Polymeric substrate having an etched-glass-like surface and a microfluidic chip made of said polymeric substrate

Abstract

The present invention relates to a polymeric substrate having a glass-like surface, in particular an etched-glass-like surface and to a chip made of at least one such polymeric substrate. The present invention also relates to a method of providing a polymeric substrate with an etched-glass-like surface. Moreover, the present invention relates to a kit for manufacturing a chip using such polymeric substrate. Moreover, the present invention relates to the use of a polymeric substrate having a glass-like surface, in particular an etched-glass-like surface for manufacturing a chip.

Claims

1. A microfluidic chip comprising a polymeric substrate that has been modified to have a first etched-glass-like surface having a roughness of >3 nm and a second substrate that is bonded to the first etched-glass like surface, wherein at least one of the first or second substrates has at least one channel, groove, recess or hole that forms a conduit at an interface between said substrates; wherein said modified surface consists essentially of a single surface layer of a SiO.sub.x film, wherein x is from 1 to <2, or wherein said modified surface consists essentially of a surface layer of a sulfonated teterafluoroethylene based fluoropolymer-copolymer (nafion) or poly[1-(trimethylsilyl)-1-[propyne] (PTMSP).

2. The microfluidic chip of claim 1, wherein said modified surface consists essentially of a single surface layer of a SiO.sub.x film, wherein x is from 1 to <2.

3. The microfluidic chip of claim 1 that comprises a surface modification consisting essentially of a single surface layer of a SiO.sub.x film, wherein x is from 1 to <2; wherein said single surface layer was formed by a process comprising coating a surface of the polymeric substrate with an SiO.sub.x precursor and converting said SiO.sub.x precursor into SiO.sub.x.

4. The microfluidic chip of claim 1, wherein said surface modification consists of a single surface layer of a SiO.sub.x film, wherein x is from 1 to <2.

5. The microfluidic chip of claim 1, wherein said modified surface consists essentially of a single surface layer of a SiO.sub.x film, wherein x is from 1 to <2 that has been further treated with a surface layer of a sulfonated teterafluoroethylene based fluoropolymer-copolymer (nafion) or poly[1-(trimethylsilyl)-1-[propyne] (PTMSP) and/or further treated by exposing the surface of the polymeric substrate to at least one of a plasma treatment, reactive ion etching, or UV-ozone treatment.

6. The microfluidic chip of claim 1, wherein said modified surface consists essentially of a surface layer of a sulfonated teterafluoroethylene based fluoropolymer-copolymer (nafion) or poly[1-(trimethylsilyl)-1-[propyne] (PTMSP).

7. The microfluidic chip of claim 1, further comprising treating the polymeric substrate with at least one of a plasma treatment, reactive ion etching, or UV-ozone treatment.

8. The microfluidic chip according to claim 1, wherein at least one of the first or second polymeric substrates is selected from the group consisting of a polyolefin, a polyether, a polyester, a polyamide, a polyimide, a polyvinylchloride, a polyacrylate, and mixtures thereof.

9. The microfluidic chip according to claim 1, wherein at least one of the first or second polymeric substrates is selected from the group consisting of an acrylnitrile-butadien-styrene, a cyclo-olefin-polymer, a cycloolefin copolymer, a polymethylene-methacrylate, a polycarbonate, a polystyrole, a polypropylene, a polyvinylchloride, a polyamide, a polyethylene, a polyethylene-terephthalate, a polytetrafluoro-ethylene, a polyoxymethylene, a thermoplastic elastomer, a thermoplastic polyurethane, a polyimide, a polyether-ether-ketone, a polylactic acid, a polymethylpentene, and mixtures thereof.

10. The microfluidic chip according to claim 1, wherein at least one of the first or second polymeric substrates comprises an inorganic material.

11. The microfluidic chip according to claim 1, wherein at least one of the first or second polymeric substrates comprises an inorganic material selected from the group consisting of a carbon black, a metal oxide and a semiconductor.

12. The microfluidic chip according to claim 1, wherein at least one of the first or second the polymeric substrates contains a metal oxide selected from the group consisting of SiO.sub.2, Al.sub.2O.sub.3, TiO.sub.2, ZrO.sub.2, and Fe.sub.2O.sub.3 or semiconductor selected from the group consisting of ZnS, SdS and SdSe.

13. The microfluidic chip of claim 1, wherein the second substrate is a solid substrate.

14. The microfluidic chip of claim 1, wherein the second substrate is a flexible foil.

15. The microfluidic chip of claim 1, wherein the second substrate is a polymeric substrate, a plastic film or a glass substrate.

16. The microfluidic chip of claim 1, wherein the conduit at the interface between said substrates extends from one edge of the substrate to another edge of the substrate or from one hole to another hole of a substrate allowing flow-through of liquid through said conduit.

17. The microfluidic chip according to claim 1, wherein the conduit has a dimension of <500 m.

18. The microfluidic chip according to claim 1, wherein the conduit has a dimension of <200 m.

19. The microfluidic chip according to claim 1, wherein the conduit is filled with a matrix suitable for at least one of the analysis, detection, separation and transport of analytes.

20. The microfluidic chip of claim 1, wherein said polymeric substrate has not been treated with detergent, exposed to chemicals other than those forming a single surface layer of an SiO.sub.x film, or activated by endowing it with functional chemical groups.

21. The microfluidic chip of claim 1, wherein said polymeric substrate has not been treated with a polyelectrolyte.

22. The microfluidic chip according to claim 1, wherein the hydrophilicity of the etched-glass-like surface is characterized by a water contact angle of less than 50.

23. The microfluidic chip of claim 1, wherein said at least one surface has at least one property selected from the group consisting of a surface charge defined by a zeta potential <0 V for pH >2, a porosity characterized by pores ranging from 0.5 nm to 50 nm, and an inner surface of >100 m.sup.2g.sup.1.

24. The microfluidic chip of claim 1, wherein the modified etched-glass-like surface on the first substrate has a roughness of >3 nm and mimics the surface of glass in at least one property selected from the group consisting of chemical content, chemical composition, chemical structure, homogeneity, morphology, porosity, hydrophilicity, surface energy affinity, adsorption affinity, surface functionality, chemical surface reactivity, physical surface reactivity, zeta potential and surface charge.

25. A method for making the microfluidic chip of claim 1, comprising: a) coating a surface of a first polymeric substrate with an SiO.sub.x-precursor, converting the SiO.sub.x-precursor to SiO.sub.x to form a first coated substrate having a single surface layer of SiO.sub.x, wherein x ranges from 1 to <2, thus modifying the surface of the first coated substrate to form a substrate having an etched-glass-like surface; and/or b) coating a surface of the first polymeric substrate with a polymer thin film having at least one property selected from the group consisting of increased intrinsic roughness, increased intrinsic porosity and increased hydrophilicity to form a first coated substrate, and/or c) exposing the surface of the polymeric substrate to at least one of a plasma treatment, reactive ion etching, or UV-ozone treatment; contacting the first coated substrate having an etched-glass-like surface with a second substrate to form a microfluidic chip having a channel between the first and second substrates, wherein the channel has a flow dimension of <500 m, and wherein the etched-glass-like surface has a roughness of >3 nm.

26. The method of claim 25, comprising a), wherein the SiO.sub.x precursor is selected from the group consisting of: i) alkoxy- or alkyl-chlorosilane, SiX.sub.4, trisiloxane compound Si.sub.3O.sub.2X.sub.6, X being, independently, at each occurrence OR or halogen, R being C.sub.1-C.sub.20 alkyl; ii) polysilazane [Si(H).sub.2N(H)].sub.n, n being selected from 3 to 10,000; iii) polyorganosilazane [Si(R).sub.2N(R)].sub.n, R being alkyl, n being selected from 3 to 100,000; and iv) a sol-gel containing SiO.sub.x particles having a diameter of from 1 to 10 m suspended in a solvent-based matrix wherein the solvent is an alcohol.

27. The method of claim 25, comprising b), wherein the film is deposited by a physical deposition method selected from the group consisting of thermal deposition, electron beam deposition, sputtering, chemical vapour deposition, electroless plating, electrochemical deposition, spray coating, dip coating, gas-phase deposition, roll-to-roll deposition, screen printing, doctor blading, wet coating, dynamic coating, and a combination thereof.

28. The method of claim 25, comprising a), wherein the coating contains at least one of SiN.sub.3, Al.sub.2O.sub.3, B.sub.2O.sub.3, TiO.sub.2, Na.sub.2O, CaO, K.sub.2O, SO.sub.3, MgO, and Fe.sub.2O.sub.3.

29. A microfluidic chip comprising a single surface layer of a SiO.sub.x film, wherein x is from 1 to <2, said microfluidic chip comprising: a first polymeric substrate surface modified by at least one process selected from the group consisting of plasma treatment, reactive ion etching, and UV-Ozone treatment, wherein the first etched-glass-like surface has a roughness of >3 nm, wherein the first polymeric substrate comprises a sulfonated tetrafluoroethylene based fluoropolymer-copolymer with perfluorovinylether groups terminated with sulfonate groups incorporated into a tetrafluorethylene backbone or poly[1-(trimethylsilyl)-1-[propyne] (PTMSP) and forms a portion of a surface of at least one closed conduit; wherein said microfluidic chip is produced by a method comprising: modifying the surface of the first polymeric substrate by wet-coating it with SiO.sub.2 sol-gel, and treating the modified first polymeric substrate with Ar/O.sub.2 plasma for a time and under conditions which increase its surface hydrophilicity to one characterized by a water contact angle <50 and to a surface roughness to >3 nm.

30. A microfluidic chip comprising: at least one modified first polymeric substrate having a first etched-glass-like surface, wherein the first etched-glass-like surface comprises at least one modification selected from the group consisting of: 1) a single surface layer of a SiO.sub.x film, wherein x is from 1 to <2, 2) a single surface layer of a SiO.sub.x film, wherein x is from 1 to <2 formed by a process comprising coating a surface of the polymeric substrate with an SiO.sub.x precursor and converting said SiO.sub.x precursor into SiO.sub.x, and 3) a first polymeric substrate surface modified by at least one process selected from the group consisting of plasma treatment, reactive ion etching, and UV-Ozone treatment, and wherein the first polymeric substrate comprises a sulfonated tetrafluoroethylene based fluoropolymer-copolymer with perfluorovinylether groups terminated with sulfonate groups incorporated into a tetrafluorethylene backbone or poly[1-(trimethylsilyl)-1-[propyne] (PTMSP) and forms a portion of a surface of at least one closed conduit; wherein said microfluidic chip is produced by a method comprising: modifying the surface of the first polymeric substrate by wet-coating it with SiO.sub.2 precursor, converting the SiO.sub.2 precursor to SiO.sub.2, and treating the modified first polymeric substrate with Ar/O.sub.2 plasma for a time and under conditions which increase its surface hydrophilicity to one characterized by a water contact angle <50 and to a surface roughness to >3 nm.

31. The method of claim 26, wherein said polyorganosilazane in iii) has the formula [Si(R).sub.2N(R)].sub.n, R being C.sub.1-C.sub.20 alkyl.

Description

(1) In the following, reference is made to the figures, wherein

(2) FIG. 1 shows transmission curves of (a) SiO.sub.2-layers of different thickness evaporated on Zeonor 1060R and (b) Zeonor 1060R and glass as reference. The transparency is not strongly dependant on layer thickness (5 nm to 100 nm),

(3) FIG. 2 shows results of durability tests of SiO.sub.2 layer on Zeonor 1060R deposited with different methods. The contact angle is more stable on low value in the case of E-gun deposition.

(4) FIG. 3 shows an example image of a substrate having appropriate channels therein, with channel dimensions before and after coating with SiO.sub.2.

(5) FIG. 4 shows a scheme for applying silane and siloxane as a liquid-SiO.sub.2 precursor, preceded by plasma treatment, such as oxygen, or H.sub.2O, or others,

(6) FIG. 5 shows examples of liquid SiO.sub.2 precursors, a) Tetraethyl orthosilicate (TEOS) b) Octachlorotrisiloxane (OTCS), and c) Hydroxymethyltriethoxysilane (TTBS-OH)

(7) FIG. 6 shows an AFM image (topography) of a polymeric substrate coated with TEOS in accordance with the present invention; the surface roughness is 0.5 nm, illustrating a homogeneous coating,

(8) FIG. 7 shows the results of XPS to confirm the presence of SiO.sub.2 which has been silanized with TEOS on the substrate surface,

(9) FIG. 8 shows a bonding scheme of a well plate b and a channel plate c, both plates having been coated a in accordance with the present invention and comprising a glass-like and/or an etched-glass-like surface. The result is a bonded chip d.

(10) FIG. 9 shows an optical microscope image of a cross section of a bonded conduit formed by two substrates that have been treated in accordance with the present invention and have been provided with a glass-like, in particular an etched-glass-like surface; there is no deformation of the structure that can be seen in the optical microscope image, hence, the dimensions of the channel/conduit do not change upon coating and bonding.

(11) FIG. 10 shows an optical microscope image of conduits formed via bonding of two substrates comprising surfaces treated in accordance with the present invention. The channels are filled with a liquid of dark color. No leakages of the liquid can be discerned,

(12) FIG. 11 shows the structure of a liquid precursor, perhydropolysilazane,

(13) FIG. 12 shows an AFM-image of a substrate coated with perhydropolysilazane after NH.sub.4OH vapor treatment, indicating a homogeneous coating,

(14) FIG. 13 shows an optical microscope image of conduits coated with perhydropolysilazane and treated with NH.sub.4OH vapor. The coating does not change the dimensions of the conduits on a m scale,

(15) FIG. 14 shows a FTIR absorption spectrum of a COC substrate coated with poly[1-(trimethylsilyl)-1-propyne] (PTMSP), the COC background spectrum has been subtracted; the presence of the coating is identified by the characteristic presence of absorption of chemical groups of PTMSP,

(16) FIG. 15 shows Kelvin probe force microscopy scans of (a) PTMSP film, (b) glass, (c) COC, and (d) COC coated with polysilazane, charged by 1 s voltage pulses applied to standard conducting AFM probe in contact to the samples. The strong charging (white spots) in (a) and (d) indicates, that water ions can penetrate into the volume of the films, similar to the charging observed on the glass film (b). The uncoated COC film (c) is not charged strongly,

(17) FIG. 16 shows AFM topography images of a untreated PMMA substrate (a), and after Ar/O.sub.2 plasma treatment (b); the surface roughness increases significantly upon plasma treatment (from 4 nm to 25 nm RMS). The receding water contact angle on (b) is much lower (<10) than the advancing contact angle (50), indicating strong roughness,

(18) FIG. 17 shows an electrophoretic separation of DNA 7500 analyte obtained with a PMMA chip in accordance with the present invention (a) and the results of an electrophoretic separation using a conventional glass chip for comparison (b),

(19) FIG. 18 shows an electrophoretic separation of Bovine Serum Albumine analyte obtained with COC chips which is wet-coated with SiO.sub.2 sol-gel in accordance to the present invention (a) and an electrophoretic separation using a conventional glass chip for comparison (b).

(20) FIG. 19 shows an optical microscope image of conduits treated with Ar/O2 plasma. The treatment allows a successful bonding.

(21) FIG. 20 shows an optical microscope image of conduits treated with Ar plasma/UV-Ozone cleaner treatment. The treatment allow a successful bonding.

(22) FIG. 21 shows an (a) optical microscope image of a cross section of a COP bonded conduit formed by two substrates that have been treated with Ar plasma/UV-Ozone cleaner, there is no deformation of the structure that can be seen in the optical microscope image, hence, the dimensions of the channel/conduit do not change upon treatment and bonding. FIG. 21(b) shows an electrophoretic separation of DNA 7500 analyte obtained with a COP chip treated with Ar plasma/UV-Ozone cleaner.

(23) FIG. 22 shows AFM topography scans of (a) Ar plasma/UV-Ozone cleaner treatment on PMMA and (b) on COP; the surface roughness increases significantly upon plasma treatment to (a) 16 nm rms roughness, and (b), to 7 nm rms roughness.

(24) FIG. 23 shows an SEM image of a COP assay substrate

(25) FIG. 24 shows contact angle measured in difference fields of an assay substrate that have been treated in accordance with the present invention, repeated in different days after treatment, in particular a TiO.sub.2 filled COP substrate treated with Ar/O2 plasma show after 114 days a contact angle below 65. The untreated TiO.sub.2 filled COP substrate have a contact angle of 110-120.

(26) FIG. 25 shows (a) three 80 mm profilometer scan lines along the x-direction, offset in the y-direction by 5 mm, on a COP slide after treatment. The undulations in height are all below 1 m on 1 mm range, and show (b) the contact angle measured in difference fields of an assay substrate that have been treated in accordance with the present invention, repeated in different days after treatment, in particular a COP substrate coated with SiO2 thin film show after 115 days a contact angle below 30.

(27) FIG. 26 shows AFM topography scans of (a) molded wells in a plastic substrate, and (b), the same plastic substrate after evaporation of 20 nm SiO.sub.2. Morphology and roughness of the wells are not affected by the SiO.sub.2 evaporation,

(28) FIG. 27 shows AFM topography scans of 11 m.sup.2 of a COC substrates after Ar/O2 plasma treatment, the roughness is increasing from 0.8 nm rms of the bare substrate to 6 nm rms after the treatment.

(29) FIG. 28 shows an XPS spectrum of (a) an untreated and (b) an Ar/O2 plasma treated COC flow cytometry chip. The data confirm that the treatment did not change the chemical composition of the polymeric substrate,

(30) FIG. 29 shows AFM topography scans of (a) 1010 m.sup.2, and (b), of 1 m.sup.2 area of COC substrates before and after TEOS coating.

(31) FIG. 30 shows an XPS spectrum of (a) an untreated and a (b) TEOS coated COC flow cytometry chip. The data confirm the presence of SiO.sub.2 on the substrate surface of the chip coated with TEOS,

(32) FIG. 31 shows contact angle versus time measured on a COC flow cytometry chip which have been treated in accordance with the present invention, in particular (a) an Ar/O2 plasma treated chip and (b) a COC substrate coated with TEOS. In (a) the contact angle stay below 45 for 60 days and in (b) the contact angle stay below 40 for 65 days.

(33) FIG. 32 shows a comparison of the Zeta potential as a function of pH for different glass surfaces to bare polymer surfaces and glass-like surfaces on polymer substrates. The IEP (isoelectric point), the pH below which the Zeta-potentials are negative, can be seen to be shifted below pH 3 for the glass-like surfaces,

(34) FIG. 33 shows a comparison of the Zeta potential as a function of pH for PMMA and COP surfaces to an etched glass surface and a SiO2 sol-gel covered surface, as well as the effect of SDS on the different surfaces. While SDS strongly affects the Zeta potentials on the bare PMMA and COP surfaces, the effect is much weaker for the SiO2 sol-gel covered substrate, similar to the behaviour observed for the etched glass surface,

(35) FIG. 34 shows the chemical structure for nafion,

(36) FIG. 35 shows a comparison of the Zeta potential as a function of pH for COP, COP covered by a layer of nafion, and of a Si-Wafer covered by a 400 nm layer of SiO.sub.2.

(37) Furthermore, reference is made to the following examples, which are given to illustrate, not to limit the present invention:

EXAMPLES

(38) In the following examples, the order in which the steps are listed typically is the order in which these steps are performed for the experiment.

Example 1

COC Substrate Coated by Evaporated SiO2 Film

(39) (FIGS. 1-3) Substrate: COP (Zeonor 1060R) Coating: 20 nm SiO.sub.2 by thermal evaporation optical transparency is higher than 85% (see FIG. 1) water contact angle (after 12 h): 5, stabilizes at about 40 (see FIG. 2) the channel dimensions of the substrate are hardly modified by the coating procedure which indicates that the channels are not totally filled or blocked with SiO.sub.2 (see FIG. 3).

Example 2

PMMA Substrates Coated with TEOS Films

(40) (FIGS. 4-10)

(41) A general scheme for SiO.sub.2 precursor coating is shown in FIG. 4, structural formulas for some liquid precursors are given in FIG. 5. substrate: PMMA channel plate and well plate (Diakon CMG 302) Coating material: TEOS Topography: 0.5 nm roughness (see FIG. 6) Composition: XPS clearly shows Si picks at the substrate surface (FIG. 7) Bonding at 70 C. The bonding scheme is shown in FIG. 8. Bonding was successful with no deformation of the channels and no leakage of fluids when filling the channels. (see FIG. 9 and FIG. 10).

Example 3

PMMA Substrates Coated with PHPS Films

(42) (FIGS. 11-13) Substrate: PMMA channel plate and well plate (Diakon CMG 302) Coating material: Perhydropolysilazane (PHPS) (see FIG. 11) Post-treatment: NH.sub.4OH vapour Water contact angle: 15 Topography: 1.7 nm roughness (see FIG. 12), channels dimensions are not modified (see FIG. 13) and channels are not filled up or blocked. Bonding at 70 C. Bonding was successful.

Example 4

PMMA Substrate Coated with PTMSP Film

(43) (FIG. 14-15) Substrate: PMMA channel plate and well plate (Delpet 70NH) Coating material: Poly[1-(trimethylsilyl)-1-propyne] (PTMSP) PTMSP solution in Toluene Coating Procedure: Spraying, Characterisation: Contact angle: advancing: 107, receding: 79, the difference in the two angles indicates a rough and porous surface; FTIR: PTMSP signals are clearly identified (see FIG. 14); Profilometer: conduits dimensions are not modified, increased roughness is observed with AFM (FIG. 16); charging: PTMSP film can be charged (by biased-probe induced charging) with water ions just like glass (see FIGS. 15a and b). Bonding at 70 C. Bonding was successful.

Example 5

PMMA Substrate Treated with Ar/O2 Plasma

(44) (FIG. 16, 17) Substrate: PMMA channel plate and well plate, (Delpet 70NH) Treatment: Ar/O.sub.2 Plasma Characterisation: Contact angle: 50 (advancing), receding: <10; AFM: 25 nm rms roughness (see FIG. 16); Bonding at 70 C. Bonding was successful. An increased roughness and hydrophilicity mimicking that of glass was achieved. Electrophoretic separation: successful DNA separation (see FIGS. 17a, and 17b separation on glass chip for comparison). A chip in accordance with the present invention provides the same electrophoretic separation of a DNA latter containing DNA molecules up to 7500 Dalton as achieved with glass chip. The gel used for the electrophoretic separation is based on polyacrilamide in 120 mM Tris-Tricine (pH 7.7-8). Dissolved detergents are SDS and LDS as well as fluorescent dye if staining occurs on chip.

Example 6

Wet Coating of COP Chip with SiO2 Sol-Gel

(45) (FIG. 18) Material: COP channel and well plate (Zeonor 1060R) Treatment: the substrate is exposed to chloroform vapor for few minutes before bonding. Bonding at 70 C. Bonding was successful. Wet-coating: SiO.sub.2 sol-gel Electrophoretic separation: good protein separation (see FIGS. 18a, and 18b separation on glass chip for comparison). A chip in accordance with the present invention provides the same electrophoretic separation of Bovine Serum Albumin (a protein) in different concentrations (e.g. 500 g/ml, 1000 g/ml, 2000 g/ml) as achieved with glass chip. Molecular weight marker containing 6 proteins (29 kDa, 45 kDa, 66 kDa, 97 kDa, 116 kDa, 200 kDa). The gel used for the electrophoretic separation is based on polyacrylamide in 120 mM Tris-Tricine (pH 7.7-8). Dissolved detergents are SDS and LDS as well as fluorescent dye if staining occurs on chip.

Example 7

COP Substrate Treated with Ar/O2-Plasma

(46) Substrate: COP channel and well plate (Zeonor 1060R) Treatment: Ar/O.sub.2 Plasma Bonding at 85 C. and at constant pressure Bonding was successful. The obtained chip has no bonding voids and no channel deformation (FIG. 19).

Example 8a

COP Substrate Treated with Ar Plasma/UV-Ozone

(47) Substrate: COP channel and well plate (Zeonor 1060R) Treatment: Ar plasma/UV-Ozone cleaner Bonding at 85 C. and at constant pressure Bonding was successful. (FIG. 20). Bonding force: 300N for 20 s were applied. The samples did not detach.

Example 8b

PMMA Substrate Treated with Ar-Plasma/UV-Ozone

(48) Substrate: PMMA channel and well plate (PMMA Delpet 70NH) Treatment: Ar plasma/UV-Ozone Bonding at 85 C. and at constant pressure Bonding was successful. FIG. 21 shows an (a) optical microscope image of a cross section of a COP bonded conduit formed by two substrates that have been treated with Ar plasma/UV-Ozone; there is no deformation of the structure that can be seen in the optical microscope image, the dimensions of the channel/conduit do not change upon treatment and bonding. FIG. 21(b) shows an electrophoretic separation of DNA 7500 analyte obtained with a COP chip treated with Ar plasma/UV-Ozone.

Example 9

COP-Substrate Treated with SiO2 Sol-Gel

(49) Substrate: COP channel and well plate (Zeonor 1060R)

(50) Coating: Coating material: SiO2 sol-gel Coating Procedure: Spraying Post treatment: O2-plasma

(51) Bonding at 87 C. Bonding was successful, the obtained chip has no bonding voids and no channel deformation.

Example 10

PMMA-Substrate Treated with SiO2 Sol-Gel

(52) Substrate: PMMA channel and well plate (PMMA Delpet 70NH)

(53) Coating: Coating material: SiO2 sol-gel Coating Procedure: Spraying Post treatment: O2-plasma Bonding at 85 C. was successful. The obtained chip has no bonding voids and no channel deformation.

Example 11

(54) Application of Substrates According to the Present Invention for Assay Applications, such as Genome Sequencing

(55) Transferring assay chemistries which have been developed for glass substrate often requires the addition of detergents to ensure the wetting of the hydrophobic plastic surface or to avoid the sticking of proteins or other biomolecules on these hydrophobic surfaces. Adding such detergents may negatively impact the performance of the assay, since such substances can lead to denaturation of proteins or other biomolecules. Large protein molecules can easily loose their functionality in the presence of detergents or other surface active substances. Providing surfaces with glass like properties ensures easy transfer of such assays to plastic consumables. It should also be mentioned that Cells or cell fragments are most easily damaged or destroyed by exposure to detergents or hydrophobic plastic surfaces. Any of the above examples represent cases, where the protein or biomolecule, large protein assemblies or cells and cell fragments are exposed to none native conditions in which they may behave very differently from within their natural environment. A glass like coating in accordance with the present invention with the right pH and ion concentration in the buffer can minimize such negative influence

(56) The substrates in accordance with the present invention can also be used to be applied in a genome sequencing assay. Conventionally, such genome sequencing is performed in a silicon chip having 50 million wells of a defined diameter and depth, wherein each well is filled with a polystyrene bead decorated with DNA. Consequently, if such structure is to be manufactured using the substrates in accordance with the present invention, the same requirements apply for the substrate in accordance with the present invention: The wells must be arranged at a defined distance from each other (FIG. 23); the substrate must have a defined smoothness, hydrophilicity and low cost. Typically, in one example, the smoothness of the substrate is 1 m on 1 mm, and the contact angle is between 20 and 50.

(57) The following treatment was performed:

(58) COP Treated with Ar/O.sub.2 Plasma Substrate: COP (Zeonor 1060R) with and without TiO2 filling material Treatment: Ar/O.sub.2-plasma

(59) FIG. 24 shows contact angle measured in difference fields of an assay substrate that have been treated in accordance with the present invention, repeated in different days after treatment. In particular a TiO.sub.2 filled COP substrate treated with Ar/O.sub.2 plasma shows a contact angle below 65 after 114 days. The untreated TiO.sub.2 filled COP substrate has a contact angle of 110-120.

(60) COP Treated with SiO2 Substrates: COP (Zeonor 1060R) with and without TiO2 filling material, Coating: 20 nm SiO.sub.2 by thermal evaporation and sputtering

(61) Profilometer measurements are shown in FIG. 25 which makes it clear that the differences in height are rather small and are within a range of approximately 300 nm. At the same time, the contact angle remains stable in various positions over a considerable amount of time, i.e. up to 115 days, at least.

(62) FIG. 26 shows AFM topography scans of (a) molded wells in a plastic substrate, and (b), the same plastic substrate after evaporation of 20 nm SiO2. Morphology and roughness of the wells are not affected by the SiO2 evaporation

Example 12

Application of Substrates According to the Present Invention for Flow Cytometry

(63) The substrates according to the present invention can also be used for flow cytometry applications. In this respect, they need to fulfil the following requirements: there must be no cell adhesion, there must be a hydrophilicity with a contact angle between 20-50 degrees, and there must be pressure durability. Furthermore, there must not be the possibility of air bubble formation during the loading of the chip.

(64) Substrate Treatment with Ar/O2 Plasma

(65) :Substrates: COC half-channel plates (Topas 8007 X10)

(66) Treatment: Ar/O2 plasma

(67) Bonding at 75 C. at constant pressure was successful.

(68) FIG. 27 shows AFM images of the surface treated in accordance with this example according to the present invention. The roughness was increased after the Ar/O2 treatment from 0.8 rms to 6 rms, showing that the same treatment can be apply on different polymeric material obtaining the same surface morphology.

(69) FIG. 28 shows an XPS spectrum of (a) an untreated and a Ar/O2 plasma treated (b) COC flow cytometry chip sample to confirm that the treatment did not change the chemical composition of the polymeric substrate.

(70) Substrate Treatment with TEOS

(71) Substrates: COC half-channel plates (Topas 8007 X10)

(72) Coating material: TEOS

(73) Coating Procedure: Dipping

(74) Post-treatment: O2 Plasma

(75) Bonding at 76 C. at constant pressure was successful.

(76) FIG. 29 shows substrate surfaces before and after the treatment according to the present invention, and FIG. 30 shows the corresponding XPS measurements.

(77) It can be seen that the surface morphology did not change (same rms on a 11 m.sup.2) while the XPS confirmed the presence of SiO2 on the surface of the treated sample.

(78) The long-time stability of the treatments according to the present invention have also been measured as can be seen in FIGS. 30a and b which basically show that the contact angle of the COC plates (Topas 8007 X10) stays below 50 for extended periods of time, thus proving that the methods in accordance with the present invention will produce substrates that can be used for commercial purposes and are also amenable to multi-use-applications.

Example 13

(79) The substrates in accordance with the present invention can also be characterized to have glass-like behaviour by measuring the respective zeta potential. As can be seen in FIG. 32, the glass-like substrates in accordance with the present invention have zeta potential curves that have the same shape and values as a glass surface. More specifically, the IEP (isoelectric point), the pH below which the Zeta-potentials are negative, can be seen to be shifted below pH 3 for the glass-like surfaces.

(80) Furthermore, as can be seen in FIG. 33, the surfaces/substrates in accordance with the present invention have the same behaviour as glass, when in contact with SDS. More specifically, the zeta potential is not much affected by the presents of SDS, whereas for substrates not treated in accordance with the present invention, the influence of SDS is much bigger. More specifically FIG. 33 shows a comparison of the Zeta potential as a function of pH for PMMA and COP surfaces to an etched glass surface and a SiO2 sol-gel covered surface, as well as the effect of SDS on the different surfaces. While SDS strongly affects the Zeta potentials on the bare PMMA and COC surfaces, the effect is much weaker for the SiO2 sol-gel covered substrate, similar to the behaviour observed for the etched glass surface.

Example 14

(81) One possibility for a surface treatment in accordance with the present invention is to treat the polymeric substrate with nafion which is a sulfonated tetrafluoroethylene based fluoropolymer-copolymer. It is a ionomer which forms ion-exchange membranes. It has a highly specific conductance for protons in solution and allows a proton conduction due to the formation of water channels. The structure of nafion and the mechanistic details of its behaviour can be inspected in FIG. 34 which show the chemical structure of nafion as well as a scheme to explain the behaviour of nafion in and towards water.

(82) FIG. 35 shows the zeta potential of a substrate in accordance with the present invention that has been coated with nafion. The nafion coated substrate shows a very negative zeta potential which is even more negative than glass.

(83) This makes substrates in accordance with the present invention that have been treated with nafion obtaining a glass-like surface.

(84) The features of the present invention disclosed in the specification, the claims and/or in the drawings may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.