REPORT - Europa...Summary/Important results The LOD are always given for 300 seconds. It is a...

REPORT

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D11.2 :Demonstration of “Lab-on-chip for environmental testing” Demonstrator 2 (Final report)

Date : 04/03/2015 Revision : Version 0

N / Réf. : DRT/LETI/DTBS/STD/LISA 15-042

V / Réf :

Participants see diffusion list for CEA

Name Function Date Signature

Authors Demo 2: J. Hue, G. Nonglaton Project leader, WP leader

04/03/2015 04/03/2015

Approval Demo 2 : J.M. Dinten

Laboratory supervisor

Diffusion list

IPT : C. Baum, T. Bastuck, A. Sauer-Budge MRS/MIN : X. Li DOL : P. Steinman PLS : S. Hamm IQS : S. Borros CET : P. Lacharmoise VTT : H. Hakalathi, J.Okkonen DUR : M. Graf COA : V. Villari PAN : R. Joachimi, J. Perl, J. Loerzer GIN : M. Känsäkoski CEA : G. Nonglaton, T. Bordy, M. Darboux, M. Domenes, C. Fontelaye, D. Lauro, F. Perraut, R. Rousier

Confidential

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Modifications Date Version

Glossary LOD = Limit Of Detection AB = antibodies BSA = Bovine Serum Albumin PMT or PM : Photomultiplier Tube or photomultiplier

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Summary/Important results The LOD are always given for 300 seconds. It is a comparison between PBS and Substance P diluted in PBS. The initial objective is 0.5 ng/ml. First of all, the LOD with the CEA chamber and a 1 mm PMMA substrate is experimentally determined between 1 ng/ml and 5 ng/ml. The extrapolation of the LOD leads to 3 ng/ml. This achievement is huge progress. Indeed, the LOD is rather low and this value is repeatable. It outlines that the surface preparation and the tripod deposition is well controlled, that the platform P2 is well designed and the fluidic protocol is compatible with the experiments. These results are also linked to quality of the biological product: the tripod and the antibody which are bought from 2 academic French partners. This point is essential. It has been observed that the substance P has to be prepare no more than one week before the detection, otherwise a degradation of the substance P is observed and the detection level is modified. It could lead to false conclusions. Therefore, from the beginning of the ML2 project, the CEA has been able to develop: - a surface preparation on PMMA, which is R2R compatible, - an “open platform P2” to follow in live the substance P detection - a fluidic protocol, which allows the detection (including the preparation and the cleaning of the fluidic circuit and the fluidic chamber). (Open platform means that the ML2 had the possibilities to send R2R hardware to be tested on the platform). Many parameters can be optimized to reach the initial objective for the LOD. During the M24 meeting, the reviewers suggest that this point is not the first priority due to the remaining time that CEA can dedicate to the ML2 project. The surface preparation parameters could be : the tripod concentration and drop number, the antibody concentration even if a first optimization have been achieved as it illustrated in this report and in the report “Report03_functionnalizationR2R” (October 2014). In this report, key parameters for the repeatability have been identified like the relative humidity during the surface preparation, the incubation time, the delay between the solution preparation and their uses, etc… Other parameters can be also optimized like: - the flow rate, - the optical power (without damaging the hybridization area. We are almost at the maximum, even if have this adjustment is not discussed in this report) - the location of the hybridization areas (i.e. close to the PMT), - the stray light, - the hybridization temperature, - the electronic adjustment, - the spot size, - the PMMA substrate shape. It has been shown, that with a CEA chamber, a 1 mm PMMA substrate or a 500 microns PMMA substrate lead to similar LOD (i.e. between 1 ng/ ml and 5 ng/ml). It also the same result with an IPT chamber with 375 microns PMMA substrate and a 160 microns substrate where the hybridization areas are located. Thanks to these LOD on PMMA, the next goal is to get similar LOD with a “full PMMA chamber”. This task is accomplished with the IPT support, which sends us the 3 PMMA parts of the fluidic chamber: a top PMMA cover, a bottom PMMA covers and a PMMA gasket with glue on each side to gather the 2 PMMA covers. IPT designs in discussion with CEA a chamber holder to receive a full PMMA chamber in using Dolomite connectors. This IPT chamber is compatible to be tested on the P2 platform. Without pumping, it is possible to assemble the IPT chamber. In using, as syringe, it has been shown that the IPT chamber is water tightness (PMMA cover thicknesses: 160/180 microns, PMMA gasket: 300 microns or 450 microns). Unfortunately, due to chamber deformation, the water tightness is not reliable, when the liquid pumping is used. When the watertightness is reached with such camber (160/180-300-160/180), the extrapolated LOD is between 300 and 400 ng/ml. It is also suspected that the chamber deformation due to the pumping has also an impact on the light guiding. It might explain why the LOD increases from 5 ng/ ml to 300 ng/ml.

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CEA showed that similar LOD (i.e. between 1 ng/ml and 5 ng/ ml) is obtained with a full PMMA chamber with a Dolomite connector, when the thickness of the chamber is 375 microns for one side and 160/180 microns for the other side. The hybridizations areas are on the 160/180 microns PMMA substrate. In replacing a 375 microns PMMA by a 250 microns PMMA, the water tightness is not insured when a Dolomite connector is used.. Therefore, CEA is in the process to glue the tube to the top PMMA cover. Then, CEA will achieve an hybridization and determine with the LOD. IQS and CEA collaborate concerning the surface preparation of PMMA sample to put the tripods on the PMMA. Today, we have 2 general means to prepare the PMMA surface: -Method A: CEA prepares the PMMA surface and puts the tripod on in this surface preparation, -Method B: IQS prepares the PMMA and CEA puts the tripods on the IQS surface preparation. With the “first surface preparation” developed by IQS, the LOD is close to ~ 50 ng/ml. This preparation is based on a PFM coating (PFM = pentafluorophenyl methacrylate), which lasts 25 minutes. With the “second surface preparation” developed by IQS, the LOD is between 10 ng/ml and 25 ng/ml (and close to 10 ng/ml). This preparation is also based on a PFM coating. But the time is now only 10 or even 5 minutes and the LOD is better. According IQS, this solution is R2R compatible. IQS has also developed a third surface preparation in using a corona system. CEA has not been yet evaluated the LOD of this technic (in progress). Now, with the P2 platform, it becomes possible to use the small electrovalves supplied by Dolomite and the Bartels pump. Indeed, the suitable electronic and the suitable control command have been developed. CEA has successfully tested a small piezoelectric pump from Bartels during hybridization with an IPT chamber (375 microns and 160/180 microns for the 2 covers and 450 microns for the PMMA gasket). The LOD is similar with a peristaltic pump or with the Bartels pump: Bartels pump: 5 ng/ml < LOD < 10 ng/ml Peristaltic pump: 1 ng/ ml < LOD < 5 ng/ ml

Flow rate: ~ 1000 l/minute The Dolomite electrovalves have been tested with a fluid circulation, but they are not implemented yet during an hybridization. To continue to achieve the current comparison concerning the LOD with various PMMA thicknesses and/or various surface preparations, a complex fluidic circuit fully automatized will not be implemented. It needs a complete revision of fluidic protocol and it will be time consuming. Nevertheless, the fluidic geometry to answer to our problem has been designed. A suitable final fluidic circuit has been already shown in D8.2 If we have time, the next step is to introduce 2 Dolomite electrovalves before and after the chamber to keep the antibodies in the fluidic chamber during the “antibody step” in order to see if the LOD remains similar (today, a manual step is used: the tube is pinched manually during this step. It is the only manual intervention). Indeed, the tubes diameters, which have to be used for the chamber, for the pump(s) and for the electrovalves are different. It will disturb a little bit our fluidic protocol and time is needed for the implementation. The platform P2 has been mechanically upgraded. Now, the emission source can be moved in X, Y and Z directions. With this upgrade, it is becomes possible to test the Polyscale source on P2 with the Dolomite connector. Indeed, with the former and initial configuration (Polyscale source in the horizontal direction, i.e. parallel to the fluidic chamber), the Dolomite connector prevents to locate the Polyscale source in front the fluidic chamber. Therefore, we decided to turn the Polyscale source from 90° (Polyscale source on P2 in the vertical direction, i.e. perpendicular to the fluidic chamber). This upgrade has an additional advantage: the adjustment between the hybridization areas and the emission sources are optimized for each fluidic chamber. We have tested a Polyscale source for the excitation. The maximum optical power density is limited by the thermal management. Compare to the CEA source (which is not R2R compatible), a factor 10 is missing (in the best case, for the structured areas which are close to the edges) Moreover, it is important to eliminate the stray light generated by the Polyscale source. The level of stray light with the Polyscale source is 3 orders more important than the stray light which is generated with the CEA source.

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At least, a cover can be added around the PMMA output and the PMT entrance (gain of factor 100). A part of the remaining factor can be gained in putting a full cover around the Polyscale source except around the structured area. Unfortunately, a factor 10 is still missing. It is probably be due to way of propagation of the incident light inside the LGP slide an then inside the PMMA substrate. Although, there is an optical filter in front of the PMT, this stray light is not a favorable point for such source. Concerning EE2 detection, the platform P2 is operational to achieve the optical tests with the fluidic CEA chamber. The first tests have been achieved with microscope glass slide. The LOD, which has been determined on glass substrate, is 30000 times higher than the initial objective. Moreover, it needs 30 minutes to reach this result (instead of 5 minutes). Now, CEA is able to put the structure to detect EE2 on PMMA substrate (like for the substance P? we begin with a 1 mm PMMA substrate. Therefore, on PMMA it has been decided to modify the link strength between the anrtibody and the surface preparation to detect a larger signal decrease, when the EE2 will be in the fluid solution which is above the sensor. A new experimental campaign can be forecasted, but this task is not the first priority.

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Outline

1. Introduction p 8 2. CEA Work during the remaining time of the ML2 project (forecasts at M24) p 8 3. Report outline p 9 4. Current LOD on PMMA for the substance P (LETI fluidic chamber and associated fluidic protocol chamber with the surface LETI process) p 9 5. Data and discussion concerning the LOD for the substance P with a “full” PMMA R2R compatible chamber (“IPT” chamber) p 15 6. Tests of the IQS preparations on PMMA substrates for the substance P detection p 18 7. Mechanical improvements on P2: possible to adjust the emission source in 3 directions p 22 8. Polyscale excitation source: optical and electrical characterization p 24 9. Control command: upgrade concerning the Dolomite electrovalves and the Bartels pump p 37 10. Hybridization with a Bartels pump p 40 11. Dolomite electrovalves p 40 12. Proposal of fluidic circuits with Dolomite electrovalves and Bartels pump p 40 13. EE2 detection p 41 14. Slopes and standard deviations: automatic process from experimental excel files generated during the (substance P) detection p 41 15. Conclusion p 42

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Annexes Annex 1: General views of P2 p 45 Annex 2: Shelf bracket for the Polyscale test on P2 p 46 Annex 3: R2R Excitation module LGP solutions proposed by Polyscale p 47 Annex 4: Results and discussion of the first tests in June 2014 concerning the LGP (Light guide plate) from Polyscale p 49 Annex 5 : Additional experimental data concerning the Polyscale source p 54 Annex 6: How the LOD is evaluated and extrapolated if necessary? p 56 Annex 7: Piezoelectric Bartels pump p 57 Annex 8: Output light fluorescence versus the PMMA thickness p 60 Annex 9: Report 1 concerning the comparison between IQS and CEA surface preparation (Batch in November/December 2014) p 61

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1. Introduction After the M24 meeting at Barcelona (October 2014), it appears that due to the complexity of the demonstrator 2, it is unrealistic to propose a full R2R solution for a commercial prototype. Nevertheless, few parts can be pushed to see their degrees of compatibility with a R2R process to determine, where are the show stoppers for these elements in R2R, which may be produced in R2R (excitation source, fluidic chamber,…) or adapted with a R2R process (like the peak and place elements: Dolomite electro valve, Bartels pump). The reviewers suggest that the objectives of the demonstrator 2 have to be refined. In November 2014, to follow the reviewer’s suggestions, CEA send to the Prime (i.e. C. Baum/IPT), the following program where the CEA objectives are refined. 2. CEA Work during the remaining time of the ML2 project (forecast at M24) The demonstrator 2 is complex. Indeed, many functions have to be insured to lead to a detection of a given identified substance in a liquid. From the state of the art inside the ML2 project, it appears that all the functions inside the demonstrator 2 cannot be insured in R2R, as it has been accurately described during the consortium meeting in Barcelona (M24). Inside the ML2 project, CEA has developed an open platform called P2 to test various (R2R) components supplied by the ML2 partners and to develop a suitable protocol to coat the adequate probes on various plastic sheets. The plastic sheets are the compulsory materials used for the R2R process. CEA is also responsible to develop the “probes immobilization process” on plastic sheet. This process has to be compatible in R2R. These probes allow detecting the pathogen. The detection is based on existing probes. In the meantime, CEA develops a suitable fluidic protocol to get the detection on P2. Therefore, the open platform P2 is regularly used to set up the probe process, but also the fluidic protocol, which is a part of the detection process. P2 is regularly improved. More details It is dangerous for the health to drink water with a microcystin concentration above 1 ng/ml. To minimize the cost of development, the substance P has been chosen to mimic the detection of the microcystin. In a former program, CEA has shown that the behaviour during the detection of the substance P and the detection of the microcystin are very similar, when adequate probes are deposited on a glass slide. Moreover, the safety rules are less constraining, when the development is achieved with the substance P. The development cost is not linked to the targets (the substance P or the microcystin). The development cost is linked to the probes materials, which are essential for the detection. The “probe quality” is essential to have a chance to get an interesting level of detection. It is the case for the substance P. To achieve the detection of the substance P, 2 kinds of biological materials are necessary “to build the probes”. They are bought from 2 Academic French partners. In the ML2, at M24, the detection level of the substance P reached, with a 1 mm thick PMMA sheet (plastic sheet suitable for the detection), is between 25 ng/ml and 50 ng/ml for a detection time of 5 minutes, with a CEA fluidic chamber (CEA chamber means that : one side is in PMMA, the other components are : a PDMS gasket and the other side in alumina. This side is also used as chamber holder). This level is typically 100 times above the forecasted target (i.e.0.5 ng/ml in 5 minutes). In the ML2 project, the CEA has also to develop the detection of the EE2. For this product, the CEA had no experience. CEA bought the suitable and commercially available materials to build an EE2 probe based on commercial biological materials. The first step is to achieve detection on a glass substrate, especially to validate the “building” probe. At M24, for the EE2, the detection reached on glass slide is about, 30000 times above the target value (after 30 minutes instead of 5 minutes). In both cases (substance P and EE2), the detection is achieved with the same P2 platform. Only the fluidic protocol and the detection methods are different (and obviously the targets and the probes). This point illustrates the versatility of the open platform P2. In Barcelona, it has been shown that during the 24 first months, CEA used about 63 men months (86%) for a cost of 0.89 MEuros (81%) (Initial total forecasted manpower = 73 men month for a total cost of 1.1 MEuros). Therefore, after the Barcelona meeting, the reviewers suggested that during the remaining time, CEA push all the actions, which are directly dedicated to the development of R2R components. The limit of detection should not be the priority. Therefore, CEA will try to mainly develop a robust protocol for the substance P. Indeed, for the limit of detection of EE2, CEA is too far from the objectives. Moreover, at that stage, for the EE2, only detection on glass substrate has been obtained.

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As reminder, it is indicated that inside the demonstrator 2, the following components might be R2R compatible: -the fluidic chamber achieved with 3 PMMA sheets, supplied by IPT (potentially fully R2R), and equipped with a Dolomite connector. One of the PMMA substrates of the chamber will be functionalized. The fluorescent probes are incorporated inside the functionalization area -the excitation light module to generate a fluorescent light thanks to the interaction with the dye probes (which is the signature to detect the researched pathogen). Polyscale proposed a solution that might be compatible with the R2R technic. This solution has to be characterized at CEA. -the “miniature electrovalves” supplied by Dolomite and the miniature Bartels “pumps” might be “peak and place” components; Therefore, during the remaining time, CEA will focus its efforts on these items. During the next 6 months (M24-M30), CEA is also ready to test the components that will be supplied by the ML2 partners. For these various tests, CEA will (mainly) use the platform P2. During the M24 meeting, CEA has indicated that for functionalized chambers in R2R process, CEA will not buy (pay) the reagents (i.e. the probes and the antibodies). 3. Report outline Reminder: the demonstrator 2 is fully functional (see deliverable D8.2) with fluidic protocols (hybridization, cleaning and preparation). Obviously, the demonstrator 2 can be improved. But, due to the remaining time that CEA can dedicate to the ML2 project, the objectives are firstly oriented towards the potential R2R components. Therefore, the outline of the deliverable is the following: - New data concerning the LOD on PMMA for the substance P detection (“CEA” chamber) - Data and discussion concerning the LOD for the substance P with a “full” PMMA R2R compatible chamber (“IPT” chamber) - Test of the IQS surface preparation on PMMA for the substance P detection - Mechanical improvements, which are compulsory to test the “R2R “components on P2 - Polyscale source: optical and electrical characterization - Control command upgrade to use the Dolomite electro-valve and the Bartels pumps - Proposal of fluidic circuits with a Dolomite fluidic electrovalves and Bartels pumps - EE2 detection - Slopes and standard deviations: automatic process from experimental excel files generated during the (substance P) detection - Optical calculations: simple photometric software 4. Current LOD on PMMA for the substance P (LETI fluidic chamber and associated fluidic protocol chamber with the surface LETI process) In this paragraph, detection means “specific detection” and the time for the detection is 600 seconds (300 seconds for the reference line and 300 second for the signal line). The spot are square (typically 2,5 mm x 2,5 mm). For these tests, one side of the chamber is in alumina with a PDMS gasket of 380 microns. The other side of the chamber is a PMMA substrate, with a one millimeter thickness. The functionalization is achieved on a PMMA substrate with a “LETI” process, which seems R2R compatible. This process is already described in the “Report03_functionnalizationR2R” (October 2014). The fluidic protocol, which is used, is already described inside D8.2. (Comparison between the PBS signal and the substance diluted in PBS). Inside the deliverable D8.2, it is mentioned that the reached LOD is between 1000 ng/ml and 3000 ng/ml (29/09/2014).

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At the M24 meeting In Barcelona, CEA indicated that the new LOD is between 25 ng/ml and 50 ng/ml. In this deliverable D11.2, CEA asserts that the LOD for the substance P, with the “LETI” process and a 1 mm PMMA thickness inside the CEA chamber, is between 1 ng/ml and 5 ng/ml. This experimental detection is obtained, as usual, after 5 minutes (experimental data). The process and the measurement are reproducible with the same LOD value. This detection result means a gain of 10 between M24 and M30 (the upper limit for the detection decrease from 50 ng/ml to 5 ng/ml) The initial objective is: detection of 0.5 ng/ml in 5 minutes. An experimental factor 10 is missing (5 ng/ml compare to 0.5 ng/ml). The LOD extrapolation with the detection of 5 ng/ml leads to : ≈ 2.9 ng/ml (ML2-64-03), coming from an average on 3 spots Spot 1: LOD extrapolated ≈ 3.7 ng/ml Spot 2: LOD extrapolated ≈ 3.2 ng/ml Spot 4: LOD extrapolated ≈ 1.8 ng/ml (Best result) Reminder: The initial objective was a LOD of 0.5 ng/ml in 300 seconds on a plastic with a R2R compatible process. Less than an order of magnitude is missing (typically a factor 4 or 6). According to the reviewers’ suggestion, due to the remaining time, an improvement of the LOD is not the first priority. Maybe, an attempt to decrease the LOD will be achieved in increasing the spot size (5 mm x 5 mm), or in searching the flow rate, which leads to the best LOD, etc… It also possible to get a better LOD in adjusting the current parameters like the tripod and the antibody concentration (see the figure 4.2 and 4.3), the drop number, etc…. It has also been highlighted during the Polyscale characterization that even with the usual excitation source, stray light remains detected by the PMT. Indeed, a part of this stray light can be rejected in protecting the fiber entrance and the PMMA light output in adding a cover around the taper fiber entrance and the PMMA edge (i.e. to plunge in darkness the PMMA light output and the optical fiber in the region where the optical coupling of the fluorescent light takes place, see the figure 4.1). It can have an impact on the LOD. Therefore, it appears that many parameters can be optimized to get the best LOD but at that stage it is not the first priority.

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Possibility to add a cover around the optical fiber and the PMMA edge (fluorescent light output) Figure 4.1

The figure 4.2 is an interesting summary concerning the LOD on PMMA. These results are obtained with the CEA fluidic chamber. The functionalizations are achieved on a 1 mm PMMA thickness. It illustrates the progresses, which have been achieved concerning the fluid protocol and the repeatability of the CEA functionalization process. Many key parameters for the functionalization process have been identified to reach a repeatable process to graft the probes on a PMMA substrate. It means that the LETI surface process for the detection is now well stabilized and controlled, that the platform P2 works well and that the fluidic protocol is well defined and controlled. To get, this results, it is absolutely necessary to prepare the fluidic circuit, mainly with BSA, as it is already accurately described in D8.2. Many parameters can be optimized to get a better LOD.

Dark chamber

PMMA slide

During the

measurements

Black cover

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AB concentration = 50 g/ml Spot 4

Flow rate ≈ 1000 l/minute At a given substance P concentration, as a first order, when the slope ratio [Substance P/ PBS]/PBS is

high, lower (i.e. better) is the LOD best tested conditions = 50 M for the tripod concentration The LOD becomes better when the tripod concentration increases:

For a tripod concentration of 40 M, the LOD is between 25 ng/ml and 50 ng/ml

For a tripod concentration of 50 M, the LOD is between 1 ng/ml and 5 ng/ml [Extrapolation at 3 ng/ml] (We have not tested a higher tripod concentration since the first results indicate that the fluorescence of the spot size reach a steady state value see figure 4.3)

CEA has developed a modular functionalization process (probe concentration, drops number,…), which allows a LOD for the substance P between 1 ng/ml and 5 ng/ml in less than 300 seconds

Figure 4.2

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Tests of various substance P concentrations

for various tripod concentrations (10 M, 20 M, 40 M, 50 M)

20 µM; 20 drops,ML2-62-02

10 µM; 20 drops,ML2-62-03

40 µM; 20 drops,ML2-62-01

50 uM; 20 drops, ML2-64-03

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The fluorescence intensities are clearly a function of the peptidic tripod concentration.

These data are not achieved on the best process that is now currently used at CEA

Figure 4.3. : Effect of the peptidic tripod concentration on fluorescence intensities

(Extracted from the CEA report for the ML2 project: Report on roll-to-roll compatible chemical

functionalization written by G. Nonglaton 14/05/2014)

It has also been shown that LOD is better with a flow rate of 1000 l/minute than a flow rate of

500 l/minute.

It has been shown that the LOD is better with an antibody concentration of 25 g/ml or 50 g/ml than

for an antibody concentration of 75 g/ml or 100 g/ml for a substance P concentration of 100 ng/ml (see figure 4.4).

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Optimization of the antibody concentration to get the best LOD

(Tripods concentration: 50 M, drop number = 20)

Figure 4.4 Be careful: -During these various experiments, it has been shown that the substance P has to be prepared no more than 1 week in advance before the detection experiment with the platform P2. The detection level evolves with the preparation time of the substance P (i.e. the LOD undergoes degradation with time) -The detection level, which has been obtained, is clearly linked to the buffer. In our case, the buffer is PBS. -It has been observed, that on the 1 mm PMMA substrate, the surface preparation which have been developed at CEA lead to a different fluorescent level according the hybridization side. The hybridization spots, which are on the side protected by “a blue sheet”, have a fluorescence level which is multiplied by 3 compare to the same hybridization spots which are on the side protected by a transparent sheet. CEA did not verify if it has an impact on the LOD but CEA decided to choose the side protected with a blue sheet for the hybridization.

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5. Data and discussion concerning the LOD for the substance P with a “full” PMMA R2R compatible chamber (“IPT” chamber) We are in the process to compare the LOD of the substance P with the CEA process : inside the LETI chamber and inside the IPT chamber. The LOD tests are achieved with the platform developed in ML2 for the demonstrator 2. As you will see, the thickness of the PMMA substrate will has a high impact on the experiments Starting point These tests imply that: 1-we are able to assemble the 3 parts of the IPT chamber plus the Dolomite connector (manual assembly) A PMMA substrate with 2 holes for the liquid circulation, input and output for the liquid (figure 5.2)

Figure 5.1 : Top layer of the IPT chamber A PMMA substrate of a given thickness to define the fluidic chamber height (figure 5.1)

Figure 5.2 : Gasket of IPT chamber (300 microns and then 450 microns) A PMMA substrate without holes

Figure 5.3 : Bottom layer, Dolomite connectors and IPT holder compatible with Dolomite connectors and the platform P2

Bottom PMMA layers

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With the CEA chamber, the hybridization areas are on the top of the chamber. With the IPT chamber, the hybridization areas are on the bottom of the chamber. 2- the water tightness is insured 3-the IPT chamber is compatible with the platform P2 (mechanically, fluidic…) We will not detail the part of this work. It is not very interesting even if it is time consuming to prepare and achieve these tasks. IPT send us a holder for the full PPMA chamber with a compatibility with the Dolomite connector (see Figure 5.4, definition by IPT and CEA and designed by IPT). The Dolomite fluidic connector is linked to our fluidic circuit to be coupled to our platform P2.

Dolomite connector and IPT holder for the “full PMMA chamber” (If possible compatible with a “R2R” process”)

Figure 5.4

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LETI fluidic chamber : one side in alumina + a PDMS gasket of 380 microns in PMMA + a PMMA substrate (possible to use various thicknesses, most of the tests area achieved with a 1 mm PMMA substrate) IPT fluidic chamber : Both sides in PMMA (one with a thickness of 160/180 microns) + a PMMA gasket of 300 microns + a PMMA substrate (possible to use various thicknesses for this top layer) For the LOD tests with both chambers, the fluidic protocol remains the same and the LETI surface preparation also. The IPT send us 2 batches of IPT chamber (see Figure 5.5).

IPT delivery in Oct. 2014 IPT delivery in Jan.2015

Thicknesses of PMMA with holes 160/180 microns 160/180 microns

Thicknesses of PMMA without holes 160/180 microns 160/180 microns

Thicknesses of PMMA gasket 300 microns 450 microns

Typically : 20 parts of each in October 2014 70 parts of each in January 2015 The supplier references of these substrates send by IPT is unknown. The PMMA gasket defined the chamber height. It can have an impact on the LOD. It has impact on the AB quantity that is used to prepare the sample for the detection. We have similar LOD with a 450 microns gasket or a 300 microns gasket (even it seems slightly better with a 300 microns: only one experiment see the result below)

1 ng/ml < LOD (300 microns gasket) < 5 ng/ml 5 ng/ ml < LOD (450 microns gasket) < 10 ng/ml

Figure 5.5

IPT chamber: thicknesses of the various parts It appears that it is not possible to achieve a substance P detection below 300 ng/ml, with 2 PMMA substrates of 160/180 microns (thickness) and a Dolomite connectors. Moreover, due to an insufficient watertightness, these experiments are not repeatable (sometimes, it is even impossible to get a fluid circulation) Therefore, it has been decided to experimentally determine, what are the smaller PMMA thicknesses, which allow getting similar detection level that has been obtained with the CEA chamber and a 500 microns or a 1000 microns PMMA substrate in using a Dolomite connector (see figure 5.6 : namely LOD < 5 ng/ml)

PMMA thickness where the hybridization areas are

located

Experimental LOD (CEA chamber)

1 mm (Top) 1 ng/ml <LOD < 5 ng/ml (LOD extrapolated ≈ 2.9 ng/ml)

500 microns (Top) LOD < 5 ng/ml

375 microns This experiment will be probably skipped since with an IPT chamber:

375 m/450um-Gasket/160-180 um 1 ng/ml < LOD <5 ng/ml

160 microns Forecasted with an adhesive gasket (≈ 260 microns)

Experimental time: 300 seconds (PBS) + 300 seconds (Substance P diluted in PBS)

Thickness effect: any major difference has been detected with the CEA chamber between 500 microns and 1000 microns for the PMMA substrate

Figure 5.5

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It is possible to use a 160/180 microns PMMA substrate for one side of the IPT chamber. At that stage, a “full PMMA chamber” with a (see Figure 5.6): -160/180 microns PMMA bottom side (side where the hybridization is achieved) -375 microns PMMA top side where are located the 2 holes for the liquid -a Dolomite connector allows to 1 ng/ml < LOD < 5 ng/ml We are not able to always insure the watertightness with the Dolomite connector, when the PMMA bottom side thickness is equal to 250 microns. We are in the process to glue the tube on the 250 microns PMMA to experimentally determine if we are able to successfully achieve an hybridization. When the thickness substrate decreases, many difficulties may appear. They might be due to a lack of substrate rigidity. Indeed, at the beginning, the water tightness of the IPT chamber (160 microns/ 300 microns/ 160 microns) has been tested in using a syringe to inject the liquid. Unfortunately, in using a (peristaltic) pump, the chamber is too flexible and a deformation occurs during the pumping. Therefore, it has many impacts. The first one is a loss of water tightness with the experimental time with a Dolomite connector and the second one is a deformation of the chamber. The deformation of the chamber has a high impact on the light guide, hence on the LOD. Therefore, we try to experimentally determine, what are the minimum PMMA thicknesses for a full PMMA chamber with the IPT holder (with or without the Dolomite connector, see figure 5.6). Indeed, for a R2R process, it is easier to have a thinner PMMA substrate. Unfortunately, thin substrates lead to a loss of rigidity. A compromise has to be found. A possibility could be to laterally reinforce the chamber support in order to minimize the chamber deformation. It cannot guarantee that it will be enough.

Report


Chamber

Connector

Top (thickness

in µm)

Gasket (in µm)

Bottom (thickness in µm)

Experimental LOD (ng/ml)

Hybridization side

IPT

Dolomite

160/180 (Drilled by

IPT)

300 (PMMA)

160/180

Estimation : between entre 300-400 ng/ml from an experimental

data at 1000 ng/ml

IPT

Dolomite

160/180 (Drilled by

IPT)

300 (PMMA)

160/180

3 additional attempts have been achieved without success

Conclusion : difficulties to guarantee the watertightness with Dolomite

connector

It is not a reliable solution

Hybridization

side

IPT Dolomite 500

(Drilled by CEA)

300 (PMMA)

500 LOD slightly inferior to 10 ng/ml

IPT

Dolomite 500

(Drilled by CEA)

300 (PMMA)

160/180 1 ng/ml <LOD < 5 ng/ml

IPT Dolomite 375

(Drilled by CEA)

450 (PMMA)

160/180 1 ng/ml <LOD < 5 ng/ml

IPT

Tube with glue Dolomite connector

cannot be used due to watertightness

reliability

250 (Drilled by

CEA)

450

(PMMA)

160/180 Forecasted

IPT


cannot be used due to watertightness reliability

250 (Drilled by

CEA)

450

(PMMA)

250 It will depend of the tests

described above

IPT


cannot be used due to watertightness reliability

160/180

160/180 It will depend of the other tests

Hybridization side

CEA

1000 380

PDMS

Alumina with dark teflon

1 ng/ml <LOD < 5 ng/ml

CEA

500 380

PDMS Alumina with dark

teflon 1 ng/ml <LOD < 5 ng/ml

CEA

375 380

PDMS Alumina with dark

teflon

CEA

160 260 ? Tape

Alumina with dark teflon

In red: forecasted experiments which have to be done (or at least a part of it)

Figure 5.6: Various chamber configurations and the status of the result

Report


6. LOD on PMMA for the substance P (LETI chamber with the IQS process) For the surface preparation of PMMA, where the tripods lie, 2 main options exist. First of all, CEA prepares the PMMA substrate (step 1) and CEA adds the tripod (step 2) Secondly, IQS prepares the PMMA substrate (step 1) and CEA adds the tripods (step 2). When, IQS prepares the PMMA substrate, CEA has to add the tripods in less than one week, otherwise the IQS surface preparation becomes useless. It is the reason why the experiments with IQS are the first priorities if we want avoiding wasting the samples and our time. It explains why, the work concerning the full PMMA chamber (see previous paragraph) is not finished yet. This collaboration is important for Leticia Fernández, who is in thesis at IQS. 6.1 Evaluation of the IQS surface preparation: “first” IQS surface preparation (December 14) Acomparison between the IQS and the LETI process to immobilize the probes on PMMA 1 mm thickness It has been announced during the M24 meeting in Barcelona. The IQS process allows detecting the substance P, but the experimental LOD is 10 times higher than the LETI LOD:

“First” IQS versus LETI surface preparation for the probe functionalization (substance P) Figure 6.1

This result is fully detailed inside the report entitled “Probe immobilization tests on IQS coated PMMA and evaluation for the substance P detection using the demo 2”. This report is reproduced in the annex 9 of this deliverable. The conclusion of this report is the following: The IQS solution using pentafluorophenyl methacrylate (PFM) activated ester has been evaluated to graft amino-modified biomolecules on PMMA substrates. The grafting of amino-modified biomolecules is performed by spotting. The validation is achieved in measuring the fluorescence. The presence of the peptidic tripod was demonstrated. The fluorescence intensities obtained with the IQS surface preparation is divided by 2 compared to the fluorescence intensities obtained with the CEA surface preparation. The functionality of the grafted probes on IQS PMMA is validated with our standard displacement immunoassay since the substance P is detected. However, with the IQS surface preparation, the experimental limit of detection is ≈ 50 ng/ml. It is 10 times worse than the LOD, which is measured with the CEA surface preparation. In conclusion, the IQS protocol, which is roll-to-roll compatible, can be used for the demo 2 application. The CEA protocol, which is also roll-to-roll compatible gave better results (initial fluorescence and LOD). But the IQS protocol can be further optimized and adapted to the CEA application. The next step remains the transfer of the functionalization protocol to IPT and its adaptation to roll-to-roll. At that stage, the CEA protocol is selected.

Coating duration of PFM = 25 minutes. The conditions of the “first” IQS surface preparation are not R2R compatible

LETI chamber IPT PMMA thickness = 1 mm

Experimental LOD

IQS surface preparation ~ 50 ng/ml

LETI surface preparation 1 ng/ml < LOD < 5 ng/ml

Report


6.2 Evaluation of the IQS surface preparation: “second” and “third” IQS surface preparation (December 14) A report concerning these experiments is in progress at CEA. The fluorescence levels of the 4 samples, which have been received at CEA, are gathered in the figure 6.2. The first three samples (“second” IQS surface preparation) have been treated by PFM as the “first” IQS preparation. The conditions, and mainly the coating time have been decreased from 25 minutes to 10 or 5 minutes. It appears that the “second” conditions is more appropriate for a R2R process and lead to a better LOD (LOD is divided by factor which is between 2 or 5, closer to 5) The third IQS surface preparation uses Corona. The LOD of corona has not been evaluated yet. The scanner fluorescence level of the “second” IQS preparation has been decreased compare to the “first” IQS preparation (Ifluo (first surface preparation) ≈ 1.6 Ifluo (Second surface preparation))

PMMA thickness Surface treatment Scanner fluorescence (V = 110 V)

LOD

1000 m PFM (10 minutes) Voltage = 1.5 ?

≈ 16000 10 ng/ml < LOD < 25 ng/ml

1000 m PFM (5 minutes) Voltage = 1.2 ?

≈ 14000 10 ng/ml < LOD < 25 ng/ml

500 m PFM (5 minutes) Voltage = ?

≈ 5000 or 14000 Has to be done

500 m * Corona ≈ 1000 Has to be done

* : It is not possible to use 1000 m substrate with the corona treatment

“Second” and “Third” IQS versus LETI surface preparation for the probe functionalization (substance P)

Figure 6.2

“Second” IQS versus LETI surface preparation for the probe functionalization (substance P)

PMMA thickness = 1000 m Figure 6.3

LETI chamber

IPT PMMA thickness = 1000 m

Experimental LOD

IQS surface preparation ~ 10 ng/ml

LETI surface preparation 1 ng/ml < LOD < 5 ng/ml

Report


7. Mechanical improvements on P2: possible to adjust the emission source in 3 directions Due to the Dolomite fluidic connector, which insures the input and the output of the IPT chamber, it was not possible to test the Polyscale source with the IPT chamber. Moreover, up to know, if was not possible to adjust the coincidence between the light source and the functionalized area in Z-direction (height). It should not be the case, but sometimes few variations have been observed with the forecasted spotting map and the reality. Therefore, it has been proposed to have a light source which can be mechanically adjusted:

- In X-direction (focusing : distance between the source and the functionalization areas) - In Y direction (localization of the spots in horizontal direction) - In Z-direction (localization of the spots in vertical direction)

With the IPT chamber, the Dolomite connector prohibits to adjust the Polyscale source as close as possible

Figure 7.1 : Drawings of the Polysacle source and the CEA chamber (Old version) Figure 6.1b

Old version with the Polyscale source which lies horizontally (impossible to put with IPT chamber with the Dolomite connector see figure 7.3)

Figure 7.2

Report


Figure 7.3: Possible to have full manual adjustments for the light source Possible to test the Polyscale source vertically due to the Dolomite connector (current version)

For this new setup the flat angle bracket has been modified (see Annex 2). With these 3 translation stages (There are identical) : 1 full rotation = 250 microns Thanks to these mechanical modifications, it becomes possible to associate the IPT chamber and the Polyscale source on P2 (see Figure 7.4)

Figure 7.4 : IPT chambreer wth the Dolomite connector and the Polyscale source on P2

With this new configuration, the Polyscale source will be tested vertically and not horizontally as it was the case, up to know. (Otherwise it is impossible due the fluidic Dolomite connector

Location for the Dolomite

connector

IPT chamber support (1) with the Dolomite connector (2) Polyscale source (3) vertically mounted

3

1

2

3 2

1

Report


8. Polyscale excitation source: optical and electrical characterization 8.0 Polyscale source (See Annex 3) The geometry of the Polyscale source is described in the Annex 3. 8.1 First characterization campaign in June 2014 (LED in serial, see Annex 4) The result of this campaign is described in the annex 4. Polyscale used 2 LED simultaneously for one spot. Unfortunately, this 2 LEDS are plugged in serial. Therefore, it limits the optical power that can be extracted from these LED with the current driver, which is implemented on P2. Indeed, this current driver allows delivering up to 5V. In adding the threshold voltage of the 2 LEDs, it limits the maximum current that can delivered (typically to 35 mA) It is the reason why CEA asks to Polyscale to supply us, 2 LED which are plugged in parallel to supply more current to this 2 LED in order to get more light on hybridization areas. 8.2 Second characterization campaign in February 2015 Polyscale gave to CEA new LED on PCB at M24 (29 October 2014). These LED are now arranged in parallel. Therefore, in plugging them on the P2 platform , more optical power is expected (first tests in June 2014 : typical optical power between 1 mw and 2 mw, see annex 5). Polyscale supplied to LETI the following items: Cyan LED 2 PCB with 4 LED on each PCB (on one PCB, 2 LED does not emit light) Green LED 2 PCB with 4 LED on each PCB 2 BEF = Bright enhanced field (structured area to redirect the light on the functionalized area) The data sheet DS105.pdf contains the LED specifications of LED used by Polyscale. Green LED supplied by Polyscale: LXZ1-PM01 Cyan LED supplied by Polyscale: LXZ1-PE01 Polyscale did not know the full part number designation of the product that they send us (Very often, this information is missing). First characterizations outside the metallic frame At that stage, the new PCB LED has been verified (outside the metallic frame which support the structured PMMA)

Measurement of the forward voltage :

Green LED LXZ1-PM01

Forward voltage (V) Cyan LED cyan LXZ1-PE01

Forward voltage (V)

1 2.71 1 2.86

2 2.83 2 2.91

3 2.54 3 3.15

4 2.57 4 Does not work

Figure 8.1: Forward voltage of the 2 LEDs plugged in parallel

Report


Experimental forecasted plan

New PCB with LED in parallel on the mechanical support and plug

Optical power without optical guide (LED output)

Optical power in front each structured area (i.e. one side of the guide is damaged in June

2014, see annex 4)

Hide 3 structured areas to use only one spot during the hybridization to exploit one spots

If it is possible try to light hybridization areas

Data extracted from the datasheet DS105.pdf corresponding to the Polyscale LEDs In this data sheet, it can be found that (for Junction temperature = 25°C)

Part number Minimum luminous flux at 500 mA

Minimum luminous flux at 700 mA

LXZ1-PM01 (Green LED)

Between 80 and 104 lm (Between 136 mW/sr and 177 mW/sr)

Typical p = 530 nm

Typical 2= 125°C


(In bracket, the corresponding mW/sr if the LED only irradiates at 530 nm, V(530 nm) =0.86, 1 lm = 1/[683 x V(lambda)] W.sr

-1)

Figure 8.2

Part number Minimum luminous flux at 500 mA Minimum luminous flux at 700 mA

LXZ1-PE01 (Cyan LED)


Typical p = 505 nm

Typical 2= 125°C


(in bracket, the corresponding mW/sr if the LED only irradiates at 505 nm, V (505 nm) = 0.40 Figure 8.3

The absolute maximum current, which can be used in these LEDs, is close 1000 mA (if a suitable cooling is achieved) absolute DC forward current (see page 7 of the datasheet) The maximum temperature of the LED junction is 150°. For the green LED, the dominant or peak wavelength is between 520 nm and 540 nm and for the cyan LED the dominant or peak wavelength is between 490 and 510 nm. The LED supplied by Polyscale does not look like the LED shown in page 9 of the Datasheet. CEA thought that Polyscale removes the LED packaging. Polyscale confirms that the LED packaging has not been removed.

Report


Figure 8.4 : Packaging drawing extracted from the data sheet

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Optical characterizations in continuous mode in using the current LED driver which is plugged on P2 8.2a. First step for the optical power characterizations: the LED are plugged on an electronic breadboard In this paragraph, the optical power is measured with an optical power meter in assuming that the wavelength is 532 nm for the green LED and 505 nm for the cyan LED. The LEDs are plugged on an electronic breadboard without a specific cooling. The measurements of the optical power corresponds to the power at the LED output (ie without LGP)

Figure 8.5: Electronic Breadboard The results are gathered in the following figures

Current (%) 100% = 700 mA

(The calibration between the percentage and the current is

not fully linear)

Optical power (mW) Just above the LED

Optical power (mW) After the smoke

20% (~ 30 mA) 24 mW 20 mW

25 % (~ 80 mA) 32 mW

30% (~ 120 mA) 55 mW 47 mW

40% (~ 220 mA) 98 mW 69 mW

50% (typically 330/340 mA, i.e. 170 mA in each LED)

115 mW 75 mW

55% 75 mW

60% Smoke ?!

As the LEDs are in parallel, the current is typically divided in 2 in each LED

Figure 8.6: Cyan PCB N°3 (2 LEDs in parallel)

Cyan PCB N°4 (The LED are not working when they arrived at LETI not tested

Report


Current (%) 100% = 700 mA


not fully linear)

Cyan N°1 Optical power (mW)


20% 14 mW 11 mW

25 % 25 mW 23 mW

30% 36 mW 32 mW

40% 53 mW 59 mW

50% 62 mW 86 mW

Figure 8.7: Cyan PCB N°1 and Cyan PCB N°2

Current (%) 100% = 700 mA


not fully linear)



20% 11 mW 8 mW

25 % 23 mW 17 mW

30% 32 mW 26 mW

40% 45 mW 39 mW

50% 48 mW 47 mW

55% 47 mW

Figure 8.8: Green PCB N°1 and PCB N°2

Current (%) 100% = 700 mA


not fully linear)



20% 10 mW 9 mW

25 % 20 mW 21 mW

30% 26 mW 26 mW

40% 35 mW 40 mW

50% 45 mW 49 mW

55% 47 mW 51 mW

Typically, the light is “eye detected” from a current of 3.3 mA (typically 16% on the driver scale).

Figure 8.9: Green PCB N°3 and PCB N°4

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8.2b. Second step for the optical power characterizations: through the PMMA light guide plate (i.e. the LGP) The PCBs with the green LEDs are introduced in the Polyscale metal frame on each side of the optical guide, according the following figure 8.9:

Figure 8.10: Metal frame orientation

Optical guide

with 4 structured

areas ( )

LED 3 PCB 4

LED 4 PCB 3

LED 2 PCB 1

LED 1 PCB 2

One LED of the PCB 2 is gone from the PCB during the optical guide mounting

This side of the guide has been damaged during the first optical campaign in June 2014 (see annex 4)

Report


To be compatible with the Dolomite connector (fluidic), the Polyscale source is placed vertically (see figure 8.11). Initially, when the IPT Chamber was not designed and forecasted, the Polyscale source was placed horizontally.

Figure 8.11 : IPT chamber with the Dolomite connector and the Polyscale source on P2

From November 2014, the optical source (LETI or Polyscale) can be moved in the 3 directions (X, Y and Y). Indeed, CEA has installed an additional stage to make the adjustment in height. An additional bracket metal with a translation stage has been installed.

Report


In figure 8.12, the optical power, which goes out from the structured area of the optical guide is measured. With the current setup and adjustments, it is the (maximum) green light, which can irradiate the functionalized area to trig the fluorescence.

Output optical power Without optical filter

Sensor size : 10 mm x10 mm Structured area : 5 mm x 5 mm

Yield

LED 1 (PCB N°2) For the yield see Figure 8.8)

4.8 mW (40%) 6.7 mW (50%)

4.8/39 = 0.12 6.7/47 = 0.14

LED 2 (PCB N°1) [then when LED is removed] For the yield see Figure 8.8)

4.0 mW (40%) 5.0 mW (50%)

4.0/45 = 0.089 5.0/48 = 0.10

LED 3 (PCB N°4) 1.9 mW (40%) 2.2 mW (50%)

1.9/40.5 = 0.047 2.2/49 = 0.045

LED 4 (PCB N°3) 1.5 mW (40%) 2.0 mW (50%)

1.5/35 = 0.043 2.0/45 = 0.044

The output power is lower when the structured area far from the LED

Figure 8.12 : Output optical power on 10 mm x 10 mm (sensor size) (from 5 mm x 5mm structured area)

A (complete) opaque cover is put on the PMMA guide except on the LED 1 Figure 8.13

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First cover : clear square opening ~ 5 mm x 5 mm second cover : clear square opening ~ 3 mm x 3 mm to simulate the spots, which are currently tested on the demo 2 (typically 2.5 mm x 2.5 mm square shape) Optical power output without optical filter : 5 mm x 5 mm At “d= 0 cm” At “d=1 cm” 3.1 mW (40%) 3.8 mW (50%) 1.5 mW (50%) 3.5 mm x 3.5 mm At “d= 0 cm” At “d=1 cm” 1.6 mW 0.8 mW at least an optical power of 12 mW is needed to get the same LOD on the demo 2 (1 mW/mm

2 x 3.5 x 3.5 = 12 mW)

(5/3)2 ≈ 2.0

3.8/1.6 ≈ 2.4 1.5/0.8 ≈ 1.9 With an optical filter XF1074 : 3.5 mm x 3.5 mm d=0 cm + (thickness filter =5.5 mm) 0.4 mW (yield after filtering 0.4/1.6 = 0.25) At least an optical power of 12 mW is needed (1 mW/mm

2 x 3.5 x 3.5 = 12 mW)

to get the same LOD on the demo 2

The light output on 10 mm x 10 mm (sensor size) is not sufficient since the typical incident optical power with our traditional LED (after an optical filtering) is more than 1 mW/mm

2

Additional characterization of the LED 1 with an opaque cover (clear opening 3.5 mm x 3.5 mm) without optical filter

Current (%) 100% = 700 mA

Optical power output After filtering

20% 0.16 mW

25% 0.37 mW

30% 0.53 mW

40% 0.78 mW

50% 0.97 mW

60% 1.03 mW

65% 1.3 mW

70% Power decrease

Without cover : 5.6 mW

Figure 8.14 : Optical power output after filtering (3.5 mm x 3.5 mm)

Typical optical incident power density used after optical filtering with our traditional LED: between 1 mW/mm

2 and 1.4 mW/mm

2

Maximum output power density with the PMMA guide: 0.11 mW/mm

2

Conclusion of these characterizations (in continuous mode) A typical factor 10 is missing (more accurately 13). It is possible to increase the current inside the “Polyscale” LED if there is an additional cooling or a heat dissipator (maximum current used: typically 500 mA 250 mA in each LED and they could be used up to 1000 mA with a suitable thermal management)

Report


8.2c. Characterizations with the BEF We used 2 BEF simultaneously. The BEF pattern is located on the smooth side. P(I =50%) with the BEF, clear opening =3.5 mm x 3.5 mm 0.56 mW without BEF P(I=50%) = 0.97 mW 0.97/0.53 ≈ 1.8 Conclusion It is not efficient to use the BEF. It is more efficient to decrease the distance between the structured areas and the functionalized areas. 8.2d. Additional characterizations These data have acquired on the “Cyan PCB n°3” with the current driver and the LED on the breadboard (i.e. without additional cooling): 70%, I = 500 mA OK 80%, I = 580 mA current decrease at 580 mA and then at 140 mA

Current (%) 100% = 700 mA

Optical output power

LED Voltage Current inside the 2 LED

20% 4.6 mW 2.40 V 30 mA

25% 8.7 mW 2.48 V 70 mA

30% 11.7 mW 2.50 V 110 mA

40% 21.9 mW 2.54 V 200 mA

50% 28.2 mW 2.53 V 290 mA

60% 30.6 mW 2.51 V 350 mA

70% 30.6 mW 2.45 V 480 mA

75% 30.7 mW 2.43 V 540 mA

Figure 8.15: Additional characterization with the current driver As shown in the figure 8 of the datasheet, above a thermal temperature of 80°C, the luminous flux drops (see Figure 8.16).

Ud ≈ 2.6 V

I ≈ 330 mA

Report


Figure 8.16: Relative Light output vs Thermal pad temperature, test current at 500 mA

8.2e. In driving the current inside the LED with a voltage To be sure that the current driver has no effect on the maximum current that can be injected inside the 2 LED in parallel, an experiment with a voltage source has been achieved. A resistance, which can dissipate at least 10 watts, is used. The current is measured inside the circuit. For a current of 600 mA, the LEDs light decreases at a similar value that value measured with the current driver. To be sure that the current limitation is due to the thermal budget, we did the same experiment in cooling the LED with fan. Therefore, it becomes possible to get an optical power of at last 63 mW (LED light output) for an associated current of 720 mA. For I = 730 mA, our optical power meter is saturated at 90 mW. A dedicated optical sensor measures an optical output power of 250 mW for current of 730 mA (sum of the current inside the 2 LEDs). With our current driver, it is not possible to deliver more than 700 mA. Typically, at 350 mA (sum of the current inside the 2 LEDs, assumption: 175 mA in each LED), the maximum output light (without the PMMA guide) for the cyan LED, measured at LETI, is 86 mW (see figure 8.7).

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8.2f. Conclusion From our experimental data we determine what might be the maximum optical power with an appropriate thermal management (i.e. with the best thermal management)

Figure 8.17 From the graph above, which is extracted from the data sheet, at 1000 mA in each LED, if the temperature remains the same, it leads to : 175 mA Relative luminous flux = 0.4 (P measured at LETI for 2 LEDs) 1000 mA relative luminous flux = 1.5 Expected value at 1000 mA under the same temperature: 86 mW x 1.5/0.4 ≈ 320 mW (factor x 3.75) This expected value is probably overestimated since it is assumed that the temperature junction remains at 25°C. For a (maximum) typical yield factor of 10% (yield factor of the guide + structured are to redirect the light through the functionalized areas see figure 8.12), it means an output light, without filtering of: 320 mW x 10% = 32 mW on a 5 mm x 5 mm structured area. For a structured area of 2.5 mm x 2.5 mm, the incident output light is divided by 4: 8 mW. An additional yield of 25% has to be taken into account for the optical filtering; it leads to a final incident optical power of 2 mW and to an optical density of ≈ 0.3 mW/mm

2.

On the platform 2, a typical power density between 1 and 1.2 mW/mm

2 is used to get the current data.

Therefore, even with a suitable thermal management (and in R2R, it could be difficult), a factor 3 to 4 will be missing with the “Polyscale optical light source” (for the structured areas which are close to edge where the Polyscale LED are implemented). The missing factor is even higher with the green LED ( there is an additional factor 1.8 = 86/47), which have the suitable wavelength for the substance P detection but an optical power which is lower. 3 or 4 x 1.8 a factor 5 or 7 is missing. The cross talking between the spot have been taken into account since today they are not insulated. 8.2g. Is it possible to send an higher current during only a pulse of 200 ms ? On the platform 2, the emission light is a succession of pulses : Pulse length = 200 s Frequency = 0.2 HZ (i.e. time between 2 pulses ≈ 5 seconds) When, the current percentage is 80%, the optical power drops (see figure 8.18). Therefore, the optical power cannot increase in reducing with pulse conditions which are necessary for the detection on P2

Report


0

0,2

0,4

0,6

0,8

1

1,2

1,4

0

200

400

600

800

1000

1200

1400

0 50 100 150 200 250 300 350 400

Vo

ltag

e O

f C

EA'

sou

rce

(V

)

Vo

ltag

e O

f P

oly

scal

es'

so

urc

e (

V)

Time (s)

40 %

50 %

60 %

70 %

80 %

100 %

Red curve : CEA source

Blue curve : Polyscale source

As observed in continuous mode, between 70% and 80% of the total driver current, the optical power of the Polyscale source drops due to thermal management.

This problem does not appear with the CEA emission source

Illustration of thermal management problem to get higher optical power with the Polyscale source Figure 8.18

This experiment has been done in measuring the autofluorescence of a pristine PMMA substrate (and also the stray light). During this experiment, it has been observed that the optical power detected with the PMT is typically 1000 times more with the Polyscale source compare to the CEA source. It means that a lot of stray light is detected when the Polyscale source is used. The experiments which are associated to the stray light are gathered in the annex 5

8.2h. Summary The “Polyscale light source” is interesting and compact solution. With the LED and the LGP that Polyscale send us, CEA has estimated what could be the incident optical power on the structured area. The conclusion is that at least a factor 10 is missing CEA experimentally shows that it is possible to get more optical power if an appropriate thermal management is achieved. The optimistic estimation indicates that a typical factor 3 or 4 is missing in assuming an appropriate thermal management and a current of 1000 mA in each LED (and only for the spots which are close the LED) to reach the same optical performance that we already have on P2 with the CEA LED. The optimistic simulation assumes that the cooling allows the junction temperature to remain at 25 °C, which is obviously not the case today.

Report


In anyway, as it is illustrated in the annex 5, the Polyscale source generates a lot of stray light which have to be eliminated. A part of the solution is to add an optical cover in front the PMT to put the PMMA light output in darkness as it is illustrated in figure Moreover, the output light which are not generated directed towards the hybridization area have to be removed as it is done for instance in figure 8.13 in adding a complete cover except on the structured area. 9. Control command: upgrade concerning the Dolomite electrovalves and the Bartels pump The control command of the platform 2 has been upgraded to have the possibility to use simultaneously: -10 Dolomite electrovalves, - 2 Bartels pumps.

The former pinch valve are not anymore compatible with the Tygon since the Tygon is not enough flexible. Moreover, the Dolomite valves are smaller than the usual electrovalves

Figure 9.1: Dolomite electrovalve compare to the former pinch electrovalve initially implement on P2

The voltage drives the membrane amplitude and the frequency drives the number of oscillations. At that stage, to modify the flow rate, it has been decided to work at constant frequency and to modify the voltage to change the flow rate (This way should be compatible with the needed flow rate during the experiments).

Figure 9.2: Bartels pump (piezoelectric pump 2 parameters: voltage and frequency)

Former EV on P2

Dolomite EV on P2

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Figure 9.3 : Control command window for the frequency pump

The frequency pump is a parameter which is not

modified during the experiments.

(2 Bartels pumps)

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9. Control command: upgrade concerning 1-Possibility to open or close 10 Dolomite electrovalves (i.e. possibility to implement 10 Dolomite electrovalves on the fluidic circuit) 2- Possibility to choose the flow rate of 4 pumps: 2 peristaltic pumps and 2 piezoelectric Bartels pumps

Figure 9.4: Control command window to define the sequence file parameters This new control command is now operational.

When the Bartels pumps are not used during the automatic process (i.e. not connected to the platform P2), the text “Nan” has to be entered in the sequence file. It indicates to the software that the pump is not used during the automatic process. (It is the way to handle the potential errors concerning this point)

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10. Hybridization with a Bartels pump The control command was necessary to achieve an attempt of hybridization with Bartels pump. CEA has successfully tested a small piezoelectric Bartels pump during an hybridization with an IPT chamber (375 microns and 160/180 microns for the 2 covers and 450 microns for the PMMA gasket). The LOD is similar with a peristaltic pump or with the Bartels pump: Bartels pump: 5 ng/ml < LOD < 10 ng/ml Peristaltic pump: 1 ng/ ml < LOD < 5 ng/ ml

Flow rate: ~ 1000 l/minute

To get a flow rate of 1000 l/min, the flowing parameters have been used: Frequency = 40 Hz Amplitude = 100 V Alternative voltage function: SRS 11. Dolomite electrovalves (used during hybridization) The Dolomite Electrovalves work well. They have been used with a pump. The next step is to implement to electrovalves with a Bartels pump to replace the current manual pinches during the “antibody step”. 12. Proposal of fluidic circuits with Dolomite electrovalves and Bartels pump : towards a full automatic process A fluidic circuit, which could fully automatize, might use a derivative circuit as already described in the specification report. This derivative circuit is compulsory to remove the air between the various solutions. To have a “compact and simple” fluidic circuit, we have in mind to use a manifold (see Figure 12.1)

Has to be evaluated air bubbles generation ?

Figure 12.1: Manifold (For many inputs or outputs) Nevertheless, such complex fluidic circuit impacts the fluidic protocol and obviously the various times of the sequence. Moreover, many tube sizes avec to be used to implement together: - the CEA and IPT chamber - the Bartels pump - the Dolomite electrovalves - 5 or 6 inputs of fluids (see figure 12.1) Therefore, it is not reasonable to completely modify the fluidic circuit while the “surface preparation” and the thicknesses of PMMA for IPT chamber have not been tested. It is also reasonable to wait the potential tests that should be done to evaluate the future IPT chamber that should be done in R2R

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13. EE2 detection Concerning EE2 detection, the platform P2 is operational to achieve the optical tests with the fluidic CEA chamber. The first tests have been achieved with microscope glass slide. The LOD are 30000 times higher than the initial objective. Moreover, it needs 30 minutes to reach this result (instead of 5 minutes). Now, CEA is able to put the structure to detect EE2 on PMMA substrate (like for the substance P? we begin with a 1 mm PMMA substrate). On PMMA it has been decided to modify the link strength between the antoboby and the surface preparation to detect a larger signal decrease when the EE2 will be in fluid solution which above the sensor. A new experimental campaign can be forecasted to determine the LOD. This task is not the first priority. 14. Slopes and standard deviations: automatic process from experimental excel files generated during the (substance P) detection We have implemented a software to process the experimental data in order to get :

- The slope and the standard deviation of the PBS reference - The slope and the standard deviation of the substance p signal

This software in Matlab reads “the text.File” (or the excel file) which are generated by our Labview software. Therefore, we can quickly access to the extrapolated the LOD, if the substance P is detected for a given substance P concentration (see Annex 6). We have experimentally seen that the extrapolation is better if the test is achieved close to the LOD. We have also seen that the extrapolation is interesting to compare sample together if the experiments are achieved with the substance P concentration. It is interesting tool. A future step, which is not forecasted, might be to implement this algorithm to indicate, during a continuous experiment, that a concentration threshold for the substance P has been exceeded.

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15. Conclusion The LOD are always given for 300 seconds. It is a comparison between PBS and Substance P diluted in PBS. The initial objective is 0.5 ng/ml. First of all, the LOD with the CEA chamber and a 1 mm PMMA substrate is experimentally determined between 1 ng/ml and 5 ng/ml. The extrapolation of the LOD leads to 3 ng/ml. This achievement is huge progress. Indeed, the LOD is rather low and this value is repeatable. It outlines that the surface preparation and the tripod deposition is well controlled, that the platform P2 is well designed and the fluidic protocol is compatible with the experiments. These results are also linked to quality of the biological product: the tripod and the antibody which are bought from 2 academic French partners. This point is essential. It has been observed that the substance P has to be prepared no more than one week before the detection, otherwise a degradation of the substance P is observed and the detection level is modified. It could lead to false conclusions. Therefore, from the beginning of the ML2 project, the CEA has been able to develop: - a surface preparation on PMMA, which is R2R compatible, - an “open platform P2” to follow in live the substance P detection - a fluidic protocol, which allows the detection (including the prrepataion and the cleaning of the fluidic circuit and the fluidic chamnber). (Open platform means that the ML2 had the possibilities to send R2R hardware to be tested on the platform). Many parameters can be optimized to reach the initial objective for the LOD. During the M24 meeting, the reviewers suggest that this point is not the first priority due to the remaining time that CEA can dedicate to the ML2 project. The surface preparation parameters could be : the tripod concentration and drop number, the antibody concentration even if a first optimization have been achieved as illustrated in this report and in the report “Report03_functionnalizationR2R” (October 2014). In this report, key parameters for the repeatability have been identified like the relative humidity during the surface preparation, the incubation time, the delay between the solution preparation and their uses, etc… Other parameters can be also optimized like: - the flow rate, - the optical power (without damaging the hybridization area. We are almost at the maximum, even if have this adjustment is not discussed in this report) - the location of the hybridization areas (i.e. close to the PMT), - the stray light, - the hybridization temperature, - the electronic adjustment, - the spot size, - the PMMA substrate shape. It has been shown, that with a CEA chamber, a 1 mm PMMA substrate or a 500 microns PMMA substrate lead to similar LOD (i.e. between 1 ng/ ml and 5 ng/ml). It also the same result with an IPT chamber with 375 microns PMMA substrate and a 160 microns substrate where the hybridization areas are located. Thanks to these LOD on PMMA, the next goal is to get similar LOD with a “full PMMA chamber”. This task is accomplished with the IPT support, which sends us the 3 PMMA parts of the fluidic chamber: a top PMMA cover, a bottom PMMA covers and a PMMA gasket with glue on each side to gather the 2 PMMA covers. IPT designs in discussion with CEA a chamber holder to receive a full PMMA chamber in using Dolomite connectors. This IPT chamber is compatible to be tested on the P2 platform. Without pumping, it is possible to assemble the IPT chamber. In using, as syringe, it has been shown that the IPT chamber is water tightness (PMMA cover thicknesses: 160/180 microns, PMMA gasket : 300 microns or 450 microns). Unfortunately, due to chamber deformation, the water tightness is not reliable, when the liquid pumping is used. When the watertightness is reached with such camber (160/180-300-160/180), the extrapolated LOD is between 300 and 400 ng/ml. It is also suspected that the chamber deformation due to the pumping has also an impact on the light guiding. It might explain why the LOD increase from 5 ng/ ml to 300 ng/ml.

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CEA showed that similar LOD (i.e. between 1 ng/ml and 5 ng/ ml) is obtained with a full PMMA chamber with a Dolomite connector, when the thickness of the chamber is 375 microns for one side and 160/180 microns for the other side. The hybridizations areas are on the 160/180 microns PMMA substrate. In replacing a 375 microns PMMA by a 250 microns PMMA, the water tightness is not insured when a Dolomite connector is used.. Therefore, CEA is in the process to glue the tube to the top PMMA cover. Then, CEA will achieve an hybridization and determine with the LOD. IQS and CEA collaborate concerning the surface preparation of PMMA sample to put the tripods on the PMMA. Today, we have 2 general means to prepare the PMMA surface: -Method A: CEA prepares the PMMA surface and puts the tripod on in this surface preparation, -Method B: IQS prepares the PMMA and CEA puts the tripods on the IQS surface preparation. With the “first surface preparation” developed by IQS, the LOD is close ~ 50 ng/ml. This preparation is based on a PFM coating (PFM = pentafluorophenyl methacrylate), which lasts 25 minutes. With the “second surface preparation” developed by IQS, the LOD is between 10 ng/ml and 25 ng/ml (and close to 10 ng/ml). This preparation is also based on a PFM coating. But the time is now only 10 or even 5 minutes and the LOD is better. According IQS, this solution is R2R compatible. IQS has also developed a third surface preparation in using a corona system. CEA has not yet been evaluated the LOD of this technic (in progress). Now, with the P2 platform, it becomes possible to use the small electrovalves supplied by Dolomite and the Bartels pump. Indeed, the suitable electronic and the suitable control command have been developed. CEA has successfully tested a small piezoelectric pump from Bartels during hybridization with an IPT chamber (375 microns and 160/180 microns for the 2 covers and 450 microns for the PMMA gasket). The LOD is similar with a peristaltic pump or with the Bartels pump: Bartels pump: 5 ng/ml < LOD < 10 ng/ml Peristaltic pump: 1 ng/ ml < LOD < 5 ng/ ml

Flow rate: ~ 1000 l/minute The Dolomite electrovalves have been tested with a fluid circulation, but they are not implemented yet during an hybridization. To continue to achieve the current comparison concerning the LOD with various PMMA thicknesses and/or various surface preparations, a complex fluidic circuit fully automatized will not be implemented. It needs a complete revision of fluidic protocol and it will be time consuming. Nevertheless, the fluidic geometry to answer to our problem has been designed. A suitable final fluidic circuit has been already shown in D8.2 If we have time, the next step is to introduce 2 Dolomite electrovalves before and after the chamber to keep the antibodies in the fluidic chamber during the “antibody step” in order to see if the LOD remains similar (today, a manual step is used: the tube is pinched manually during this step. It is the only manual intervention). Indeed, the tubes diameters, which have to be used for the chamber, for the pump(s) and for the electrovalves are different. It will disturb a little bit our fluidic protocol and time is needed for the implementation. The platform P2 has been mechanically upgraded. Now, the emission source can be moved in X, Y and Z directions. With this upgrade, it is becomes possible to test the Polyscale source on P2 with the Dolomite connector. Indeed, with the former and initial configuration (Polyscale source in the horizontal direction, i.e. parallel to the fluidic chamber), the Dolomite connector prevents to locate the Polyscale source in front the fluidic chamber. Therefore, we decided to turn the Polyscale source from 90° (Polyscale source on P2 in the vertical direction, i.e. perpendicular to the fluidic chamber). This upgrade has an additional advantage: the adjustment between the hybridization areas and the emission sources are optimized for each fluidic chamber. We have tested a Polyscale source for the excitation. The maximum optical power density is limited by the thermal management. Compare to the CEA source (which is not R2R compatible), a factor 10 is missing (in the best case, for the structured areas which are close to the edges) Moreover, it is important to eliminate the stray light generated by the Polyscale source. The level of stray light with the Polyscale source is 3 orders more important than the stray light which is generated with the CEA source.

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At least, a cover can be added around the PMMA output and the PMT entrance (gain of factor 100). A part of the remaining factor can be gained in putting a full cover around the Polyscale source except around the structured area. Unfortunately, a factor 10 is still missing. It is probably be due to way of propagation of the incident light inside the LGP slide an then inside the PMMA substrate. Although, there is an optical filter in front of the PMT, this stray light is not a favorable point for such source. Concerning EE2 detection, the platform P2 is operational to achieve the optical tests with the fluidic CEA chamber. The first tests have been achieved with microscope glass slide. The LOD, which has been determined on glass substrate, is 30000 times higher than the initial objective. Moreover, it needs 30 minutes to reach this result (instead of 5 minutes). Now, CEA is able to put the structure to detect EE2 on PMMA substrate (like for the substance P? we begin with a 1 mm PMMA substrate. Therefore, on PMMA it has been decided to modify the link strength between the EE2 and the surface preparation to detect a larger signal decrease, when the EE2 will be in the fluid solution which is above the sensor. A new experimental campaign can be forecasted, but this task is not the first priority.

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Annex 1: General views of P2

This platform has been built to be versatile: to test various configurations and protocols, to test various (R2R) components. This platform can be adapted: - to answer to various questions

Figure A-1: Platform 2 (P2)

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Annex 2: Shelf bracket for the Polyscale test on P2

Figure A2-1: Modification to insure the mechanical compatibility with the IPT chamber (Drawings)

Figure A2-2: Mechanical interface for the Polyscale excitation module (with P2)

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17

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6

Carré de 10,7 centré sur la pièce

Ø2 traversant

17,5

41

17

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Chambrage pour vis M4 CHC

35

7,2

Chambrage pour vis M2 CHC

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Annex 3: R2R Excitation module LGP solutions proposed by Polyscale Solution 1: Light Guide Plate = LGP (S. Hamm) Polyscale proposes to replace the current LED solution by the solution described in this annex. IPT and CEA discussed to define an excitation module compatible with the functional spot size currently used on P2.

Figure A3-1: Drawing of an excitation module with a light guide plate

Polyscale has already achieved the first version of this excitation module (see figure A3-2)

Polyscale achievement of the excitation module with a Light Guide Plate (LGP) - June 2014 Figure A3-2

CEA has in mind: - to optically characterize this module - to simulate the optical power of this source with the current LED solution (if it is possible) and to measure the associated LOD (or the effect of the incident power on the LOD), - to achieve hybridization with the “R2R excitation module” (if the mechanics and electronics are compatible with P2). This excitation module can be tested with or without excitation filter (at least an excitation filter in front of one spot). Polyscale and CEA have designed a mechanical piece to withstand the Polyscale excitation module (see annex 2).

First test results are reported in the annex 12. For new tests, Polyscale has ordered a new PCB for the LEDs to compatible with the electronic of P2.

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Solution 2: without light guide plate (S. Hamm) Polyscale proposes another R2R compatible solution. CEA did not receive this source.

Figure A3-4: Drawing of an excitation module with a BEF and without a light guide plate (drawing)

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Annex 4

Results and discussion of the first tests in June 2014 concerning the LGP (Light guide plate) from Polyscale

Result of the 1st characterization of the Polyscale excitation module with CEA platform 2 (P2)

Conclusion: To go further and to be fully compatible with the power supply of P2 for a given spot, the LED of the LGP have to be electrically plugged in parallel. This task is in progress at Polyscale. Nevertheless, few characterizations and observations have been done. They are reported below.

Various observations 1-At that stage, the following point is not a big deal Height of the PLS light source (i.e. grating height) [to be in coincidence with the functionalized areas] Smaller altitude of the grating ≈ 44 mm Wished altitude: 42.5 mm (or 45 mm in the center) 3 options to correct this point : -With a new LGP modified the altitude of the grating -With this current grating modified “the metallic flat angle bracket” Drilling at another location Another possibility: to do another shelf bracket piece with a hole located 1.5 mm lower (i.e. hole at lower height)

-to adopt a new strategy to move the emission source in front of the fluidic chamber (Finally, CEA has adopted this solution since this solution generates many advantages as the fact to to test the Polyscale source on P2, with or without the fluidic Dolomite connector and to have the possibility to adjust the current emission source just in front the hybridizations areas)

2- Use this excitation module to detect the Rhodamine 6G or/and to follow an antibody decrease

Use the future excitation module to detect the Rhodamine 6G diluted in water or/and to follow an antibody decrease (CEA has already bought Rhodamine 6G, which has to be diluted in water) It could be apply on real spot sizes since the mechanical adaptation has been achieved.

Now, this solution became obsolete since the CEA process to detect the substance P is very robust.

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With our software, the LED current is driven with a current percentage

-Green visible light appears for 17% -From 20%, the emitted light does not increase It correspond to a current between 30 mA and 35 mA Explanation: the driver, which is used on the test platform P2, can at least deliver 700 mA when the LED threshold voltage is 2.8 V (case of the usual LED module used by CEA). The maximum voltage is 5 V. Therefore, the remaining (5-2.8)V =2.2 V allow delivering up to 700 mA. For the excitation Polyscale module: Each grating is irradiated by 2 LEDs’, which are plugged in series. Therefore, the LEDs in serial lead to a threshold voltage of 2 x 2.3 = 4.6 V The remaining 0.4 V only leads to a maximum current of 35 mA.

LED driven by the software

LED switch on

1 2 2 4 3 3 4 1

At this stage, this permutation is not a real problem.

Figure A4-1 : LED control command

λPeak ≈ 498 nm (i.e. at I ≈ 35 mA)

Figure A4-2 : LED spectrum

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Spectrum Of LED's polyscale

Plot1

Plot2

Front view

Front view

LED number = LED which is switched on

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LED switch on

LED 1 (mW) LED 2 (mW) LED3 (mW) LED 4 (mW)

1 1.5 (1.1) 0.71 1.5 1.3 2 1.35 3 (2.5) 1.3 1 3 1 0.9 1.3 (1.3) 1 4 0.4 0.26 0.7 1.6 (1.6)

(In bracket, optical power with the excitation time inside the software.

Other measurements: Continuous irradiation)

Figure A4-4 : Optical power (with the light sensor calibration at 515 nm) and cross talk evaluation

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LED 1; Po = 20 %

LED 2; Po = 20 %

LED 3; Po = 20 %

LED 4; Po = 20 %

a) Warmup time : typically 15 seconds

b) The stability of the output signal is not so good with LGP LEDs compare to the LED which currently

equip P2 It could be due to the current value, which is low during these tests.

LED 1 LED 2 LED 3 LED 4

A= Average (V)_ G=0.53 V With the LGP

0.98 0.89 0,89 0,89

SD= Standard deviation (V) With LGP

0.1729 0.0484 0.238 0.0467

SD/A 17,7% 5,5% 2,7% 5,4%

SD/A with current CEA LED (spot size = 5 mm)

≈ 0.3%

Figure A4-3: Fluorescence data versus time with the Round PMT

(PMMA slide of 1 mm with a set point at 20% for each group of 2 LEDs)

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Jean (CEA) Stephan (Polyscale) [22/07/2014] 1) We have mechanically opened the excitation module. We should be able to remove and change PCB, where the LEDs are located (cf. Figure A12-4) ==> Therefore, we are waiting for the 4 new PCB (2 in cyan, 2 in green) that you prepare Finally, CEA receive this LED at M24 meeting in Barcelona

Figure A4-4 : Mechanical support of the Polyscale excitation module

(PCB for the LED : on the side)

2) One LED has been damaged (strange: only one LED although the same current should pass through the

2 LEDs) ==> collateral damage on one side of LGP and on the small wedge (see photo A4-5).(*)

One lateral side of the LGP + the small wedge is damaged resulting from a LED failure Figure A4-5 : Mechanical support of the Polyscale excitation module + Lateral view of the LGP

3) Next steps 3a) we will replace the PCB with new one (LED connected in parallel, 2 LED per gratings) 3b) we will test the PCB electronically and optically without the LGP (to avoid the LGP damage) 3c) if everything is OK, we will add the LGP for the optical characterization 3d) and then, if possible to see what is the behavior during an antibody decrease when the PLS excitation module is used (other possibility, to detect a liquid solution with Rhodamine 6G inside)

Damage on one side of the LGP

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(*)-May be you can achieve another similar LGP since one side is slightly damaged due a LED failure

P.S. : For discussion: The cross talk between the functionalized areas might disturb the measurements (cf. photo A4-6).

Figure A4-6: More light in the grating areas but the light is everywhere an additional opaque mask with suitable openings could be a solution

The light insulation might be easier for you if the distance between the 4 functionalized areas is increased (2 gratings on the right are the same location and the 2 others close to the other side ==> symmetry center: center of the optical LGP). [If the gratings are well separated, we will have a drawback: 2 functionalized areas will far from the PMT sensor ==> unfortunately, they will lead to a higher LOD] Therefore, with a larger distance between the functionalized areas, it will be easier for you to get insulation between the various functionalized areas, For instance in sharing the current piece with 4 gratings in 4 similar smaller pieces with one grating on each. You can have "black paint" around each piece (see discussion with CETEMMSA) At that stage, it appears that if the light is mainly directed towards the functionalized areas, the excitation light can be found everywhere (cf. figure A4-6). May be smaller LGP for each guide might be the solution to irradiate the suitable zone. If this light is not redirected parallel to the PMMA light guide, a huge amount of stray light will be detected by the PMT.

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Annex 5: Additional data concerning the Polyscale source: how to decrease the stray light

With the configuration shown in figure A5-1, the PMT detects 1000 times more light through a PMMA substrate illuminated with a Polyscale source compare to lighting with the CEA emission source. To decrease the gap between these 2 measurements: 1-a cover is added on the PMT in order to put the PMT entrance and the PMMA light output in darkness. Now ratio drops from 1000 to 13 (the same cover is put with the CEA emission source and small drop is also observed).

See figure 4.1 2- A better cover is added on the Polyscale source itself (see Figure A5-2). With this cover, the ratio drops from 13 to 4 (see Figure 4.5) We have determined that the optical power density between the 2 sources is about 13 (for a Green LED), but the source size is different. Size of the Polyscale source: 5 x 5 = 25 mm

2

Size of the CEA source: 2.5 x 2.5 x (/4) ≈ 5 mm2

In taking into account the source size : It means a ratio of 4/5 = 0.8 for the optical power detected by the PMT 0.8 = optical power generated by the Polyscale source/ optical power generated by the CEA source If we use the measurements at the output of the structured area, the expected ratio is 1/13 ≈ 0.08. We expect a ratio of 1 and we have a ratio of 0.8/0.08 = 10 (i.e. with the Polyscale source, we detect with the PMT a light power that it 10 times the expected power). It is probably be due to the generation and the way of propagation of the incident light inside the LGP slide. Although, there is an optical filter in front of the PMT, this stray light is not a favorable point for such source

Figure A5-1 : a « general » protection Figure A5-2 : «full» dark protection

(Improvement)

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+fullDARkG= 0,7 V(extrapolé)

Excitation CEA+CacheDiff

+filtreG= 0,7 V

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80 %100 %

Figure A5-3 : Comparison between the Polyscale source and the CEA source

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Annex 6: How the LOD is evaluated and extrapolated if necessary? The methodology, which is presented, is questionable. But this methodology has been applied to compare the experiments together. The goal is to have an objective and quantitative criterion to determine, what are the best conditions (fluidic protocol, optical configuration, functionalization method, optical parameters…), which lead towards the smallest quantification limit (QL) but above all towards the smallest detection limit (DL). DL is smaller than QL (i.e. DL < QL). The quantification limits are evaluated during 300 seconds (i.e. for a window of 300 seconds) and the tests are typically achieved during 300 seconds. 1-First of all, the slope of the substance P (SP) is evaluated in V/s in fitting the experimental data a by linear line. The fitted data are subtracted to the experimental data. Then, the standard deviation of this result is

evaluated (P). 2-If it is possible, the slope of the buffer (SB) is evaluated in V/s in fitting the experimental data by a linear

line. If this slope is negative, SB is equal to 0 (SB =0) and B = 0.

If SB > 0, the standard deviation of SB is evaluated (B). These data are obtained on a 300 seconds window: - For the substance P - For the buffer before the substance P (i.e. the reference line) (The best case is to have the substance P diluted in the same buffer than the buffer used for the reference line)

How to evaluate the standard deviation of the difference between the 2 slopes (substance P and buffer) ?

If the 2 noises are uncorrelated: uncorrelated t = (B2 + P

2)1/2

If the 2 noises are correlated: correlated t = (B + P) > (B2 + P

2)1/2

The worst case will be assumed: the noises are correlated. The symbol for the total standard deviation is t Therefore, the quantification limit (QL) is given by: The time tQ , which is necessary to quantify the test concentration C test (i.e. the concentration used during the tests, which usually last 300 seconds) is given by:

tQ = 3 t /(Sp - SB) This time will be converted in a concentration, which represents the minimum concentration which is able to quantify in 300 seconds, according this (questionable) formula:

CQL = tQ x Ctest / ttest = 3 t /(Sp - SB) x Ctest / ttest (1) It is correct to compare various CQL when they are obtained with the same ttest and the Ctest. We have many tests where ttest = 300 seconds and Ctest = 1000 ng/ml. For instance for ttest = 300 seconds and Ctest = 1000 ng/ml, if tQ = 30 seconds, it leads to CQL =100 ng/ml When CQL is ≈ 0.1 Ctests, Ctests have to be decreased. From the formula (1), 3 CQL can be evaluated:

-CQL (SP) = 3 P/(Sp) x Ctest / ttest

only the substance P parameters are taken into account. The “buffer line” is “perfect” (SB = 0, B = 0)

-CQL (SP, B) = 3 P/(Sp -SB) x Ctest / ttest

The buffer line is almost perfect (SB , B = 0)

-CQL (real) = 3 t/(Sp -SB) x Ctest / ttest

In taking into account the buffer parameters (SB, B) The calculated concentrations are independent of the PMT gain (ratio between the standard deviation and the slope, which are obtained from data store with the same PMT gain). (In this document LOD = CQL (real))

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Annex 7: Piezolectric Bartels pump

They have been ordered in April 2014. They only arrive at CEA at the beginning of September 2014.

Figure A7-1: Bartels pump : smaller than the current CEA peristaltic pumps on P2

ADVANTAGES -VERY SMALL (ALSO for the power supply plug and play) : similar to the Dolomite pumps -R2R possible in pick and place -The flow rate is compatible with the demonstrator 2 -Self-priming -The flow rate has been tested during 45 hours and the ageing effect is almost insignificant compare to the ageing observed with the Dolomite pumps (see Figure A7-2) -Typical maximum flow rate ≈ 7 ml/min (tested alone not on P2) DRAWBACKS - The flow rate is not reversible DRAWBACK MAINLY DURING THE ADJUSTEMENT Additional test with PBS has to be done before to be implemented on P2, to determine if the salt inside the solution is a show stopper or not the salt is not show stopper Hybridization has been achieved with this pump and it works well.

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Figure A7-2: Bartels pump: Study concerning the flow rate stability with deionized (and filtered) water

Figure A7-3: Flow rate versus the voltage amplitude at constant frequency (20 Hz)

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y = 0,0071x - 0,1823 R² = 0,9944

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Figure A7-4 : Power supply and control command for Bartels pumps The company, which sells this product, continuously decreases the size of the control command

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Annex 8: Output light fluorescence versus the PMMA thickness

The output fluorescent light, from 3 PMMA slides with 3 different thicknesses, functionalized with a similar a quantity of fluorescent tripods, have been tested. The initial fluorescence, obtained with the same process, has been measured with a scanner at 532 nm. The offset (signal without the tripod fluorescence) is insignificant to compare the results except when an additional slide is used with the 500 microns thickness. The conclusion is: There is almost no difference for a PMMA thickness of 1 mm and 500 microns on P2 (96 %) On P2, the fluorescence output signal is typically of 60% with 200 microns compare to a 1 mm thickness. (Experimental data obtained with the same optical power and the same PMT and the same geometrical configuration. It is more difficult to keep the substrate flat for a 200 microns thickness It is probably the reason why have a degradation of the detected signal ?)

Plot 1 Plot 2 Plot 4

Slide Thickness Voltage

(V) Fluorescence

level (a.u.) Corrected

voltage (V) Voltage (V)

Fluorescence level (a.u.)

Corrected voltage (V)

Voltage (V) Fluorescence

level (a.u.) Corrected

voltage (V)

ML2-62-01

1 mm 1,35 32929 1,32 1,51 35025 1,14 1,88 44714 1,81

ML2-62-08

500 µm (+ 500 µm)

1,29 33653 1,29 1,49 39833 1,27 1,52 46554 1,52

500 µm 1,33 33653 1,33 1,52 39833 1,30 1,61 46554 1,61

ML2-62-07

200 µm (+1 mm)

0,76 33664 0,76 1,03 34634 0,77 1,18 39832 1,01

200 µm 0,74 33664 0,74 1,05 34634 0,78 1,16 39832 0,99

Final result

Ratio [500 µm/1mm] 0,96

Ratio [200 µm/1mm] 0,59 probably due a bending effect (see the comment below)

Figure A8: Output light fluorescence versus the PMMA thickness (Functionalized areas with tripod for the substance P)

Now, we have shown that, with 160 microns PMMA substrate (location of the hybridization areas) associated with a 375 microns, the LOD remains stable compare to a 500 microns or a 1000 microns PMMA substrate. Therefore, we are convinced that he ratio 200 microns/1 mm reported in figure A8 is due to the light guide bending. It is true that a thinner substrate could be easily bent and decoupled the light before reaching the PMT. But we do not see any other objective reason, why a thinner substrate does not give the same level of output fluorescent light. Indeed, the guiding light is achieved by total internal reflection (under the assumption that the substrate quality and specially the surface roughness is independent of the substrate thickness).

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Annex 9 Report 1 concerning the comparison between IQS and CEA surface preparation

(Batch in November/December 2014)

ML² Project Report on roll-to-roll compatible chemical functionalization

Date : 2014/05/14 Revision :

N / Ref. : V / Ref :

Participants: G. Nonglaton, C. Fontelaye, J. Hue

Name Function Date Version

Editor(s): G. Nonglaton Surface chemistry Responsible

2014/05/14

Examine and approve: J. Hue CEA Project Leader

2014/05/27

Diffusion list:

Confidential

Report


FORMER EVENTS

Nature of the modifications Date Version Corrections by JH Corrections by G. Marchand

14/06/03 14/06/04

V02 V03

CONTENTS

In order to graft amino-modified biomolecules on plastic substrates, we have evaluated different solutions compatible with roll-to-roll process:

1. The efficiency of a glutaraldehyde modified PolyEthylenImine layer (CEA). 2. The grafting of amino-modified tripode on plasma activated PMMA by coupling

with EDC (CEA) 3. The IQS solution using pentafluorophenyl activated ester.

The grafting of amino-modified biomolecules was measured by fluorescence.

KEY WORDS

Surface chemistry, peptide immobilization, fluorescence, PMMA, roll-to-roll, displacement immunoassay

CONTENTS

1 Introduction 63

2 Material and methods 64

2.1 Protocols for chemical grafting 64

2.2 Tripod spotting 64

2.3 Static antibodies incubation 65

2.4 Static substance P incubation 65

2.5 Surface characterisation 65

3 Results and discussion 65

3.1 PEI/GA protocol 65

3.2 PFM protocol 69

3.3 The EDC/NHS coupling 71

3.4 Displacement immunoassay on PMMA Erreur ! Signet non défini.

4 Conclusion 76

5 References 77

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1 Introduction In order to chemically modify the plastic substrates, we have evaluated different protocols. The PEI-GA protocol was inspired from a publication of Feyssa et al.

1 First, a polyethyleneimine (PEI) layer was

deposited onto oxygen plasma activated plastic substrate and further cross-linked with glutaraldehyde (GA) to provide an amine-reactive aldehyde surface (Figure 1). Then, the aldehyde functions react with the amine function of terminal lysine of the peptidic tripod leading to a stable Schiff base. Thus the peptidic tripod is immobilized.

Figure 1. Schematic of the peptidic tripod immobilization using PEI-GA modified surface

The EDC/NHS coupling is inspired from a publication of Lee et al.2 First, the plastic substrate is activated

with oxygen plasma to create hydroxyl and carboxyl group.3 Then, the amino-modified biomolecules are

immobilized via the coupling of amino groups with carboxyl groups using the well-known carbodiimide EDC in combination with N-hydroxysuccinimide (NHS).

4

Figure 2. Schematic of the peptidic tripod immobilization using of EDC/NHS coupling

An optimized version of this protocol was developed without NHS.

Figure 3. Schematic of the peptidic tripod immobilization using of EDC/NHS coupling

The PFM protocol was developed and proposed by Borròs et coll. from IQS.5 A pentafluorophenyl

methacrylate (PFM) polymer is deposited by plasma polymerization (Figure 4) or spray-coating (Figure 5). This polymer with activated ester is used to immobilize the amino-modified molecule. The reaction between an ester and an amine leads to a stable amide function.

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Figure 4. Schematic of the peptidic tripod immobilization using plasma polymerized PFM surface

Figure 5. Schematic of the peptidic tripod immobilization using spray coated PFM surface

These protocols were chosen to be integrated in a roll-to-roll process. They were evaluated for the grafting of biomolecules. The grafting of fluorescent amino-modified peptidic tripod is revealed by reading the fluorescence with a scanner.

2 Material and methods Glass slides are commercially available. COC substrates are provided by TOPAS (5013 grade, thickness = 1010 µm – 1120 µm). PMMA substrates are supplied by Polyscale (Mitsubishi batch: July 2013, thickness

≈ 1040 m). PC substrates by Polydis (thickness ≈ 1000 µm)

2.1 Protocols for chemical grafting

2.1.1 PEI-GA protocol

The substrate is activated using remote RF oxygen plasma (200 sccm O2 flow, 450 W, 300 s) with commercial equipment (MVD100 from Applied MST, San José, US). Polyethylenimine 50% (w/v) aqueous solution (PEI) and glutaraldehyde (GA) solution (Grade II, 25% in H2O) was purchased from Sigma Aldrich. PEI solution was prepared as follows: 100 mg/mL aqueous PEI was diluted with PBS1x to obtain 20 mg/mL of PEI solution. Substrates were then left in PEI solution for 20 min at ambient temperature, before being rinsed in phosphate buffered saline 1x (the resultant 1x PBS has a final concentration of 10 mM PO4

3-, 137

mM NaCl, and 2.7 mM KCl) and water for 5 min each and dried under nitrogen. Substrates were then left in GA solution for 20 min at ambient temperature, before being rinsed in water for 5 min and dried under nitrogen.

2.1.2 EDC/NHS coupling

The substrates are activated using remote RF oxygen plasma (200 sccm O2 flow, 450 W, 300 s) with commercial equipment (MVD100 from Applied MST, San José, US). N-hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was purchased from Sigma-Aldrich.

2.1.3 PFM protocol

The substrates are coated by IQS according to their protocol.

2.2 Tripod spotting

NH2-peptidic tripod were used (IRCOF, LQ14 : Ac-Lys-(R6G-isonip-WS)-Ser-Ser-Lys-Arg-Pro-Ala-Pro-Gln-Gln-Phe-Phe-Gly-Ala-Met-NH2 and LQ12 : Ac-Lys-Ser-Ser-Lys(Dde)-Arg-Pro-Ala-Pro-Gln-Gln-Phe-Phe-Gly-Ala-Met-NH2). LQ14 is used as the reference tripod (equipped with fluorescent dye). LQ12 is used as a control tripod (without fluorescent dye). The tripod immobilisation was performed by spotting using a commercial ultra-low volume dispensing system (SciFlexarrayer S3 from Scienion AG, Berlin, Germany). A dispense capillary, PDC2030 from Scienion, was used.

For PEI-GA protocol two patterns of 10 x 10 spots containing 5 drops of 300 pL of the solution containing 10 µM LQ14 tripod in HCOOK 2M (Sigma Aldrich, 99%) and two patterns of 10 x 10 spots with a distance between each spot of 250 um, containing 5 drops of 300 pL of the solution

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containing 10 µM LQ12 tripod in HCOOK 2M were spotted on slide and left overnight at 4 °C. The slides were quickly rinsed with DI water, washed with a 0.2% SDS solution for 5 min under gentle agitation, quickly rinsed with DI water and finally washed with DI water for 5 min under gentle agitation before drying by centrifugation. The typical spot diameter is about 200 um.

For EDC/NHS coupling two patterns of 10 x 10 spots containing 5 or 10 drops of 300 pL of the solution containing 10 µM LQ14 tripod, 10 mM NHS, 5 mM EDC in 50mM MES buffer and two patterns of 10 x 10 spots with a distance between each spot of 250 um, containing 5 drops of 300 pL of the solution containing 10 µM LQ12 tripod, 10 mM NHS, 5 mM EDC in 50mM MES buffer were spotted on slide and left 15 or 60 min at room temperature or 37°C. The slides were quickly rinsed with DI water, washed with a 0.2% SDS solution for 5 min under gentle agitation, quickly rinsed with DI water and finally washed with DI water for 5 min under gentle agitation before drying by centrifugation. The typical spot diameter is about 250 um.

2.3 Static antibodies incubation

The recognition with antibodies was performed in PBS 1X at different concentrations. The tripods were incubated with this mixture using a Geneframe sealing system and a cover slip was placed over the solution. The slides were left at room temperature for 1 h. Then after a washing step with PBS 1x + Tween 20 0.1% and PBS 1X (5 min each under orbital shaking), the slides were spin dried and finally analysed by fluorescence.

2.4 Static substance P incubation

The recognition with substance P was performed in PBS 1X at different concentrations. The tripods were incubated with this mixture using a Geneframe sealing system and a cover slip was placed over the solution. The slides were left at room temperature for 1 h. Then after a washing step with PBS 1X + Tween 0.1% and PBS 1X (5 min each under orbital shaking), the slides were spin dried and finally analysed by fluorescence.

2.5 Surface characterisation

2.5.1 Contact angle

Contact angle measurements were obtained using a sessile drop method. Drop of water with a volume of 1µL is deposited at three different locations of each sample at ambient temperature. The angle formed between the liquid/solid interface and the liquid/vapor interface is measured using a commercial drop-shape analysis system (Digidrop, GBX instrument).

2.5.2 Fluorescence

Fluorescence measurements were performed with a microarray scanner (LS ReloadedTM

from TECAN, Männedorf, Switzerland) with a spatial resolution of 6 µm. This scanner is equipped with two channels for simultaneous scanning with two lasers (Cy3 and Cy5). Substrates functionalised with Cy3-labelled biomolecules were scanned at 532 nm. The values of Focus Gain and PMT were usually fixed at respectively 140 and 110. Then raw data were analysed using GenePix software.

3 Results and discussion

3.1 PEI/GA protocol

The PEI/GA protocol is composed of 3 steps: 1) Treatment by oxygen plasma, 2) PEI coating 3) GA coating.

By measuring the contact angle with a drop of water, we are able to monitor the modification of the physicochemical surface properties. Bare 1000 µm and 1500 µm PMMA and 750 µm and 1000 µm PC slides are mostly hydrophobic and contact angles of 77± 3°, 85± 3°, 95± 3°, and 91± 3° were respectively measured (Figure 6Erreur ! Source du renvoi introuvable.). These measurements are consistent with

contact angle values of PMMA and PC substrates found by Rucker et al.6 After oxygen plasma, a

significant decrease was observed with a contact angle of 29±1°, 27±1°, 12±1°, 11±1° respectively on 1000 and 15000 PMMA and 750 and 1000 PC. These measurements are also consistent with contact angle values of PMMA and PC substrates after oxygen plasma found in the literature.

7 After PEI coating,

the water contact angles tend to slightly increase around 30°. PEI surfaces are known be to be rather

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hydrophilic. And 30° is consistent to PEI surfaces.8 After GA coating, the water contact angles continue to

increase to 40°.

Figure 6. Contact angle

After the tripod spotting, the fluorescence of the fluorescent tag rhodamine-6G labeling the tripod is evaluated with a scanner at 532nm. We compare the fluorescence obtained with the PEI/GA protocol on PC and PMMA substrates with the fluorescence obtained with the CEA2 protocol (see report 01 functionalization) on glass slide. The results are shown in Figure 7. PMMA gives 2.5 to 4 times more fluorescence than PC. Nevertheless the fluorescence measurements are 1.5 to 2 times less intense with PMMA than with GS (glass slide Gold seal).

If we compare the fluorescence images of GS coated with CEA2 and PMMA coated with PEI/GA, we observe a higher background of PEI/GA coated PMMA than CEA2 coated GS. We have compared the fluorescence images of GS and PMMA each coated with PEI/GA (Figure 8). The background and spot fluorescence intensities are similar.

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Figure 7. Fluorescence intensities on PC and PMMA substrates using PEI/GA protocol and glass slide coated

with CEA2 chemistry

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Figure 8. Fluorescence intensities on PMMA substrates and glass slide coated with PEI/GA layer

The results demonstrate that background fluorescence is not due to the substrate but it is due to PEI/GA coating layer. We have tested different solutions to decrease the fluorescence background: 1) a treatment with sodium borohydride (NaBH4) to reduce the Schiff base formed after the reaction with amines and aldehydes to amine,

9

2) a Bovin Serum Albumine (BSA) solution or powdered non-fat milk as blocking agents to avoid the non-specific adsorption of fluorescent tripod (Figure 9).

After NaBH4 reduction treatment, the background signal strongly decreases but in the meantime the signal decreases. BSA or powdered non-fat milk passivation treatment has no significant effect. Finally, we have tested the localization of PEI by spotting instead of dipping method. The results do not improve the signal to noise ratio ((S-B)/B). We have compared the background fluorescence of a bare PMMA substrate with PEI/GA coated PMMA substrate: the PEI/GA coated is about 50 times more fluorescent. In order to improve the fluorescence intensity of the spots, we have evaluated the effect of the concentration of tripod (from 0.1nM to 100µM) and the effect of the number of drops per spot. After

PMMA GS

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spotting, the substrates are treated with NaBH4 and then the fluorescence is observed. The fluorescence image is reported in Figure 10. The signal to noise ratio as function of the tripod concentration and number of drops is reported in Figure 11.

Figure 11. Signal to noise ratio as function of the tripod concentration and number of drops

In Figure 10 and Figure 11, a significant effect of the concentration and the number of drops per spots is observed. The more concentrated solution of tripod we use the higher fluorescence intensities we get. The same result is observed in increasing the number of drops. Nevertheless a saturation effect at signal to noise ratio of 50 is clearly observed after 50µM. It seems that above the number of drops or the tripod concentration, we cannot get more fluorescence than this ratio. This can be explained by a quenching of the fluorescence due to the proximity of the Rhodamine carried by the immobilized tripod. The effect of PEI concentration on the fluorescence was also evaluated (Table 1). The images show that the fluorescence decreases when less concentrated PEI solution is used.

Table 1. Fluorescence images of PEI/GA coated PMMA with various concentration of PEI.

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3.2 PFM protocol

The PFM protocol simply consists in a coating step with a pentafluorophenyl activated methacrylate polymer on substrate. This polymer was initially deposited by plasma polymerization (pp-PFM). Then IQS has developed a spray coating deposition process (spray-PFM).

3.2.1 Plasma polymerization process

First, we compared the fluorescence obtained with pp-PFM protocol on PMMA substrates with the fluorescence obtained with the CEA2 protocol on glass slide. A 10µM tripod solution is spotted on pp-PFM coated PMMA and after rinsing the fluorescence of the rhodamine-6G tag is read using a scanner at 532nm. The results on several PMMA are shown in Figure 12. The fluorescence measurements are about 4 times less intense with pp-PFM coated PMMA than with CEA2 coated GS.

For pp-PFM, we observe strong background fluorescence. This background is mainly due to the PFM polymer coating as it is observed in the case of PEI/GA protocol. Indeed, we have compared the background fluorescence of a bare PMMA substrate with PFM coated PMMA substrate: the PFM coated is more than 20 times more fluorescent (Figure 13). After spotting the fluorescent tripod, the fluorescence slightly increases. As suggested by IQS, we dipped the PMMA substrate in amino ethanol solution as a blocking agent. Unfortunately, this treatment has no significant effect on background fluorescence.

Figure 12. Fluorescence intensities of PFM coated PMMA and CEA2 coated glass slides

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In conclusion, the fluorescence intensities on pp-PFM PMMA are relatively low compare to our glass standard. Moreover, the background is significantly higher. It is a drawback for the limit of detection. Finally, due to the fact that the plasma polymerization process is not compatible with a roll-to-roll process since this process needs vacuum, IQS decided to stop this way and developed a spray coating deposition process (spray-PFM).

3.2.2 Spray coating process

The spray coating process was evaluated with polycarbonate (PC) and polymethyl-methacrylate (PMMA) substrate. A 50 µM tripod solution is spotted on spray-PFM coated PC and PMMA and after rinsing the fluorescence of the rhodamine-6G tag is read with a scanner at 532nm (Figure 14). In using a spray coating process, it leads to 4 times more fluorescence on PMMA than on PC. The background is about 7 times less important with PMMA than PC.

Figure 13. Background fluorescence of PMMA

substrates

Figure 14. Fluorescence intensities of spray-PFM coated PC and PMMA

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In comparison with pp-PFM on PMMA, the spray-PFM protocol gives 6 times more fluorescence (Figure 15). In addition, the noise of spray-PFM PMMA is about 10 times lower than pp-PFM PMMA. This result was confirmed with a second batch of pp-PFM coated PMMA. Furthermore, if we compare spray-PFM PMMA with CEA2 GS protocol, the fluorescence intensities is slightly higher and the noise is similar. We have tried to increase the fluorescence by spotting a 100 µM tripod solution instead to 50 µM, but the fluorescence was divided by 1.7. This result is probably due to a quenching effect due to the proximity of the dyes. In conclusion, the spray-PFM protocol developed by IQS is a good candidate for the PMMA functionalization to immobilize the peptidic tripod.

3.3 The carbodiimide coupling

3.3.1 The carbodiimide coupling with N-hydroxysuccinimide additive (EDC/NHS coupling)

The simplicity of this protocol and its compatibility with a R2R is interesting. Indeed, the activation by oxygen plasma is perfectly compatible with a roll-to-roll process. Another advantage is the absence of chemical coating, which gives some fluorescence background. The oxygen plasma on PMMA creates carboxylic groups at the surface. These groups can be coupled with amine using EDC/NHS.

Figure 16.Principle of EDC/NHS coupling

Figure 15. Comparison between spray-PFM and pp-PFM PMMA and CEA2-GS protocols

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EDC is working in the 4.0-6.0 pH range. EDC reacts with carboxylic acid to yield activated ester. N-hydroxysuccinimide (NHS) will react with the activated ester reacts to create a more reactive amine- product. Then the amino modified peptidic tripod will easily react to form the amide bond. Different conditions were evaluated for the EDC/NHS coupling: with or without oxygen plasma activation, 15min or 60 min of immobilization, at 25°C or 37°C. We compared the fluorescence obtained after spotting the peptidic tripod. The results are plotted in Figure 17.

Figure 17. Fluorescence intensities of tripod immobilized on PMMA by coupling

The fluorescence intensities are more than 2 times higher with oxygen plasma activation than without activation. There is no significant effect of temperature and time. More experiments should be performed to reduce the time. Different types of buffers were tested (Figure 18). MES buffer (4-morpholino-ethanesulfonic acid) is a suitable reaction buffer for EDC/NHS coupling. Phosphate buffers like PBS 1mM are compatible with the reaction chemistry. Buffers with amine function like Tris 0.1M (tris(hydroxymethyl)methylamine) or carboxylic function like citrate 0.1M or carbonate 0.1M have also been tested.

Figure 18. Effect of buffer

If we compare the results obtained, we observe that PBS 1mM pH7.4 gives higher fluorescence than MES 50mM pH4.5 and the other buffers. Tris 0.1M and carbonate 0.1M buffers give the lowest fluorescence, possibly due to their pH (respectively 8.0 and 9.6) and the presence of amine and carboxylic function. In spite of the presence of carboxylic function, Citrate 0.1M pH5.5 buffer gives slightly more fluorescence than MES 50mM pH4.5 buffer. Theoretically, the reaction with EDC and NHS is most efficient at pH 4.5-7.2 and

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the pH of the MES buffer is maybe too low. An evaluation of different pH MES buffers should be done in the future. In parallel, we have investigated the effect of the addition of MeOH, NaCl or BSA (Figure 19).

Figure 19. Effect of the buffer composition

Addition of 0.15M NaCl leads to the best results. Addition of 3% MeOH and 1% BSA allows also increasing the fluorescence. But when 1% BSA and 0.15M NaCl are added, the fluorescence slightly decreases. Finally, we look at the concentration of the tripod solution and the number of drops (Figure 20). As expected, 100µM with 3 drops gives the best fluorescence. Due to the tripod cost, we did not tried more concentrated than 100µM.

Figure 20. Effect of concentration of tripod solution and number of drops

Thus we have determined the best conditions of spotting (3 drops, 100µM in PBS1x at 25°C during 15min) on PMMA substrate activated with oxygen plasma. At the same time, we have investigated the functionalization of COC with a direct coupling (Figure 21).

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Figure 21. Fluorescence images and intensities of PMMA and COP substrates spotted with fluorescent tripod (LQ14) and non-

fluorescent tripod (LQ12).

The fluorescence signal is comparable on PMMA and on COP but the background fluorescence is 5 times lower.

3.3.2 Immunoassay using GeneFrame

We have tested the displacement immunoassay on PMMA. After the immobilization of tripod, the substrates are incubated with antibodies, followed by a second incubation with substance P. After each step, the fluorescence intensities are measured. We have tested different concentrations of tripod (1, 10 and 100µM) and two concentrations of antibody (25 and 50µg/mL). The results are reported in Figure 22 and Figure 23. The antibody incubation leads to a fluorescence decrease due to the dye quenching. The substance P is detected when the fluorescence increases.

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Figure 22. Fluorescence intensities at different steps of the displacement immunoassay for 50µg/mL of Ab

Figure 23. Fluorescence intensities at different steps of the displacement immunoassay for 25µg/mL of Ab

For 50µg/mL of antibodies the substance P is clearly detected. It is not the case with only with 25µg/mL.

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3.3.3 Immunoassay using ML2 platform

We have tested the displacement immunoassay on PMMA in using the ML2 platform P2 (or P1). We have also determined a limit of detection (LOD) for a circulation of substance P during 300 s for different protocols. Few results are compiled in Erreur ! Source du renvoi introuvable..

Table 2. Results coming from the ML2 platform

Protocol Roll-to-roll compatible

Detection on geneFrame

Detection on ML2 platform

Limit of Detection

1 CEA2-GS No Yes Yes 2 PEI/GA-PMMA Yes No NA 3 ppPFM-PMMA No No NA 4 sprayPFM-PMMA Yes NA Yes 5 Coupling-PMMA Yes Yes Yes 50 ng/mL

4 Conclusion If we compare the signal-to-noise ratio obtained with the three different functionalization protocols on PMMA (PEI/GA, pp-PFM, spray-PFM and coupling), we can see that the direct coupling and spray-PFM gives the best results (Figure 24).

Figure 24. Comparison on the different functionalization protocols

More investigation should be performed to optimize the conditions on flat PMMA substrates.

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5 References 1. Feyssa, B.; Liedert, C.; Kivimaki, L.; Johansson, L.-S.; Jantunen, H.; Hakalahti, L., Patterned Immobilization of Antibodies within Roll-to-Roll Hot Embossed Polymeric Microfluidic Channels. PLoS ONE 2013, 8 (7), e68918. 2. Lee, J. H.; Choi, H. K.; Chang, J. H., Optimization of biotin labeling of antibodies using mouse IgG and goat anti-mouse IgG-conjugated fluorescent beads and their application as capture probes on protein chip. J. Immunol. Methods 2010, 362 (1-2), 38-42. 3. Chai, J. N.; Lu, F. Z.; Li, B. M.; Kwok, D. Y., Wettability interpretation of oxygen plasma modified poly(methyl methacrylate). Langmuir 2004, 20 (25), 10919-10927. 4. (a) Piehler, J.; Brecht, A.; Geckeler, K. E.; Gauglitz, G., Surface modification for direct immunoprobes. Biosens Bioelectron 1996, 11 (6-7), 579-590; (b) Yang, J. M.; Lin, H. T.; Lai, W. C., Properties of modified hydroxyl-terminated polybutadiene based polyurethane membrane. J. Membr. Sci. 2002, 208 (1-2), 105-117; (c) Zhang, S.; Bian, Z.; Gu, C.; Zhang, Y.; He, S.; Gu, N.; Zhang, J., Preparation of anti-human cardiac troponin I immunomagnetic nanoparticles and biological activity assays. Colloids Surf., B 2007, 55 (2), 143-148; (d) Jackeray, R.; Zainul Abid, C. K. V.; Singh, G.; Jain, S.; Chattopadhyaya, S.; Sapra, S.; Shrivastav, T. G.; Singh, H., Selective capturing and detection of Salmonella typhi on polycarbonate membrane using bioconjugated quantum dots. Talanta 2011, 84 (3), 952-962. 5. Cifuentes, A.; Borros, S., Comparison of Two Different Plasma Surface-Modification Techniques for the Covalent Immobilization of Protein Monolayers. Langmuir 2013, 29 (22), 6645-6651. 6. Rucker, V. C.; Havenstrite, K. L.; Simmons, B. A.; Sickafoose, S. M.; Herr, A. E.; Shediac, R., Functional Antibody Immobilization on 3-Dimensional Polymeric Surfaces Generated by Reactive Ion Etching. Langmuir 2005, 21 (17), 7621-7625. 7. Tang, L.; Lee, N. Y., A facile route for irreversible bonding of plastic-PDMS hybrid microdevices at room temperature. Lab Chip 2010, 10 (10), 1274-1280. 8. (a) Dacarro, G.; Cucca, L.; Grisoli, P.; Pallavicini, P.; Patrini, M.; Taglietti, A., Monolayers of polyethilenimine on flat glass: a versatile platform for cations coordination and nanoparticles grafting in the preparation of antibacterial surfaces. Dalton Trans. 2012, 41 (8), 2456-2463; (b) Yan, L.; Huck, W. T. S.; Zhao, X.-M.; Whitesides, G. M., Patterning Thin Films of Poly(ethylene imine) on a Reactive SAM Using Microcontact Printing. Langmuir 1999, 15 (4), 1208-1214. 9. Xia, B.; Dong, C.; Lu, Y.; Rong, M.; Lv, Y.-z.; Shi, J., Preparation and characterization of chemically-crosslinked polyethyleneimine films on hydroxylated surfaces for stable bactericidal coatings. Thin Solid Films 2011, 520 (3), 1120-1124.

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