OPTICAL DETECTION METHOD

Abstract

A process for detecting the sensitivity of one or more polymers and/or of one or more mixtures of polymers to a compound, including the steps of exposing a plurality of individualized micro-deposits including the polymer(s) and/or the mixture(s) of polymers to the compound, and detecting, by interferometry, a variation in appearance of an assembly of micro-deposits exposed to the compound and or a variation in the dimensions and/or refractive index of at least one of the micro-deposits exposed to the compound, linked to an interaction between the polymer(s) and or the mixture(s) of polymers and the compound.

Claims

1. A process for detecting sensitivity of one or more polymers and/or of one or more mixtures of polymers to a compound, including the steps consisting in: exposing a plurality of individualized micro-deposits including the polymer(s) and/or the mixture(s) of polymers to the compound, and detecting by interferometry a variation in the appearance of a set of micro-deposits exposed to the compound and/or variation in the dimensions and/or refractive index of at least one of the micro-deposits exposed to the compound, linked to an interaction between the polymer(s) and/or the mixture(s) of polymers and the compound.

2. The process as claimed in claim 1, the thickness of the micro-deposits being chosen to allow the formation of an interference pattern when illuminated with a suitable light.

3. The process as claimed in claim 1, wherein a largest dimension of the micro-deposits being between 10 m and 100 m.

4. The process as claimed in claim 1, the thickness of each micro-deposit passing through only one maximum.

5. The process as claimed in claim 1, each micro-deposit having a convex outer surface.

6. The process as claimed in claim 1, detection by interferometry of a variation in the volume of at least one micro-deposit being performed, this variation in volume taking place under the effect of an interaction between the polymer(s) and/or the mixture(s) of polymers and the compound.

7. The process as claimed in claim 6, the detection being performed after magnification of the interference pattern.

8. The process as claimed in claim 1, including the reconstruction of the volume of the micro-deposit from the localization of the interference fringes.

9. The process as claimed in claim 1, the micro-deposits being arranged in the form of raster dots.

10. The process as claimed in claim 1, the micro-deposits being arranged in macro-patterns.

11. The process as claimed in claim 1, the micro-deposits being located on a reflective support.

12. The process as claimed in claim 1, the micro-deposits being located on a hydrophobic support.

13. The process as claimed in claim 1, the compound being an enzyme.

14. The process as claimed in claim 1, the polymer(s) being biopolymers.

15. The process as claimed in claim 1, the micro-deposits including a resin.

16. A device for performing the process according to claim 1, including: a support, a plurality of individualized micro-deposits borne by the support, the thickness of the micro-deposits being chosen to allow the formation of an interference pattern when illuminated with a suitable light, each micro-deposit including at least one polymer.

17. The device as claimed in claim 16, the thickness of the micro-deposits being between 100 nm and 1500 nm.

18. The device as claimed in claim 16, the largest dimension of the micro-deposits being between 10 m and 100 m.

19. The device as claimed in claim 16, the thickness of each micro-deposit passing through only one maximum.

20. The device as claimed in claim 16, each micro-deposit having a convex outer surface.

21. The device as claimed in claim 16, the micro-deposits being arranged in the form of raster dots.

22. The device as claimed in claim 16, the micro-deposits being arranged in macro-patterns.

23. The device as claimed in claim 16, the support being reflective.

24. The device as claimed in claim 16, the support being hydrophobic.

25. The device as claimed in claim 16, the polymer(s) being degradable with an enzyme or a set of enzymes.

26. The device as claimed in claim 16, the polymer(s) being biopolymers.

27. The device as claimed in claim 16, the micro-deposits including a resin.

28. An assembly including a device as defined in claim 16 and a compound interacting with said polymer to lead to a variation in the dimensions and/or refractive index of the micro-deposits.

29. A process for manufacturing a device as defined in claim 16, including the deposition by inkjet printing onto the support of dots of an ink containing the polymer(s) to form the micro-deposits.

30. An ink for performing the process as claimed in claim 29, including: polymers, a binder, and an organic cosolvent chosen to allow the formation of a convex-shaped micro-deposit during drying of the ink.

Description

[0164] The invention may be understood more clearly on reading the detailed description that follows, of nonlimiting examples of implementation thereof, and also on examining the attached drawing, in which:

[0165] FIG. 1 represents in top view an example of a device according to the invention,

[0166] FIG. 2 represents schematically in enlarged scale a macro-pattern,

[0167] FIG. 3 represents a detail of the formation of the micro-deposits,

[0168] FIG. 4 illustrates the formation of interference fringes on the micro-deposits,

[0169] FIG. 5 illustrates the use of the device for detecting a hydrolysis reaction,

[0170] FIG. 6 illustrates the effect of the compound on the micro-deposits,

[0171] FIGS. 7 and 8 illustrate enzymatic activity tests performed using devices according to the invention, and

[0172] FIG. 9 is a comparison of the enzymatic activity at various incubation durations, obtained using a device according to the invention.

[0173] FIG. 1 shows an example of a device 1 according to the invention, including a support 10 which is, for example, a silicon wafer that has been made hydrophobic, on which are formed macro-patterns 20 in the form, for example, of squares placed with respective spacings D.sub.X and D.sub.Y in directions X and Y, corresponding to those of the wells of a conventional well plate.

[0174] The side D of a macro-pattern 20 measures, for example, between 3 and 5 millimeters and the values D.sub.X and D.sub.Y are, for example, between 3 and 5 mm also.

[0175] Each macro-pattern 20 which has a millimetric size is formed from a raster of micro-deposits 21, these micro-deposits being close enough to give with the naked eye a uniform appearance to each macro-pattern 20. For example, the separation d.sub.x in the X direction and the separation d.sub.y in the Y direction are between 50 and 340 micrometers, better between 50 and 100 micrometers, the diameter d of a micro-deposit 21 being, for example, between 10 and 100 micrometers and its height h (which corresponds to the thickness) between 100 nm and 1500 nm.

[0176] In the example under consideration, the micro-deposits 21 are deposited by inkjet printing with a uniform spacing. The ink has a formulation that is suited to the formation of a convex outer surface.

[0177] The micro-deposits 21 allow, due to the difference in refractive index with the support 10, and also to the reflective nature of the support 10, the formation of interference fringes such as Newton rings, as illustrated in FIG. 4. These interference patterns may be observed by microscope by illuminating the device 1 with white or monochromatic light.

[0178] The device 1 may be used by depositing on each macro-pattern 20 a drop G of a test compound, as illustrated in FIG. 5. Depending on whether or not a degradation reaction of the polymer by hydrolysis takes place, the change in appearance of the macro-pattern 20 may be observed with the naked eye.

[0179] FIG. 5 shows at the top in the center the situation in which there is no reaction between the enzyme and the polymer, the hydrolysis reaction being zero, and at the bottom on the left the case where the hydrolysis is partial, or even total at the bottom on the right, which allows detection of the enzyme. This qualitative approach may be completed by a quantitative approach by observation of the interference patterns.

[0180] Specifically, in the case of a partial hydrolysis, the modification of the interference patterns which may be detected by interferometry makes it possible to calculate the volume of the micro-deposit degraded by the hydrolysis reaction. In the case of a total hydrolysis, the micro-deposits are completely destroyed.

EXAMPLES

Example 1

[0181] Manufacture of a Detection Device

[0182] A device according to the invention for testing various enzymes or mixtures of enzymes with respect to a polymer was prepared according to the manufacturing process described above.

[0183] First Step: Preparation of the Ink

[0184] A first aqueous ink formulation suitable for inkjet printing was prepared according to the specificities below, in which the polymer used is arabinoxylan:

TABLE-US-00002 Mono-polymer ink (formulation 1) Variant 1 Variant 2 Arabinoxylan Megazyme-wheat 5 mg/mL 3 mg/mL Arabinoxylan-Medium viscosity 22 cSt-323 Kd molecular weight Melamine formaldehyde (resin) 0.025 mg/mL 0.015 mg/mL Resimene AQ-7551 sold by INEOS Melamines DMSO CAS 67-68-5 10% vol 10% vol Poly(allylamine) hydrochloride 0.004 mg/mL 0.004 mg/mL PAH sold by Polyscience, Mw-120000-200000 g/mol Hydrochloric acid HCl 0.01 mM 0.01 mM

[0185] The viscosity of the ink obtained according to variant 1 of formulation 1 is 17 mPa.Math.s (at the ejection temperature of 20-30 C.) and the surface tension is of the order of 60 N/m (at the ejection temperature).

[0186] In the case of variant 1 of formulation 1, the polymer/resin ratio is 200 (5/0.025).

[0187] In the case of variant 2 of formulation 1, the polymer/resin ratio is also 200 (3/0.015).

[0188] Below, a second formulation in which the polymer used is beta-glucan, and a third formulation in which the polymers used are arabinoxylan and beta-glucan (ink with a 50%/50% mixture of polymers), are proposed as illustration of the invention:

TABLE-US-00003 Mono-polymer ink (formulation 2) Variant 1 Variant 2 Beta-glucan 7 mg/mL 3 mg/mL Melamine formaldehyde (resin) 0.025 mg/mL 0.015 mg/mL Resimene AQ-7551 sold by INEOS Melamines DMSO CAS 67-68-5 10% vol 10% vol Poly(allylamine) hydrochloride 0.004 mg/mL 0.004 mg/mL PAH sold by Polyscience, Mw-120000-200000 g/mol Hydrochloric acid HCl 0.01 mM 0.01 mM

[0189] In the case of variant 1 of formulation 2, the polymer/resin ratio is 280 (7/0.025).

[0190] In the case of variant 2 of formulation 2, the polymer/resin ratio is also 200 (3/0.015).

TABLE-US-00004 Multi-polymer ink (formulation 3) Variant 1 Variant 2 Arabinoxylan Megazyme-wheat 2.5 mg/mL 1.5 mg/mL Arabinoxylan-Medium viscosity 22 cSt-323 Kd molecular weight Beta-glucan 2.5 mg/mL 1.5 mg/mL Melamine formaldehyde (resin) 0.025 mg/mL 0.015 mg/mL Resimene AQ-7551 sold by INEOS Melamines DMSO CAS 67-68-5 10% vol 10% vol Poly(allylamine) hydrochloride 0.004 mg/mL 0.004 mg/mL PAH sold by Polyscience, Mw-120000-200000 g/mol Hydrochloric acid HCl 0.01 mM 0.01 mM

[0191] In the case of variant 1 of formulation 3, the polymer/resin ratio is 200 ((2.5+2.5)/0.025).

[0192] In the case of variant 2 of formulation 3, the polymer/resin ratio is also 200 ((1.5+1.5)/0.015).

[0193] Second Step: Modification of the Support

[0194] A silicon wafer is used as support.

[0195] In order to obtain convex-shaped deposits and to avoid spreading of the droplets, the upper face of the wafer is treated with C.sub.4F.sub.8 to be made hydrophobic.

[0196] Third Step: Inkjet Printing

[0197] The ink prepared beforehand according to variant 1 of formulation 1 was loaded into an inkjet printer and ink dots are printed on the support, as raster dots.

[0198] The printing parameters are: [0199] ink ejection temperature: room temperature (20 C.-30 C.), [0200] ejection volume of the droplets (per micro-deposit): 65 pL 5 pL, [0201] micro-deposit thickness: less than 1 m, [0202] ejection speed of the droplets: 5 to 6 m/s, and [0203] macro-pattern: square matrix with a side length of 4 cm with spacing between two micro-deposits in the macro-pattern equal to 70 m

[0204] Fourth Step: Thermal Crosslinking of the Ink Deposits

[0205] The support onto which the liquid ink dots were deposited is placed at 130 C. for 10 to 180 minutes until crosslinking of the resin and solidification of the micro-deposits are complete.

[0206] Use of the Detection Device

[0207] The device thus manufactured was used to study the kinetics of degradation of the polymer present in the deposits, namely arabinoxylan (AX), by enzymatic solutions of commercial xylanase each having a different level of activity.

[0208] First, the device was immersed in water at 37 C. for 2 hours, and then immersed in an aqueous solution of BSA (bovine serum albumin) at 0.25 g/L at 37 C. for 1 hour, in order to passivate the surface between the polymer deposits, which improves the measurement sensitivity.

[0209] In this example, the protein used as passivation agent is BSA. However, the invention is not limited to this particular case.

[0210] For example, proteins other than BSA may be used as passivation agent, such as ovalbumin or lysozyme.

[0211] The concentration range of passivation agent in the bath in which the device is immersed is preferentially between 0.1 g/L and 1 g/L.

[0212] Xylanase solutions of different concentrations were prepared, having enzymatic activities ranging from 0.23 nkat/mL (dilution to 102 400test No. 2) up to 14.8 nkat/mL (dilution to 1600test No. 8).

[0213] A drop of 7 L of each of these solutions is deposited on a corresponding macro-pattern. As control, a drop of acetate buffer is deposited on another pattern.

[0214] After incubation for 60 minutes at room temperature, rinsing with water and drying of the device, different results for each concentration were observed with the naked eye (cf. table below).

TABLE-US-00005 Dilution factor (relative Result Enzymatic to the observed Test activity commercial (with the No. (nkat/mL) xylanase solution) naked eye) 1 0 (control) 2 0.23 102 400 3 0.46 51 200 4 0.92 25 600 + 5 1.85 12 800 ++ 6 3.7 6400 +++ 7 7.4 3200 +++ 8 14.8 1600 +++

[0215] This test made it possible to estimate a sensitivity threshold approximately located at 0.92 nkat/mL (i.e. 6.410.sup.3 nkat).

Example 2

[0216] FIG. 7 represents a photograph of an assembly of macro-patterns according to the invention which has been placed in contact with different concentrations of xylanase (14.8-0.23 nkat/mL) for 60 minutes after hydration by immersion in water for 2 hours at 37 C. followed by passivation for 1 hour in a solution of BSA at 0.25 g/L at 37 C.

[0217] This photograph reveals the detection limit with the naked eye for a dilution factor of 25 600.

Example 3

[0218] FIG. 8 represents an image matrix of micro-deposits, chosen randomly from the macro-patterns exposed to different concentrations of enzyme for quantifying the action of the enzyme on AX. Xylanase is suspended at concentrations of between 0.45 and 14.6 nkat/mL for different exposure times.

[0219] Representation of the degradation kinetics is given in FIG. 8. It shows that the sensitivity threshold is between 1.83 and 0.91 nkat/mL for a 30-minute incubation. In the case of an incubation for twice as long (60 minutes), this threshold is between 0.45 and 0.91 nkat/mL.