DEM1 chips with optical nano- struct. Deliverable 07 · Grant agreement no: 646260 R2R Biofluidics Deliverable 7.1 – DEM1 chips with optical nano-struct. 3 of 8 1 Aim of the Deliverable

DEM1 chips with optical nano-

struct.

Deliverable 07.1

DOCUMENT HISTORY

Version Date Status of document / Reason of change (responsible person)

1 2017-05-29 Document created (JR, GSB)

2 2017-05-30 Internal review (JR)

3 2017-05-31 Review comments addressed (GSB)

4 2017-06-01 Formal check (BNN)

5 2017-06-01 Final deliverable submission (JR)

R2R Biofluidics Identifier R2R Biofluidics_D07.1_ DEM1 chips with optical nano-struct._r0_FINAL

Submission date May 31st, 2017

Actual date of submission June 1st, 2017

Responsible partner 11 – GSB (former: 6 – Greiner BioOne Diagnostics)

Internal reviewer

Formal check

01 – JOANNEUM RESEARCH

3 - BNN

Confidentiality public

Ref. Ares(2017)2761661 - 01/06/2017

Grant agreement no: 646260

R2R Biofluidics Deliverable 7.1 – DEM1 chips with optical nano-struct. 2 of 8

Table of contents

1 AIM OF THE DELIVERABLE D07.1 ........................................................................................................................ 3

2 CHIPS BASED ON GENSPEED® DESIGN ................................................................................................................. 3

2.1 BRIEF DESCRIPTION OF THE GENSPEED® DETECTION SYSTEM ......................................................................................... 3

2.1.1 The components of the system ..................................................................................................................... 3

2.1.2 The GENSPEED® test chip surface ................................................................................................................. 5

2.2 ENHANCEMENT OF THE LIGHT OUT-COUPLING ONTO THE PHOTODIODES ............................................................................ 5

2.2.1 Fabrication of the optical structures ............................................................................................................. 5

2.2.2 Chip Assembling ............................................................................................................................................ 6

2.2.3 Performance of optical structures ................................................................................................................. 7

2.2.4 Outputs ......................................................................................................................................................... 7

3 CONCLUSION ...................................................................................................................................................... 8

4 OUTLOOK ........................................................................................................................................................... 8

List of figures

Figure 1: The components of the GENSPEED® system. (A) The GENSPEED® R2 device. (B) Its tray with a

commercial GENSPEED® test chip and (C) a schematic drawing of the commercial GENSPEED® chip showing

inlet, reaction channel and waste reservoir. ..................................................................................................... 4

Figure 2: Chemiluminescence based ss-DNA detection within the capillary-force driven microfluidic

channel. (1) Capturing biotinylated ss-DNA targets with surface bound ss-DNA probes over DNA

hybridization. (2) Introduction of an enzyme solution (streptavidin-HRP) through the microfluidic channel.

(3) Following the washing of the unbound enzymes, introduction of a chemiluminescent solution to

generate (4) a chemiluminescent signal over the enzymes bound to the biotinylated ss-DNA targets. .......... 4

Figure 3: The commercial GENSPEED® chips. (C) Photograph of commercial GENSPEED® chips. .................... 5

Figure 4: R2R imprinted optical structures on PET foil ...................................................................................... 5

Figure 5: The imprinted optical structures. (The imprinted optical structures at the bottom part of GBO®

foils, which were produced using R2R technology at the partner JR. ............................................................... 6

Figure 6: A test chip assembled by means of ultrasonic bonding technique. GENSPEED® chip cover as the

top part and the foil printed with ss-DNA on the top and the optical structures on the bottom surface as the

bottom part. ...................................................................................................................................................... 6

Figure 7: GENSPEED® results using the demo-solution. Blue bars represent the intensities measured with a

GENSPEED® chip with the imprinted optical structures, while the lower red bars represent the intensities

measured with standard GENSPEED® chip without imprinted structures; This gives a clear indication for

signal enhancement due to imprinted optical structures (Each experiment was performed three times) ..... 7

Figure 8: JR chip measurements with demo solution. A data set measured with Dem1 chips with optical

structures. (average of ten measurements). Each data was represented as a mean value ± its standard

deviation. ........................................................................................................................................................... 8



1 Aim of the Deliverable D07.1

The GENSPEED test chip by GENSPEED Biotech GmbH is an in-vitro diagnostic chip for point of care

applications. This deliverable describes the work that has been done regarding the implementation of optical

structures into this chip to increase light out-coupling and thus improve device performance.

Originally, the microfluidic chips included in the GENSPEED test kits are composed of two components: a

polystyrene (PS) bottom foil and an injection molded microfluidic top part. After surface coating of both

components using a proprietary surface modification procedure based on plasma polymerization, up to 10

stripes of different capture probes are printed onto the foil using capillary printing technology. Foil and

microfluidic top part are bonded together by means of ultrasonic welding. The resulting microfluidic architecture

includes an inlet-port, a reaction channel and a waste reservoir also serving as capillary pump. During the

assay procedure, sample and reagents are dispensed into the inlet and sucked through the channel into the

waste reservoir via capillary forces defined by the architecture of both, the reaction channel and waste

reservoir.

Within the work of this deliverable, the PS bottom foil was equipped with optical structures that prevent total

internal reflection at the foil-air boundary of the generated luminescence and by that improve outcoupling and

guidance of light towards the photodetector. The most promising structures (simulated in WP 2) were triangular

structures (see Del 2.3). The shape factor is defined by the ratio of the lattice constant and the height of the

structure.

2 CHIPS BASED on GENSPEED® DESIGN

2.1 Brief description of the GENSPEED® detection system

2.1.1 The components of the system

The GENSPEED® R2 device combines optoelectronic detection with automated microfluidic dispensing. It

includes a cartridge containing the assay-reagents (Fig.1A) and a tray into which the GENSPEED® test chip

is inserted (Fig.1B). The GENSPEED® test chip has a capillary-force driven microfluidic channel system

(Fig.1B). As shown in Fig.1C, the GENSPEED® chip is composed of two parts: a foil with single stranded (ss)-

DNA probes and microfluidic top part, with an inlet, a reaction channel and a waste reservoir.



Figure 1: The components of the GENSPEED® system. (A) The GENSPEED® R2 device. (B) Its tray with a commercial

GENSPEED® test chip and (C) a schematic drawing of the commercial GENSPEED® chip showing inlet, reaction channel and waste reservoir.

As demonstrated in Fig.2, the detection principle (e.g. rapid testing of MRSA1) is based on a chemiluminescent

detection technique. Based on this technique, labelled target ss-DNAs are captured by surface bound probe

ss-DNAs (Fig.2-1). Following the introduction of enzymes in the microfluidic channel (Fig.2-2), the

chemiluminescent substrate is added (Fig.2-3) in order to trigger a chemiluminescent reaction located at the

captured target ss-DNAs (Fig.2-4). The chemiluminescent signal is then detected on a photodiode-array

underneath the chip.

Figure 2: Chemiluminescence based ss-DNA detection within the capillary-force driven microfluidic channel. (1)

Capturing biotinylated ss-DNA targets with surface bound ss-DNA probes over DNA hybridization. (2) Introduction of an enzyme solution (streptavidin-HRP1) through the microfluidic channel. (3) Following the washing of the unbound enzymes, introduction of a chemiluminescent solution to generate (4) a chemiluminescent signal over the enzymes bound to the biotinylated ss-DNA targets.

1 Horse-radish peroxidase.



2.1.2 The GENSPEED® test chip surface

As can be seen in Fig.3, the chip surface has seven defined ss-DNA prints (blue stripes) for the specific

detection of MRSA.

Figure 3: The commercial GENSPEED® chips. (C) Photograph of commercial GENSPEED® chips.

2.2 Enhancement of the light out-coupling onto the photodiodes

2.2.1 Fabrication of the optical structures

The out-coupling of the chemiluminescence signal to the photodiode array was improved by imprinting micron-

scaled optical structures at the bottom foil of the chips. The fabrication of the optical structures via R2R-UV-

NIL was already described in Del 4.4. Figure 4 shows a picture of the R2R imprinted optical structures.

Figure 4: R2R imprinted optical structures on PET foil

While these structures (Figure 4) were printed in the first run onto PET substrates, the finally required substrate

is PS. Therefore, the herein described approach used batch UV nanoimprint lithography for the fabrication of

the optical structures. The optical structures were directly imprinted on the bottom side of the foil (unmodified



side) with a PDMS mold into a UV-resist. The UV resist was specially developed for this project by micro resist

technology to match the refractive index of polystyrene and thus avoid further optical losses. The capillary

printing2 of ss-DNA was done on the top surface of the foil. For the capillary printing, the polystyrene was

modified with a plasma polymerisation process to enhance the adhesion of the capture molecules. Figure 5

shows the manually imprinted optical structures on the modified PS foil.

Figure 5: The imprinted optical structures. (The imprinted optical structures at the bottom part of GBO® foils3, which

were produced using R2R technology at the partner JR.

2.2.2 Chip Assembling

In order to perform the chip assembling, the bottom foil printed with ss-DNA on the top and the optical

structures on the bottom surface was bonded to the GENSPEED® microfluidic top part via ultrasonic bonding.

Fig. 6 shows an assembled chip with the optical structures (imprinted at the bottom side).

Figure 6: A test chip assembled by means of ultrasonic bonding technique.4 GENSPEED® chip cover as the top part and

the foil printed with ss-DNA on the top5 and the optical structures on the bottom surface as the bottom part.

2 The ss-DNA printing was done in GENSPEED-Biotech GmbH. 3 The chip assembling was performed in GENSPEED-Biotech GmbH 5 The top surface of the foils was modified by plasma-polymerization to facilitate ss-DNA immobilization.



2.2.3 Performance of optical structures

The measurements were performed with the commercial GENSPEED® R2 device using a solution for

GENSPEED MRSA demonstration purposes. This demo-solution contains the specific oligonucleotides that

simulate the presence of the mecA gene and Staphylococcus aureus in the sample and thus gives a positive

result in the GENSPEED MRSA test procedure. 20 µl of this demo-solution were dispensed into the inlet part

of the chip and subsequently the assay procedure (as described in Fig.2) was automatically performed in the

GENSPEED® R2 device. The raw data was analyzed both using a MATLAB4 code and the software ORIGIN

by calculating the integrated photocurrent measured at each photodiode (=’Pixel’ in Figure 7). In the MATLAB

data evaluation, the data was corrected for both background and Gaussian signal distribution on the

photodiode array. The plots show the mean value ± corresponding standard deviation.

2.2.4 Outputs

Figure 7: GENSPEED® results using the demo-solution. Blue bars represent the intensities measured with a

GENSPEED® chip with the imprinted optical structures, while the lower red bars represent the intensities measured with standard GENSPEED® chip without imprinted structures; This gives a clear indication for signal enhancement due to imprinted optical structures (Each experiment was performed three times)

Figure 7 shows results from measurements with demo-solution using GENSPEED® chips with imprinted

optical structures (blue bars) and standard GENSPEED® chips without such optical structures (average of

three measurements). As can be deduced from these measurements, the signal intensities measured on the

photodiodes were significantly increased by enhancing the out-coupling of the light via the imprinted optical

structures.

4 The MATLAB code was provided by GENSPEED-Biotech GmbH.



3 Conclusion

This deliverable, dealing with the integration of the optical outcoupling structures in the existing Genspeed

chip, demonstrates the whole production chain of the R2R Biofluidics consortium. The optical structures were

first simulated with a FDTD simulation tool (JR) and subsequently fabricated with grey scale laser lithography

to generate a master (JR). This master was used to create a large area shim (InMold) for consecutive R2R UV

imprinting (JR). The UV imprinting resist was especially designed and produced to match the refractive index

of the desired substrate (MRT). The capillary printing of the target molecules as well as US bonding was done

at partner GENSPEED Biotech.

The first measurements indicate a clear signal enhancement. However, to sophistically quantify the signal

enhancement more data have to be generated.

4 Outlook

In a next step, the fluidic part of the GENSPEED chip is replaced with R2R imprinted microfluidic channels,

opening new perspectives for high throughput/low cost production in detection systems based on combined

optical detection and microfluidics. The microfluidic channels were, according to the process chain described

for the optical structures, simulated, mastered, stepped up to shim dimensions and imprinted with R2R UV

nanoimprinting. We could already demonstrate an assembled chip consisting of imprinted microfluidic

channels and optical outcoupling structures on the counter foil. The functionality of the fully assembled Dem1

structures with R2R compatible design was proven with the response to DEMOSOLUTION® (10

measurements). The measurement data contained the characteristic peak signals, which were successfully

identified by the GENSPEED detection algorithms (see figure 8).

Figure 8: JR chip measurements with demo solution. A data set measured with Dem1 chips with optical structures. (average of ten measurements). Each data was represented as a mean value ± its standard deviation.

Documents

DEM1 chips with optical nano- struct. Deliverable 07 · Grant agreement no: 646260 R2R Biofluidics Deliverable 7.1 – DEM1 chips with optical nano-struct. 3 of 8 1 Aim of the Deliverable