Sviluppo di sistemi di analisi ambientali

basati sull'utilizzo dell’alga fotosintetica

verde Chlamydomonas reinhardtii

Workshop NEMO – 21 November 2017, AdR1

Viviana Scognamiglio

IC - CNR AdR1

Rome - Italy

viviana.scognamiglio@mlib.ic.cnr.it

Roma

Trieste

BariFunctional and structural

characterisation of biological

molecules and organisms for

biosensing application

Design, synthesis, and

characterisation of novel

artificial molecules bioinspired

to natural molecules

Biosensor development,

prototyping, and application in

agrifood, environmental,

security, and biomedical fields

IC - CNR AdR1

Via Salaria km 29,3

00015 Monterotondo

Rome - Italy

viviana.scognamiglio@mlib.ic.cnr.it

Gruppo Biosensor

Attività di ricerca

Biosensori e nanotecnologie per il controllo ambientale ed agroalimentare

Area tematica

☒ Ambiente

☒ Agrobio

Referente Attività

Viviana Scognamiglio

http://www.mlib.cnr.it/v4/baa_ic/

Competenze

- Sviluppo di nano e micro-sistemi di biosensori ottici ed elettrochimici per il rilevamento

di contaminanti ambientali e per il controllo della sicurezza e della qualità

agroalimentare;

- Caratterizzazione strutturale/funzionale di proteine e microorganismi mediante tecniche

spettroscopiche (spettroscopia di fluorescenza, dicroismo circolare);

- Progettazione e sintesi di nuove molecole artificiali bioinspirate a molecole naturali;

- Marcatura di biomolecole con sonde fluorescenti o mediatori elettroattivi,

funzionalizzazione con nanomateriali (nanoparticelle di Prussian Blue, Carbon Black,

grafene) e immobilizzazione su substrati ad hoc (array di microelettrodi in oro, elettrodi

stampati in plastica o carta, supporti ITO).

Our research activity - the exploitation

of Chlamydomonas reinhardtii as:

- biopharm of added value compounds for nutraceutics

and cosmeceutics

- monitoring system for microgravity and ionizing

radiations in space research

- Biological recognition elements for biosensor

development

Lambreva M.D., Russo D., Polticelli F., Scognamiglio V., Antonacci A., Zobnina V., Campi G., Rea G. (2014) Structure/function/dynamics of Photosystem IIplastoquinone binding sites. Current Protein and Peptide Science 15(4): 285–295.Rea, G., Polticelli, F., Antonacci, A., Scognamiglio, V., Katiyar, P., Kulkarni, S.A., Johanningmeier, U., Giardi, M.T. (2009) Structure-based design of novel Chlamydomonasreinhardtii D1-D2 photosynthetic proteins for herbicide monitoring. Protein Science, 18(10):2139-51Zobnina, V., Lambreva, M. D., Rea, G., Campi, G., Antonacci, A., Scognamiglio, V., Giardi, M.T., Polticelli, F. (2016). The plastoquinol–plastoquinone exchangemechanism in photosystem II: insight from molecular dynamics simulations. Photosynthesis Research, 1-16Janssen, P.J.D., Lambreva, M.D., Plumeré, N., Bartolucci, C., Antonacci, A., Buonasera, K., Frese, R., Scognamiglio, V., Rea, G. (2014) Photosynthesis at the forefront of asustainable life. Frontiers in Chemistry: Agricultural Biological Chemistry. Front. Chem. 2:36. doi:10.3389/fchem.2014.00036

Biosensor Advantages

Selectivity at biological levels

Tuning sensitivity

General toxicity

High throughput analyses

Small size and low cost

High speed

V Scognamiglio et al. (2013) Nanotechnology in glucose monitoring: advances and challenges in the last 10 years. Biosensors and Bioelectronics

47: 12-25.

Antonacci A. et al. Commercially available (bio)sensors in the agrifood sector. In Biosensors for sustainable food: new opportunities and technical

challenges. Editors: Scognamiglio V. et al. (2016) Comprehensive Analytical Chemistry Series (CAC) (Elsevier)

V Scognamiglio et al. (2010) Biosensors for effective environmental and agrifood protection and commercialization: from research to market.

Microchimica Acta 170: 215-225

• Natural biological entity

• Bioinspired synthetic material

biocomponent

• optical

• electrochemical

• thermometric

• piezoelectric

• magnetic

transducer

• Physico-chemical parameters variation

• Signal output

electronics

target analytes

A biosensor is a device able to provide

quantitative and qualitative analytical

information by employing a biological

recognition element intimately associated

with a physicochemical detector component

(IUPAC Thevenot DR, Tóth K, Durst RA, Wilson GS (1999)

Electrochemical Biosensors: Recommended Definitions and

Classification. Pure and Applied Chemistry 71:2333-2348)

atrazine

0,01 0,1 1 10 100 10000

50

100

150

200

250

300

350

Chlamydomonas reinhardtii

Kautsky fluorescence

transient curve

Time (s)

0,0 2,0x10-8

4,0x10-8

0,4

0,6

0,8

1,0

No

rma

lise

d 1

-Vj

Atrazine (M)

Chlamydomonas reinhardtii wild-type

resistant mutant strains

sensitive mutant strains

Rea, G., Polticelli, F., Antonacci, A.,

Scognamiglio, V., Katiyar, P., Kulkarni, S. A.,

Giardi, M. T. (2009). Structure‐based design

of novel Chlamydomonas reinhardtii D1‐D2

photosynthetic proteins for herbicide

monitoring. Protein Science, 18(10), 2139-

2151.

Rea, G., Antonacci, A., Polticelli, F., Giardi,

M. T., Lambreva, M., Pastorelli, S.,

Scognamiglio, V., Giardi M.T. (2011).

Computational biology, protein engineering,

and biosensor technology: a close

cooperation for herbicides monitoring.

INTECH Open Access Publisher.

Giardi, M. T., Scognamiglio, V., Rea, G., Rodio,

G., Antonacci, A., Lambreva, M.,

Johanningmeier, U. (2009). Optical

biosensors for environmental monitoring

based on computational and

biotechnological tools for engineering

the photosynthetic D1 protein of

Chlamydomonas reinhardtii. Biosensors and

Bioelectronics, 25(2), 294-300.

Biosensor development

2007-2010. FILAS. BIO-NUTRA2009-2011. EU FP7-SME-2008-1. SENSBIOSYN ID: 2325222009-2011. EU FP7-SME-2008-1. BEEP-C-EN, ID: 2320822009-2012. FILAS. BIOAL2010-2012. FILAS. MICROBIOSIS2006-2009. EU FP6. NUTRA-SNACK2006-2009. ASI 2005 MoMa

2008-2010. ETB-2007-34. MULTIBIOPLAT

2005-2011. MIUR. AGROBIOSENS

2005-2009. MISE. MULTITASKS

Scognamiglio et al. (2009) Chlamydomonas reinhardtii genetic variants as probes for fluorescence sensing system in detection of pollutants. Analitycal& Bioanalitycal Chemistry 394(4): 1081-1087

Giardi, M.T., Scognamiglio, V., Rea, G., Rodio, G., Antonacci, A., Lambreva, M., Pezzotti, G., Johanningmeier, U. (2009) Optical biosensors for environmental monitoring based on computational and biotechnological tools for engineering the photosynthetic D1 protein of Chlamydomonas reinhardtii. Biosensors and Bioelectronics 25(2):294-300

Scognamiglio V et al. (2012) Towards an integrated biosensor array for simultaneous and rapid multi-analysis of endocrine disrupting chemicals. Analytica Chimica Acta. 751: 161-70

Scognamiglio V et al. (2012) A new embedded biosensor platform based on Micro-Electrodes Array (MEA) technology. Sensors & Actuators B: Chemical. 176: 275–283.

Screen printed electrodes

Gold micro electrode array (MEA)

A whole-cell optical bioassay for the detection of CWA simulants

exploiting the green photosynthetic algae Chlamydomonas reinhardtii

Plant efficiency analyser

(PEA) Hanatech Instruments

Chlamydomonas reinhardtii

In collaboration with Prof. Arduini,

Prof. Danila Moscone,

Prof. Giuseppe Palleschi,

Tor Vergata University

A. Antonacci, M. D. Lambreva, F. Arduini, D. Moscone, G. Palleschi, V. Scognamiglio*. A whole cell optical bioassay for the

detection of chemical warfare mustard agent simulants. Sensors and actuators B: Chemical 257, 658-665

0,01 0,1 1 10 100 1000

0,0

0,2

0,4

0,6

0,8

1,0

2,5 mM0,8 mM

0,0 0,5 1,0 1,5 2,0 2,5

0,2

0,4

0,6

0,8

FV/F

M

bis2CEA (mM)

No

rma

lise

d F

I

Time (ms)

bis2CEA

0,2 mM

Bis2CEA

y = y = 0.75±0.01 - 0.20±0.01 x (R2 = 0.9854)

LOD = 50 µM control atrazine bis2CEA 2CEES arsenic copper ALL

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

FV/F

M

Interference studies

Poly(e-Caprolactone)

nanocapsulated atrazine ALGAE AS BIOSENSOR FOR NANO-HERBICIDES

in collaboration with:

Prof. Leonardo Fernandes Fraceto

Dr. Francesca Di Pippo

0 10 20 30 40 50

0,0

2,0x106

4,0x106

6,0x106

8,0x106

1,0x107

1,2x107

Cell

count (c

ell

n°

mL

-1)

Time (h)

IL

IL + Atrazine

IL + NANO

IL + NANOATRA

IL + NANO+ATRA

0 20 40 60 80 100

0,38

0,40

0,42

0,44

0,46

0,48

0,50

0,52

0,54

1 -

Vj

Analyte (nM)

C. reinhardtii IL

+ atrazine

+ nanoatrazine

Fraceto, L. F., Grillo, R., De Medeiros, G. A., Scognamiglio, V., Rea, G., Bartolucci, C. (2016). Nanotechnology in Agriculture: which innovationpotential does it have? Frontiers in Environmental Science, 4, 20.A. Antonacci, F. Arduini, V. Scognamiglio* (2018). Vanguard nano(bio)sensor technologies fostering the renaissance of agriculture, in: EmergingTrends in Agri-Nanotechnology: Fundamental and Applied Aspects, Edt. H.B. Singh, S. Mishra, L. F. Fraceto, R. de Lima. CABI, UK (in stampa, inuscita nel 2018).Amina Antonacci, Fabiana Arduini, Danila Moscone, Giuseppe Palleschi, Viviana Scognamiglio*. Nanostructured (Bio)Sensors For SmartAgriculture. Trends in Analytical Chemistry (in press) doi.org/10.1016/j.trac.2017.10.022

D1/D2

protein

D1 biomimetic

peptide

QB BINDING POCKET MIMICKING PEPTIDE

•Grafting of the QB pocket D1 residues 211-280

•Preserving sticking interactions

Phe211 on helix D and Phe274, Leu275, Trp278, Pro279 on helix E

•Increasing solubility

substituting six Phe with Tyr, four Ala with Ser (213, 233, 250,

and 263), eliminating residues 276 to 280 and adding three Lys at

both the N- and C-terminal ends

200 210 220 230 240

-9000

-6000

-3000

0

3000

6000

9000

12000

15000

[]

(d

ec c

m2 /

dm

ol re

s)

Wavelength (nm)

D1pepMut

300 320 340 360 380 400

0,2

0,4

0,6

0,8

1,0 D1PepMut

No

rma

lise

d F

I

Wavelength (nm)

Circular dichroism and Fluorescence spectroscopy

Binding studies towards QB and atrazine

Scognamiglio, V. et al. (2013). Design and biophysical characterization of atrazine-

sensing peptides mimicking the Chlamydomonas reinhardtii plastoquinone binding

niche. Phys. Chem. Chem. Phys. 15(31), 13108-13115.

Prof. Fabio Polticelli

0 2000 4000 6000 8000 10000 12000

0,75

0,80

0,85

0,90

0,95

1,00

Norm

alis

ed F

I

[Atrazine] (nM)

100 200 300 400 500 600

0,82

0,84

0,86

0,88

0,90

0,92

0,94

Norm

alis

ed F

I

[Atrazine] (nM)

Immobilisation of enzymes, proteins, and microorganisms

by electrospray ionisation (esi)

Chlamydomonas reinhardtiiLaccase

In collaboration

with

Dr. Pietro

Calandra

In collaboration with

Dr. Lorenzo Avaldi

Dr. Paola Bolognesi

Projects in progress

Eranetmed – RQ3-2016

“Climate/demographic change”

and Environment

Project title:

Integrated nanotechnologies for

sustainable sensing water and sanitation

Project acronym:

NanoSWS

Dr. Chrysi LaspidouDr. Fabiana Arduini

Prof. Giuseppe Palleschi

Prof. Aziz Amine

Samira El Haouat

Prof. Hong-Ying

Dr. Mohammad Amine

EUAlgae Action No. ES1408

(European Network for Algal-Bioproducts),

European COST programme.

Grant Holder: Fundación IMDEA Energía

(Spain)

Responsible IC: Viviana Scognamiglio

Working Group 3: Refining of Microalgae Into

Its Value Components

(http://eualgae.eu/working-groups/wg-3/)

Working Group 4: Valorisation of

Intermediates and Subproducts

(http://eualgae.eu/working-groups/wg-4/)

Academic collaborations

ISM – CNR (Rome, Italy, Dr. Lorenzo Avaldi)

IRSA – CNR (Rome, Italy, Dr. Francesca Di Pippo)

IIT, Genova (Dr. Mariantonietta Parracino)

UNESP, Sorocaba (Brasile, Prof. Leonardo Fraceto)

La Sapienza University (Rome, Italy, Prof. Alessio Valletta)

“Tor Vergata” University (Rome, Italy, Prof. Fabiana Arduini, Prof. Giuseppe Palleschi)

University Hassan II of Casablanca (Morocco, Prof. Aziz Amine)

University Of Thessaly (Greece, Dr. Chrysi Laspidou)

Industrial collaborations

Biosensor s.r.l (Rome, Italy)

Microsis s.r.l. (Rome, Italy)

Tecnosens (Rome, Italy)

Etatron (Rome, Italy)

DAS (Palombara S, RM - Italy)

NanoImmunotech (Spain)

DropSens (Spain)

Watec (Marocco)

Puratis (Switzerland)

2017 publications:

A. Antonacci, F. Arduini, D. Moscone, G. Palleschi, V. Scognamiglio*. Nanostructured (bio)sensors for smart

agriculture. TrAC, Trends in Analytical Chemistry (in press)

A. Antonacci, M. D. Lambreva, F. Arduini, D. Moscone, G. Palleschi, V. Scognamiglio*. A whole cell optical

bioassay for the detection of chemical warfare mustard agent simulants. Sensors and actuators B: Chemical

257, 658-665.

Arduini, F., Cinti, S., Scognamiglio, V., Moscone, D., Palleschi, G. (2017). How cutting-edge technologies

impact the design of electrochemical (bio) sensors for environmental analysis. A review. Analytica Chimica

Acta 959, 15-42.

Arduini, F., Cinti, S., Scognamiglio, V., Moscone, D. (2017). Paper Based Electrochemical Devices in

Biomedical Field: Recent Advances and Perspectives. In Comprehensive Analytical Chemistry (Vol. 77, pp.

385-413). Elsevier.

S. Cinti, V. Scognamiglio, D. Moscone, F. Arduini. Efforts, challenges, and future perspectives of graphene-

based (bio)sensors for biomedical applications. In: Graphene Bioelectronics. Ed: Ashutosh Tiwari. ISBN:

9780128133491. https://doi.org/10.1016/B978-0-12-813349-1.00006-8

A. Antonacci, F. Arduini, V. Scognamiglio* (2018). Vanguard nano(bio)sensor technologies fostering the

renaissance of agriculture, in: Emerging Trends in Agri-Nanotechnology: Fundamental and Applied Aspects,

Edt. H.B. Singh, S. Mishra, L. F. Fraceto, R. de Lima. CABI, UK (in press).

Viviana Scognamiglio

Gabriele Favaretto Loredana Caccavale

Amina AntonacciJelena Vasiljević