimization and applied to the

following experiments.

Another important test using this filter was made in order to evaluate the amount of light still reach-

48

ing the photodiode, when a channel was filled with black ink. The figure A.2 in Appendix shows the

acquisition of photocurrent in function of time, measured at 520 nm. A black ink channel yield an output

photocurrent of order ≈ 10−11A, being two orders of magnitude higher compared to the dark photocur-

rent measured. With this experiment an expected result would be an output photocurrent of ≈ 10−13A,

since the channel was filled with black ink and subjected to an incident light beam with 1000x less inten-

sity. The possible contributions to higher photocurrent values acquired may be sourced on the scattered

light occured in the PDMS structure and black ink interaction with channel surfaces. Furthermore, the

black ink channel acquisitions were irregular, these values suggest that the black ink was not completely

opaque when a small volume was introduced in the channel, causing the incident and scattered light to

reach the detector.

In each experiment accomplished, the essential steps in protocol were followed:

• Dark Photocurrent vs Bias Voltage (V);

• Dark Photocurrent vs time;

• ND 3 photocurrent acquisition and beam alignment;

• Photocurrents (in function of time and spectrum) acquisition of an empty PDMS channel;

• Photocurrents acquisition while (time) and after (spectrum) flowing APTES;

• Photocurrents acquisition while (time) and after (spectrum) flowing AuNPs;

• Photocurrents acquisition of channel filled with black ink;

• Dark Photocurrent vs time;

These steps were followed to observe if the spectrum of an empty channel and of APTES filled channel

were almost coincident, since the air’s index of refraction is ≈ 1.00 and APTES is 1.43 (at 20 C). [56]

The next immobilization of AuNPs for 30 min was performed and its spectrum was acquired. As expected

the spectrum shows a decrease in photocurrent when compared to the APTES, however this decrease is

not uniform, by which for smaller wavelengths from 450 nm and higher ones from 600 nm, the spectra

are coincident, as shown in figure A.3 in Appendix. Through spectra acquisitions it was possible to

calculate the absorbance values, using photocurrent acquisitions of the flown APTES as reference. The

calculations were corrected with photocurrent values from black ink acquisition and plotted in figure 3.27.

As seen in the figure below, it is not similar to the initial absorbances calculated reported by spectroscopy

value in figure 3.26. This experiment was however subjected to a different approach: a ND 3 filter was

used, AuNPs were flown at 1 µL/min for only 30 min and the calculated values were corrected with

a black ink channel. There is no evident plasmonic peak in this figure, only a higher value is spotted

at 555 nm. Although the raw data seen in figure A.3 in Appendix, shows differences in photocurrent

intensities from APTES to AuNPs, whereas a theoretical absorbance of 0.12 is expected, the analysis of

data yields a value of 0.21. This can be explained by the correction with black ink, that is subtracted to

both acquisitions I0 and Isample so, when transmittance is calculated by the ratio of the two, it yields a

49

lower value than the ones obtained without the ink correction. Hence, the absorbance value of a lower

transmittance results in a higher value. To illustrate this explanation, the transmittance calculated with

ink correction for the plasmon wavelength is considered, estimated at Tcorrected=0.81, whereas for the

non-corrected transmittance, Tnotcorrected= 0.87. The respective absorbances are approximately 0.21 and

0.14 .

Figure 3.27: A 30 min immobilization assay, with the respective absorbances calculated and corrected

with black ink at the end of the experiment.

In order to enhance signal acquisition and avoid black ink described problems, two strategies were

performed: flowing the black ink into the channel at 0.3 µL/min and to fabricate a glass Al barrier, with

a row of squared holes with area smaller than the photodiode ones. The figure 3.28, represented below,

demonstrates the prepared setup for acqusition. The Al was manually aligned and the confirmation

for the alignment was given by aligning the light beam onto the Al hole, acquiring its photocurrent

with picoammeter. Two manual alignments were involved: the alignment of the barrier on top of the

photodiode dye and the alignment of the channel on top of the barrier. This setup was used in the

following experiments.

Figure 3.28: PDMS channels on top of a photodiode dye, with Al barrier beneath.

Several experiments made using this setup involved different time-windows, 30 min, 45 min and 50 min

of AuNPs, using ND3 as well. All these experiments were not successful probably due to the misaligments

between the Al barrier and the photodiode. One of the was performed in order to investigate if by stopping

50

and pausing the flow, then flowing the AuNPs again, would be any aggregation occurring meanwhile that

could be detected. For that reason, an assay was performed in which the AuNPs were flown for 20 min,

stopped for 15 min and flown again for more 30 min. The total time of the assay was about 65 min and

was performed using ND 3 filter, the Al barrier and absorbance calculations used the black ink correction.

In figures 3.29 and 3.30 it is possible to identify clearly the LSPR of the AuNPs in different times of the

experiment. The absorbances showed are calculated accordingly to the photocurrent values displayed in

figures A.6 and A.7 in the Appendix section.

Figure 3.29: Spectrum of plasmonic peak after 20 min of immobilizing AuNPs.

Figure 3.30: Spectrum of plasmonic peak after 30 min of immobilizing AuNPs.

In figure 3.29 the LSPR peak is at 525 nm, instead of the expected 520 nm, with an absorbance value

of ≈ 0.64 . The small shift in wavelength value obtained can be explained by a possible local change in

medium originated by a local volume of APTES. After stopping for 15 min and flowing 30 min of AuNPs

this possible local volume dispersed, since the peak was now registered at 520 nm. Higher absorbance

51

values calculated in this experiment can be explained by the layers’ formation inside the channel, which

increased the interparticle interactions of AuNPs, thus increasing the absorbance registered at LSPR

peak. In addition, during this assay the photocurrent vs time was acquired at plasmon wavelength

and the related transmittance values were calculated. Instead of using the APTES spectrum raw data

as reference for each wavelength, the average photocurrent of the 10 min flowing APTES was used.

The average value was then used as reference to calculate transmittance values for each time of the 20

and 30 min of flowing AuNPs. Figures 3.31 and 3.32 display the behaviour through time of AuNPs

immobilization process. Furthermore, in figure A.7 in the Appendix section is given the comparison

between photocurrents in each step of the experiment.

Figure 3.31: Transmittance over time during 20 min of immobilizing AuNPs.

Figure 3.32: Transmittance over time during 30 min of immobilizing AuNPs.

At 520 nm, the respective absorbance values at the end of the 20 and 30 min flowing are 0.60 and

0.81, respectively. Furthermore, the 20 min curve demonstrates that from 500 s to 800 s ( 8 min to 13

52

min) there is a ≈ 15 % decrease in transmittance from initial value of measured with APTES solution

at the beginning of the experiment. This similar decreasing behaviour is in accordance with what was

already seen in Figure 3.1, in Olympus Microscope assays. This transmittance curve differs from the

results showed in Figure 3.1 at the beginning of the assay, since the transmittance value at 0 s is not

1. Moreover, the final transmittance value of 20 min assay is 0.54, which is the same transmittance

value that the 30 min starts with, suggesting that the 15 min stop between flowing did not affect the

environment inside the channel. At the beginning of the assay in figure 3.31, the transmittance suffered

an abrupt variation in the 100 s of flowing, possibly due to the presence of local air bubbles that remained

in the sensor area of detection, being removed by the movement of the liquid itself.

Other experiments were made in order to reproduce this results in a 75 min time-window assay, al-

though they were not successful. One of the experiments made was the PBS washing experiment using the

referred conditions, the photocurrent values obtained for each wavelength of the immobilization, followed

by washing were coincident, implying the strength of the electrostatic interaction of AuNPs and APTES.

However, the absorbance value of the LSPR was not possible to identify. In the experiments performed,

the setup used with the Al proven to be as much challenging as crucial to the success of the assay. The

two manual alignments made are potentially the source of error introduced in the acquisition, a part from

other non-controlled factors such as the surface chemistry performed on the channel surfaces. The suc-

cessful acquisition represented in figure 3.26 with no light barrier, no ND filter and using photocurrents

from the initial empty channel as reference was the landmark for comparison. These conditions were

not repeated since the device would increase its dark current, affecting the following acquisitions, and

also the scattered light would contribute to a higher signal output. Hence, the further conditions used,

such as ND 3 filter, the ink correction and the barrier to protect from scattered light are justified, but

demanding to be reproducible. Whilst the experiments made, the system showed great sensitivity, since

the obtained spectra did not present a smooth curve, instead a ripple behaviour, showing that there is

still noise introduced in the acquisition.

3.3.2 LSPR detection in microfluidics using photoconductors

The photoconductor experiments were engaged to investigate if the results described in the previous

section would still be confirmed using these devices instead. The protocol of the immobilization followed

the previously described steps: 10 min of surface silanization (APTES) and flowing the AuNPs at 1

µL/min. Due to the operating nature of these devices, an external voltage was applied in the ranges of [10-

30] V. The ensuing results build a path on the optimization of LSPR acquisition using photoconductors.In

order to obtain a higher order of magnitude signal from the experiment, in comparison with the dark and

black ink photocurrents seen in Appendix figures A.8, one of the first experiments using these devices

was to perform a 30 min immobilization of AuNPs, using the Al barrier aligned to the photoconductor

dye as well. This experiment aimed to compare with described results using photodiodes, but also to

inquire if the tradeoff between acquiring higher output signal and the scattered light interference was also

significant. Therefore, a full light beam ( ND 0 filter) and black ink correction data were used at the end

53

of the experiment. Depicted in figure 3.33 is the absorbance spectrum of the AuNPs immobilized after 30

min, where it is possible to identify the plasmonic peak at 535 nm with a ≈ 0.20 value of absorbance. The

photocurrent spectra are dispayed in figure A.11 in Appendix, used for the absorbance calculation, and

characterizing the typical photoconductive response of this device to an incident light beam. The shift

from 520 nm to 535 nm may suggest that several layers of AuNPs were built due to a possible presence

of APTES local volume, upon the photoconductor acquisition area.

Figure 3.33: Calculated absorbance spectrum over 30 min immobilization of AuNPs in photoconductor,

operating on applied 10 V.

The calculated absorbance values are lower compared to those seen in spectrum acquired by spec-

trophotometer (figure 3.26), specifically those in the lower range wavelengths, due to the coincident

photocurrents acquisition in the 400 nm range, displayed also in figure A.11 in Appendix. Additionally,

similar results are found in 30 min immobilization experiment using photodiodes, in figure 3.27, where

the peak was possibly identified at 555 nm. Both present lower values for low wavelengths and the LSPR

appears to be shifted to higher ones as well. Nevertheless, these two experiments fall appart, not on the

immobilization protocol, but on the acquisition device, where the only possible comparison between these

is the AuNPs immobilization steps and the respective LSPR acquisition protocol.

The photocurrent values over the assay time were obtained, as display in figure A.12 in Appendix section,

thus allowing the respective transmittance values calculation, as illustrated in figure 3.34. The initial

transmittance at the beginning of the experiment should start at 1, being the APTES the reference,

having a 100% transmittance. The presented curve shows a few discrepancies with prior results regarding

the initial value and the unexpected variance after 200 s of flowing AuNPs solution. The latter possibly is

due to the local change in refractive index of an air bubble introduced in the channel that may caused the

increase in transmittance. As seen in Figure 3.14, where it is shown a image of a whole channel, the area

near the inlet accumulated a higher density of aggregates compared to the outlet. Thence, it is possible

that when the tube with AuNPs was introduced in the channel, some particles may already interacted

with APTES. This particular incident is corroborated due to the use of the first photoconductor in the

54

used dye, which was aligned closely to the inlet of the channel.

Figure 3.34: Transmittance calculated values of 30 min immobilization acquired in photoconductor,

operating on applied 10 V.

Still in the same experiment, another interaction was put to the test. After flowing the AuNPs

immobilization, Bovine Serum Albumine (BSA) 4% solution was flown for 10 min at 0.5 µL/min flow

rate, according to previous work at INESC-MN. The yield absorbance spectrum is shown in figure 3.35,

where it is easily identified the plasmonic peak at 525 nm wavelength with an absorbance value of ≈

0.56. The curve obtained is analogous to the one obtained in figure 3.26 using photodiodes, although

the presented values are higher for the interaction of BSA and the AuNPs. These higher values can be

originated by the increased hindrance to light passage which originated low photocurrents acquisition,

since the protein interaction and binding to the AuNPs occurred throughout the channel surfaces. Thus,

when the photocurrent was acquired it presents a lower value than the initial yielded from the AuNPs

immobilization. Apparently, the adsorption of BSA on the AuNPs did not introduce a variation in the

refraction index, by which a shift to higher wavelengths would have been seen.

55

Figure 3.35: Comparison of absorbance spectrum of BSA spectrum after immobilization of AuNPs in a

channel, operating on applied 10 V.

The use of full light beam incident on photoconductor in the experiments performed had an increased

dark photocurrent outcome. The long exposures to light in each step acquisition protocol causes the

increase in the dark photocurrent of the photoconductor throughout the experiment. This fact gains

greater importance, since the output photocurrents of the assay are affected with this factor. Hence, the

following experiments were once more executed using ND 3 filter to avoid this occurrence.

The upcoming experiment was accomplished using the above described protocol for 75 min, ND 3

filter and also the Al barrier aligned below the PDMS channels. For this experiment, an external voltage

of 30 V to the photoconductors dye was applied. Figure 3.36 depicts the LSPR peak obtained in this

assay, where an absorbance value for the plasmonic wavelength calculated was ≈ 0.19 at 532 nm.

Figure 3.36: Spectrum of absorbances acquired after a 75 min immobilization assay. The calculated

values were corrected with black ink filled channel used as baseline.

The same effect was seen in figure 3.27, although a smaller shift in wavelength is noticed. The obtained

56

spectrum of absorbances presents a similar curve to the one obtained in UV-Vis Spectroscopy, but the

absorbance values differ greatly, for lower and higher wavelengths. In this scenario, the negative values

take place since there is an overlap and opposite behaviour of the APTES and AuNPs spectra for higher

wavelengths than 600 nm, depicted in figure A.13 in Appendix section. Although the PDMS channel

presents the usual roseate colour, visible at naked-eye, the spectrum obtained does not reassures the

success of the experiment. The absorbance values calculated were always based on the APTES and black

ink spectrums acquisition also shown in figure A.13, analogous of the calculations met in photodiodes

acquisition. Here, it is demonstrated that using these devices, issues regarding the spectrum acquisition

for lower and higher wavelengths persist, which ultimately influences the calculation of the absorbance

spectrum. Since the protocol for immobilization was not changed, and the acquisition steps were thor-

oughly performed similarly using both photodetectors with due differences, this may be dependent on:

the photoconductor acquisition or due to the local area occurrences within the channel, as explicited in

the previous section.

While immobilization of the AuNPs was executed, the photocurrent in function of time was acquired at

the same wavelength, 520 nm, as explicited in figure A.14 in the Appendix section, used for the trans-

mittance calculations. The final calculated transmittance value for 75 min flowing, showed in figure 3.37

is ≈ 0.75, which relates to an absorbance value of ≈ 0.29.

Figure 3.37: Evolution of calculated transmittance over 75 min immobilization of AuNPs in photocon-

ductor.

These values suggest that the LSPR peak intensity would be at 520 nm, since it reveals a higher

absorbance value than the one obtained by the spectra. This fact also may indicate that the acquisition of

each spectrum was not successful as hypothesized previously. Moreover, the transmittance curve presents

a wave-like shape, where shoulders are in evidence. In particular, this behaviour was present in some of

the experiments, possibly due to the eccentric local flow in the sensing area over the photoconductor.

There were several challenges faced to align the Al barrier, so that the performed experiments could

be reproducible. In order to simplify the alignment between the photoconductor and the barrier and to

57

improve quality of the output data, a new barrier was fabricated. This TiW enclosed a new deposition

patterning at one edge to aid the process of alignment, where a stripe corresponding to the photoconductor

layout would match (if well aligned). The aligments performed using this barrier were undifferentiated

from the ones using Al. A difference is pointed out on the chosen material for the new barrier, since the

present alloy was accomplished to be more dense and opaque than the Al firstly used. In figure 3.38 it is

shown the whole setup with the two alignments were accomplished and held still with scotch tape.

Figure 3.38: PDMS channel aligned with new barrier, TiW, on top of photoconductor dye.

Several 75 min AuNPs immobilization experiments were made using the new fabricated barrier, with

an applied external voltage of 30 V and ND 3 filter. For lower range wavelengths (400 nm) and higher

ones (600 nm) the photocurrent measurements for each immobilization step overlapped. This fact can

be related to the photoconductor EQE, illustrated in figure 3.39:

Figure 3.39: EQE for a-Si:H photoconductor for electrode distance of 10 µm and its dependency on bias

voltage. [57]

For a bias voltage of 30 V, the EQE decrease for higher wavelengths. Even for ≈ 450 nm the EQE

is around 17%, whereas for 600 nm the EQE is 4%. In addition, in figure 3.40 it is explicited the

amount of output photocurrent of the device per incoming photon of a given wavelength (and energy):

photoconductor responsitivity. The decreasing in responsivity for higher bias voltage is dependent on

electrode distance (Lg). Despite the device used in this work possesses an electrode distance of ≈ 200

µm, a higher value in comparison with those studied in this figure, it is still possible to address the

importance of these a-Si:H photoconductor characteristics. Henceforth, the results also suggest that

possibly the quantum efficiency of the device is not optimized for the applied bias voltages and for these

58

wavelengths.

Figure 3.40: Responsivity of a-Si:H photoconductor and dependency on electrode spacing. [57]

None of the performed experiments was successful with the new barrier, as the acquired photocurrent

showed a high degree of variation in each acquisition value, seen in figure A.15 in the Appendix section,

ultimately resulting in no LSPR peak detection. The photocurrent values obtained recursively revealed a

scattered acquisition spectra, however in some assays there was no overlap of the spectrum acquisitions,

and only the scattered values were acquired with no typical shape of the photoconductive response. This

scattered acquisition may the result of an increase in defect density due to the increase of light exposure.

This would reduce the efficiency in the conversion of incident light into photocurrent.

59

60

4Conclusions and Future Challenges

Contents

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

4.1 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

61

The latest discoveries on physical properties of AuNPs have revolutionized LoC systems in microflu-

idic devices, for biomarkers detection. The use of PoC systems is becoming a contemporary necessity,

since an accurate and early diagnosis dictates the probability for a successful therapeutic. The use of

AuNPs extends the possibilities not only for theragnostics (diagnostics and therapeutics), on which its

thermal properties are used for localized cell treatments, but also for imagiologic and detection purposes.

Therefore, AuNPs have been showing promising applications for PoC devices, for its simplicity, sensitivity

and speed. They also have endured optimization processes for biormarker detection limit on blood and

serum samples, for early disease diagnosis. [58] [59] So far the optical characteristics and morphology of

these particles have built the foundations for different signaling systems, from chemiluminescence and

fluorescence to colorimetry.

The focus of this work was on colorimetric detection of LSPR in a PDMS microfluidic chip. For that

purpose, the immobilization process started with surface activation on UV-O and Plasma Cleaner for

sealing purposes. Plasma Cleaner proven to activate more efficiently and homogenously the PDMS and

glass surfaces, avoiding the encountered initial leakage problems. After the sealing process, the surface

functionalization was performed using APTES, in order to silanize with positive charges (amine groups)

the channel surfaces. The adsorption of citrate stabilized AuNPs to APTES modified channel surfaces,

by electrostatic interaction, have been shown to be stable and longing. Washing experiments with PBS at

a high concentration revealed the strength of amine groups binding with citrate on the particles’ surface.

The LSPR wavelength confirmation for the used AuNPs was given by UV-Vis spectroscopy, which

allowed a theoretical approach on the flow rate used and the time-window of each experiment. Experi-

mentally, the insertion of AuNPs at 1 µL/min in the silanized channel yielded immobilization of these

particles after approximately 10 min, visible at naked eye. The theoretical calculated time-window of

135 min needed to acquire an absorbance value of 0.02 as the minimum detectable absorbance value

was then surpassed. This may be explained since we have hypothesized one monolayer of AuNPs on a

surface, which is very irrealistic and unlikely, but for the calculations had to be considered for simplic-

ity. However, these studied parameters proven to be successful in order to reach the absorbance value

predicted previously by the spectrophotomete, using the same flow rate and lower time-windows of 20,

30 and 75 min were as successful as the initial admitted. The image acquisition on microscope showed

that the immobilization of AuNPs started from the center to the edges of the channel, indicating that

the Brownian motion affects the dispersion stability of this colloidal solution. Due to this motion, the

sedimentation of AuNPs was caused not by gravimetric forces, but due to colloidal aggregation originated

by interparticle collisions. By analyzing the theoretical parameters for mass transport in laminar flow

regime, the high values for PeS and PeH implied the depletion zone for the electrostatic interaction was

much thinner than the channel and much thinner that the sensing area (Wc channel surface), respectively.

The particles were flown through the channel without diffusion very far, being collected electrostically

on the depletion zone of the sensing area. This theoretical approach was based on having one surface of

the channel (Wc) as sensing region, but reasonably, all surfaces formed a depletion zone. These Pe values

characterized the minimum JD number of particles reaching a channel surface for a given assay time and

since shorter time-windows were needed, indicating a higher value for JD in the performed experiments.

62

The environment inside the channel was later visualized using SEM, through the abrupt de-sealing

of PDMS from the glass surface. This allowed an analysis to the recursive appearance of blue-coloured

”spots” while flowing AuNPs. These ”blue spots” were conceived to be substantial aggregates of AuNPs,

different sized and randomly distributed in the channel, in which by further analysis it was positively

confirmed. With the use of this technique, it was possible to obtain 20 nm resolution images of the glass

surface areas, with prior Tantalum deposition. Although the usual methods seen for the visualization

of AuNPs morphology and organization are AFM and Transmission Electron Microscopy, the performed

SEM visualization of the immobilized AuNPs was still successful. Some questions arose regarding the

de-sealing process affecting the initial environment, by which conclusions taken from the glass surface

visualization would be compromised. However, as referred formerly, several studies confirm particle

aggregation of AuNPs in many assay conditions.

The LSPR detection in a microfluidic channel was performed using 200 µm sq. photodiodes and

photoconductors, aligned below of the microfluidic chip. The experiments were subjected to several mod-

ifications, since many issues regarding scattered light as source of noise emerged in current acquisitions.

Both setups used were coupled with two fabricated light barriers, in order to decrease the incoming

scattered light. The inherent problems regarding the barriers were mainly about manual misalignments

between the barriers and the devices, also between the barriers and the PDMS channels. In addition,

the possible misaligments of the light beam and the whole setup were also considered. Throughout the

assays, the acqusition system proven to be very sensitive, embedding noise not only from light, but also

from external sources, such as the introduction of liquids in the channel by the syringe pump and from

the acquisition room itself. Furthermore, a black ink was used, as a measure of the amount of light still

reaching the photodetector, to fill the channel at assay terminus so that the output data could be cor-

rected afterwards. The black ink purchased revealed high hydrophobic behaviour towards the channels’

surfaces, causing a non-homogenous spreading in the channel. These factors arise as the black ink was

chosen for data baseline correction throughout experiments, since it revealed some problems regarding

acquisition reproducibility, which was far from being accomplished. Therefore, the spectra obtained in

each experiment diverged in shape and in value. The figure A.4 in Appendix demonstrates the compar-

ison between the spectrum acquisition made previously seen in figure A.3 and another empty channel

filled only with black ink. Moreover, it is shown that while acquiring the correspondent photocurrent of

a black ink-filled channel I(t), the ink was removed partially in the channel to the outlet, due to own

hydrophobicity towards the PDMS and glass surfaces, as seen in figure A.5 also in Appendix section.

Since the ink was not flown and only initially introduced, the removal occurred repeatedly along the

experiments, implying that the ink itself appeared to build no adhesion to channel surfaces. Moreover,

by observing figure A.4 and its values, it proposes the possibility that the interaction between the ink

and the AuNPs differs from the ink filled channel, due to the fact that the channel filled with AuNPs

has less available ”free path” than the latter. Then, when the channel with particles is filled with ink

local irregularites are created that allow the light to enter and scatter inside the channel, thus increasing

photocurrent acquisition. This fact was visible in considerable experiments, where the spectrum and

current acquisitions were skewed, ultimately causing high variations in data analysis.

63

The AuNPs spectrum of absorbances acquired in spectrophotometer and photodiode, seen in figure

3.26, represent the acquisition capability of this photodetector, on which no ND filter and light barrier were

used. The success of the following experiments was compared with this result and evaluated accordingly.

In photodiodes, the plasmonic peak was successfully acquired by immobilizing the AuNPs in the 75 min

(figure 3.26), 20 min and 30 min assays (figures 3.29 and 3.30, respectively), with conclusive absorbances

calculated. In many experiments performed, the output spectra were analogous to the obtained figure

3.27, where the peak was poorly evident and the spectrum boundaries do not follow the observed trend

seen in figure 3.26 . So far, in order to obtain a successful absorbance spectra spectra, a full-wavelength

decrease is needed in photocurrent acquisition, compared with the photocurrent acquisition from the

APTES measurement, as e.g. see Appendix figures A.6 and A.7. In some experiments the overlap of

both photocurrent spectra (APTES and AuNPs) can be explained with the results obtained in SEM

acquisitions. There were different sized and randomly spaced aggregates of AuNPs, regardless the de-

sealing experiment being abrupt and possibly introduced a chaotic environment. Let it be assumed that

inside the channel, prior to the de-sealing, there were aggregates with different size formed dispersed

through the surfaces inside the channel. This random and spaced aggregates can coincide with the

location of the photodiode area, where locally the volume itself is not filled with JD number of particles.

This fact suggests that when the spectrum acquisition begins for higher and finishes for lower wavelengths,

the resident AuNPs and the remained APTES interactions contribute to the overlap of the two spectra.

Although, for the wavelength range of biological interest (500-540) nm of this AuNPs size, the contribution

of the dispersed AuNPs increases, due to the LSPR. As claimed, the typical absorbance spectrum of

AuNPs is possible to obtain using photodiodes, therefore the unsuccessful experiments were possibly a

result from misalignments of the barrier, between the device and the PDMS channels; possibly due to

the immobilization chemistry itself, which may have caused the variations seen in acquisitions.

Exploiting the usage of photoconductors, the LSPR was acquired only at 30 min (figure 3.33) and

75 min (figure 3.36) experiments, although not as accurately as using photodiodes. The overal results

obtained showed differences in spectrum acquisition from the one seen in figure 3.26 using photodiodes.

For lower and higher wavelengths the absorbances values are lower than expected, due to photocurrent

acquisition overlapping. This overlap may suggest that the AuNPs when immobilized with APTES

may not display a predominant absorbance effect, resulting in a similar photocurrent acquired from

APTES. In addition, different bias voltages were used in order to analyse the increase in photoacquisition

sensitivity, in which 30 V yielded poor results in terms of LSPR peak acquisition. The repeated use of

incident light beam caused an increase in the electron-holes pairs generation. Since the electrons drift

faster to the positive pole than holes, an accumulation of negative charges could occurr. As the holes

move slower they cannot reach the negative pole at the same rate, the equilibrium of this process is

disrupted. Therefore, the use of a higher voltage applied intended to force the movement of these

charge carriers, since these devices have a considerable area, where electrode distance is about 200 µm.

Efforts were made in order to optimize photocurrent acquisitions in immobilization assays. In fact, the

fabricated Al barrier proven to be useful in these acquisitions, has allowed the detection of the LSPR

peak. Furthermore, the use of TiW barrier, microfabricated afterwards, could not be proven to be as

64

useful, since the photoconductors acquisitions showed high variability in each acquisition. This scattered

acquisition may suggest that persistent photoconductivity (PPC) occurred. Due to intense recursive

experiments, the excess of light incident on these devices increased the defect density in a-Si:H, in which

reduced the efficiency to convert light into photocurrent. Also the increase in dark current identified

throughout experiment was a consequence of this effect. To minimize the developed defects, an annealing

process may be performed, where the photoconductors are put in the oven at 165C for 20 min and left

overnight. Unfortunately, this procedure could not be done, since the photoconductors dye is glued to

a plastic support. Furthermore, in the majority of the experiments, the output colouration of a channel

with AuNPs immobilization performed was a naked-eye visible roseate. However, the yield data acquired

from it did not come into agreement, since the absorbances calculated were lower than 0.29 . Still,

unsuccessful experiments may also be the output of several factors, from misalignments in the setup, the

used bias voltages that may not be optimized for these acquisitions and the possible occurrences of local

aggregation to accumulation of liquid/ air bubbles may also explain these results.

Through achieved experiments, the LSPR peak was obtained not at 520 nm as confirmed initially,

but for longer wavelengths, and the absorbance values showed variability. These shifts may suggest a

change in the local refractive index, caused by a certain volume of the remained APTES flown previously,

as seen in similar work. [49] [60] Whereas the variation of the absorbance calculated suggest the possible

AuNPs non-controlled layer formation in channel surface that contribute to the increase in absorbance,

while a lower value of absorbance may suggest non-homogenous adsorption of AuNPs on channel surface.

The BSA experiment performed in a microfluidic channel using photoconductor presented an absorbance

value increased compared to the previous acquired value for the AuNPs immobilization, as expected,

although the shift is not representative, where the change in refractive index was not visible. The factors

involving protein-nanoparticle interaction were targeted in several studies. In literature, studies were

made regarding the interaction between AuNPs and BSA protein in [61], [62], [63]. Focusing on [63],

Chaudhary, A. et al. investigated the kinetic binding of this protein to citrate stabilized AuNPs among

other shapes and surface modifications through fluorescence quenching. One of the findings yielded that

AuNPs showed the higher order of binding constant, since surface functionalization and morphology play

a key role in this adsorption process. Therefore, the higher absorbances registed can be related to the

affinity encountered on BSA interaction on the surface of the AuNPs.

These devices show differences in operation, characterization values but also in fabrication time, and

associated costs, by which photodiodes are more expensive to fabricate. Current values obtained from

photodiodes for dark current and black ink channel revelead to be much lower than the ones obtained

with photoconductors. Additionally, photodiodes presented near 100% EQE, with unitary gain, while

photoconductors presented lower values for EQE but higher gain values. The response time of each

device was also considered in the experiments, on which photodiodes presented a response time lower

than photoconductors. All these factors described are influential in the decision of which type of device

should be advised when detecting the plasmonic peak. Although photoconductors are relatively more

affordable to fabricate, there are more promising results with photodiode experiments, since they have

shown higher reproducibility and an accurate detection of the LSPR peak.

65

Finally, this work defines the first steps towards the development of a photodetectors setup for LSPR

in a microfluidic biochip. Further improvements should be considered when using these devices, in respect

of scattered light exclusion method, possibly by using a suitable baseline correction (different black ink

wwith the advantages of an adhesive behaviour). Also, the alignments issues should be adressed, not

only for light beam but for the barriers as well. Aiming at future biological challenges not performed

in this work, the optical detection of LSPR shift by photodetectors, as a result of protein-binding, is

still under study. Further experiments concerning immunologic assays through colorimetric detection in

photodiodes should be implemented.

66

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[2] O. Louis, Catherine; Pluchery, Gold Nanoparticles for Physics, Chemistry and Biology. Imperial

College Press, 2012, vol. 1.

[3] W. Haiss, N. T. K. Thanh, J. Aveyard, and D. G. Fernig, “Determination of size and concentration

of gold nanoparticles from UV-Vis spectra,” Anal. Chem., vol. 79, no. 11, pp. 4215–4221, 2007.

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71

72

AAppendix

Contents

A.1 Photodiodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

A.2 Photoconductors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

A.3 Photodetectors Runsheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

73

A.1 Photodiodes

Figure-of-Merit of sensor 12 in dye 7.

Figure A.1: Figure of merit of photodiode using five different ND filters.

Photocurrent acquisition at 520 nm, using ND 3 filter of a microfluidic channel filled with black ink.

This acquisition shows the typical values of black ink measurement.

Figure A.2: Photocurrent measurements with photodiode of a channel filled with black ink using ND 3

filter at wavelength 520 nm.

Spectra measurements of a 30 min AuNPs immobilization experiment, using ND3 filter. The overlap

is identified for low and higher wavelengths in all acquisitions, with black ink exception.

74

Figure A.3: Example of photocurrent values acquired through an immobilization experiment of 30 min.

The intensity photocurrent values are exhibited in the plot for APTESand AuNPs.

Spectra measurements of two microfluidic channels where the black ink was flown. This figure shows

the difference of a channel with AuNPs covered with ink and another channel only with ink inside.

Figure A.4: Spectra acquired in different channels: black ink flown at the end of the experiment, covering

the AuNPs (grey curve) and another channel filled only with ink (black curve).

Photocurrent acquisition at 520 nm wavelength of a microfluidic channel filled with black ink, using

ND 3 filter.

75

Figure A.5: Hydrophobicity of the used black ink demonstrated by the step represented in acquisition.

The local removal of the ink caused the photocurrent to increase.

Photocurrents acquisition of the first 20 min and after 30 min of flowing AuNPs, using Al barrier and

ND 3 filter.

Figure A.6: Photocurrent acquisitions of the 20 + 30 min of AuNPs immobilization, using the Al barrier.

The respective photocurrents acquisition also with ND 3 filter, Al barrier, at 520 nm, for the main

steps performed in the above experiment. In the bar graphic is depicted the last acquired value at the

end time of each measurement, in order to visualize the differences between them.

76

Figure A.7: Comparison between photocurrents acquisition in each main step of the above described

AuNPs immobilization, using the Al barrier and ND 3 filter.

A.2 Photoconductors

The figures A.8 shows the response in time, to an external applied voltage of 30 V. The initial decreasing

of photocurrent values over time is observed due the applied voltage from the software, which applies

this voltage only when the acquisition is started. In figure A.8 it is illustrated the dark photocurrent

of the used device. Whereas, in figure A.9, photocurrent acquisition is made with light beam incident

of the photoconductor, using ND3 filter. At last, in figure A.10 it is depicted the photocurrent values

for a black ink filled channel flowing at 0.3 µL/min. In all cases the acquisition was made until a

plateau of photocurrent stabilization was achieved. In each case, the last value is showed near the curve,

demonstrating that the photocurrent values for the dark photocurrent and the black ink channel do not

differ and are one order of magnitude lower than the photocurrent yield from the ND 3 filtered beam. This

fact could be problematic, since the immobilization yield signal could be embedded and not identified.

77

0 50 100 150 200

5,0x10-12

1,0x10-11

1,5x10-11

2,0x10-11

2,5x10-11

3,0x10-11

3,5x10-11

4,0x10-11

Cur

rent

Abs

olut

e V

alue

s (A

)

Time (s)

(202,407; 6,38E-12

Figure A.8: Dark photocurrent measurement over

time of a photoconductor.

0 50 100 150 200

3,5x10-11

4,0x10-11

4,5x10-11

5,0x10-11

5,5x10-11

6,0x10-11

6,5x10-11

7,0x10-11

7,5x10-11

Cur

rent

Abs

olut

e V

alue

s (A

)

Time (s)

(185,906; 3,719E-11)

Figure A.9: Photocurrent values measured of a ND

3 filtered light beam shined on top of the photocon-

ductor.

0 50 100 150 200

5,0x10-12

1,0x10-11

1,5x10-11

2,0x10-11

2,5x10-11

3,0x10-11

3,5x10-11

Cur

rent

Abs

olut

e V

alue

s (A

)

Time (s)

(200,601; 6,22E-12

Figure A.10: Photocurrent values measured of a

black ink channel using ND 3 filter .

Photoconductor acquisition of photocurrents through the 30 min assay, using the Al and no ND filter

applied. Each curve represent the usual photoresponsive curve in each step of the experiment.

78

Figure A.11: Photocurrent acquisitions in photoconductor of 30 min immobilization of AuNPs, operating

on applied 10 V.

The bar graphic below compares the last measured value in time, for each acquisition, throughout the

above showed 30 min immobilization of AuNPs experiment.

Figure A.12: Comparison of photocurrent values in each acquisition time of the main steps. These were

obtained with ND 0 filter, using Al barrier at an applied voltage of 10 V.

Photocurrent measurements using Al barrier, in a 75 min assay, using an external voltage of 30 V.

79

Figure A.13: Photocurrents acquired in a 75 min immobilization of AuNPs assay, operatinng on applied

30 V.

The graphic below compares the last measured value in each acquisition, throughout the above 75

min immobilization of AuNPs experiment, using ND 3 filter and the Al barrier.

Figure A.14: Photocurrents acquired in a 75 min immobilization of AuNPs assay, using ND 3 filter, Al

barrier and operating on applied 30 V.

Photocurrent measurements using TiW barrier, ND 3, in a 75 min assay, using an external voltage of

30 V.

80

Figure A.15: Photocurrent spectrum acquisitions of a 75 min immobilization of AuNPs, using TiW barrier

and ND 3 filter.

81

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Dep

osition

rate:D

eposition

time:1h00min50s(1.37

Å/s)

6.3

p+ a-S

i:H [1

00Å

] dep

ositi

on b

y R

F

Cond

ition

s:V=0V,T

subs=250

ºC,P=0.1Torr,P R

F=5W

,F(SiH

4)=10sccm

,F(B

2H6/H 2)=

5sc

cm

Dep

osition

time:1m

in40s(1.37

Å/s)

VISU

ALIN

SPEC

TION

7. R

esis

t coa

t and

isla

nd d

efin

ition

Da

te:

Prog.06/02

–Pho

toresis

tcoa

ting

Machine

:SVG

resis

tcoa

tera

nddevelop

ertrack2

Lasere

xposure

Machine

:Heide

lbergInstrumen

tsDire

ctW

riteLaserLith

ograph

ySystem

(DWL)

Cond

ition

s:Focus:-20

;Ene

rgyfile=85

Map

_AM

SION

Mask:__________L2n-i-paSiH____INVE

RTED

___________________________

Maskalignm

ent:(x,y)=

(70,70

) Prog.06/02

–Pho

toresis

tdevelop

men

tMachine

:SVG

resis

tcoa

tera

nddevelop

ertrack1

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckpho

toresis

tdefinition

8. n-i-p

isla

nd e

tch

via

RIE

usi

ng L

AM

D

ate:

Reactiv

eIonEtching(RIE)o

fthe

n-i-pa-Si:Hisland

sMachine

:LAM

ResearchRa

inbo

wPlasm

aEtcher

Cond

ition

s:RecipeSF6_

CHF3;P=100

mTo

rr;P=200

W;F(SF 6)=

50sccm

;F(CHF

3)=50sccm

Etchingtim

e=20

0s

MICRO

SCOPE

VISUALIN

SPEC

TIONto

n-i-pislan

dchecketch

84

9. P

hoto

resi

st s

trip

D

ate:

Material:Microstrip

Machine

:Wetben

ch

Metho

d:Im

merseth

esampleinhotm

icrostrip

for1

5min.+Rinsewith

IPA+Rinsewith

water+Drywith

compressedair.

MICRO

SCOPE

VISUALIN

SPEC

TIONto

verifycom

pleteph

otoresistre

moval

10. R

esis

t coa

t for

SiN

x vi

a de

finiti

on

Dat

e:

Prog.06/02

–Pho

toresis

tcoa

ting

Machine

:SVG

resis

tcoa

tera

nddevelop

ertrack2

Lasere

xposure

Machine

:Heide

lbergInstrumen

tsDire

ctW

riteLaserLith

ograph

ySystem

(DWL)

Cond

ition

s:Focus:-20

;Ene

rgyfile=85

Map

_AM

SION

Mask:__________L3aSiHvia____INVE

RTED

___________________________

Maskalignm

ent:(x,y)=

(70,70

) Prog.06/02

–Pho

toresis

tdevelop

men

tMachine

:SVG

resis

tcoa

tera

nddevelop

ertrack1

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckpho

toresis

tdefinition

11. S

iNx

late

ral w

all p

assi

vatio

n la

yer d

epos

ition

SiNx[200

0Å]

D

ate:

RF-PEC

VDdep

osition

ofSiNx[200

0Å]

Machine

:RF-PE

CVD

Cond

ition

s:V=0V;T

sub=

100

o C;P=10

0mTo

rr;P R

F=10W;F(SiH4)=5sc

cm;F(NH 3)=

10sccm

;F(H2)=35sccm

Dep

osition

time:1h30min

VISU

ALIN

SPEC

TION

12. S

iNx

via

and

pads

lift-

off

D

ate:

Material:Microstrip

Machine

:Wetben

ch

Metho

d:Im

merseth

esampleinhotm

icrostrip

(occasiona

lultrason

icpulses)+rinsewith

water+drywith

compressedair,checkthelift-offp

rocessinth

emicroscop

ean

drepe

atth

estep

sabo

veifincomplete.

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckfo

rclean

lift-off

13. R

esis

t coa

t for

ITO

top

cont

act d

efin

ition

Dat

e:

Prog.06/02

–Pho

toresis

tcoa

ting

Machine

:SVG

resis

tcoa

tera

nddevelop

ertrack2

Lasere

xposure

Machine

:Heide

lbergInstrumen

tsDire

ctW

riteLaserLith

ograph

ySystem

(DWL)

Cond

ition

s:Focus:-20

;Ene

rgyfile=85

Map

_AM

SION

Mask:__________L4ITOTop

Con

tact____N

ON_INVE

RTED

___________________________

Maskalignm

ent:(x,y)=

(70,70

) Prog.06/02

–Pho

toresis

tdevelop

men

tMachine

:SVG

resis

tcoa

tera

nddevelop

ertrack1

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckpho

toresis

tdefinition

85

14. I

TO to

p co

ntac

t dep

ositi

on IT

O [2

000Å

]

Dat

e:

DCM

agne

tron

Spu

tteringofITO[2

000Å]

Machine

:Alcatel

Cond

ition

s:

Depo

sitiontim

e:

Calibratio

nsample:_______________________________

VISU

ALIN

SPEC

TION

15. I

TO to

p co

ntac

t lift

-off

D

ate:

Material:Microstrip

Machine

:Wetben

ch

Metho

d:Im

merseth

esampleinhotm

icrostrip

(occasiona

lultrason

icpulses)+rinsewith

water+drywith

compressedair,checkthelift-offp

rocessinth

emicroscop

ean

drepe

atth

estep

sabo

veifincomplete.

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckfo

rclean

lift-off

16. R

esis

t coa

t for

met

allic

top

lines

def

initi

on

D

ate:

Prog.06/02

–Pho

toresis

tcoa

ting

Machine

:SVG

resis

tcoa

tera

nddevelop

ertrack2

Lasere

xposure

Machine

:Heide

lbergInstrumen

tsDire

ctW

riteLaserLith

ograph

ySystem

(DWL)

Cond

ition

s:Focus:-20

;Ene

rgyfile=85

Map

_AM

SION

Mask:__________L5AlTop

Con

tact____N

ON_INVE

RTED

___________________________

Maskalignm

ent:(x,y)=

(70,70

) Prog.06/02

–Pho

toresis

tdevelop

men

tMachine

:SVG

resis

tcoa

tera

nddevelop

ertrack1

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckpho

toresis

tdefinition

17. M

etal

lic to

p lin

es d

epos

ition

TiW

[150

Å] +

Al [

1500

Å]

Dat

e:

Magne

tron

sputterin

gofTita

nium

Tun

gsten[150

Å]and

Aluminum

[150

0Å]

Machine

:Nordiko700

0Dep

osition

con

ditio

ns:

VISU

ALIN

SPEC

TION

18. M

etal

lic to

p lin

es li

ft-of

f

Dat

e:

Material:Microstrip

Machine

:Wetben

ch

Metho

d:Im

merseth

esampleinhotm

icrostrip

(occasiona

lultrason

icpulses)+rinsewith

water+drywith

compressedair,checkthelift-offp

rocessinth

emicroscop

ean

drepe

atth

estep

sabo

veifincomplete.

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckfo

rclean

lift-off

86

19. P

rote

ctiv

e la

yer a

nd p

ad d

efin

ition

D

ate:

Prog.06/02

–Pho

toresis

tcoa

ting

Machine

:SVG

resis

tcoa

tera

nddevelop

ertrack2

Lasere

xposure

Machine

:Heide

lbergInstrumen

tsDire

ctW

riteLaserLith

ograph

ySystem

(DWL)

Cond

ition

s:Focus:-20

;Ene

rgyfile=85

Map

_AM

SION

Mask:__________L6SiNxVia____INVE

RTED

___________________________

Maskalignm

ent:(x,y)=

(70,70

) Prog.06/02

–Pho

toresis

tdevelop

men

tMachine

:SVG

resis

tcoa

tera

nddevelop

ertrack1

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckpho

toresis

tdefinition

20. P

rote

ctiv

e la

yer d

epos

ition

SiN

x [2

000Å

]

Dat

e:

RF-PEC

VDdep

osition

ofSiNx[200

0Å]

Machine

:RF-PE

CVD

Cond

ition

s:V=0V;T

sub=

100

o C;P=10

0mTo

rr;P R

F=10W;F(SiH4)=5sc

cm;F(NH 3)=

10sccm

;F(H2)=35sccm

Dep

osition

time:1h30min

VISU

ALIN

SPEC

TION

21. P

rote

ctiv

e la

yer l

ift-o

ff.

D

ate:

Material:Microstrip

Machine

:Wetben

ch

Metho

d:Im

merseth

esampleinhotm

icrostrip

(occasiona

lultrason

icpulses)+rinsewith

water+drywith

compressedair,checkthelift-offp

rocessinth

emicroscop

ean

drepe

atth

estep

sabo

veifincomplete.

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckfo

rclean

lift-off

22. P

hoto

resi

st c

oatin

g fo

r cut

ting

Dat

e:

Prog.06/02

–Pho

toresis

tcoa

ting

Machine

:SVG

resis

tcoa

tera

nddevelop

ertrack2

23. D

icin

g sa

mpl

es in

indi

vidu

als

dies

D

ate:

Machine

:Disc

oDA

D32

1DicingSaw

24. P

hoto

resi

st s

trip

D

ate:

Material:Microstrip

Machine

:Wetben

ch

Metho

d:Im

merseth

esampleinhotm

icrostrip

for1

5min.+Rinsewith

IPA+Rinsewith

water+Drywith

compressedair.

87

Constructio

nofintrinsica-Si:Hpho

tocond

uctorsfo

rmicroflu

idicche

milu

minescence

applications

Layerstack:Glass[0

.7m

m];Al[2

000Å],ia-Si:H

[500

0Å];SiNx[200

0Å]

1.

Su

bstratecleaning

Da

te_____/_____/______

Substrate:2.5cmx5cmCorning173

7Glass[0

.7m

m]

Machine

:Wetben

ch

Metho

d:Clean

inhotAlcon

ox®for2

5min.+ultrason

icfo

r5m

in.+rinsewith

water+drywith

com

pressedair

VISU

ALIN

SPEC

TIONto

detectresidue

sontheglass

2.

Alelectrode

san

dlin

esdep

osition

Da

te_____/_____/______

Magne

tron

sputterin

gofAl[20

00Å]

Machine

:Nordiko700

0De

positioncond

ition

s:se

quen

ce“Al200

0Åno

etch”

VISU

ALIN

SPEC

TION

3.

Ph

otoresistcoa

t+Electrode

definition

+Develop

men

tDa

te_____/_____/______

a. Prog.06/02

–Pho

toresis

tcoa

ting

Machine

:SVG

resis

tcoa

tera

nddevelop

ertrack

b. Electrod

eexpo

sure

Machine

:Heide

lbergInstrumen

tsDire

ctW

riteLaserLith

ograph

ySystem

(DWL)

Cond

ition

s:

Mask:_____L

1AlBottomCon

tact_________INVE

RTED

____________________

Maskalignm

ent:(x,y)=

(,)

c. Prog.06/02

–Pho

toresis

tdevelop

men

tMachine

:SVG

resis

tcoa

tera

nddevelop

ertrack

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckpho

toresis

tdefinition

APP

END

IX I

II: R

UN

SH

EETS

USE

D I

N T

HE

MIC

ROFA

BRIC

ATI

ON

OF

THE

AM

ORP

HO

US

SILI

CON

PH

OTO

SEN

SORS

17

3

III.

2.

Run

shee

t PP

Th

e pr

esen

t run

she

et d

escr

ibes

the

mic

rofa

bric

atio

n pr

oces

sing

of th

e in

tegr

ated

amor

phou

s sili

con

phot

ocon

duct

or fo

r flu

ores

cenc

e det

ectio

n.

R

espo

nsib

le: A

lexa

ndra

Pim

ente

l

Dat

e: _

_Set

embr

o 20

07

Des

crip

tion:

Stu

dy o

f the

fluo

resc

ence

resp

onse

of a

par

alle

l con

tact

a-S

i:H (5

000

Å) d

evic

e w

ith a

-

SiC:

H (1

.96µ

m) f

luor

esce

nce

filte

r.

Al e

lect

rode

s are

dep

osite

d ov

er g

lass

subs

trate

and

a-S

i:H is

land

s are

def

ined

ove

r the

ele

ctro

des.

Laye

r st

ack:

Gla

ss (1

mm

) / A

l (15

00 Ǻ

) / i

a-Si

:H (5

000 Ǻ)

/ a-

SiC:

H (1

.96 µm

) / S

iO2 (7

50 Ǻ

).

Step

2: E

lect

rode

s Dep

ositi

on

Subs

trate

: gla

ss

Mac

hine

: Nor

diko

700

0 D

epos

ition

con

ditio

ns b

y Sp

utte

ring

of A

l:

P=1k

W ;

F Ar =

50

sccm

; P =

3 m

Torr

mod

4 / f

30 /

Seq.

69; t

=___

_s

Al t

arge

t thi

ckne

ss =

150

0 Å

Dat

e: _

_/07

/200

6

Re

spon

sible

: A. P

imen

tel

Dat

e: _

_/07

/200

6

Re

spon

sible

: A. P

imen

tel

Step

1: S

ubstr

ate

Clea

ning

Su

bstra

te: 2

.5 c

m x

5cm

Cor

ning

705

9 gl

ass

Mac

hine

: wet

ben

ch

Cond

ition

s: Cl

ean

in

hot

Alc

anox

du

ring

25

min

.; ul

traso

nic f

or 5

min

; rin

se w

ith w

ater

: dry

com

pres

sed

air

APP

END

IX I

II: R

UN

SH

EETS

USE

D I

N T

HE

MIC

ROFA

BRI

CA

TIO

N O

F TH

E A

MO

RPH

OU

S SI

LIC

ON

PH

OTO

SEN

SORS

17

3

III.

2.

Run

shee

t PP

Th

e pr

esen

t run

she

et d

escr

ibes

the

mic

rofa

bric

atio

n pr

oces

sing

of th

e in

tegr

ated

amor

phou

s sili

con

phot

ocon

duct

or fo

r flu

ores

cenc

e de

tect

ion.

R

espo

nsib

le: A

lexa

ndra

Pim

ente

l

Dat

e: _

_Set

embr

o 20

07

Des

crip

tion:

Stu

dy o

f the

fluo

resc

ence

resp

onse

of a

par

alle

l con

tact

a-S

i:H (5

000

Å) d

evic

e w

ith a

-

SiC:

H (1

.96µ

m) f

luor

esce

nce

filte

r.

Al e

lect

rode

s are

dep

osite

d ov

er g

lass

subs

trate

and

a-S

i:H is

land

s are

def

ined

ove

r the

ele

ctro

des.

Laye

r st

ack:

Gla

ss (1

mm

) / A

l (15

00 Ǻ

) / i

a-Si

:H (5

000 Ǻ)

/ a-

SiC:

H (1

.96 µm

) / S

iO2 (7

50 Ǻ

).

Step

2: E

lect

rode

s Dep

ositi

on

Subs

trate

: gla

ss

Mac

hine

: Nor

diko

700

0 D

epos

ition

con

ditio

ns b

y Sp

utte

ring

of A

l:

P=1k

W ;

F Ar =

50

sccm

; P =

3 m

Torr

mod

4 / f

30 /

Seq.

69; t

=___

_s

Al t

arge

t thi

ckne

ss =

150

0 Å

Dat

e: _

_/07

/200

6

Re

spon

sible

: A. P

imen

tel

Dat

e: _

_/07

/200

6

Re

spon

sible

: A. P

imen

tel

Step

1: S

ubst

rate

Cle

anin

g Su

bstra

te: 2

.5 c

m x

5cm

Cor

ning

705

9 gl

ass

Mac

hine

: wet

ben

ch

Cond

ition

s: Cl

ean

in

hot

Alc

anox

du

ring

25

min

.; ul

traso

nic

for 5

min

; rin

se w

ith w

ater

: dry

com

pres

sed

air

APP

END

IX II

I:RU

N S

HEE

TS U

SED

IN T

HE

MIC

ROFA

BRIC

ATI

ON

OF

THE

AM

ORP

HO

US

SILI

CON

PH

OTO

SEN

SORS

174

Dat

e: _

_/07

/200

6

Re

spon

sible

: A. P

imen

tel

Step

5: P

hoto

resis

t Stri

p M

achi

ne: w

et b

ench

Su

bstra

te: g

lass

/ Al/

PR

Cond

ition

s for

Pho

tore

sist S

tripp

ing:

ho

t m

icro

strip

for

20

min

; IP

A r

inse

; D

I rin

se;

dry

com

pres

sed

air

Dat

e: _

_/07

/200

6

Re

spon

sible

: A. P

imen

tel

Step

4: E

lect

rode

s Wet

Etc

hing

Su

bstra

te: g

lass

/ Al/

PR

Mac

hine

: w

et b

ench

Co

nditi

ons f

or A

l Wet

Etc

hing

: Al E

tcha

nt

Thic

knes

s to

rem

ove:

150

0 Å

Etc

h ra

te: 1

1 Å

/s.

Ove

retc

h : u

ntil

glas

s is c

lean

. Te

mpe

ratu

re: @

RT

Dat

e: _

_/07

/200

6

R

espo

nsib

le: V

. Soa

res a

nd A

.Pim

ente

l

Step

3: P

hoto

resis

t Coa

t + B

otto

m E

lect

rode

Exp

osur

e (A

l) +

Dev

elop

men

t

3.1.

Pho

tore

sist C

oat

Mac

hine

: SV

G tr

ack

Subs

trate

: gla

ss/ A

l Ph

otor

esist

Coa

ting

cond

ition

s: 1)

Pro

g 03

/02,

SV

G h

ot p

late

(pre

treat

men

t: 60

s pre

-hea

t @ 1

10ºC

) 2)

Pro

g 06

/02,

Targ

et th

ickn

ess:

1200

nm

3.2.

Ele

ctro

des E

xpos

ure

Subs

trate

: gla

ss/A

l(150

0 Ǻ

) M

achi

ne: D

WL

Mas

k: p

palin

v

Map

: PP

Alig

nmen

t mar

k ap

pear

ing

on th

is la

yer:

(x,y

)=(1

68,5

4)

Ener

gy: _

____

_ ;

L

aser

Pow

er: _

____

_ ;

Foc

us: _

____

__

3.3.

Dev

elop

D

evel

opm

ent c

ondi

tions

: Pr

og.

05/0

2 -

pre-

heat

at

100

ºC d

urin

g 60

s +

60

s de

velo

p.

88

4.

Alelectrode

san

dlin

esW

etEtching

Da

te_____/_____/______

Material:Alum

inum

wetetcha

nt

Machine

:Wetben

ch

Metho

d:Im

merseth

esampleinth

eAlum

inum

etcha

ntand

mon

itoru

ntilun

protectedareas(with

outp

hotoresis

t)areclean

+

washthorou

ghlywith

water+drywith

com

pressedair.

MICRO

SCOPE

VISUALIN

SPEC

TIONto

verifyetch

5.

Ph

otoresiststrip

Date_____/_____/______

Material:Microstrip

Machine

:Wetben

ch

Metho

d:Im

merseth

esampleinhotm

icrostrip

for4

5min.+rinsewith

IPA+rin

sewith

water+drywith

com

pressedair.

MICRO

SCOPE

VISUALIN

SPEC

TIONto

verifycom

pleteph

otoresistre

moval

6.

Intrinsica-Si:Hdep

osition

Da

te_____/_____/______

RF-PEC

VDdep

osition

ofi-aSi:H[5

000Å]

Machine

:RF-PE

CVD

Cond

ition

s:V=0V;T s

ub=250

o C;P=10

0mTo

rr;P R

F=5W

;F(SiH4)=10sccm

De

positionrate:1

.37Å/s,Dep

osition

time:1h0

0min50

s

VISU

ALIN

SPEC

TION

7.

Ph

otoresistcoa

t+ia-Si:Hisland

definition

+Develop

men

t

Date_____/_____/______

a. Prog.06/02

–Pho

toresis

tcoa

ting

Machine

:SVG

resis

tcoa

tera

nddevelop

ertrack

b. i-a

Si:Hisland

exposure

Machine

:Heide

lbergInstrumen

tsDire

ctW

riteLaserLith

ograph

ySystem

(DWL)

Cond

ition

s:

Mask:________L2

i-aSiH____________INVE

RTED

__________________

Maskalignm

ent:(x,y)=

(,)

c. Prog.06/02

–Pho

toresis

tdevelop

men

tMachine

:SVG

resis

tcoa

tera

nddevelop

ertrack

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckpho

toresis

tdefinition

APP

END

IX II

I:RU

N S

HEE

TS U

SED

IN T

HE

MIC

ROFA

BRI

CA

TIO

N O

F TH

E A

MO

RPH

OU

S SI

LIC

ON

PH

OTO

SEN

SORS

174

Dat

e: _

_/07

/200

6

Re

spon

sible

: A. P

imen

tel

Step

5: P

hoto

resi

st S

trip

M

achi

ne: w

et b

ench

Su

bstra

te: g

lass

/ Al/

PR

Cond

ition

s for

Pho

tore

sist S

tripp

ing:

ho

t m

icro

strip

for

20

min

; IP

A r

inse

; D

I rin

se;

dry

com

pres

sed

air

Dat

e: _

_/07

/200

6

Re

spon

sible

: A. P

imen

tel

Step

4: E

lect

rode

s Wet

Etc

hing

Su

bstra

te: g

lass

/ Al/

PR

Mac

hine

: w

et b

ench

Co

nditi

ons f

or A

l Wet

Etc

hing

: Al E

tcha

nt

Thic

knes

s to

rem

ove:

150

0 Å

Etc

h ra

te: 1

1 Å

/s.

Ove

retc

h : u

ntil

glas

s is c

lean

. Te

mpe

ratu

re: @

RT

Dat

e: _

_/07

/200

6

R

espo

nsib

le: V

. Soa

res a

nd A

.Pim

ente

l

Step

3: P

hoto

resi

st C

oat +

Bot

tom

Ele

ctro

de E

xpos

ure

(Al)

+ D

evel

opm

ent

3.1.

Pho

tore

sist

Coa

t M

achi

ne :

SVG

trac

k Su

bstra

te: g

lass

/ Al

Phot

ores

ist C

oatin

g co

nditi

ons:

1)

Pro

g 03

/02,

SV

G h

ot p

late

(pre

treat

men

t: 60

s pre

-hea

t @ 1

10ºC

) 2)

Pro

g 06

/02,

Targ

et th

ickn

ess:

1200

nm

3.2.

Ele

ctro

des E

xpos

ure

Subs

trate

: gla

ss/A

l(150

0 Ǻ

) M

achi

ne: D

WL

Mas

k: p

palin

v

Map

: PP

Alig

nmen

t mar

k ap

pear

ing

on th

is la

yer:

(x,y

)=(1

68,5

4)

Ener

gy: _

____

_ ;

L

aser

Pow

er: _

____

_ ;

Foc

us: _

____

__

3.3.

Dev

elop

D

evel

opm

ent c

ondi

tions

: Pr

og.

05/0

2 -

pre-

heat

at

100

ºC d

urin

g 60

s +

60

s de

velo

p.

APP

END

IX II

I:RU

N S

HEE

TS U

SED

IN T

HE

MIC

ROFA

BRI

CA

TIO

N O

F TH

E A

MO

RPH

OU

S SI

LIC

ON

PH

OTO

SEN

SORS

174

Dat

e: _

_/07

/200

6

Re

spon

sible

: A. P

imen

tel

Step

5: P

hoto

resi

st S

trip

M

achi

ne: w

et b

ench

Su

bstra

te: g

lass

/ Al/

PR

Cond

ition

s for

Pho

tore

sist S

tripp

ing:

ho

t m

icro

strip

for

20

min

; IP

A r

inse

; D

I rin

se;

dry

com

pres

sed

air

Dat

e: _

_/07

/200

6

Re

spon

sible

: A. P

imen

tel

Step

4: E

lect

rode

s Wet

Etc

hing

Su

bstra

te: g

lass

/ Al/

PR

Mac

hine

: w

et b

ench

Co

nditi

ons f

or A

l Wet

Etc

hing

: Al E

tcha

nt

Thic

knes

s to

rem

ove:

150

0 Å

Etc

h ra

te: 1

1 Å

/s.

Ove

retc

h : u

ntil

glas

s is c

lean

. Te

mpe

ratu

re: @

RT

Dat

e: _

_/07

/200

6

R

espo

nsib

le: V

. Soa

res a

nd A

.Pim

ente

l

Step

3: P

hoto

resi

st C

oat +

Bot

tom

Ele

ctro

de E

xpos

ure

(Al)

+ D

evel

opm

ent

3.1.

Pho

tore

sist

Coa

t M

achi

ne :

SVG

trac

k Su

bstra

te: g

lass

/ Al

Phot

ores

ist C

oatin

g co

nditi

ons:

1)

Pro

g 03

/02,

SV

G h

ot p

late

(pre

treat

men

t: 60

s pre

-hea

t @ 1

10ºC

) 2)

Pro

g 06

/02,

Targ

et th

ickn

ess:

1200

nm

3.2.

Ele

ctro

des E

xpos

ure

Subs

trate

: gla

ss/A

l(150

0 Ǻ

) M

achi

ne: D

WL

Mas

k: p

palin

v

Map

: PP

Alig

nmen

t mar

k ap

pear

ing

on th

is la

yer:

(x,y

)=(1

68,5

4)

Ener

gy: _

____

_ ;

L

aser

Pow

er: _

____

_ ;

Foc

us: _

____

__

3.3.

Dev

elop

D

evel

opm

ent c

ondi

tions

: Pr

og.

05/0

2 -

pre-

heat

at

100

ºC d

urin

g 60

s +

60

s de

velo

p. A

PPEN

DIX

III

: RU

N S

HEE

TS U

SED

IN

TH

E M

ICR

OFA

BR

ICA

TIO

N O

F TH

E A

MO

RPH

OU

S SI

LIC

ON

PH

OTO

SEN

SORS

17

5

Step

8: i

a-S

i:H Is

land

RIE

Su

bstra

te: g

lass

/ Al /

i a-

Si:H

/ PR

M

achi

ne: L

AM

R

eact

ive

Ion

Etch

ing

(RIE

) con

ditio

ns:

Rec

ipe

6 Th

ickn

ess t

o re

mov

e: ~

5000

Å

Etch

rate

of i

a-S

i:H: 1

5.3

Å/s

Et

ch ti

me:

___

_ s+

100%

Ove

rEtc

h

Act

ual e

tch

time:

Ove

retc

h un

til g

lass

is c

lean

.

Dat

e: _

_/07

/200

6

R

espo

nsib

le: V

. Soa

res a

nd A

. Pim

ente

l

Dat

e: _

_/07

/200

6

R

espo

nsib

le: V

. Soa

res a

nd A

. Pim

ente

l

Step

7: P

hoto

resi

st C

oat +

i a-

Si:H

Isla

nd E

xpos

ure

+ D

evel

opm

ent

7.1.

Pho

tore

sist

Coa

t: R

epea

t ste

p 3.

1.

7.2:

i a-

Si:H

Isla

nd E

xpos

ure

Subs

trate

: gla

ss/ A

l/ i a

-Si:H

/ PR

Mac

hine

: DW

L M

ask:

ppi

slin

v M

ap: P

P A

lignm

ent m

ark

appe

arin

g on

this

laye

r: (x

,y)=

(168

,174

) Ex

posu

re C

ondi

tions

: En

ergy

: __

La

ser P

ower

: ___

mW

Fo

cus:

__

Offs

ets:

X=_

__

Y

=___

sc

. __

7.3.

Dev

elop

: Rep

eat s

tep

3.3

Step

6: P

EC

VD

Dep

ositi

on o

f i a

-Si:H

Dat

e: _

_/07

/200

6

Res

pons

ible

: J.P

. Con

de a

nd A

. Pim

ente

l

Subs

trate

: gla

ss/ A

l/ M

achi

ne: R

F-U

HV

C

ondi

tions

for i

a-S

i:H (5

000 Ǻ

) PEC

VD

dep

ositi

on:

V=

0 V

T Sub

= 2

50 ºC

P=

0.1

Tor

r P R

F =

5 W

F S

iH4 =

10

sccm

d tar

get:

5000

Å

r dep

ositi

on:

0.73

Å/s

D

epos

ition

Tim

e: 1

h00m

in50

s St

art @

___

h___

min

Fi

nish

@ _

__h_

__m

in

APP

END

IX I

II: R

UN

SH

EETS

USE

D I

N T

HE

MIC

ROFA

BRIC

ATI

ON

OF

THE

AM

ORP

HO

US

SILI

CON

PH

OTO

SEN

SORS

17

5

Step

8: i

a-S

i:H Is

land

RIE

Su

bstra

te: g

lass

/ Al /

i a-

Si:H

/ PR

M

achi

ne: L

AM

Re

activ

e Io

n Et

chin

g (R

IE) c

ondi

tions

: Re

cipe

6

Thic

knes

s to

rem

ove:

~50

00 Å

Et

ch ra

te o

f i a

-Si:H

: 15.

3 Å

/s

Etch

tim

e: _

___

s+10

0% O

verE

tch

A

ctua

l etc

h tim

e: O

vere

tch

until

gla

ss is

cle

an.

Dat

e: _

_/07

/200

6

R

espo

nsib

le: V

. Soa

res a

nd A

. Pim

ente

l

Dat

e: _

_/07

/200

6

R

espo

nsib

le: V

. Soa

res a

nd A

. Pim

ente

l

Step

7: P

hoto

resi

st C

oat +

i a-

Si:H

Isla

nd E

xpos

ure

+ D

evel

opm

ent

7.1.

Pho

tore

sist C

oat:

Repe

at st

ep 3

.1.

7.2:

i a-

Si:H

Isla

nd E

xpos

ure

Subs

trate

: gla

ss/ A

l/ i a

-Si:H

/ PR

Mac

hine

: DW

L M

ask:

ppi

slinv

M

ap: P

P A

lignm

ent m

ark

appe

arin

g on

this

laye

r: (x

,y)=

(168

,174

) Ex

posu

re C

ondi

tions

: En

ergy

: __

La

ser P

ower

: ___

mW

Fo

cus:

__

Offs

ets:

X=_

__

Y

=___

sc

. __

7.3.

Dev

elop

: Rep

eat s

tep

3.3

Step

6: P

ECVD

Dep

ositi

on o

f i a

-Si:H

Dat

e: _

_/07

/200

6

Res

pons

ible

: J.P

. Con

de a

nd A

. Pim

ente

l

Subs

trate

: gla

ss/ A

l/ M

achi

ne: R

F-U

HV

Co

nditi

ons f

or i

a-Si

:H (5

000 Ǻ

) PEC

VD

dep

ositi

on:

V=

0 V

T Sub

= 2

50 ºC

P=

0.1

Tor

r P R

F =

5 W

F S

iH4 =

10

sccm

d tar

get:

5000

Å

r depo

sitio

n: 0

.73

Å/s

Dep

ositi

on T

ime:

1h0

0min

50s

Star

t @ _

__h_

__m

in

Fini

sh @

___

h___

min

89

8.

Intrinsica-Si:Hisland

etchby

RIEwith

LAM

Date_____/_____/______

Reactiv

eionetching(RIE)o

fthe

ia-Si:Hisland

sMachine

:LAM

ResearchRa

inbo

wPlasm

aEtcher

Cond

ition

s:RecipeSF6_

CHF3;P=100

mTo

rr;P=200

W;F(SF 6)=

50sccm

;F(CHF

3)=50sccm

Etchingtim

e=20

0s

MICRO

SCOPE

VISUALIN

SPEC

TIONto

verifyetch

9.

Ph

otoresiststrip

Date_____/_____/______

Material:Microstrip

Machine

:Wetben

ch

Metho

d:Im

merseth

esampleinhotm

icrostrip

for4

5min.(occasio

nalultrason

icpulses)+rinsewith

IPA+rin

sewith

water+

drywith

com

pressedair.

MICRO

SCOPE

VISUALIN

SPEC

TIONto

verifycom

pleteph

otoresistre

moval

10

. Ph

otoresistcoa

t+pad

viadefinition

+Develop

men

t

Date_____/_____/______

a. Prog.06/02

–Pho

toresis

tcoa

ting

Machine

:SVG

resis

tcoa

tera

nddevelop

ertrack

b. Padviaforliftoff.

Machine

:Heide

lbergInstrumen

tsDire

ctW

riteLaserLith

ograph

ySystem

(DWL)

Cond

ition

s:

Mask:___________L

3SiNxVia________INVE

RTED

___________________

Maskalignm

ent:(x,y)=

(,)

c. Prog.06/02

–Pho

toresis

tdevelop

men

tMachine

:SVG

resis

tcoa

tera

nddevelop

ertrack

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckpho

toresis

tdefinition

11. SiNxpa

ssivationlayerd

eposition

Da

te_____/_____/______

RF-PEC

VDdep

osition

ofSiNx[200

0Å]

Machine

:RF-PE

CVD

Cond

ition

s:V=0V;T

sub=

100

o C;P=10

0mTo

rr;P R

F=10W;F(SiH4)=5sc

cm;F(NH 3)=

10sccm

;F(H2)=35sccm

De

positionrate:0

.7Å/s,D

eposition

time:47m

in

VISU

ALIN

SPEC

TION

APP

END

IX I

II: R

UN

SH

EETS

USE

D I

N T

HE

MIC

ROFA

BRIC

ATI

ON

OF

THE

AM

ORP

HO

US

SILI

CON

PH

OTO

SEN

SORS

17

5

Step

8: i

a-S

i:H Is

land

RIE

Su

bstra

te: g

lass

/ Al /

i a-

Si:H

/ PR

M

achi

ne: L

AM

Re

activ

e Io

n Et

chin

g (R

IE) c

ondi

tions

: Re

cipe

6

Thic

knes

s to

rem

ove:

~50

00 Å

Et

ch ra

te o

f i a

-Si:H

: 15.

3 Å

/s

Etch

tim

e: _

___

s+10

0% O

verE

tch

A

ctua

l etc

h tim

e: O

vere

tch

until

gla

ss is

cle

an.

Dat

e: _

_/07

/200

6

R

espo

nsib

le: V

. Soa

res a

nd A

. Pim

ente

l

Dat

e: _

_/07

/200

6

R

espo

nsib

le: V

. Soa

res a

nd A

. Pim

ente

l

Step

7: P

hoto

resis

t Coa

t + i

a-Si

:H Is

land

Exp

osur

e +

Dev

elop

men

t

7.1.

Pho

tore

sist C

oat:

Repe

at st

ep 3

.1.

7.2:

i a-

Si:H

Isla

nd E

xpos

ure

Subs

trate

: gla

ss/ A

l/ i a

-Si:H

/ PR

Mac

hine

: DW

L M

ask:

ppi

slinv

M

ap: P

P A

lignm

ent m

ark

appe

arin

g on

this

laye

r: (x

,y)=

(168

,174

) Ex

posu

re C

ondi

tions

: En

ergy

: __

La

ser P

ower

: ___

mW

Fo

cus:

__

Offs

ets:

X=_

__

Y

=___

sc

. __

7.3.

Dev

elop

: Rep

eat s

tep

3.3

Step

6: P

ECVD

Dep

ositi

on o

f i a

-Si:H

Dat

e: _

_/07

/200

6

Res

pons

ible

: J.P

. Con

de a

nd A

. Pim

ente

l

Subs

trate

: gla

ss/ A

l/ M

achi

ne: R

F-U

HV

Co

nditi

ons f

or i

a-Si

:H (5

000 Ǻ

) PEC

VD

dep

ositi

on:

V=

0 V

T Sub

= 2

50 ºC

P=

0.1

Tor

r P R

F =

5 W

F S

iH4 =

10

sccm

d tar

get:

5000

Å

r depo

sitio

n: 0

.73

Å/s

Dep

ositi

on T

ime:

1h0

0min

50s

Star

t @ _

__h_

__m

in

Fini

sh @

___

h___

min

APP

END

IX II

I:RU

N S

HEE

TS U

SED

IN T

HE

MIC

RO

FAB

RIC

ATI

ON

OF

THE

AM

OR

PHO

US

SILI

CO

N P

HO

TOSE

NSO

RS

176

Step

10:

Pho

tore

sist

Coa

t +Pa

ds E

xpos

ure

+ D

evel

opm

ent

10.1

. Pho

tore

sist

Coa

t: R

epea

t ste

p 3.

1.

10.2

: Ele

ctri

c Le

ads E

xpos

ure

Subs

trate

: gla

ss/A

l/ a-

Si:H

/i a

-Si:H

/ M

achi

ne: D

WL

Mas

ks: p

padn

inv

M

ap: P

P A

lignm

ent m

ark

appe

arin

g on

this

laye

r: (x

,y)=

(168

,294

) Ex

posu

re C

ondi

tions

: En

ergy

: __

La

ser P

ower

: ___

mW

Fo

cus:

__

Off

sets

: X=_

__

Y

=___

sc

. __

10.3

. Dev

elop

: Rep

eat s

tep

3.3

Dat

e: _

_/07

/200

6

R

espo

nsib

le: V

. Soa

res a

nd A

.Pim

ente

l

Step

9: P

hoto

resi

st S

trip

Su

bstra

te: g

lass

/ Al /

i a-

Si:H

/ PR

M

achi

ne: w

et b

ench

Ph

otor

esis

t Stri

ppin

g co

nditi

ons:

ho

t mic

rost

rip fo

r 30

min

; IPA

rins

e; D

I rin

se; d

ry

com

pres

sed

air

Dat

e: _

_/07

/200

6

R

espo

nsib

le: A

. Pim

ente

l

APP

END

IX II

I:RU

N S

HEE

TS U

SED

IN T

HE

MIC

ROFA

BRI

CA

TIO

N O

F TH

E A

MO

RPH

OU

S SI

LIC

ON

PH

OTO

SEN

SORS

176

Step

10:

Pho

tore

sist

Coa

t +Pa

ds E

xpos

ure

+ D

evel

opm

ent

10.1

. Pho

tore

sist C

oat:

Repe

at st

ep 3

.1.

10.2

: Ele

ctric

Lea

ds E

xpos

ure

Subs

trate

: gla

ss/A

l/ a-

Si:H

/i a

-Si:H

/ M

achi

ne: D

WL

Mas

ks: p

padn

inv

M

ap: P

P A

lignm

ent m

ark

appe

arin

g on

this

laye

r: (x

,y)=

(168

,294

) Ex

posu

re C

ondi

tions

: En

ergy

: __

La

ser P

ower

: ___

mW

Fo

cus:

__

Offs

ets:

X=_

__

Y

=___

sc

. __

10.3

. Dev

elop

: Rep

eat s

tep

3.3

Dat

e: _

_/07

/200

6

R

espo

nsib

le: V

. Soa

res a

nd A

.Pim

ente

l

Step

9: P

hoto

resi

st S

trip

Subs

trate

: gla

ss/ A

l / i

a-Si

:H /

PR

Mac

hine

: wet

ben

ch

Phot

ores

ist S

tripp

ing

cond

ition

s:

hot m

icro

strip

for 3

0 m

in; I

PA ri

nse;

DI r

inse

; dry

co

mpr

esse

d ai

r

Dat

e: _

_/07

/200

6

Re

spon

sible

: A. P

imen

tel

90

12. SiNxpa

dslift-off

Date_____/_____/______

Material:Microstrip

Machine

:Wetben

ch

Metho

d:Im

merseth

esampleinhotm

icrostrip

for4

5min.(occasio

nalultrason

icpulses)+rinsewith

water+drywith

compressedair.

MICRO

SCOPE

VISUALIN

SPEC

TIONto

che

ckfo

rclean

lift-off

91

92