Copolymer of N-(2-hydroxypropyl) methacrylamide and carboxybetaine metacrylamide, polymer brushes

Abstract

The present invention relates to the preparation and use of copolymers composed of N-(2-hydroxypropyl) methacrylamide (HPMAA) and carboxybetaine methacrylamide (CBMAA). The invention further describes polymer brushes having structure I
SR-polymer(I) wherein S is a substrate; R is a residue of a polymerization initiator or a RAFT agent bound to the substrate; and polymer is the copolymer of N-(2-hydroxypropyl) methacrylamide and carboxybetaine methacrylamide Furthermore, production of these polymer brushes, containing random or block copolymers grafted to or from a substrate is described. The copolymer brushes are suitable for protecting substrates from deposition and/or adhesion of biological substances, and/or against thrombus formation. The brushes functionalized by covalent attachment of bioactive substances to CBMAA monomer units are particularly suitable for specific interaction with target biological substances which is not affected by nonspecific deposition of non-target compounds.

Claims

1. A substrate with a polymer brush having the structure I:
SR-polymer(I) wherein S is a substrate; R is a residue of a polymerization initiator or a RAFT agent bound to the substrate; and polymer is a copolymer of N-(2-hydroxypropyl) methacrylamide and carboxybetaine methacrylamide, wherein the molar fraction of carboxybetaine methacrylamide monomer units is up to 40 mol %.

2. The substrate with the polymer brush according to claim 1, which is selected from polymer brush containing a random copolymer of N-(2-hydroxypropyl) methacrylamide (HPMAA) and carboxybetaine methacrylamide (CBMAA) of structure II:
SR-poly(HPMAA-co-CBMAA)(II), or a block copolymer composed of a poly(N-(2-hydroxypropyl) methacrylamide) block (polyHPMAA) and a poly(carboxybetaine methacrylamide) block (polyCBMAA) of structure III:
SR-polyHPMAA-b-polyCBMAA(III).

3. The substrate with the polymer brush according to claim 1 wherein the copolymer in a dry state has a thickness of 1 nm to 100 nm.

4. A method for preparation of the substrate with the polymer brush according to claim 2 wherein the copolymer of N-(2-hydroxypropyl) methacrylamide and carboxybetaine methacrylamide is a random copolymer, characterized in that R-poly(HPMAA-co-CBMAA) is prepared by living radical polymerization in a solution comprising mixture of HPMAA and CBMAA monomers, polymerization initiator or a RAFT agent with a functional moiety R, and the R-poly(HPMAA-co-CBMAA) is subsequently attached to the substrate via R.

5. A method for preparation of the substrate with the polymer brush according to claim 2, wherein the copolymer of N-(2-hydroxypropyl) methacrylamide and carboxybetaine methacrylamide is a random copolymer, characterized in that a polymerization initiator or RAFT agent with a functional moiety R is covalently bound to a substrate surface and subsequently random copolymer of N-(2-hydroxypropyl) methacrylamide and carboxybetaine methacrylamide is grafted from the surface in a solution comprising mixture of HPMAA and CBMAA monomers.

6. A method for preparation of the substrate with the polymer brush according to claim 2 wherein the copolymer of N-(2-hydroxypropyl) methacrylamide and carboxybetaine methacrylamide is a block copolymer, characterized in that a polymerization initiator or RAFT agent with a functional moiety R is covalently bound to a substrate surface, then the block copolymer of polyHPMAA-b-polyCBMAA is polymerized from the surface in a solution comprising HPMAA monomer and in a solution comprising CBMAA monomer, respectively.

7. The substrate with the polymer brush according to claim 1, wherein the molar fraction of carboxybetaine methacrylamide monomer units is up to 30 mol %.

8. A bioanalytical device comprising the polymer brush according to claim 1, wherein the device surface is the substrate S of the polymer brush.

9. The bioanalytical device of claim 8, wherein the device is a sensor for direct detection of analytes or sensor for multi-step detection of analytes.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1 shows as an example of a polymer brush a statistic copolymer poly(HPMAA-co-CBMAA) grafted from a surface of a golden substrate. R is the residuum of the ATRP initiator -merkaptoundecyl bromoisobutyrate covalently bound to the golden surface, m is the number of HPMAA monomeric units, and n is the number of CBMAA monomeric units randomly distributed in the copolymer, o is the number of all monomeric units in the polymer chain.

(2) FIG. 2 shows the dependence of the percentage of CBMAA monomeric units in the brush of poly(HPMAA-co-CBMAA) on the percentage of CBMAA monomer in the mixture of HPMAA and CBMAA monomers present in the polymerization solution. The copolymer was grafted from the golden surface of the SPR chip by ATRP according to the Example 4 from the polymerization solutions containing various concentrations of CBMAA to HPMAA monomers.

(3) FIG. 3 shows the decrease in concentration of ionized carboxyl groups (the intensity of the band of valence vibration CO at 1607 cm.sup.1) in the polyCBMAA brush grafted from SPR chip after the NHS/EDC activation measured by FTIR GASR spectroscopy (Example 9). (1) polyCBMAA; (2) polyCBMAA, NHS/EDC activation (12 min), PBS (60 min).

EXAMPLES

Example 1

(4) Preparation of Poly(HPMAA-co-CBMAA) by RAFT Polymerization in Solution

(5) In a Schlenk flask equipped with magnetic stirrer bar, monomers N-(2-hydroxypropyl) methacrylamide (HPMAA, 4.3 g, 30 mM, 85 mol %), carboxybetaine methacrylamide (CBMAA, 1.3 g, 5.2 mM, 15 mol %) and chain transfer agent 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid (CTP, 35 mg, 0.124 mM) were dissolved in 45 ml of DMAc and deoxygenated by four freeze-pump-thaw cycles. Subsequently, 1 ml (0.051 mM) of the initiator 2,2-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride solution (VA-044, 16.4 mg, 1 ml DMAc) was added and another one freeze-pump-thaw cycle was performed. The flask containing the pink solution was filled with argon and placed into the oil bath (50 C.) to start the polymerization. After 24 h, the polymerization was quenched by exposing the reaction mixture to air and liquid nitrogen. The polymerization solution was dialyzed against deionized water for 46 h using a SpectraPor 3 membrane (MWCO 3500 Da) and freeze-dried to yield a pink solid. The conversion was 80% as determined from .sup.1H NMR spectroscopy. The obtained polymer was characterized by SEC M.sub.n=66000 g.Math.mol.sup.1, =1.35.

Example 2

(6) Preparation of PolyHPMAA-b-polyCBMAA by RAFT Polymerization in Solution

(7) Preparation of the first block polyHPMAA as a macroRAFT agent: In a Schlenk flask equipped with magnetic stirrer bar, HPMAA monomer (3.1 g, 21.7 mM), CTP (50 mg, 0.178 mM) were dissolved in 17.8 ml of DMAc and deoxygenated by four freeze-pump-thaw cycles. Subsequently, 100 l (0.357 mM) of the initiator V-501 solution (100 mg, 1 ml DMAc) was added and another one freeze-pump-thaw cycle was performed. The flask containing the pink solution was filled with argon and placed into the oil bath (70 C.) to start the polymerization. After 8 h, the polymerization was quenched by exposing the reaction mixture to air and liquid nitrogen. The polymerization solution was dialyzed against deionized water for 46 h using a SpectraPor 3 membrane (MWCO 3500 Da) and freeze-dried to yield a pink solid. The conversion was 38% as determined from .sup.1H NMR spectroscopy. The obtained polymer was characterized by SEC M.sub.m=6500 g.Math.mol.sup.1, =1.1.

(8) The preparation of second block and the extension of polyHPMAA macroRAFT agent to polyHPMAA-b-polyCBMAA was performed as follows. The above noted polyHPMAA (M.sub.n=6500 g mol.sup.1, =1.1) was employed as a macroRAFT agent. In a Schlenk flask equipped with magnetic stirrer bar, 125 mg (0.019 mM) of polyHPMAA was dissolved in 4 ml of acetate buffer. Then, CBMAA monomer (690 mg, 2.8 mmol) was added and the mixture deoxygenated by four freeze-pump-thaw cycles. Subsequently, 100 l (0.004 mM) of the initiator V-501 solution (10 mg, 1 ml acetate buffer) was added and another one freeze-pump-thaw cycle was performed. The polymerization mixture was allowed to polymerize for 6 h. After that, the polymerization was quenched by exposing the reaction mixture to air and liquid nitrogen. The polymerization solution was dialyzed against deionized water for 72 h using a SpectraPor 3 membrane (MWCO 3500 Da) and freeze-dried to yield a pink solid. The conversion was 63% as determined from .sup.1H NMR spectroscopy. The obtained polymer was characterized by SEC M.sub.n=35 000 g.Math.mol.sup.1, =1.2.

Example 3

(9) Preparation of Poly(HPMAA-co-CBMAA) Brush by Grafting to Gold

(10) The copolymer poly(HPMAA-co-CBMAA), M.sub.n=66 000 g.Math.mol.sup.1, =1.35 was prepared by RAFT polymerization according to the Example 1. The procedure lead to the copolymer terminated with dithiobenzoate functional group (DTB) which was a residue of chain transfer agent 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid used for the RAFT polymetization. Gold-coated glass substrate was rinsed twice with ethanol and deionized Milli-Q water, blow-dried with nitrogen, and cleaned in a UV/Ozone cleaner for 20 min. For the copolymer grafting to gold surface poly(HPMAA-co-CBMAA)-DTB was dissolved in dimethylformamide (DMF) 3.5 ml and subsequently 2 ml of dichloroethane (DCE) was dropped until cloudy solution appeared at the final DMF/DCE volume ratio of 65/35%. Immediately after cleaning, the substrate was immersed in the solution of poly(HPMAA-co-CBMAA)-DTB (100 mg in 5.5 ml of DMF/DCE). After 4 days in dark, the substrate was taken out of the solution, washed successively with DMF and water and stored in water.

Example 4

(11) Preparation of Poly(HPMAA-co-CBMAA) Brush by Grafting from Gold

(12) A glass plate was coated with a gold layer in vacuum. The plate was rinsed with ethanol and water, dried, and cleaned 20 min in UV/Ozone cleaner. The plate was immersed into the 0.1 M solution of initiator -merkaptoundecyl bromoisobutyrate in ethanol for 24 h in dark. Into the Schlenk flask containing CuCl (35 mg), CuCl.sub.2 (10.5 mg) and Me.sub.4Cyclam (121 mg) 7 ml of de-gassed methanol was added and the catalytic mixture was mixed until fully dissolved. In another Schlenk flask 3.1 g of the monomer mixture containing 15 mol % CBMAA and 85 mol % HPMAA was dissolved, while cooled with ice, in 12 ml of de-gassed water and 5 ml of ethanol. After dissolution, the catalyst solution was added to the flask under argon. Homogeneous polymerization mixture was dosed under argon into the second Schlenk flask with an inserted plate coated with the initiator and the mixture was polymerized for 120 min at 30 C. Then the plate was rinsed with water. The same procedure was used to prepare the brushes in solutions comprising mixtures of CBMAA and HPMAA monomers at molar ratios of CBMAA to HPMA of 7:93, 3:7, 3:2, and 4:1. The molar ratios of CBMAA to HPMA in the copolymer brushes as determined by FTIR-GASR spectroscopy were similar as those in the polymerization feed (see FIG. 2) indicating a random character of the copolymerization. The thickness of the surface polymer layer determined by ellipsometry was 30 nm.

Example 5

(13) Preparation of Poly(HPMAA-co-CBMAA) Brush by Grafting from Glass

(14) A glass plate was rinsed with ethanol and water, dried, and finally cleaned 20 min in UV ozone cleaner. The plate was immersed for 3 h to 1 mM solution of initiator 11-(trichlorsilyl)undecyl-2-bromo-2-methylpropanoate in dry toluene. After that, the plate was rinsed with toluene, acetone, ethanol, and water, and dried. 7 ml of de-gassed methanol was added into a Schlenk flask containing CuCl (35 mg), CuCl.sub.2 (10.5 mg) and Me.sub.4Cyclam (121 mg) and the catalytic mixture was mixed until fully dissolved. In the second Schlenk flask 2.9 g of the monomer mixture containing 15 mol % CBMAA and 93 mol % HPMAA was dissolved, while cooled with ice, in 12 ml of de-gassed water and 5 ml of ethanol. After that the catalyst solution was added to the flask under argon The polymerization mixture was added under argon into the third Schlenk flask containing the plate coated with the initiator, and the grafting of the copolymer was performed for 120 min at 30 C. Then, the plate was rinsed with water.

Example 6

(15) Preparation of Poly(HPMAA-co-CBMAA) Brush by Grafting from Nano-Particles (NP) of -Polycaprolactone (PCL)

(16) The solution of -bromo isobutyric acid, 0.15 M, N-hydroxysuccinimide (NHS), 0.05 M, and EDC, 0.2 M, in water was left 7 min at 25 C. and then transferred to suspension of hollow poly(-caprolactone) nano-particles (diameter 150 nm, 5.510.sup.9/ml), and the suspension was mixed through the night. Then the suspension was dialyzed 12 h at the room temperature through the membrane Spectra/Pore (6 000 through 8 000 Da). CuBr.sub.2 (8.1 mg), 2,2-dipyridyl (145 mg) and 5.7 g of the monomer mixture containing 15 mol % CBMAA and 85 mol % HPMAA was dissolved, while cooled with ice, in 10 ml of water was degassed by bubbling argon for 1 h. Then CuCl (37 mg) was added under argon and the solution was bubbled with argon for further 30 min. This solution was added to 5 ml of suspension of nano-particles with the bound initiator, and the system was polymerized under argon at 30 C. The size distribution of nano-particles before and after modification was determined using the method of quasi-elastic dispersion. The thickness of the polymer brush was controlled by the polymerization time (30 min to 150 min) in the range from 20 nm to 200 nm. The coverage of nano-particles with the poly(HPMAA-co-CBMAA/15 mol %) brush prevented the aggregation of nano-particles upon incubation with undiluted blood plasma.

Example 7

(17) Preparation of Poly(HPMAA-co-CBMAA) Brush by Grafting from Silicon Coated with Anchoring Intermediate Layer of Polydopamine (PDA)

(18) A silicon plate was cleaned by sonication in methanol and water, immersed for 10 min in the mixture of 25% ammonia, 30% hydrogen peroxide, and water (1:1:5, v/v/v) heated to 70 C. Then it was rinsed with water and ethanol, dried and cleaned 2 h in the UV/Ozone cleaner. Then the plate was inserted into the opened Petri dish containing the solution of 2 mg of dopamine hydrochloride per ml of air saturated 10 mM Tris-HCl buffer, pH 8.5. After 3 h of gentle mixing, the plate was rinsed with water, sonicated in water 15 min and dried. The plate with the layer of PDA (13 nm) was immersed into the solution of 0.24 M triethylamine at 0 C., put into the shaker and the solution of 2-brom-2-methylpropanoyl bromide (0.24 M in 10 ml of tetrahydrofuran) was added at 0 C. After 3 min, the plate was removed and rinsed successively with tetrahydrofuran, ethanol, and water, and dried. Degassed ethanol (30 ml) was transferred under argon into the Schlenk flask containing CuBr (57.3 mg), CuBr.sub.2 (17.7 mg) and Me.sub.4Cyclam (122.7 mg). The blue solution of this catalyzer was transferred to the second Schlenk flask containing 4.5 g of the monomer mixture of CBMAA (15 mol %) and HPMAA (85 mol %). The polymerization solution was transferred to the reactor containing the silicone plate coated with the anchoring layer of polydopamine, and left to polymerize 2 h at 30 C. The plates were then rinsed with ethanol and water. The presence of polymer brush of poly(HPMAA-co-CBMAA/15 mol %) was checked by FTIR-IRRAS spectroscopy and the thickness of the layer determined by ellipsometry was 20 nm.

Example 8

(19) Antithrombogenic Characteristics of Poly(HPMAA-co-CBMAA/15 mol %)

(20) The surface of a glass plate coated with poly(HPMAA-co-CBMAA/15 mol %) brush according to the Example 5 was immersed in the citrated blood plasma from a healthy donor mixed with 1M solution of CaCl.sub.2 in water. The turbidity was measured at 405 nm. In comparison with untreated glass, the re-calcification time was extended 8-times (40 min vs. 5 min). Thrombogenicity of this surface was further assessed by monitoring of coagulation kinetics of whole blood. Whole blood from a healthy donor collected into citrate was mixed with a 1 M solution of CaCl.sub.2 in water (final concentration 0.02 M) and immediately applied to the surface of the sample (100 l). At time intervals of 5, 15 and 30 min, the blood coagulation was stopped by adding 3 ml of distilled water. After 5 min, 200 ml of the sample was collected and the amount of released hemoglobin from erythrocytes not caught in the blood clot was determined from absorbance at 540 nm. The size of the blood clot is inversely proportional to the absorbance value. While on the surface prepared according to the Example 2, there was no significant decrease in hemoglobin levels even after 30 min, on the untreated glass surface the decrease occurred already after 5 min, and after 30 min more than 85% of erythrocytes were captured in the blood clot.

Example 9

(21) Binding of Biorecognition Substances to Poly(HPMAA-co-CBMAA) Brushes Grafted from SPR Chips

(22) The brushes of poly(HPMAA-co-CBMAA) copolymers containing 7 mol %, 15 mol % and 30 mol % of CBMAA, and the brushes of polyCBMAA and polyHPMAA homopolymers were grafted from gold surface of SPR chips using the procedure described in the Example 4. SPR chips coated with the polymer brush were rinsed with water, 10% acetic acid, water, nitrogen blow dried, and inserted into a flow cell of the SPR sensor instrument. Then they were activated in situ for 20 min with the solution of NHS, 0.1 M, and EDC, 0.05 M, in water. The solution was replaced with borate buffer, 10 mM, pH 8.5, and after 15 min the activated chips were reacted with a solution of a biorecognition substance (antibody against E. coli, 50 g anti-E. coli/ml, 100 g streptavidin/ml in the borate buffer, or amino-modified oligonucleotide probe, 11-mer, 4 M). The functionalized chips were sequentially rinsed with borate buffer (10 mM, pH 8.5, 50 min) containing 150 mM NaCl and 10 mM imidazole, NaOH (2 mM, 3 min) and ethanolamine (1 M, 5 min). Finally, the chips were rinsed with water and the amount of immobilized ligand was determined by SPR.

(23) TABLE-US-00001 TABLE 1 The amount of the bound antibody anti-E. coli, streptavidin, and amino-modified oligonucleotide probe on polyCBAA, poly(HPMAA-co-CBMAA), polyHPMAA, and polyCBMAA. Bound ligand [ng/cm.sup.2] Polymer brush Anti-E. coli Streptavidin ON-probe polyCBAA* 153.2. 136.8 30.9 polyHPMAA 0.0 0.0 N/A Poly(HPMAA-co- 20.6 N/A 2.9 CBMAA/7 mol %) Poly(HPMAA-co- 200.1 30.1 18.6 CBMAA/15 mol %) Poly(HPMAA-co- 362.2 60.8 30.6 CBMAA/30 mol %) PolyCBMAA 450.8 242.0 31.5 *Delivered from the University of Washington, Seattle, USA

(24) Table 1 demonstrates the dependence of the binding capacity for ligands on the content of CBMAA in the copolymer

Example 10

(25) Binding of Surface Antigen of Hepatitis B (HBsAg) Virus to Poly(HPMAA-co-CBMAA) Brush Grafted from SPR Chip

(26) Poly(HPMAA-co-CBMAA/15 mol %) brush grafted from the SPR chip according to the Example 4 was activated for 10 min by water solution of EDC, 0.2 M, and NHS, 0.05 M. Then the surface was rinsed shortly with acetate buffer (10 mM, pH 5) and HEPES buffer (10 mM, pH 7.5) and the solution of 25 g HBsAg/ml HEPES was added. The mixture was allowed to react for 10 min and then the plate was transferred to PBS buffer. The SPR chip prepared by such procedure was used for the direct detection of antibodies to the hepatitis B virus in sera of patients diluted with PBS to 10%. Positive and negative sera of patients were resolved and the relative titer of antibodies against the hepatitis B virus was determined.

(27) The fluorescent biosensor with the bound HBsAg prepared by the same procedure and combined with a fluorescently labeled secondary antibody was used for the detection of anti-HBsAg in saliva samples by SPR enhanced fluorescence. The limits of detection of this method achieved 10 pM in this model example.

Example 11

(28) Resistance of Poly(HPMAA-co-CBMAA) Brushes to Non-Specific Fouling from Blood Plasma and Foodstuffs Before and After Binding Biorecognition Substances

(29) Brushes of poly(HPMAA-co-CBMAA) copolymers containing 7 mol %, 15 mol % and 30 mol % of CBMAA, and brushes of polyCBMAA and polyHPMAA were grafted from the gold surface of SPR chips using the procedure described in Example 4 and functionalized by attachment of antibody against E. coli (anti-E. coli) according to Example 9 (see Table 1 for the amount of the attached antibody). The surfaces coated with the brushes without the attached anti-E. coli and the surfaces after the functionalization were exposed to undiluted citrate blood plasma and undiluted extracts of the foodstuffs for 10 min at 25 C. The fouling was determined by SPR (Table 2).

(30) TABLE-US-00002 TABLE 2 Fouling from undiluted blood plasma and undiluted food extracts. A - Unmodified brushes; B - Brushes functionalized with anti-E. coli. Fouling [ng/cm.sup.2] Blood plasma Milk Spinach Cucumber Hamburger Salad A - Non-functionalized surfaces PolyCBAA* 6.8 0.0 4.8 0.0 0.0 0.0 PolyHPMAA 0.0 0.0 0.0 0.0 N/A 0.0 Poly (HPMAA-co-CBMAA/ 0.0 0.0 0.0 0.0 N/A 0.0 7 mol %) Poly(HPMAA-co-CBMAA/ 2.9 0.0 0.5 0.3 0.0 0.2 15 mol %) Poly(HPMAA-co-CBMAA/ 10.0 0.6 1.2 1.4 0.0 1.4 30 mol %) PolyCBMAA 11.1 27.9 2.7 2.1 N/A 2.4 B. Functionalized surfaces PolyCBAA* 20.2 8.0 62.9 8.1 2.2 4.3 PolyHPMAA 0.0 0.0 0.0 0.0 N/A 0.0 Poly (HPMAA-co-CBMAA/ 2.6 0.0 N/A 0.0 N/A 0.0 7 mol %) Poly(HPMAA-co-CBMAA/ 8.5 1.7 3.4 0.0 0.0 0.0 15 mol %) Poly(HPMAA-co-CBMAA/ 16.2 6.8 4.2 2.8 N/A 3.5 30 mol %) PolyCBMAA 25.4 30.5 12.2 3.4 N/A 4.9 *Delivered from the University of Washington, Seattle, USA

(31) Table 2 documents that the excellent antifouling properties of polyHPMAA brush are not affected when exposed to the conditions of the functionalization. The fouling remained below the limit of detection of the SPR method (0.3 ng/cm.sup.2) in all the tested media.

Example 12

(32) SPR Detection of Pathogenic Bacteria in Food Samples Using Poly(HPMAA-co-CBMAA) Brush Grafted from SPR Chip

(33) Pathogenic bacteria E. coli O157:H7 (deactivated with heat shock) in the buffer solution and 100% extract from hamburger and lettuce were detected using SPR four-channel sensor. SPR chip covered with poly(HPMAA-co-CBMAA/15 mol %) and functionalized with the respective antibodies (channel 1-3:anti-E coli, channel 4: anti-Salm) were prepared according to the Example 9. The amounts of the immobilized antibodies were 244 ng/cm.sup.2 for anti-E. coli (channel 1-3) and 204 ng/cm.sup.2 in the reference channel with immobilized antibody to Salmonella spp., anti-Salm (channel 4). The detection of bacteria was performed in three steps. In the direct detection step I the bacteria E. coli O157:H7 at the concentration of 1.510.sup.7 cfu/ml were detected in PBS buffer (channel 1), in the extract from hamburger (channel 2) and in the extract from lettuce (channels 3 and 4). The time of incubation was 10 min at the flow of 30 l/min and then the sensor was rinsed 10 min with PBS. In the step II, the secondary biotinylated antibody to E. coli (5 g/ml) was circulated in all channels for 15 min to confirm specificity of the binding of the bacteria to immobilized antibodies. In the Step III, streptavidin (100 g/ml,) was circulated for 15 min to increase the response of the sensor. The shifts of the resonance wavelength displayed for each step separately are shown in Table 3.

(34) TABLE-US-00003 TABLE 3 Responses of the sensor (shift of the resonance wavelength in nm) upon detection of E. coli O157:H7 to poly(HPMAA-co-CBMAA/15%) in Steps I., II., and III. Channels 1-3 (measuring) were functionalized anti-E. coli, channel 4: reference anti-Salm. Response of SPR sensor [nm] Channel 2 - anti- Channel 3 - anti- Channel 1 - anti- E. coli/detection E. coli/detection Channel 4 (ref.) - anti- E. coli/ detection in extract in extract Salm/detection Detection step in PBS from hamburger from lettuce in PBS I. Direct detection of E. coli 0.8 1.1 0.9 0.0 O157:H7 II. Detection of biotinylated 5.5 5.6 6.3 0.0 antibody anti-Ecoli III. Detection of streptavidin 11.1 11.5 12.0 0.1

Example 13

(35) SPR Detection of MicroRNA Using Poly(HPMAA-co-CBMAA/15 mol %) Brush Grafted from SPR Chip

(36) SPR chips were coated with poly(HPMAA-co-CBMAA/15 mol %) brush (thickness about 40 nm in hydrated condition) and functionalized by the covalent attachment of amino-modified oligonucleotide probes using the optimized NHS/EDC chemistry (probeNdmiR-122, sequence 5-Am-MC12-CA AAC ACC ATT G-3, 4 M in 10 mM borate buffer, pH 8.0, 30 min, flow 7.5 l/min, 25 C.) according to the Example 9. MicroRNA (miR-122) was detected by the biosensor at the concentration of 100 nM in PBS. To regenerate the biosensing surface, the captured miRNA molecules were released from the immobilized oligonucleotide probes by NaOH (6 mM, 5 min) and the measurement was repeated after re-injecting the miRNA solution, The sensor response did not change significantly, even after four measuring/regeneration cycles.

Example 14

(37) SPR Detection of DNA Oligonucleotides Using Poly(HPMAA-co-CBMAA/15 mol %) and PolyCBMAA Brushes Grafted from SPR Chip

(38) SPR chips were coated with poly(HPMAA-co-CBMAA/15 mol %) or polyCBAA brushes and functionalized by the covalent attachment of streptavidin using NHS/EDC chemistry (50 g/ml of streptavidin in 10 mM borate buffer, pH 8.0, 20 min, flow 20 l/min, 25 C.) according to the Example 9. The biotinylated oligonucleotide probes were bound to the immobilized streptavidin through noncovalent streptavidin-biotin interaction in solution of the probe (200 nM in PBS) The number of probes bound to one molecule of streptavidin was set at an average value of 3.1 for the surface with poly(HPMAA-co-CBMAA/15 mol %) and 3.2 for polyCBAA. A response of the biosensor was detected after injecting solution of target DNA oligonucleotides (200 nM in PBS, 15 min, 30 ml/min, 25 C.). An average number of the captured DNA oligonucleotide per one immobilized probe was 0.9 on poly(HPMAA-co-CBMAA/15 mol %) and 0.8 on polyCBAA.