Terpolymer and polymer brushes for use against non-specific adsorption of substances from biological media

Abstract

A random terpolymer of N-(2-hydroxypropyl) methacrylamide, carboxybetaine methacrylamide and sulfobetaine methacrylamide, and a polymer brush and to a functionalized polymer brush containing this terpolymer are disclosed. The random terpolymer increases the resistance of the substrate surface to non-specific adsorption of substances from biological media and/or to non-specific interaction with biological media components, and is suitable for use in the form of a polymer brush, for example in sensors or membranes.

Claims

1. Random terpolymer of N-(2-hydroxypropyl) methacrylamide, carboxybetaine methacrylamide and sulfobetaine methacrylamide.

2. The random terpolymer according to claim 1, containing: 0.1 mol % to 40 mol % of sulfobetaine methacrylamide; 0.1 mol % to 50 mol % of carboxybetaine methacrylamide; and N-(2-hydroxypropyl) methacrylamide forming balance to 100 mol %.

3. Polymer brush having structure I
S—R—poly(HPMAA-co-CBMAA-co-SBMAA)   (I) wherein S is a substrate having a surface suitable for binding of polymerization initiators; R is a residue of a polymerization initiator selected from the group consisting of ATRP initiators, SET-LRP initiators and RAFT agents; poly(HPMAA-co-CBMAA-co-SBMAA) is a random terpolymer according to claim 1; — is a covalent bond.

4. Functionalized polymer brush having structure II
S—R—poly(HPMAA-co-CBMAA-co-SBMAA)—F   (II) wherein S is a substrate having a surface suitable for binding of polymerization initiators; R is a residue of a polymerization initiator selected from the group consisting of ATRP initiators, SET-LRP initiators and RAFT agents; poly(HPMAA-co-CBMAA-co-SBMAA) is a random terpolymer according to claim 1; — is a covalent bond; F is at least one functional entity covalently bound to the random terpolymer by amide bond formed between —NH— group of the functional entity and —C(O)— group of carboxybetaine methacrylamide.

5. The functionalized polymer brush according to claim 4, wherein the functional entity F is selected from the group consisting of proteins, antibodies, peptides, nucleic acids, oligonucleotides.

6. The polymer brush according to claim 3, wherein the thickness of layer of the random terpolymer in the polymer brush is 10 nm to 500 nm, preferably 20 nm to 300 nm, as measured by ellipsometric method in water.

7. A method for preparation of the functionalized polymer brush according to claim 4, comprising the steps of: subjecting the carboxybetaine carboxyl groups in a polymer brush to a reaction with activation agents for activation of the carboxyl groups to form active ester groups; subjecting the active ester groups to a reaction with amino group of the functional entity to form an amide covalent bond; subjecting the unreacted active ester groups to reaction with (2-aminoethoxy)acetic acid and/or glycine.

8. A method for increasing the resistance of a surfaces against non-specific adsorption of substances from biological media and/or against non-specific interactions with biological media components comprising the step of applying the random terpolymer according to claim 1 on the surface.

9. A method for increasing the resistance of substrate surface against non-specific adsorption of substances from biological media and/or against non-specific interactions with biological media components, comprising the step of grafting the polymer brush according to claim 3 on the substrate surface.

10. A method for specific and selective interactions with target analytes, selected from proteins, peptides, nucleic acids, oligonucleotides, comprising the step of contacting the target analytes with the functionalized polymer brush according to claim 4.

11. A sensor, characterized in that it comprises the polymer brush according to claim 3.

12. The sensor according to claim 11, which is selected from surface plasmon resonance sensors, quartz crystal microbalances and fluorescent sensors.

13. A membrane, characterized in that it comprises the polymer brush according to claim 3.

14. The functionalized polymer brush according to claim 4, wherein the thickness of layer of the random terpolymer in the polymer brush is 10 nm to 500 nm, preferably 20 nm to 300 nm, as measured by ellipsometric method in water.

15. The functionalized polymer brush according to claim 5, wherein the thickness of layer of the random terpolymer in the polymer brush is 10 nm to 500 nm, preferably 20 nm to 300 nm, as measured by ellipsometric method in water.

16. A method for increasing the resistance of substrate surface against non-specific adsorption of substances from biological media and/or against non-specific interactions with biological media components, comprising the step of grafting the functionalized polymer brush according to claim 4 on the substrate surface.

17. A sensor, characterized in that it comprises the functionalized polymer brush according to claim 4.

18. The sensor according to claim 17, which is selected from surface plasmon resonance sensors, quartz crystal microbalances and fluorescent sensors.

19. A membrane, characterized in that it comprises the functionalized polymer brush according to claim 4.

20. A membrane, characterized in that it comprises the functionalized polymer brush according to claim 5.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0035] FIG. 1: Line (1) shows a typical IRRAS spectrum of the poly[HPMAA(70 mol %)-co-CBMAA(15 mol %)-co-SBMAA(15 mol %)] brush on gold. The characteristic absorption bands of the groups of individual components are marked: Am I and Am II for amide bands of the chain, —OH for the band of the hydroxyl group of HPMAA, —COO— for the band of the ionized carboxyl group of CBMAA, —SO.sub.3— for the bands of the ionized sulfo group of SBMAA.

Line (2) in FIG. 1 shows PM-IRRAS spectra of terpolymer prepared in solution by RAFT polymerization as described in Example 3.

[0036] FIG. 2 is a response pattern of SPR sensor with a chip with gold surface coated with the polymer brush, according to Examples 4 and 5.

[0037] FIG. 3 is a response pattern of SPR sensor upon detection of E. coli on the functionalized terpolymer brush, according to Example 6.

[0038] FIG. 4: Detection of SARS-Cov-2 spiked in cell cultured medium using poly[HPMAA(77 mol %)-co-CBMAA (20 mol %)-co-SBMAA(3 mol %)] functionalized QCM platform with anti- SARS-Cov-2 antibodies (Example 8).

[0039] FIG. 5: Detection of 1×10.sup.5 PFU/mL SARS-CoV-2 spiked in cultured cell medium using QCM sensor based on short peptide-functionalized poly[HPMAA(77 mol %)-co-CBMAA (20 mol %)-co-SBMAA(3 mol %)] surface (Example 9). The figure shows the detection response (black line) and blank response (red).

EXAMPLES

Example 1: Preparation of a Polymer Brush on Gold

[0040] A glass plate with a vapour-deposited gold layer was cleaned in a UV ozone generator for 10 minutes, then immediately rinsed with water and ethanol. The plate was then immersed in a 0.1 mM solution of ω-mercaptoundecyl bromoisobutyrate initiator in ethanol for 48 hours at room temperature without exposure to light. The catalyst mixture was prepared in a Schlenk flask by dissolving CuCl (28 mg), CuCl.sub.2 (8.4 mg) and Me4cyclam (96.8 mg) in degassed methanol (2 mL) under a nitrogen atmosphere and sonicated for 5 minutes until complete dissolution. In a second Schlenk flask, the monomers SBMAA (138 mg, 3 mol %), CBMAA (572 mg, 15 mol %) and HPMAA (1.847 mg, 82 mol %) were dissolved in degassed water (16.9 mL) and methanol (0.1 mL). Examples of additional batches for the preparation of surfaces with a different percentage of components are given in Table 1. After dissolution, the catalyst mixture was added to the monomer mixture under nitrogen. The homogeneous polymerization mixture was added to the reactor with a plate coated with initiator under nitrogen. The polymerization was carried out in the closed reactor for 2 hours at room temperature. The plate was then rinsed with water and stored in phosphate buffer (pH 7.4) at 4° C. before further use.

[0041] The chemical structure of the terpolymer brush was verified by infrared reflection absorption spectroscopy (PM-IRRAS). FIG. 1 shows a typical IRRAS spectrum for one embodiment of the terpolymer brush.

TABLE-US-00001 TABLE 1 Selected examples of batches for the preparation of terpolymer with different content of individual monomers Composition of polymerization batch [mol %] Composition of polymerization batch [mg] HPMAA CBMAA SBMAA HPMAA CBMAA SBMAA 84.5 15.0 0.5 1.903 572 23 82.0 15.0 3.0 1.847 572 138 80.0 15.0 5.0 1.802 572 230 75.0 15.0 10.0 1.689 572 460 70.0 15.0 15.0 1.576 572 690 55.0 15.0 30.0 1.239 572 1.379

Example 2: Preparation of a Polymer Brush on Glass

[0042] A glass plate was rinsed with acetone, sonicated for 20 minutes in 50% methanol at 20° C. and then sonicated for another 20 minutes in chloroform. The plate was then rinsed with water, air dried and cleaned for 4 minutes in a UV ozone generator. After cleaning, the plate was dehydrated for 1 hour at ≥90° C. in the presence of silica gel and immediately then immersed for 2 hours in a 3 mM solution of the initiator (MeO).sub.3—Si—(CH.sub.2).sub.11—Br in anhydrous n-heptane at room temperature. The plate was then rinsed with ethanol and water and placed in the reactor to polymerize. Another procedure for preparing the polymer brush is analogous to Example 1 above. The catalyst mixture was prepared in a Schlenk flask by dissolving CuCl (28 mg), CuCl.sub.2 (8.4 mg) and Me4cyclam (96.8 mg) in degassed methanol (2 mL) under nitrogen atmosphere and sonicated for 5 minutes until complete dissolution. In a second Schlenk flask, the monomers SBMAA (138 mg, 3 mol %), CBMAA (572 mg, 15 mol %) and HPMAA (1,847 mg, 82 mol %) were dissolved in degassed water (16.9 mL) and methanol (0.1 mL). (Other examples of batches are the same as the examples of batches listed in Table 1). After dissolution, the catalyst mixture was added to the monomer mixture under nitrogen. The homogeneous polymerization mixture was added to the reactor with a plate with attached initiator under nitrogen. The polymerization was carried out in the closed reactor for 2 hours at room temperature. The plate was then rinsed with water and stored in phosphate buffer (pH 7.4) at 4° C. before further use.

Example 3: Preparation of Terpolymer in Solution by RAFT Polymerization

[0043] The terpolymer (pSBMAA 3 mol. %; pCBMAA 20 mol. %; pHPMAA 77 mol. %) in solution was prepared using modified RAFT (Reversible Addition Fragmentation Transfer) polymerization procedure described previously (C. Rodriguez-Emmenegger, B. V. K. J. Schmidt, Z. Sedlakova, V. Šubr, A. Bologna Alles, E. Brynda, C. Barner-Kowollik, Macromolecular Rapid Communications 2011, 32, 958).

[0044] Briefly, monomers—N—(2-hydroxypropyl)methacrylamide (HPMAA; 344.0 mg; 77 mol. %), carboxybetaine methacrylamide (CBMAA; 151.2 mg; 20 mol. %) and sulfobetaine methacrylamide (SBMAA; 27.4 mg; 3 mol. %)—and initiator 4,4-azobis(4-cyanopentanoic acid) (0.504 mg; 0.45 mM) were dissolved in 4 ml of acetic acid buffer (0.27 M acetic acid and 0.73 M sodium acetate, pH 5.2) in a Schlenk flask. In a second glass vial, a solution of RAFT agent 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (100 mg; 5 mg/ml) in 20 ml of 0.05 M sodium hydroxide was prepared. Both freshly prepared solutions were mixed in the Schlenk flask in an ice bath and the mixture was purged with nitrogen and stirred for 1 hour. Then, the polymerization mixture was heated at 75° C. for 10 hours in an oil bath. The reaction was stopped via rapid cooling and exposure to air. The polymerization solution was then dialyzed against Milli-Q water for 72 hours using SpectraPor 3 membrane (MWCO: 3500 Da), ultrapure water was changed several times. After the dialysis, the solution was freeze-dried to yield a solid product. The chemical structure of the terpolymer was verified by infrared spectroscopy (FIG. 1, line (2)). The infrared spectra contained characteristic bands of all components (amide: 1642 cm.sup.−1, 1528 cm.sup.−1, carboxyl: 1723 cm.sup.−1, 1594 cm.sup.−1, sulfo: 1200 cm.sup.−1, 1033 cm.sup.−1).

Example 4: Bacterial Antibody Binding

[0045] A poly[(HPMAA(80 mol %)-co-CBMAA(15 mol %)-co-SBMAA(5 mol %)] terpolymer brush was prepared on the gold layer of the chip according to Example 1. The chip was rinsed with water, inserted into the SPR sensor and carboxyl groups of CBMAA were activated for 20 minutes at a flow rate of 10 μL/min with a solution of 0.1M NHS and 0.5M EDC in deionized water to the active ester. After activation, the level of sensor response for the surface in water was recorded. 50 μg/mL of bacterial anti-Salmonella antibody was dissolved in borate buffer (10 mM, pH 8.5), infused into the microfluidic system of the SPR sensor at a rate of 30 μL/min and covalently bound by reaction with the activated brush (20 minutes). After immobilization of the antibody, the level of sensor response in water was again recorded. The amount of bound antibody was determined from the difference between the sensor response in water before antibody injection and the response in water after immobilization was completed (FIG. 2, the ‘immobilization level’ corresponds to 206 ng/cm.sup.2).

Example 5: Functionalization Capacity and Resistance to Non-Specific Adsorption after Binding of Functional Entities

[0046] According to Example 1, poly(HPMAA-co-CBMAA-co-SBMAA) terpolymer brushes were prepared containing (a) 84.5 mol % HPMAA, 15 mol % CBMAA, 0.5 mol % SBMAA, (b) 82 mol % HPMAA, 15 mol % CBMAA, 3 mol % SBMAA and (c) 55 mol % HPMAA, 15 mol % CBMAA, 30 mol % SBMAA on the gold layer. Furthermore, two already known reference antifouling surfaces were prepared, showing a high degree of resistance to non-specific bonds and at the same time sufficient functionalization capacity—a copolymer containing 85 mol % HPMAA and 15 mol % CBMAA (WO2016177354A3) and a polyCBAA homopolymer. The following is a procedure for the preparation of these polymers:

poly[HPMAA(85 mol %)-co-CBMAA(15 mol%)]

[0047] A glass plate with a vapour-deposited gold layer was cleaned in a UV ozone generator for 10 minutes, then immediately rinsed with water and ethanol. The plate was then immersed in a 0.1 mM solution of ω-mercaptoundecyl bromoisobutyrate initiator in ethanol for 48 hours at room temperature without exposure to light. The catalyst mixture was prepared in a Schlenk flask by dissolving CuCl (28 mg), CuCl.sub.2 (8.4 mg) and Me4cyclam (96.8 mg) in degassed methanol (2 mL) under a nitrogen atmosphere and sonicated for 5 minutes until complete dissolution. In a second Schlenk flask, the monomers CBMAA (572 mg, 15 mol %) and HPMAA (1,914 mg, 85 mol %) were dissolved in degassed water (16.9 mL) and methanol (0.1 mL). After dissolution, the catalyst mixture was added to the monomer mixture under nitrogen. The homogeneous polymerization mixture was added to the reactor with a plate coated with initiator under nitrogen. The polymerization was carried out in the closed reactor for 2 hours at room temperature. The plate was then rinsed with water and stored in phosphate buffer at 4° C. before further use.

polyCBAA

[0048] A glass plate with a vapour-deposited gold layer was cleaned in a UV ozone generator for 10 minutes, then immediately rinsed with water and ethanol. The plate was then immersed in a 0.1 mM solution of ω-mercaptoundecyl bromoisobutyrate initiator in ethanol for 48 hours at room temperature without exposure to light. The catalyst mixture was prepared in a Schlenk flask by dissolving CuCl (28 mg), CuCl.sub.2 (8.4 mg) and Me4cyclam (96.8 mg) in degassed methanol (2 mL) under a nitrogen atmosphere and sonicated for 5 minutes until complete dissolution. In a second Schlenk flask, the monomer CBMAA (3,590 mg) was dissolved in degassed water (16.9 mL) and methanol (0.1 mL). After dissolution, the catalyst mixture was added to the monomer mixture under nitrogen. The homogeneous polymerization mixture was added to the reactor with a plate coated with initiator under nitrogen. The polymerization was carried out in the closed reactor for 2 hours at room temperature. The plate was then rinsed with water and stored in phosphate buffer (pH 7.4) at 4° C. before further use.

[0049] The above-described polymer brushes were tested for resistance to non-specific adsorption: (a) activated and deactivated surface, (b) activated and functionalized surface after deactivation. The following procedure was analogous for all brushes. The chip with brush was rinsed with water, inserted into the SPR sensor and activated at a flow rate of 10 μL/min for 20 minutes with a solution of 0.1M NHS and 0.5M EDC in deionized water. In case (a) the surface was further washed with borate buffer (10 mM, pH 8.5, flow rate 30 μL/min) for 25 minutes; in case (b) the surface was washed with borate buffer (10 mM, pH 8.5, flow rate 30 μL/min) containing a specific dissolved functional entity with primary amines for 25 min. The following entities were immobilized on the following surfaces:

poly[HPMAA(84.5 mol %)-co-CBMAA(15 mol %)-co-SBMAA(0.5 mol %)]: amino-terminal DNA oligonucleotide (4 μM solution);
poly[HPMAA(82 mol %)-co-CBMAA(15 mol %)-co-SBMAA(3 mol %]: anti-bacterial anti-Salmonella antibody (50 μg/mL solution);
poly[HPMAA(55 mol %)-co-CBMAA(15 mol %)-co-SBMAA(30 mol %)]: poly L-lysine (50 μg/mL solution);
poly[HPMAA(85 mol %)-co-CBMAA(15 mol %)]: anti-bacterial anti-Salmonella antibody (50 μg/mL solution), or amino-terminal DNA oligonucleotide (4 μM solution), or poly L-lysine (50 μg/mL solution);
polyCBAA: anti-bacterial anti-Salmonella antibody (50 μg/mL solution), or amino-terminal DNA oligonucleotide (4 μM solution), or poly L-lysine (50 μg/mL solution).

[0050] Furthermore, in both cases (a) and (b), the same procedure was used for all surfaces. The brush was deactivated by reaction with 1M (2-aminoethoxy)acetic acid (30 min, flow rate 10 μL/min) and then incubated in PBS (10 mM phosphate buffer+150 mM NaCl+2.7 mM KCl, pH 7.4) for achieving equilibrium and constant sensor response. Then, 100% human blood plasma was injected at a rate of 30 μL/min at 25° C. for 10 minutes, followed by rinsing with PBS. After rinsing with PBS for 10 minutes, higher ionic strength PBS (10 mM phosphate buffer+750 mM NaCl+2.7 mM KCl, pH 7.4) was injected for 5 minutes to wash away the electrostatically adsorbed non-specific deposit, and finally the entire surface was relaxed again in PBS. The level of non-specific adsorption was determined from the difference between the sensor response in PBS before the blood plasma injection and 10 minutes after the end of the injection. The level of non-specific adsorption after washing in higher ionic strength PBS was determined from the difference of the sensor response in PBS before blood plasma injection and 10 minutes after the end of washing in higher ionic strength PBS. The results are shown in FIG. 2, Table 2 and Table 3.

[0051] FIG. 2 demonstrates that even at a concentration of 15 mol % CBMAA in the terpolymer, a sufficient amount of functional element (206 ng/cm.sup.2 anti-Salmonella) binds to the surface, while achieving high resistance to non-specific adsorption—non-specific adsorption from undiluted human plasma was 0 ng/cm.sup.2.

TABLE-US-00002 TABLE 2 Non-specific adsorption from undiluted blood plasma after activation and deactivation and activation/covalent binding of anti-Salmonella antibody and deactivation. Non-specific adsorption from undiluted blood plasma [ng/cm.sup.2] poly[HPMAA(82 mol %)- co-CBMAA(15 mol %)- poly[HPMAA(85 mol %)- co-SBMAA(3 mol %)] polyCBAA co-CBMAA(15 mol %)] After activation/ 8.1 66.1 20.8 deactivation After activation/ 0 2.5 16.9 deactivation and washing with higher ionic strength PBS After activation/anti- 10.9 51.9 39.5 Salmonella anchoring/ deactivation After activation/anti- 4.5 8.5 40.9 Salmonella anchoring/ deactivation and washing higher ionic strength PBS

[0052] Table 2 demonstrates the improved anti-fouling properties of the poly(HPMAA-co-CBMAA-co-SBMAA) terpolymer brush after activation and immobilization of functional elements compared to known anti-fouling carboxy-functional surfaces. At the same time, it shows a significantly lower level of non-specific adsorption even without the need to wash away the electrostatically adsorbed non-specific deposit.

TABLE-US-00003 TABLE 3 Non-specific adsorption from undiluted blood plasma after activation and covalent attachment of positively (poly L-lysine) and negatively (amino-terminated DNA oligonucleotide) charged ligand under given functionalization conditions on terpolymer brushes with different molar proportions of individual monomer units. Non-specific adsorption from undiluted blood plasma [ng/cm.sup.2] poly[HPMAA(84.5 mol %)- co-CBMAA(15 mol %)- poly[HPMAA(85 mol %)- co-SBMAA(0.5 mol %)] co-CBMAA(15 mol %)] After activation/anchoring 1.7 4.1 of amino-terminal DNA oligonucleotides/ deactivation After activation/anchoring 0 3.0 of amino-terminated DNA oligonucleotides/ deactivation and washing with higher ionic strength PBS poly[HPMAA(55 mol %)- co-CBMAA(15 mol %)- poly[HPMAA(85 mol %)- co-SBMAA(30 mol %)] co-CBMAA(15 mol %)] After activation/poly L- 6.0 11.7 lysine anchoring/ deactivation and washing with higher ionic strength PBS

[0053] Table 3 shows the advantage of tunability of the terpolymer brush composition over a wide range of sulfobetaine component content. For different ligands, the appropriate brush composition can be evaluated on the basis of the charge distribution so that after immobilization the surface remains as neutral as possible and thus resistant to non-specific adsorption. Thus, even lower levels of non-specific adsorption can be achieved than with known functionalizable anti-fouling surfaces without the sulfobetaine component.

Example 6: Biorecognition Ability of Terpolymer Brush after Binding of Functional Entities

[0054] According to Example 1, poly[HPMAA (75 mol %)-co-CBMAA (15 mol %)-co-SBMAA (10 mol %)] was prepared on the gold surface of the chip. The chip was inserted into the SPR sensor and functionalized with anti-Escherichia coli (E. coli) antibody, anti-E. coli, and with anti-carcinoembryonic antigen (CEA) antibody, anti-CEA, according to the procedure of Example 3. The latter antibody was used as a reference to show the specificity of E. coli detection. After functionalization, the remaining active esters were deactivated for 30 minutes by reaction with a solution of 1M (2-aminoethoxy)acetic acid (30 min, flow rate of 10 μL/min) and then the chip was allowed to stabilize in PBS (10 mM phosphate buffer+150 mM NaCl+2.7 mM KCl, pH 7.4). After stabilizing the sensor response, a solution of 1×10.sup.7 CFU/mL of inactivated E. coli in PBS (15 min, flow rate of 45 μL/min) was injected onto the chip. The chip was then rinsed again with PBS until the sensor response stabilized. FIG. 3 shows a record of the SPR sensor response. The response from the reference channel (black colour) in which anti-CEA antibody was immobilized was negligible against the specific response of the SPR sensor (red colour) in which anti-E. coli antibody was immobilized.

[0055] The polymer brushes in Examples 4, 5 and 6 were prepared in the thickness range of the random polymer layer from 80 to 100 nm (thickness was verified by ellipsometry and SPR in water).

Example 7

[0056] According to Example 1, the terpolymer brush with a composition of 81 mol % HPMAA, 15 mol % CBMAA, 4 mol % SBMAA was prepared on the gold surface of the chip. The chip was rinsed with water, inserted into the SPR sensor, and the carboxyl groups of CBMAA were activated for 20 minutes at a flow rate of 10 μL/min with a solution of 0.5M EDC and 0.1M NHS in deionized water to form active ester. After activation, the level of sensor response for the surface in water was recorded. 50 μg/mL of bacterial anti-E. coli antibody in borate buffer (10 mM, pH 8.5) was soaked in the microfluidic SPR sensor system at a flow rate of 30 μL/min and immobilized with 104±25 ng/cm.sup.2 of antibody. Subsequently, the residual active esters were deactivated for 36 minutes with one of three deactivating agent solutions: (1) a solution of glycine (1M in deionized water, pH 7), or (2) a solution of (2-aminoethoxy)acetic acid (1M in deionized water, pH 7).

[0057] The surfaces thus prepared were further washed with PBS (10 mM phosphate buffer+150 mM NaCl+2.7 mM KCl, pH 7.4) for 10 minutes. Then, 100% human blood plasma was injected at a rate of 30 μL/min at 25° C. for 10 minutes, followed by washing again with PBS. After washing with PBS for 10 minutes, higher ionic strength PBS (10 mM phosphate buffer+750 mM NaCl+2.7 mM KCl, pH 7.4) was injected for 5 minutes and finally the whole surface was relaxed again in PBS. The non-specific adsorption was determined from the difference in sensor response in PBS before blood plasma injection and 10 minutes after injection. The non-specific adsorption after washing with PBS with higher ionic strength was determined from the difference between the sensor response in PBS before the injection of blood plasma and 10 minutes after the end of injection with higher ionic strength PBS.

TABLE-US-00004 TABLE 4 Deactivation of residual active esters - non-specific adsorption from undiluted blood plasma after deactivation by two deactivation reagents Non-specific adsorption from Non-specific adsorption 100% blood plasma/washing from 100% blood plasma with higher ionic strength PBS Deactivating agent (ng/cm.sup.2) (ng/cm.sup.2) Glycine 20.3 13.7 (2-aminoethoxy)acetic acid 21.2 18.1

Example 8: QCM Biosensor Based on Antibody-Functionalized Terpolymer Brush for Direct Label-Free Detection of SARS-CoV-2 in Complex Biological Media

[0058] To prepare poly[HPMAA(77 mol %)-co-CBMAA (20 mol %)-co-SBMAA(3 mol %)]-coated QCM crystals (QCM=quartz crystal microbalance), QCM crystals with a thin gold layer were cleaned in UV-ozone cleaner for 10 minutes and subsequently rinsed with ultra-pure water and ethanol. Immediately after the cleaning, the crystals were immersed in an initiator solution of a 1 mM ω-mercaptoundecyl bromoisobutyrate in ethanol to form a self-assembled monolayer (SAM) and were left in the solution for 3 days at room temperature in dark. Methanol and water were degassed via 6 freeze-pump-thaw cycles. The catalyst solution was prepared as follows: under the nitrogen atmosphere CuCl (151.9 mg), CuCl.sub.2 (29.4 mg), and Me4Cyclam (338.8 mg) in a Schlenk tube were mixed with 7.1 mL of degassed methanol. The mixture was sonicated for 5 minutes to dissolve all solids. In a second Schlenk tube, monomers SBMAA (0.483 g, 3 mol %), CBMAA (2.667 g, 20 mol %), or HPMAA (6.069 g, 77 mol %) were dissolved in 13.3 mL degassed water and 46.1 mL degassed methanol and stirred. After the dissolution was completed, the catalyst solution was added to the monomer solution using a gastight syringe. The polymerization mixture was added into the reactor containing the substrates coated with the initiator SAM. The polymerization was carried out for 2 hours at room temperature. Finally, the samples were washed with ultra-pure water and stored in PBS at 6° C. until used. Before the experiment, QCM crystal coated with poly[HPMAA(77 mol %)-co-CBMAA (20 mol %)-co-SBMAA(3 mol %)] was removed from storing solution, rinsed with ultrapure water, and mounted into QCM sensor with the microfluidic system. After, it was washed with PBS buffer for 5 minutes (a flow rate of 45 μl/min) and ultrapure water for 5 minutes (45 μl/min). Subsequently, brushes were activated with a freshly prepared solution of 0.1 M NHS and 0.5 M EDC for 20 minutes (7 μl/min). Afterward, the coatings were washed for 3 minutes (60 μl/min) with HEPES buffer (5 mM HEPES, pH 6). The 50 μg/mL of the anti-SARS-Cov-2 (N-protein) antibody was diluted in HEPES buffer (5 mM HEPES, pH 7) and the solution was added and left to react with activated coating for 12 minutes (15 μl/min). After, all coatings were rinsed again with HEPES buffer (5 mM HEPES, pH 6) for 10 minutes (30 μl/min) and immersed in the deactivation solution of AEAA (1M aminoethoxy acetic acid, pH 7) for 25 minutes (7 μl/min). After, the coatings were rinsed with PBS for 10 minutes (30 μl/min) to create a baseline. Afterward, the SARS-Cov-2-spiked cell medium samples (ranging from 0 (blank) to 1×10.sup.6 PFU/mL) were added and let react for 12 minutes (30 μl/min). Finally, all the coatings were rinsed with PBS buffer for 10 minutes (30 μl/min). The concentration dependency of QCM sensor response is depicted in FIG. 4.

Example 9: QCM Biosensor Based on Peptide-Functionalized Terpolymer Brush for Direct Detection of SARS-CoV-2 in Complex Biological Medium

[0059] The QCM sensor coated with poly[HPMAA(77 mol %)-co-CBMAA (20 mol %)-co-SBMAA(3 mol %)] was prepared as described in Example 2.

[0060] Before the experiment, QCM crystal coated with poly[HPMAA(77 mol %)-co-CBMAA (20 mol %)-co-SBMAA(3 mol %)] was removed from storing solution, rinsed with ultrapure water, and mounted into QCM sensor with the microfluidic system. Afterwards, it was washed with PBS buffer for 5 minutes (a flow rate of 45 μl/min) and ultrapure water for 5 minutes (45 μl/min). Subsequently, brushes were activated with a freshly prepared solution of 0.1 M NHS and 0.5 M EDC for 20 minutes (7 μl/min). Afterward, the coatings were washed for 3 minutes (60 μl/min) with HEPES buffer (5 mM HEPES, pH 6). The 50 μg/mL of a short polyK-modified peptide sequence (GSGGSG-IEEQAKTFLDKFNHEAEDLFYQS, SEQ ID NO. 1) with a high affinity to SARS-Cov-2 was diluted in HEPES buffer (5 mM HEPES, pH 7) and the solution was added and left to react with activated coating for 12 minutes (15 μl/min). After, all coatings were rinsed again with HEPES buffer (5 mM HEPES, pH 6) for 10 minutes (30 μl/min) and immersed in the deactivation solution of AEAA (1M aminoethoxy acetic acid, pH 7) for 25 minutes (7 μl/min). Then, the coatings were rinsed with PBS for 10 minutes (30 μl/min) to create a baseline. Afterwards, the 1×10.sup.5 PFU/mL SARS-Cov-2-spiked cell medium or pure unspiked cell medium (blank) sample was added and let react for 12 minutes (30 μl/min). Finally, all the coatings were rinsed with PBS buffer for 10 minutes (30 μl/min). The example of detection is depicted in FIG. 5.