A sugar-sensitive and antifouling bifunctional probe, its preparation method and application

Abstract

This invention provides a glucose-sensitive and antifouling bifunctional probe, its preparation method, and its application, belonging to the field of glucose detection technology. It addresses the problems of poor sensitivity and antifouling stability in existing biological probes used for glucose detection. The preparation method of the glucose-sensitive and antifouling bifunctional probe includes: preparing a phenylboronic acid hydrogel membrane as a glucose-sensitive functionalized probe; mixing 2-bromoisobutyric acid, 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine, and a chemical bridge containing a primary amine group, and performing an amidation reaction to obtain an ATRP initiator; mixing an antifouling monomer and the ATRP initiator, and performing an ATRP reaction to obtain an antifouling coating solution; wherein the chemical bridge containing the primary amine group is 1,3-diamino-isopropanol and / or pentaerythritol; and spin-coating the antifouling coating solution onto the surface of the phenylboronic acid hydrogel membrane of the glucose-sensitive functionalized probe to obtain a glucose-sensitive and antifouling bifunctional probe with an antifouling coating. This glucose-sensitive and antifouling bifunctional probe exhibits high sensitivity and antifouling stability.

Description

Technical Field

[0001] This invention relates to the field of glucose detection technology, specifically to a glucose-sensitive and anti-fouling bifunctional probe, its preparation method, and its application. Background Technology

[0002] Diabetes is a non-communicable disease that seriously threatens health, with its main harm lying in the occurrence of complications such as vision loss and kidney failure. Saliva collection is safe, non-invasive, and convenient, thus offering a natural advantage as a sample for disease testing. Studies have shown a positive correlation between salivary glucose and blood glucose concentrations; when blood glucose levels exceed 10-15 mmol / L, a large amount of glucose is secreted into the saliva.

[0003] In recent years, phenylboronic acid hydrogel membranes have been widely used as glucose-sensitive functionalized probes for diabetes prevention and adjuvant therapy due to their excellent glucose selectivity, wide detection range, and low cost. However, the concentration of glucose in saliva is low (μM level), only 1 / 10 to 1 / 100 of the blood glucose concentration; in addition, saliva contains a large number of impurities such as proteins (mM level), which can non-specifically adsorb onto the surface of glucose-sensitive functionalized probes, leading to inaccurate detection signals. Among these issues, the low rate and efficiency of phenylboronic acid-glucose molecule binding in complex liquid environments is the fundamental problem causing low detection sensitivity and long detection times.

[0004] Polymer molecular brushes prepared using the ATRP method can significantly reduce the non-specific adsorption of impurities such as proteins by providing a stable hydration layer. However, as an antifouling coating, the polymer molecular brush exhibits low grafting rate and poor adhesion on the surface of phenylboronic acid hydrogel membranes. Furthermore, its steric hindrance reduces the binding rate and efficiency of phenylboronic acid-glucose, leading to a decrease in the sensitivity and antifouling performance of the sugar-sensitive antifouling bioprobe. Studies have found that antifouling coatings containing two catechol groups and a single-arm polymer molecular brush demonstrate better adhesion and antifouling performance on the substrate surface. However, due to the mismatch between the number of catechol groups and the number of polymer molecular brushes, increasing the antifouling coating thickness inevitably increases steric hindrance and strong dipole interactions, thus affecting the bioprobe sensitivity and antifouling stability. Summary of the Invention

[0005] The technical problem this invention aims to solve is to provide a bifunctional glucose-sensitive and anti-fouling probe with high sensitivity, excellent anti-fouling performance, and good stability. The anti-fouling coating of this bifunctional probe possesses superior adhesion and anti-fouling properties. It not only overcomes the problem of decreased glucose sensitivity and anti-fouling performance caused by steric hindrance and strong dipole interactions in traditional anti-fouling coatings, but also maintains good anti-fouling performance for a long time, enabling sensitive detection of glucose molecules in real physiological environments.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing a sugar-sensitive-antifouling bifunctional probe, comprising: Step (1): Prepare a phenylboronic acid hydrogel membrane as a sugar-sensitive functionalized probe; Step (2): 2-bromoisobutyric acid, 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine and a chemical bridge containing a primary amine group are mixed and subjected to an amidation reaction to obtain an ATRP initiator; the antifouling monomer and the ATRP initiator are mixed and subjected to an ATRP reaction to obtain an antifouling coating solution. The chemically bridged linker containing a primary amine group is 1,3-diamino-isopropanol and / or pentaerythritol; Step (3): Spin coat the antifouling coating solution onto the surface of the phenylboronic acid hydrogel membrane of the sugar-sensitive functionalized probe to obtain a sugar-sensitive-antifouling bifunctionalized probe containing an antifouling coating.

[0007] Optionally, the thickness of the phenylboronic acid hydrogel film is 100-800 nm.

[0008] Optionally, the chemically bridged linker containing a primary amine group is pentaerythritol.

[0009] Optionally, the mass ratio of the ATRP initiator to the antifouling monomer is 1:100-500.

[0010] Optionally, the antifouling monomer is sulfonated betaine methacryloyl ester and / or carboxylated betaine methacrylate.

[0011] Optionally, in step (1), the preparation of the phenylboronic acid hydrogel membrane includes: dissolving N,N-methylenebisacrylamide, 2,2-dimethoxy-1,2-diphenyl ethyl ketone, 3-acryloylaminophenylboronic acid and acrylamide in a solvent to obtain a prepolymer solution, spin-coating the prepolymer solution onto the surface of a double-bond functionalized quartz chip, and irradiating the quartz chip coated with the prepolymer solution with an ultraviolet lamp to obtain the phenylboronic acid hydrogel membrane.

[0012] Optionally, the mass ratio of N,N-methylenebisacrylamide, 2,2-dimethoxy-1,2-diphenyl ethyl ketone, 3-acryloylaminophenylboronic acid and acrylamide is 1:1-5:10-25:18-50.

[0013] Optionally, the thickness of the anti-fouling coating is 120-350 nm.

[0014] Secondly, the present invention provides a sugar-sensitive and anti-fouling bifunctional probe, which is prepared by the above-mentioned method for preparing a sugar-sensitive and anti-fouling bifunctional probe.

[0015] Thirdly, the present invention provides the application of the above-mentioned sugar-sensitive and anti-fouling dual-function probe in the detection of salivary glucose.

[0016] The above-described solution of the present invention has at least the following beneficial effects: The above-described scheme of the present invention first prepares a sugar-sensitive functionalized probe containing a phenylboronic acid hydrogel membrane. An antifouling coating with catechol groups adapted to polymer molecular brushes is prepared using an ATRP method with chemical bridges. The antifouling coating is then spin-coated onto the surface of the phenylboronic acid hydrogel membrane to obtain a sugar-sensitive-antifouling bifunctionalized probe. In the preparation of the ATRP initiator, 2-bromoisobutyric acid, 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine, and a chemical bridge containing primary amine groups are mixed and subjected to an amidation reaction. The chemical bridge ensures that the number of catechol groups is compatible with the number of polymer molecular brushes. Different amounts of primary amine chemical bridges can generate different numbers of ATRP initiation sites, thereby controlling the number of polymer molecular brushes. Simultaneously, the chemical bridge regulates the number of TBDMS protecting groups of 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine, thereby controlling the compatibility between the number of catechol groups and the number of polymer molecular brushes, improving the adhesion and antifouling stability of the antifouling coating, and increasing detection sensitivity. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the preparation process of the ATRP initiator containing 4 catechol groups and 4 initiation sites in Example 1; Figure 2 The image shown is the AFM image of the phenylboronic acid hydrogel membrane of Example 1. Figure 3 The image shows the AFM diagram of the sugar-sensitive-antifouling bifunctional probe from Example 1. Detailed Implementation

[0018] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0019] In a first aspect, the present invention provides a method for preparing a sugar-sensitive-antifouling bifunctional probe, comprising: Step (1): Prepare a phenylboronic acid hydrogel membrane as a sugar-sensitive functionalized probe; Step (2): 2-bromoisobutyric acid, 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine and a chemical bridge containing a primary amine group are mixed and subjected to an amidation reaction to obtain an ATRP initiator; the antifouling monomer and the ATRP initiator are mixed and subjected to an ATRP reaction to obtain an antifouling coating solution. The chemical bridge containing the primary amine group is 1,3-diamino-isopropanol and / or pentaerythritol; Step (3): Spin coat the antifouling coating solution onto the surface of the phenylboronic acid hydrogel membrane of the sugar-sensitive functionalized probe to obtain a sugar-sensitive-antifouling bifunctionalized probe containing an antifouling coating.

[0020] For example, in step (1), the preparation of the phenylboronic acid hydrogel membrane includes: dissolving N,N-methylenebisacrylamide, 2,2-dimethoxy-1,2-diphenyl ethyl ketone, 3-acrylamide phenylboronic acid and acrylamide in a solvent to obtain a prepolymer solution, spin-coating the prepolymer solution onto the surface of a double-bond functionalized quartz chip, and irradiating the quartz chip coated with the prepolymer solution with an ultraviolet lamp to obtain the phenylboronic acid hydrogel membrane.

[0021] For example, the mass ratio of N,N-methylenebisacrylamide, 2,2-dimethoxy-1,2-diphenyl ethyl ketone, 3-acryloylaminophenylboronic acid and acrylamide is 1:1-5:10-25:18-50.

[0022] For example, the viscosity of the prepolymer at 25°C is 4-25 mPa·s. This viscosity range facilitates uniform spin coating and allows for controllable thickness of the phenylboronic acid hydrogel film between 100-800 nm.

[0023] For example, in step (1), the spin coating speed is 500-3000 rpm.

[0024] For example, in step (1), the wavelength λ of the ultraviolet lamp is 365nm and the irradiation time is 60-70min.

[0025] For example, in step (1), the preparation method of the prepolymer solution includes: weighing N,N-methylenebisacrylamide, 3-acrylamide phenylboronic acid and acrylamide into centrifuge tubes, adding dimethyl sulfoxide into the centrifuge tubes, wrapping with tin foil, and labeling it as solution A; weighing 2,2-dimethoxy-1,2-diphenyl ethyl ketone into centrifuge tubes, adding dimethyl sulfoxide into the centrifuge tubes, wrapping with tin foil, and labeling it as solution B; sonicating solution A and solution B respectively to completely dissolve the solids in the solution, and then mixing solution A and solution B evenly to obtain the prepolymer solution.

[0026] For example, the thickness of the phenylboronic acid hydrogel film is 100-800 nm. The viscoelasticity of the phenylboronic acid hydrogel film is positively correlated with its resistance R. When the thickness is in the range of 100-800 nm, the change in its resistance is almost constant. Therefore, the effect of viscoelasticity on the decrease in sensitivity and stability can be reduced, and the sugar sensitivity performance can be improved.

[0027] For example, in step (2), the chemical bridge containing primary amine groups is pentaerythritol. Using pentaerythritol as a bridge allows the resulting ATRP initiator to contain four catechol groups and four initiation sites (four polymer molecular brushes). On the one hand, the four catechol groups can enhance the adhesion of the antifouling coating, ensuring long-term antifouling stability; on the other hand, the four catechol groups necessarily contain four polymer molecular brushes simultaneously. By controlling the length of the molecular brushes, the antifouling coating within the thickness of this invention can achieve excellent sensitivity and antifouling stability.

[0028] For example, the molar ratio of the chemical bridge to 2-bromoisobutyric acid and 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine is 1:4-10:4-10. This molar ratio ensures complete consumption of the chemical bridge, and for schemes where the chemical bridge is pentaerythritol, it facilitates the preparation of an antifouling coating with four catechol groups and four molecular brushes.

[0029] For example, the mass ratio of the ATRP initiator to the antifouling monomer is 1:100-500.

[0030] For example, the antifouling monomer is sulfonated betaine methacryloyl ester and / or carboxylated betaine methacrylate.

[0031] For example, the temperature of the ATRP reaction is 30-40°C.

[0032] For example, in step (2), the preparation method of the ATRP initiator includes: mixing 2-bromoisobutyric acid, 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine with a solvent, adding an activator, activating the carboxyl group, and then adding a chemical bridge containing a primary amine group to carry out an amidation reaction to obtain the ATRP initiator.

[0033] For example, the activator comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) in a molar ratio of 1:1.

[0034] For example, a catalyst and ligands are also added to the ATRP reaction.

[0035] For example, the catalyst is cuprous bromide and copper bromide, and the ligand is 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane.

[0036] For example, in step (3), the spin coating speed is 500-4500 rpm.

[0037] For example, the thickness of the antifouling coating is 120-350 nm. Within this thickness range, the interactions between the antifouling polymer molecular chains are weak, preventing the formation of a dense barrier layer, thus facilitating glucose molecule penetration and binding to sugar-sensitive functionalized probes.

[0038] This invention first prepares a phenylboronic acid hydrogel membrane via surface-initiated free radical polymerization. Then, using chemical bridging polymers, an antifouling coating solution with compatible catechol groups and polymer molecular brushes is prepared via the ATRP method. This antifouling coating solution is then spin-coated onto the surface of the phenylboronic acid hydrogel membrane to obtain a sugar-sensitive and antifouling bifunctional probe. Optimizing the thickness of the phenylboronic acid hydrogel membrane reduces the influence of viscoelasticity, thereby improving sugar sensitivity. Furthermore, the compatibility between the number of catechol groups and the number of polymer molecular brushes significantly enhances the adhesion and antifouling properties of the antifouling coating, reduces its steric hindrance, and improves sensitivity.

[0039] Secondly, this invention provides a sugar-sensitive and anti-fouling bifunctional probe, which is prepared by the above-described method for preparing a sugar-sensitive and anti-fouling bifunctional probe. This sugar-sensitive and anti-fouling bifunctional probe exhibits high sensitivity and good anti-fouling stability.

[0040] Thirdly, the present invention provides the application of the above-mentioned sugar-sensitive and anti-fouling dual-function probe in the detection of salivary glucose.

[0041] The following specific embodiments further illustrate the sugar-sensitive-antifouling bifunctional probe of the present invention and its preparation method.

[0042] Example 1 (1) Weigh 3 mg of crosslinking agent N,N-methylenebisacrylamide, 34 mg of 3-acrylamide phenylboronic acid, and 55 mg of acrylamide into a 1.5 mL centrifuge tube. Add 100 μL of dimethyl sulfoxide into the same centrifuge tube and wrap it with aluminum foil. This solution is labeled as solution A. Weigh 5 mg of 2,2-dimethoxy-1,2-diphenyl ethyl ketone into a 1.5 mL centrifuge tube. Add 100 μL of dimethyl sulfoxide into the same centrifuge tube and wrap it with aluminum foil. This solution is labeled as solution B. Sonicate solutions A and B for 10 min to completely dissolve the solids. Mix solutions A and B thoroughly to obtain the desired prepolymer solution (viscosity 20 mPa·s). Spin-coat the prepolymer solution onto the surface of a quartz chip at 2000 rpm and irradiate with a UV lamp (wavelength λ = 365 nm) for 60 min to obtain the sugar-sensitive functionalized probe. The mass ratio of N,N-methylenebisacrylamide, 2,2-dimethoxy-1,2-phenylethyl ketone, 3-acryloylaminophenylboronic acid, and acrylamide was 1:1.7:11.3:18.3; the obtained sugar-sensitive functionalized probe (phenylboronic acid hydrogel membrane) had a thickness of 260 nm (e.g., Figure 2 (As shown).

[0043] (2) 2.5 mg of 2-bromoisobutyric acid and 12 mg of 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine were placed in a three-necked flask. 50 mL of dimethyl sulfoxide was added to the flask, and the system was activated at 60 °C with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) in a molar ratio of 1:1 to carry out an amidation reaction. Subsequently, pentaerythritol chemically bridged linkers were added for further amidation to obtain an ATRP initiator with four catechol groups and four initiation sites. Figure 1 As shown in the diagram, the red circle indicates the initiation site. 5.9 mg of cuprous bromide, 19.1 mg of copper bromide, 40.0 mg of 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane, and 2.5 mg of the ATRP initiator obtained above were weighed and placed in a round-bottom flask containing ethanol and water. 500 mg of sulfonated betaine methacryloyl ester was also weighed and placed in the same flask. The ATRP reaction was carried out at 30°C to obtain an antifouling coating solution with four catechol groups and four polymer molecules suitable for brushing. The mass ratio of the ATRP initiator to the antifouling monomer (sulfonated betaine methacryloyl ester) was 1:200.

[0044] (3) The antifouling coating solution obtained above is spin-coated onto the surface of the sugar-sensitive functionalized probe obtained in step 1 at 4000 rpm to obtain a sugar-sensitive-antifouling bifunctionalized probe with an antifouling coating on the surface. The thickness of the entire probe is 380 nm (e.g., ...). Figure 3As shown in the figure, since the thickness of the sugar-sensitive functionalized probe is 260nm, the thickness of the anti-fouling coating is 120nm.

[0045] Example 2 (1) Weigh 2 mg of crosslinking agent N,N-methylenebisacrylamide, 25 mg of 3-acrylamide phenylboronic acid, and 40 mg of acrylamide into a 1.5 mL centrifuge tube. Add 100 μL of dimethyl sulfoxide into the same centrifuge tube and wrap it with aluminum foil. This solution is labeled as solution A. Weigh 4 mg of 2,2-dimethoxy-1,2-diphenyl ethyl ketone into a 1.5 mL centrifuge tube. Add 100 μL of dimethyl sulfoxide into the same centrifuge tube and wrap it with aluminum foil. This solution is labeled as solution B. Sonicate solutions A and B for 10 min to completely dissolve the solids. Mix solutions A and B thoroughly to obtain the desired prepolymer solution (viscosity 16 mPa·s). Spin-coat the prepolymer solution onto the surface of a quartz chip at 1500 rpm and irradiate with a UV lamp for 60 min to obtain a sugar-sensitive functionalized probe. The mass ratio of N,N-methylenebisacrylamide, 2,2-dimethoxy-1,2-phenylethyl ketone, 3-acryloylaminophenylboronic acid and acrylamide is 1:2:12.5:20; the thickness of the obtained sugar-sensitive functionalized probe is 420 nm. (2) 2.5 mg of 2-bromoisobutyric acid and 12 mg of 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine were placed in a three-necked flask. 50 mL of dimethyl sulfoxide was added to the flask, and the system was activated at 60 °C with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) in a molar ratio of 1:1 to carry out the amidation reaction. Subsequently, 1,3-diamino-isopropanol chemical bridges were added for further amidation to obtain an ATRP initiator with two catechol groups and two initiation sites. 5.9 mg of cuprous bromide, 19.1 mg of copper bromide, 40.0 mg of 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane, and 2 mg of the ATRP initiator obtained above were weighed and placed in a round-bottom flask containing ethanol and water. 450 mg of carboxybenzene methacrylate was also weighed and placed in the same round-bottom flask. The ATRP reaction was carried out at 30°C to obtain an antifouling coating solution with two catechol groups and two polymer molecules adapted to the brush. The mass ratio of the ATRP initiator to the antifouling monomer (carboxybenzene methacrylate) was 1:225.

[0046] (3) The antifouling coating solution obtained above is spin-coated onto the surface of the sugar-sensitive functionalized probe obtained in step 1 at 3500 rpm to obtain a sugar-sensitive-antifouling bifunctionalized probe with an antifouling coating on the surface. The thickness of the antifouling coating is 220 nm.

[0047] Example 3 (1) Weigh 2.5 mg of crosslinking agent N,N-methylenebisacrylamide, 50 mg of 3-acrylamide phenylboronic acid, and 50 mg of acrylamide into a 1.5 mL centrifuge tube. Add 100 μL of dimethyl sulfoxide into the same centrifuge tube and wrap it with aluminum foil. This solution is labeled as solution A. Weigh 5 mg of 2,2-dimethoxy-1,2-diphenyl ethyl ketone into a 1.5 mL centrifuge tube. Add 100 μL of dimethyl sulfoxide into the same centrifuge tube and wrap it with aluminum foil. This solution is labeled as solution B. Sonicate solutions A and B for 10 min to completely dissolve the solids. Mix solutions A and B thoroughly to obtain the desired prepolymer solution (viscosity 25 mPa·s). Spin-coat the prepolymer solution onto the surface of a quartz chip at 1500 rpm and irradiate with a UV lamp for 60 min to obtain a sugar-sensitive functionalized probe. The mass ratio of N,N-methylenebisacrylamide, 2,2-dimethoxy-1,2-phenylethyl ketone, 3-acryloylaminophenylboronic acid and acrylamide is 1:2:20:20; the thickness of the obtained sugar-sensitive functionalized probe is 370 nm. (2) 2.5 mg of 2-bromoisobutyric acid and 12 mg of 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine were placed in a three-necked flask. 50 mL of dimethyl sulfoxide was added to the flask, and the system was activated at 60 °C with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) in a molar ratio of 1:1 to carry out an amidation reaction. Subsequently, pentaerythritol chemical bridges were added for further amidation to obtain an ATRP initiator with four catechol groups and four initiation sites. 5.9 mg of cuprous bromide, 19.1 mg of copper bromide, 40.0 mg of 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane, and 1 mg of the ATRP initiator obtained above were weighed and placed in a round-bottom flask containing ethanol and water. 500 mg of sulfonated betaine methacryloyl ester was also weighed and placed in the same flask. An ATRP reaction was carried out at 30°C to obtain an antifouling coating solution with four catechol groups and four polymer molecular brushes. The mass ratio of the ATRP initiator to the antifouling monomer (sulfonated betaine methacryloyl ester) was 1:500.

[0048] (3) The above-obtained antifouling coating solution is spin-coated onto the surface of the sugar-sensitive functionalized probe obtained in step 1 at 1500 rpm to obtain a sugar-sensitive-antifouling bifunctionalized probe. The thickness of the antifouling coating is 350 nm.

[0049] Example 4 (1) Weigh 1.5 mg of crosslinking agent N,N-methylenebisacrylamide, 15 mg of 3-acrylamide phenylboronic acid, and 45 mg of acrylamide into a 1.5 mL centrifuge tube. Add 100 μL of dimethyl sulfoxide into the same centrifuge tube and wrap it with aluminum foil. This solution is labeled as solution A. Weigh 3 mg of 2,2-dimethoxy-1,2-diphenyl ethyl ketone into a 1.5 mL centrifuge tube. Add 100 μL of dimethyl sulfoxide into the same centrifuge tube and wrap it with aluminum foil. This solution is labeled as solution B. Sonicate solutions A and B for 10 min to completely dissolve the solids. Mix solutions A and B thoroughly to obtain the desired prepolymer solution (viscosity 12 mPa·s). Spin-coat the prepolymer solution onto the surface of a quartz chip at 3000 rpm and irradiate with a UV lamp for 60 min to obtain a sugar-sensitive functionalized probe. The mass ratio of N,N-methylenebisacrylamide, 2,2-dimethoxy-1,2-phenylethyl ketone, 3-acryloylaminophenylboronic acid and acrylamide is 1:2:10:30; the thickness of the obtained sugar-sensitive functionalized probe is 280 nm. (2) 2.5 mg of 2-bromoisobutyric acid and 12 mg of 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine were placed in a three-necked flask. 50 mL of dimethyl sulfoxide was added to the flask, and the system was activated at 60 °C with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) in a molar ratio of 1:1 to carry out an amidation reaction. Subsequently, 1,3-diamino-isopropanol chemical bridges were added for further amidation to obtain an ATRP initiator with two catechol groups and two initiation sites. 5.9 mg of cuprous bromide, 19.1 mg of copper bromide, 40.0 mg of 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane, and 5 mg of the ATRP initiator obtained above were weighed and placed in a round-bottom flask containing ethanol and water. 500 mg of sulfonated betaine methacryloyl ester was also weighed and placed in the same flask. An ATRP reaction was carried out at 30°C to obtain an antifouling coating solution with four catechol groups and four polymer molecular brushes. The mass ratio of the ATRP initiator to the antifouling monomer (sulfonated betaine methacryloyl ester) was 1:100.

[0050] (3) The above-obtained antifouling coating solution is spin-coated onto the surface of the sugar-sensitive functionalized probe obtained in step 1 at 2500 rpm to obtain a sugar-sensitive-antifouling bifunctionalized probe. The thickness of the antifouling coating is 305 nm.

[0051] Comparative Example 1 In this comparative example, an antifouling coating was not prepared on the surface of the sugar-sensitive functionalized probe.

[0052] Weigh 3 mg of crosslinking agent N,N-methylenebisacrylamide, 34 mg of 3-acrylamide phenylboronic acid, and 55 mg of acrylamide into 1.5 mL centrifuge tubes. Add 100 μL of dimethyl sulfoxide to the same centrifuge tube and wrap it with aluminum foil. This solution is labeled as solution A. Weigh 5 mg of 2,2-dimethoxy-1,2-diphenyl ethyl ketone into a 1.5 mL centrifuge tube. Add 100 μL of dimethyl sulfoxide to the same centrifuge tube and wrap it with aluminum foil. This solution is labeled as solution B. Sonicate solutions A and B for 10 min to completely dissolve the solids. Mix solutions A and B thoroughly to obtain the desired prepolymer solution. Spin-coat the prepolymer solution onto the surface of a quartz chip at 2000 rpm and irradiate with UV light for 60 min to obtain a sugar-sensitive functionalized probe.

[0053] Comparative Example 2 A bifunctional sugar-sensitive and antifouling probe was prepared by placing an antifouling material inside the sugar-sensitive probe: (1) The quartz chip was ultrasonically treated in a piranha solution (98% H2SO4:30% H2O2=7:3) for 10 min to remove organic matter, then rinsed with distilled water and dried under nitrogen. Then, at room temperature, the cleaned quartz chip was immersed in a solution with a concentration of 1×10⁻⁶. -3 In a solution of 2-(2-bromoisobutyryloxy)undecylthiol (MuBiB) initiator, a self-assembled monolayer (SAM) can be formed on the surface of a quartz chip after 24 hours. Before grafting the polymer molecular brush, the MuBiB initiator-modified quartz chip is cleaned with ethanol and then dried with nitrogen gas for later use.

[0054] (2) A mixture of 10 mL ethanol and water (volume ratio 1:1) was degassed, and this process was repeated three times. Then, under a nitrogen atmosphere, the solution was transferred to a round-bottom flask containing 5.9 mg of cuprous bromide, 19.1 mg of copper bromide, and 40.0 mg of 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane. A quartz chip modified with MuBiB initiator was placed in a 50 mL specially made flat-bottom flask, and 500 mg of sulfonyl betaine methacryloyl ester was weighed and added to the flask. The mixture was then degassed, and this process was repeated three times. The prepared blue catalyst solution was then transferred to a flat-bottom flask containing a quartz chip modified with MuBiB initiator, and a surface-initiated ATRP reaction was carried out at 30 °C to obtain a polymer molecular brush.

[0055] (3) A prepolymer solution containing 3 mg of crosslinking agent N,N-methylenebisacrylamide, 34 mg of 3-acrylamide phenylboronic acid, 55 mg of acrylamide and 5 mg of 2,2-dimethoxy-1,2-diphenyl ethyl ketone was dropped onto the surface of the quartz chip with the above-mentioned growing polymer brush. The quartz chip with the prepolymer solution was irradiated with a UV lamp for 60 min, and an IPN hydrogel film was prepared by UV gel curing method.

[0056] Comparative Example 3 Direct preparation of sugar-sensitive and antifouling bifunctional probes for modifying phenylboronic acid hydrogel membranes with traditional antifouling coatings: The preparation process of the sugar-sensitive functionalized probe is the same as that in Example 1; Traditional antifouling coating preparation method: 5.9 mg of cuprous bromide, 19.1 mg of copper bromide, 40.0 mg of 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane, and 5 mg of MuBiB initiator were weighed and placed in a round-bottom flask containing ethanol and water. 500 mg of sulfonated betaine methacryloyl ester was also weighed and placed in the same flask. An ATRP reaction was carried out at 30 °C to obtain the traditional antifouling coating solution. The obtained antifouling coating solution was spin-coated onto the surface of a sugar-sensitive functionalized probe at 4000 rpm to obtain a sugar-sensitive-antifouling bifunctionalized probe with a traditional antifouling coating modified phenylboronic acid hydrogel membrane.

[0057] Comparative Example 4 The antifouling coating of the comparative sugar-sensitive-antifouling bifunctional probe contains two catechol groups and a single-arm polymer molecular brush. The preparation method is as follows: The preparation process of the sugar-sensitive functionalized probe is the same as that in Example 1; Antifouling coating preparation method: 50 mg of MBHA amino resin was dissolved in methylpyrrolidone at room temperature, and the formate ester was deprotected with 20% piperidine by mass fraction; 2.5 mg of 2-bromoisobutyric acid and the above MBHA amino resin were placed in a mixed solvent of O-benzotriazole-tetramethylurea hexafluorophosphate and methylpyrrolidone (volume ratio 1:1) and subjected to amidation reaction at 60 °C to obtain an ATRP initiator with 2 catechol groups and 1 initiation site. 5.9 mg of cuprous bromide, 19.1 mg of copper bromide, 40.0 mg of 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane, and 5 mg of the ATRP initiator obtained above were weighed and placed in a round-bottom flask containing ethanol and water. 500 mg of sulfonated betaine methacryloyl ester was also weighed and placed in the same round-bottom flask. An ATRP reaction was carried out at 30°C to obtain an antifouling coating with two catechol groups and one polymer molecular brush. The mass ratio of the ATRP initiator to the antifouling monomer (sulfonated betaine methacryloyl ester) was 1:200.

[0058] Comparative Example 5 The sugar-sensitive-antifouling bifunctional probe was prepared using the same method as in Example 1, except that pentaerythritol chemical bridges were not added in step (2).

[0059] Test case The biological probes prepared in Examples 1-4 and Comparative Examples 1-5 were loaded into a quartz crystal microbalance biosensor. Glucose solutions of 0 mg / L, 5 mg / L, 10 mg / L, 12 mg / L, 13 mg / L, 15 mg / L, 20 mg / L, 30 mg / L, 35 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, and 300 mg / L were sequentially added to the flow cell of the sensor. Frequency changes were observed, and the concentration of glucose solution corresponding to the stable frequency observed by the biosensor was recorded to obtain the detection limit. The frequency changes at different times corresponding to the addition of 500 mg / L mucin were also recorded to obtain the corresponding amount of mucin adsorbed on the probe surface. The results are shown in Table 1.

[0060] Table 1. Detection limits and antifouling stability data for different probes

[0061] As can be seen from Table 1, the sugar-sensitive-antifouling bifunctional probe prepared by the preparation method of the present invention has high detection sensitivity and antifouling stability.

[0062] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a sugar-sensitive and anti-fouling bifunctional probe, characterized in that, include: Step (1): Prepare a phenylboronic acid hydrogel membrane as a sugar-sensitive functionalized probe; Step (2): 2-bromoisobutyric acid, 3,4-bis(tert-butyl-dimethyl-siloxy)-1-phenylalanine and a chemical bridge containing a primary amine group are mixed and subjected to an amidation reaction to obtain an ATRP initiator; the antifouling monomer and the ATRP initiator are mixed and subjected to an ATRP reaction to obtain an antifouling coating solution. The chemically bridged linker containing a primary amine group is 1,3-diamino-isopropanol and / or pentaerythritol; Step (3): Spin coat the antifouling coating solution onto the surface of the phenylboronic acid hydrogel membrane of the sugar-sensitive functionalized probe to obtain a sugar-sensitive-antifouling bifunctionalized probe containing an antifouling coating.

2. The method for preparing the sugar-sensitive-antifouling bifunctional probe according to claim 1, characterized in that, The thickness of the phenylboronic acid hydrogel film is 100-800 nm.

3. The method for preparing the sugar-sensitive-antifouling bifunctional probe according to claim 1, characterized in that, The chemical bridge containing the primary amine group is pentaerythritol.

4. The method for preparing the sugar-sensitive-antifouling bifunctional probe according to claim 1, characterized in that, The mass ratio of the ATRP initiator to the antifouling monomer is 1:100-500.

5. The method for preparing the sugar-sensitive-antifouling bifunctional probe according to claim 1, characterized in that, The antifouling monomer is sulfonated betaine methacryloyl ester and / or carboxylated betaine methacrylate.

6. The method for preparing the sugar-sensitive-antifouling bifunctional probe according to claim 1, characterized in that, In step (1), the preparation of the phenylboronic acid hydrogel membrane includes: dissolving N,N-methylenebisacrylamide, 2,2-dimethoxy-1,2-diphenyl ethyl ketone, 3-acrylamide phenylboronic acid and acrylamide in a solvent to obtain a prepolymer solution, spin-coating the prepolymer solution onto the surface of a double-bond functionalized quartz chip, and irradiating the quartz chip coated with the prepolymer solution with an ultraviolet lamp to obtain the phenylboronic acid hydrogel membrane.

7. The method for preparing the sugar-sensitive-antifouling bifunctional probe according to claim 6, characterized in that, The mass ratio of N,N-methylenebisacrylamide, 2,2-dimethoxy-1,2-diphenyl ethyl ketone, 3-acryloylaminophenylboronic acid and acrylamide is 1:1-5:10-25:18-50.

8. The method for preparing the sugar-sensitive-antifouling bifunctional probe according to claim 1, characterized in that, The thickness of the antifouling coating is 120-350 nm.

9. A sugar-sensitive and anti-fouling dual-function probe, characterized in that, The sugar-sensitive-antifouling bifunctional probe is prepared by the preparation method of the sugar-sensitive-antifouling bifunctional probe according to any one of claims 1 to 8.

10. The application of the glucose-sensitive and anti-fouling bifunctional probe according to claim 9 in the detection of salivary glucose.

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