An anti-fouling super-hydrophilic gel qcm sensor and a preparation method and application thereof

Abstract

The present application relates to the technical field of sensors, and provides a kind of antifouling superhydrophilic gel QCM sensor and its preparation method and application.The present application first prepares boric acid hydrogel coating on the surface of QCM wafer, then prepares transition layer on the surface of boric acid hydrogel coating, and then obtains antifouling superhydrophilic gel QCM sensor by plasma treatment and coprecipitation deposition.The antifouling superhydrophilic gel QCM sensor prepared by the present application has good superhydrophilicity, is resistant to aging, can maintain superhydrophilic state in air for up to 480h, can maintain superhydrophilic state in water for up to 696h, has excellent protein resistance and antibacterial performance, and can realize accurate and effective monitoring of saliva sugar.

Description

Technical Field

[0001] This invention relates to the field of sensor technology, and in particular to an antifouling superhydrophilic gel QCM sensor, its preparation method, and its application. Background Technology

[0002] Protein adsorption onto material surfaces is a very common phenomenon. In analytical science, non-specific protein adsorption on sensor surfaces or analytical platforms is a serious problem affecting the analytical performance of equipment. Generally, proteins are adsorbed onto material surfaces within seconds of contact with a fluid. Proteins wet, disperse, and adsorb onto the surface, binding through chemical bonds, electrostatic interactions, and other means, ultimately attaching to the solid surface. Meanwhile, the bacterial community in the human oral cavity is highly complex, comprising approximately 1000 species. Like proteins, bacteria can reach material surfaces through van der Waals forces, electrostatics, and hydrophobic surfaces, causing specific reactions between bacteria and the material surface. This leads to non-specific adsorption onto the sensor chip surface, thereby reducing the sensor's sensitivity, specificity, and reproducibility.

[0003] Quartz crystal microbalance (QCM) is a mass sensor with advantages such as high sensitivity (its measurement accuracy can reach the nanogram level, theoretically detecting mass changes equivalent to a fraction of a monolayer or atomic layer), strong real-time measurement capability, simple integration, and low production cost. It is widely used in gas and liquid composition analysis, trace substance measurement, and thin film thickness detection. This has led to the widespread application of QCM in metrology, analytical chemistry, biology, surface / interface science, life sciences, and nanoscience, making it a powerful tool for advancing research in these fields.

[0004] QCM-based glucose detection technology detects glucose by designing and modifying a glucose-sensitive membrane on the surface of a crystal chip, which then specifically and reversibly binds to glucose. By recording the frequency shift before and after the QCM chip comes into contact with a sugar-containing solution, the glucose concentration in the solution can be continuously monitored. The absolute value of ΔF increases when glucose molecules bind to the glucose-sensitive membrane. However, when testing real saliva samples, the main challenge lies in the high complexity of saliva composition. Saliva contains a low concentration of small glucose molecules (only 1 / 100 to 1 / 50 of blood glucose) and extremely high concentrations of large proteins (71–2232 mg / L) and bacteria. When the QCM sensor is exposed to a saliva sample, the large signal generated by the non-specific adsorption of proteins and bacteria by the membrane on the chip makes it difficult to effectively identify the weak signal generated by the binding of trace amounts of glucose.

[0005] Therefore, to achieve effective and accurate detection of salivary glucose, it is urgent to develop a material with both antibacterial and antiprotein properties to solve this problem. Summary of the Invention

[0006] In view of this, the present invention provides a fouling-resistant superhydrophilic gel QCM sensor, its preparation method, and its application. The gel QCM sensor provided by the present invention has good fouling resistance and hydrophilicity, excellent antibacterial and antiprotein properties, and can achieve effective and accurate detection of saliva sugar.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] A method for preparing an antifouling superhydrophilic gel QCM sensor includes the following steps:

[0009] A boric acid hydrogel coating was prepared on the surface of a QCM wafer using a prepolymerization solution; the prepolymerization solution consisted of 3-acrylamidophenylboronic acid, N,N'-methylenebisacrylamide, acrylamide, and 2,2-dimethoxyphenyl ethyl ketone.

[0010] A transition layer solution is coated onto the surface of the boric acid hydrogel coating to form a boric acid hydrogel-transition composite layer on the surface of the QCM wafer; the raw materials for preparing the transition layer solution include carboxylated cellulose nanofibers, activators, and hyperbranched polyethyleneimine;

[0011] The boric acid hydrogel-transition composite layer is modified by plasma treatment to form a modified boric acid hydrogel-transition composite layer on the surface of the QCM wafer.

[0012] The QCM wafer with the boric acid hydrogel-transition composite modified layer attached is immersed in a coprecipitation solution for coprecipitation deposition, forming a deposition layer on the surface of the boric acid hydrogel-transition composite modified layer to obtain the antifouling superhydrophilic gel QCM sensor; the components of the coprecipitation solution include tannic acid, polyethyleneimine, polyvinylpyrrolidone and water.

[0013] Preferably, before preparing the boric acid hydrogel coating on the surface of the QCM wafer, the surface of the QCM wafer is further modified. The surface modification includes the following steps: ultrasonically treating the QCM wafer in Piranha solution, followed by a first wash and a first dry; then immersing the QCM wafer in a mixed solution of (3-aminopropyl)triethoxysilane and ethanol, followed by a second wash and a second dry; then immersing the QCM wafer in maleic anhydride solution, followed by a third wash and a third dry.

[0014] Preferably, in the prepolymerization solution, the mass fraction of 3-acrylamidophenylboronic acid is 10-30%, the mass fraction of N-N'-methylenebisacrylamide is 0.5-5%, the mass fraction of acrylamide is 61-89%, and the mass fraction of 2,2-dimethoxy-phenylethyl ketone is 0.5-4%.

[0015] Preferably, the method for preparing the boric acid hydrogel coating is a UV-assisted polymerization method; the UV-assisted polymerization method includes the following steps: dropping the prepolymerization solution onto the surface of a quartz plate, placing the QCM wafer face down on the prepolymerization solution, pressing the back of the QCM wafer, irradiating the QCM wafer with ultraviolet light, and then separating the QCM wafer with the boric acid hydrogel coating from the quartz plate.

[0016] Preferably, the mass ratio of the carboxylated cellulose nanofibers to the hyperbranched polyethyleneimine is 1:0.5-3; the activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and the mass ratio of the carboxylated cellulose nanofibers, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:0.02-0.06:0.05-0.08;

[0017] The method for preparing the transition layer solution includes: mixing carboxylated cellulose nanofibers, an activator, and water and reacting for 10–30 min; then mixing the resulting reaction solution with hyperbranched polyethyleneimine for 6–48 h; centrifuging the resulting mixture to remove the supernatant; and dispersing the centrifuged residue in ethanol to obtain the transition layer solution.

[0018] Preferably, the coating method is spin coating; the spin coating includes low-speed spin coating and high-speed spin coating performed sequentially; the low-speed spin coating has a rotation speed of 200-1000 rpm and a time of 3-10 s, and the high-speed spin coating has a rotation speed of 2500-4000 rpm and a time of 10-40 s.

[0019] Preferably, the oxygen flow rate of the oxygen plasma treatment is 10-30 sccm, the time is 3-10 min, and the power is 100-300 W.

[0020] Preferably, the mass ratio of tannic acid, polyethyleneimine, and polyvinylpyrrolidone in the coprecipitation solution is 2-6:1:5-30.

[0021] The present invention also provides a fouling-resistant superhydrophilic gel QCM sensor prepared by the preparation method described above.

[0022] The present invention also provides the application of the antifouling superhydrophilic gel QCM sensor described above in saliva sugar monitoring.

[0023] This invention provides a method for preparing an antifouling superhydrophilic gel QCM sensor. The method first prepares a boric acid hydrogel coating on the surface of a QCM wafer, then prepares a transition layer on the boric acid hydrogel coating, and finally obtains the antifouling superhydrophilic gel QCM sensor through plasma treatment and co-precipitation deposition. In this invention, the transition layer is composed of carboxylated cellulose nanofibers (CNF-C) and hyperbranched polyethyleneimine (bPEI). Cellulose nanofibers (CNF) possess unique high mechanical strength, tunable surface chemistry, and biodegradability. CNF-C not only retains the aforementioned properties of CNF but also has a large number of carboxyl groups that enhance its hydrophilicity. bPEI has a dense molecular structure and a three-dimensional spatial structure, which maintains good material permeability. This invention spin-coates the transition film synthesized from CNF-C and bPEI onto the surface of HPBA to form a boric acid hydrogel-transition composite layer (CNF-C@bPEI / HPBA), which can prevent damage to the boric acid hydrogel coating during subsequent plasma treatment.

[0024] This invention utilizes plasma treatment to modify CNF-C@bPEI / HPBA. Plasma treatment is widely recognized as a green technology, reducing chemical consumption and requiring no pretreatment of the sample for modification on any type of material. Furthermore, plasma treatment achieves very thin surface modification depths (from a few nanometers to hundreds of nanometers), minimizing its impact on the overall material. The average energy of various active particles in the plasma is higher than the bond energies of the constituent materials. Therefore, after plasma bombardment of the material surface, bond breakage occurs, generating a large number of free radicals. Upon contact with atmospheric oxygen, these free radicals undergo oxidation, forming hydrophilic groups such as -OH and -COOH. In addition, the material surface can be etched to alter its roughness. In summary, this step significantly increases the free energy of the CNF-C@bPEI / HPBA surface and reduces the surface water contact angle, thereby achieving a superwettable state.

[0025] However, plasma-modified materials often exhibit "aging" phenomena on their surfaces. This is because superhydrophilic materials have high surface energy, which gradually decreases over time, causing the surface to shift from hydrophilic to hydrophobic, ultimately leading to a reduction in surface energy. Therefore, this invention utilizes a mixed solution of tannic acid (TA), polyethyleneimine (PEI), and polyvinylpyrrolidone (PVP) via co-precipitation to address this issue. TA, a natural plant polyphenol, contains numerous hydroxyl groups, which interact with the hydrophilic polymer PVP through hydrogen bonds. Under the action of the PEI crosslinking agent, TA copolymerizes on the membrane surface, further enhancing the membrane's hydrophilicity. Simultaneously, the addition of TA reduces pore collapse caused by strong capillary forces during drying, protecting the membrane's permeability. More importantly, TA interacts with polar groups on the membrane surface, stabilizing hydrophilic groups and suspending bonds on the material surface, thus maintaining its superhydrophilicity. In summary, this invention, through the co-precipitation of TA, PE, and PVP, forms a co-deposited coating on the modified CNF-C@bPEI / HPBA surface, extending the material's superhydrophilic state.

[0026] The results of the examples show that the antifouling superhydrophilic gel QCM sensor prepared by the present invention has good superhydrophilicity and is resistant to aging. The superhydrophilic state can be maintained for up to 480 hours in air and up to 696 hours in water. It has excellent anti-protein and antibacterial properties and can achieve accurate and effective monitoring of saliva sugar. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the process for preparing the antifouling superhydrophilic gel QCM sensor in an embodiment of the present invention;

[0028] Figure 2 Water contact angle diagrams for HPBA(a), CNF-C@bPEI / HPBA(b), plasma-treated CNF-C@bPEI / HPBA(c), and co-precipitation-treated CNF-C@bPEI / HPBA(d);

[0029] Figure 3 The results of superhydrophilic aging tests of plasma-treated CNF-C@bPEI / HPBA and co-precipitation-treated CNF-C@bPEI / HPBA in air (a) and water (b);

[0030] Figure 4 The responses of HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA to BSA in saliva;

[0031] Figure 5The responses of HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA to DIA in saliva;

[0032] Figure 6 The responses of HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA to MYO in saliva;

[0033] Figure 7 The responses of HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA to KER in saliva;

[0034] Figure 8 The responses of HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA to LYS in saliva;

[0035] Figure 9 The responses of HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA to LAC in saliva;

[0036] Figure 10 The responses of HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA to FIB in saliva;

[0037] Figure 11 The responses of HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA to TRF in saliva;

[0038] Figure 12 Adsorption counts of Streptococcus mutans, Staphylococcus epidermidis, and Escherichia coli on HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA membranes;

[0039] Figure 13 SEM images of the effects of HPBA(i), plasma-treated CNF-C@bPEI / HPBA(ii), and co-precipitated CNF-C@bPEI / HPBA(iii) membranes on different bacteria.

[0040] Figure 14 The response of a superhydrophilic gel QCM sensor to different concentrations of glucose in 50% saliva to prevent contamination. Detailed Implementation

[0041] This invention provides a method for preparing an antifouling superhydrophilic gel QCM sensor, comprising the following steps:

[0042] A boric acid hydrogel coating was prepared on the surface of a QCM wafer using a prepolymerization solution; the prepolymerization solution consisted of 3-acrylamidophenylboronic acid, N,N'-methylenebisacrylamide, acrylamide, and 2,2-dimethoxyphenyl ethyl ketone.

[0043] A transition layer solution is coated onto the surface of the boric acid hydrogel coating to form a boric acid hydrogel-transition composite layer on the surface of the QCM wafer; the raw materials for preparing the transition layer solution include carboxylated cellulose nanofibers, activator, hyperbranched polyethyleneimine, water, and ethanol;

[0044] The boric acid hydrogel-transition composite layer is modified by plasma treatment to form a modified boric acid hydrogel-transition composite layer on the surface of the QCM wafer.

[0045] The QCM wafer with the boric acid hydrogel-transition composite modified layer attached is immersed in a coprecipitation solution for coprecipitation deposition, forming a deposition layer on the surface of the boric acid hydrogel-transition composite modified layer to obtain the antifouling superhydrophilic gel QCM sensor; the components of the coprecipitation solution include tannic acid, polyethyleneimine, polyvinylpyrrolidone and water.

[0046] This invention uses a prepolymer solution to prepare a boric acid hydrogel coating (denoted as HPBA) on the surface of a QCM wafer. This invention does not have special requirements for the QCM wafer; any QCM wafer well-known to those skilled in the art can be used. In a specific embodiment of this invention, the QCM wafer is AT-cut type, with a nominal frequency of 5MHz and a preferred diameter of 2.3cm. In this invention, before preparing the boric acid hydrogel coating on the surface of the QCM wafer, it is preferable to first modify the surface of the QCM wafer. The surface modification preferably includes the following steps: ultrasonically treating the QCM wafer in a Piranha solution, followed by a first wash and a first dry; then immersing the QCM wafer in a mixed solution of (3-aminopropyl)triethoxysilane (APS) and ethanol, followed by a second wash and a second dry; then immersing the QCM wafer in a maleic anhydride solution, followed by a third wash and a third dry; in this invention, the Piranha solution is preferably obtained by mixing 96wt% sulfuric acid and 30wt% hydrogen peroxide in a volume ratio of 7:3; the ultrasonic treatment time is preferably 5–20 minutes. The first washing is preferably water washing; the volume ratio of APS to ethanol in the APS and ethanol mixed solution is preferably 100 μL: 50 mL; the soaking time of the QCM wafer in the APS and ethanol mixed solution is preferably 6–48 h, more preferably 12 h, and the soaking temperature is preferably room temperature; the second washing is preferably ethanol washing; the solvent of the maleic anhydride solution is preferably N,N′-dimethylformamide, and the concentration of the maleic anhydride solution is preferably 0.02 g / mL; the soaking time of the QCM wafer in the maleic anhydride solution is preferably 6–48 h, more preferably 12 h, and the temperature is preferably room temperature; the first drying, second drying, and third drying are all preferably nitrogen blowing. This invention cleans the chip surface by ultrasonic treatment with Piranha solution, resulting in the production of hydroxyl groups on the surface; soaking in the APS and ethanol mixed solution causes the growth of amino groups on the chip surface; and soaking in the maleic anhydride solution causes the growth of polymerizable double bonds on the chip surface.

[0047] In this invention, the prepolymer solution comprises 3-acrylamidophenylboronic acid (3-APB), N,N'-methylenebisacrylamide (BIS), acrylamide (AM), and 2,2-dimethoxyphenyl ethyl ketone (DMPA). In the prepolymer solution, the mass fraction of 3-acrylamidophenylboronic acid is preferably 10-30%, more preferably 18%; the mass fraction of N,N'-methylenebisacrylamide is preferably 0.5-5%, more preferably 2%; the mass fraction of acrylamide is preferably 61-89%, more preferably 78%; and the mass fraction of 2,2-dimethoxyphenyl ethyl ketone is preferably 0.5-4%, more preferably 2%. 3-acrylamidophenylboronic acid serves as the host for binding glucose molecules, N,N'-methylenebisacrylamide is a crosslinking agent, acrylamide is a monomer for synthesizing boric acid hydrogels, and 2,2-dimethoxyphenyl ethyl ketone is a photoinitiator.

[0048] In this invention, the method for preparing the boric acid hydrogel coating is preferably an ultraviolet (UV) pressure-assisted polymerization method. The UV pressure-assisted polymerization method preferably includes the following steps: adding the prepolymer solution to the surface of a quartz plate, placing the QCM wafer face down on the prepolymer solution, pressing the back of the QCM wafer, and irradiating the QCM wafer with UV light. Then, the QCM wafer with the boric acid hydrogel coating is separated from the quartz plate. The method for pressing the back of the QCM wafer is preferably using a pressing machine. This invention does not have special requirements for the pressing pressure; it is sufficient to press the QCM wafer firmly. The wavelength of the UV light is preferably 365 nm, and the irradiation time is preferably 10–60 min, more preferably 30 min. The method for separating the QCM wafer with the boric acid hydrogel coating from the quartz plate is preferably: immersing the combined wafer and quartz sheet obtained after UV irradiation in distilled water for 1 hour to separate the QCM wafer with the boric acid hydrogel coating from the quartz plate. After obtaining the QCM wafer with the boric acid hydrogel coating, the present invention preferably rinses the QCM wafer with distilled water repeatedly and then dries it with nitrogen.

[0049] After obtaining a boric acid hydrogel coating on the surface of the QCM wafer, the present invention coats a transition layer solution onto the surface of the boric acid hydrogel coating to form a boric acid hydrogel-transition composite layer on the surface of the QCM wafer. In this invention, the raw materials for preparing the transition layer solution include carboxylated cellulose nanofibers (CNF-C), an activator, and hyperbranched polyethyleneimine (bPEI); the mass ratio of the carboxylated cellulose nanofibers to the hyperbranched polyethyleneimine is preferably 1:0.5-3, more preferably 1:1; the molecular weight of the hyperbranched polyethyleneimine is preferably 25,000; the activator preferably includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the mass ratio of the carboxylated cellulose nanofibers, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is preferably 1:0.02-0.06:0.05-0.08, more preferably 1:0.04:0.06.

[0050] In this invention, the preferred method for preparing the transition layer solution includes: mixing carboxylated cellulose nanofibers, an activator, and water and reacting for 10–30 min, preferably 15 min; then stirring and mixing the resulting reaction solution with hyperbranched polyethyleneimine for 6–48 h, preferably 12 h; centrifuging the resulting mixture, removing the supernatant, and dispersing the centrifuged residue in ethanol to obtain the transition layer solution. In this invention, the carboxylated cellulose nanofibers and the activator are first reacted to activate the carboxyl groups, which is beneficial for improving the reaction efficiency with hyperbranched polyethyleneimine. After the activated carboxylated cellulose nanofibers and hyperbranched polyethyleneimine are mixed, a condensation reaction occurs, generating a white flocculent substance. The centrifugation speed is preferably 10,000 rpm, and the centrifugation time is preferably 10 min. The preferred ratio of carboxylated cellulose nanofibers to ethanol is 2 g: 1 mL.

[0051] In this invention, the coating method for the transition layer solution is preferably spin coating; the spin coating preferably includes sequential low-speed spin coating and high-speed spin coating; the rotation speed of the low-speed spin coating is preferably 200-1000 rpm, more preferably 500 rpm, and the spin coating time is preferably 3-10 s, more preferably 5 s; the rotation speed of the high-speed spin coating is preferably 2500-4000 rpm, more preferably 3000 rpm, and the spin coating time is preferably 10-40 s, more preferably 30 s; preferably, the transition layer solution is dropwise added to the surface of the boric acid hydrogel, and then spin coating is performed; the drop volume of the transition layer solution is preferably 10-30 μL / cm. 2 More preferably 22 μL / cm 2After spin coating, a transition layer is formed on the surface of the boric acid hydrogel, that is, a boric acid hydrogel-transition composite layer (denoted as CNF-C@bPEI / HPBA) is obtained on the surface of the QCM wafer; after obtaining the boric acid hydrogel-transition composite layer, the present invention preferably rinses the QCM wafer with the boric acid hydrogel-transition composite layer attached repeatedly with distilled water and then dries it with nitrogen.

[0052] After forming a boric acid hydrogel-transition composite layer on the surface of the QCM wafer, the present invention modifies the boric acid hydrogel-transition composite layer by plasma treatment, thereby forming a modified boric acid hydrogel-transition composite layer on the surface of the QCM wafer. In the present invention, the oxygen flow rate of the oxygen plasma treatment is preferably 10-30 sccm, more preferably 20 sccm, the time is preferably 3-10 min, more preferably 5 min, and the power is preferably 100-300 W, more preferably 200 W; in a specific embodiment of the present invention, oxygen plasma is used to bombard the boric acid hydrogel-transition composite layer.

[0053] After forming a boric acid hydrogel-transition composite modified layer on the surface of the QCM wafer, the present invention immerses the QCM wafer with the boric acid hydrogel-transition composite modified layer attached in a coprecipitation solution for coprecipitation deposition, forming a deposition layer on the surface of the boric acid hydrogel-transition composite modified layer to obtain the antifouling superhydrophilic gel QCM sensor. In the present invention, the components of the coprecipitation solution include tannic acid (TA), polyethyleneimine (PEI), polyvinylpyrrolidone (PVP), and water, wherein the average molecular weight of the polyvinylpyrrolidone is preferably 5800; the mass ratio of tannic acid, polyethyleneimine, and polyvinylpyrrolidone in the coprecipitation solution is preferably 2-6:1:5-30, more preferably 4:1:10; the volume ratio of tannic acid to water is preferably 20 mg:30 mL; the temperature of the coprecipitation deposition is preferably room temperature, and the time is preferably 10-15 h, more preferably 12 h; after the coprecipitation deposition is completed, the present invention preferably rinses the obtained antifouling superhydrophilic gel QCM sensor with distilled water and then dries it with nitrogen.

[0054] This invention also provides an antifouling superhydrophilic gel QCM sensor prepared by the preparation method described above; the antifouling superhydrophilic gel QCM sensor includes a QCM wafer and a boric acid hydrogel-transition composite modified layer attached to the surface of the QCM wafer and a deposition layer attached to the surface of the boric acid hydrogel-transition composite modified layer; the deposition layer is obtained by co-precipitation of tannic acid, polyethyleneimine and polyvinylpyrrolidone; the QCM wafer is preferably a surface-modified QCM wafer, and the method of surface modification will not be described in detail here.

[0055] This invention also provides the application of the antifouling superhydrophilic gel QCM sensor described above in saliva sugar monitoring; this invention does not have special requirements for the method of the application, and any method well known to those skilled in the art can be used; in a specific embodiment of this invention, it is preferable to install the antifouling superhydrophilic gel coated chip prepared by this invention into the flow cell of the QCM, use a peristaltic pump to pump the sample solution to be tested into the flow cell, use QCM data acquisition software to monitor the frequency of the chip in real time, and record the detection data after the frequency stabilizes.

[0056] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0057] The sources of the reagents and consumables used in the examples are shown in Table 1.

[0058] Table 1 Summary of reagents and consumables used in the examples.

[0059]

[0060]

[0061] Figure 1 This is a schematic diagram of the process for preparing the antifouling superhydrophilic gel QCM sensor in an embodiment of the present invention.

[0062] Example 1

[0063] (1) Surface modification of QCM wafers: QCM wafers were sonicated in Piranha solution (H2SO4 (96%, w / w) and H2O2 (30%, w / w) in a volume ratio of 7:3) for 10 min. The treated wafers were then repeatedly washed with distilled water and dried under nitrogen. The wafers were then immersed in a mixed solution of APS (100 μL) and ethanol (50 mL). After soaking at room temperature for 12 h, the wafers were rinsed with ethanol and then dried under N2. Finally, the above wafers were immersed in maleic anhydride (1 g) and N,N′-dimethylformamide (50 mL) at room temperature for 12 h. The wafers were rinsed with ethanol and dried under N2.

[0064] Borate hydrogel (HPBA) synthesis on QCM wafer surface: First, a 5 mol / L prepolymer solution was prepared, consisting of 18% 3-APB, 2% BIS, 78% AM, and 2% DMPA by mass fraction. Then, 25 μL of the prepolymer solution was pipetted onto a 10 × 10 cm quartz plate. The wafer was placed face down on the prepolymer solution, and the back of the QCM wafer was pressed firmly using a press-fit machine under appropriate force. After irradiating the wafer with a UV lamp (λ = 365 nm) for 30 min, the wafer was placed in distilled water for 1 h, causing the hydrogel-coated QCM wafer to separate from the quartz plate. Finally, the coated wafer was repeatedly rinsed with re-distilled water and dried with N2.

[0065] (2) Preparation of CNF-C@bPEI / HPBA: CNF-C (2g) was added to 40mL of water containing EDC (80mg) and NHS (120mg) and reacted for 15min. Then bPEI (2g) was added and the mixture was magnetically stirred overnight. The resulting white flocculent material was centrifuged at high speed (10000rpm, 10min), and the supernatant was collected and redispersed with ethanol (1mL) to obtain a spin-coating solution. Then, 50μL of the spin-coating solution was added dropwise onto boric acid hydrogel (HPBA) (low speed: 500rpm, 5s; high speed: 3000rpm, 30s) to obtain CNF-C@bPEI / HPBA. The wafer was repeatedly rinsed with distilled water and dried with N2 for the next modification step.

[0066] (3) Preparation of plasma-treated CNF-C@bPEI / HPBA: CNF-C@bPEI / HPBA was prepared by treating it with O2 plasma (O2 flow rate: 20 sccm, time: 5 min, power: 200 W), and then washed with distilled water and dried with N2.

[0067] (4) Preparation of coprecipitated CNF-C@bPEI / HPBA: TA (20 mg), PEI (5 mg), and PVP (50 mg) were added to 30 mL of water and dissolved by sonication to obtain a coprecipitate solution. The wafers obtained in (3) were immersed in the coprecipitate solution and reacted at room temperature for 12 h. They were rinsed with distilled water and dried with N2 for subsequent testing.

[0068] Example 2: Water Contact Angle Test

[0069] The water contact angle of the surface of the material obtained in each step of Example 1 was tested, and the results are as follows: Figure 2 As shown, Figure 2(a), (b), (c), and (d) show the water contact angle test results for HPBA, CNF-C@bPEI / HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitation-treated CNF-C@bPEI / HPBA (i.e., the final antifouling superhydrophilic gel QCM sensor), respectively.

[0070] according to Figure 2 The results show that, compared with HPBA, the increased surface roughness of CNF-C@bPEI / HPBA improves the hydrophilicity of the film surface, resulting in a significant decrease in the contact angle. The plasma-treated CNF-C@bPEI / HPBA surface has many hydrophilic groups, making it a superhydrophilic film. CNF-C@bPEI / HPBA treated with co-precipitation also maintains a good superhydrophilic state.

[0071] Example 3: Superhydrophilic aging test

[0072] This invention extends the superhydrophilic state of materials through a simple and mild coprecipitation method. In this embodiment, the changes in the water contact angle of plasma-treated CNF-C@bPEI / HPBA and coprecipitated CNF-C@bPEI / HPBA were continuously monitored. Materials with a water contact angle <10° are generally considered to be superhydrophilic; therefore, 10° is considered the critical value for superhydrophilicity. The specific operation method is as follows: the plasma-treated CNF-C@bPEI / HPBA and the coprecipitated CNF-C@bPEI / HPBA (i.e., the final antifouling superhydrophilic gel QCM sensor) from Example 1 were placed in air and water, respectively, and the water contact angle was continuously monitored. The results are shown in […]. Figure 3 .

[0073] according to Figure 3 It can be seen that, in air, the superhydrophilicity of plasma-treated CNF-C@bPEI / HPBA can be maintained for 42 hours (15.48°±6.08°), while that of co-precipitated CNF-C@bPEI / HPBA can be maintained for 480 hours (11.19°±3.06°). Calculations show that the superhydrophilic state of the material before and after co-precipitation treatment is prolonged by 11.43 times in air. Figure 3 (a) In water, plasma-treated CNF-C@bPEI / HPBA maintained its superhydrophilic state for 64 h (11.02°±1.36°), while co-precipitated CNF-C@bPEI / HPBA maintained it for 696 h (11.87°±3.36°). Calculations show that the superhydrophilic state of the materials in water was prolonged by 10.88 times before and after co-precipitation treatment. Figure 3(b) The above results confirm that co-precipitation treatment significantly delays the superhydrophilic aging of CNF-C@bPEI / HPBA films.

[0074] Example 4 Anti-protein performance test

[0075] Eight common and highly concentrated proteins found in saliva—bovine serum albumin (BSA), diamylase (DIA), myoglobin (MYO), keratin (KER), lysozyme (LYS), lactoferrin (LAC), fibrinogen (FIB), and transferrin (TRF)—were selected for antifouling performance testing. Protein solutions were prepared using these proteins in a phosphate buffer solution at pH 7.5, with concentrations of 0 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 500 mg / L, and 1000 mg / L. The specific operating procedures were as follows: CNF-C@bPEI / HPBA wafers coated with co-precipitated material were dried with nitrogen and installed in the flow cell of a QCM (Qualitative Analyzer). A single protein solution was pumped into the flow cell using a peristaltic pump, and the wafer frequency was monitored in real-time using QCM data acquisition software. After the frequency stabilized, the antifouling performance of the material was evaluated. During the experiment, a sample solution (2 mL) was injected at a flow rate of approximately 200 μL / s. Once the solution has been completely injected into the reaction chamber, turn off the peristaltic pump. After the frequency stabilizes, continue testing the next sample.

[0076] This invention uses QCM to analyze the antifouling ability of HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA wafers in Example 1 against eight common proteins in saliva. The results are as follows: Figures 4-11 As shown. From Figures 4-11As can be seen, compared with HPBA, both plasma-treated CNF-C@bPEI / HPBA and co-precipitated CNF-C@bPEI / HPBA wafers exhibited superior anti-fouling performance in the detection of eight proteins. More importantly, for BSA and DIA, when the concentration increased to 1000 mg / L, the ΔF values ​​of the co-precipitated CNF-C@bPEI / HPBA wafers were 0.43±3.01 Hz and -0.2±6.39 Hz, respectively, with a response frequency almost zero. When the concentrations of MYO, KER, LYS, LAC, FIB, and TRF were 1000 mg / L, the corresponding ΔF values ​​were 5.87±3.83 Hz, 3.17±3.51 Hz, -14.07±9.41 Hz, -8.27±6.57 Hz, -20.9±17.23 Hz, and 1.30±5.16 Hz, respectively. It can be seen that, compared with the other two membranes, CNF-C@bPEI / HPBA exhibits the best anti-protein adsorption capacity after co-precipitation treatment, and its antifouling performance is due to the superhydrophilicity and the deposition layer formed by co-precipitation.

[0077] Example 5 Antibacterial test

[0078] Anti-adhesion experiments were conducted using Streptococcus mutans (S. mutans), Staphylococcus epidermidis, and Escherichia coli (E. coli), all widely distributed in saliva and the daily environment. The specific experimental method is as follows: First, these three bacteria were stored at -80℃ and streaked overnight on bovine heart and brain infusion (BHI) agar plates. Each strain was inoculated from a BHI agar plate into 10 mL of BHI and incubated at 37℃ for 24 h (except for S. mutans). Bacteria (2400 g) were collected by centrifugation, washed twice with sterile phosphate-buffered saline (PBS), and resuspended in sterile PBS to a final concentration of 1×10⁻⁶. 7 Antibacterial assays were performed using CFU / mL. Different coated wafers were placed in 2 mL of bacterial suspension in 6-well cell culture plates and incubated for 3 h. After incubation, the specimens were removed with sterile forceps, gently rinsed with sterile PBS to remove loosely adhering bacteria, and then immersed in 2 mL of sterile PBS in a fresh 6-well cell culture plate. To quantify viable adhering bacteria, the plates were sonicated for 30 s, and 10 μL of serially diluted samples were plated in triplicate on BHI agar plates. The plates were incubated at 37 °C for 24 h or 48 h, and colony formation was calculated in units (CFU), with results recorded as CFU per mL. The entire bacterial adhesion assay was repeated in three independent assays.

[0079] Test results are as follows Figures 12-13 As shown, Figure 12Adsorption counts of Streptococcus mutans, Staphylococcus epidermidis, and Escherichia coli on HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA membranes; Figure 13 SEM images of the effects of HPBA(i), plasma-treated CNF-C@bPEI / HPBA(ii), and co-precipitated CNF-C@bPEI / HPBA(iii) membranes on different bacteria.

[0080] from Figure 12 It was found that, compared with HPBA, the plasma-treated CNF-C@bPEI / HPBA surface showed a 97.2% reduction in Streptococcus mutans, while the co-precipitation-treated CNF-C@bPEI / HPBA showed a 98.4% reduction. Similar results were observed for Staphylococcus epidermidis and Streptococcus mutans, with the co-precipitation-treated CNF-C@bPEI / HPBA exhibiting a 96.3% antibacterial adhesion rate. Furthermore, this embodiment also used Escherichia coli to study the antibacterial properties of the material, and the co-precipitation-treated CNF-C@bPEI / HPBA achieved an anti-adhesion rate of 79.5% against Escherichia coli. These results indicate that the co-precipitation-treated superhydrophilic material possesses excellent anti-adhesion capabilities against these three bacteria, including the most common pathogenic bacteria in the oral cavity.

[0081] Simultaneously, SEM was used to visually observe the amount of bacteria adhering to the material surface and the morphology of bacteria on different material surfaces; Figure 13 It can be seen that both plasma treatment and co-precipitation treatment of CNF-C@bPEI / HPBA effectively prevent bacterial adhesion to the wafer surface, with co-precipitation treatment showing the best results. These results are consistent with bacterial count data, further confirming the excellent anti-adhesion ability of co-precipitated CNF-C@bPEI / HPBA. SEM images also show that the morphology of bacteria on the surfaces of HPBA, plasma-treated CNF-C@bPEI / HPBA, and co-precipitated CNF-C@bPEI / HPBA materials did not change significantly, indicating that the antibacterial mechanism is to prevent bacteria from adhering to the material surface.

[0082] Example 6: Real Saliva Test

[0083] To fully demonstrate the antifouling performance of the co-precipitated CNF-C@bPEI / HPBA wafers, this embodiment added different concentrations of glucose to real saliva solutions. The volume ratio of saliva to PBS (pH 7.5) was 1:1, and the glucose concentrations were 0.5 mM, 1.0 mM, 1.5 mM, and 2 mM, with PBS used as a control. The antifouling superhydrophilic gel QCM sensor prepared in Example 1 was used to detect saliva solutions with different glucose concentrations. The detection method was as follows: The CNF-C@bPEI / HPBA wafers coated with the co-precipitated solution were dried with nitrogen and installed in the flow cell of the QCM. A peristaltic pump was used to pump the saliva solution into the flow cell, and the frequency of the wafers was monitored in real time using QCM data acquisition software. After the frequency stabilized, the detection data was recorded. During the experiment, a sample (2 mL) was injected at a flow rate of approximately 200 μL / s. After the solution was completely injected into the reaction cell, the peristaltic pump was turned off. After the frequency stabilized, the next sample was tested.

[0084] Test results are as follows Figure 14 As shown.

[0085] according to Figure 14 As can be seen, the absolute value of the response frequency increases significantly with the increase of glucose concentration, indicating that the sensor has good anti-fouling performance and is expected to be applied to the detection of glucose in actual saliva samples.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle 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 an antifouling superhydrophilic gel QCM sensor, characterized in that, Includes the following steps: A boric acid hydrogel coating was prepared on the surface of a QCM wafer using a prepolymerization solution; the prepolymerization solution consisted of 3-acrylamidophenylboronic acid, N,N'-methylenebisacrylamide, acrylamide, and 2,2-dimethoxyphenyl ethyl ketone. A transition layer solution is coated onto the surface of the boric acid hydrogel coating to form a boric acid hydrogel-transition composite layer on the surface of the QCM wafer. The raw materials for preparing the transition layer solution include carboxylated cellulose nanofibers, an activator, and hyperbranched polyethyleneimine. The mass ratio of the carboxylated cellulose nanofibers to the hyperbranched polyethyleneimine is 1:0.5~3. The activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. The mass ratio of the carboxylated cellulose nanofibers, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:0.02~0.06:0.05~0.

08. The method for preparing the transition layer solution includes: mixing carboxylated cellulose nanofibers, activator and water and reacting for 10-30 min; then mixing the resulting reaction solution with hyperbranched polyethyleneimine for 6-48 h; centrifuging the resulting mixture to remove the supernatant; and dispersing the centrifuged residue in ethanol to obtain the transition layer solution. The boric acid hydrogel-transition composite layer is modified by plasma treatment to form a modified boric acid hydrogel-transition composite layer on the surface of the QCM wafer. The QCM wafer with the boric acid hydrogel-transition composite modified layer attached is immersed in a coprecipitation solution for coprecipitation deposition, forming a deposition layer on the surface of the boric acid hydrogel-transition composite modified layer to obtain the antifouling superhydrophilic gel QCM sensor; the components of the coprecipitation solution include tannic acid, polyethyleneimine, polyvinylpyrrolidone and water.

2. The preparation method according to claim 1, characterized in that, Before preparing the boric acid hydrogel coating on the surface of the QCM wafer, the surface of the QCM wafer is modified. The surface modification includes the following steps: the QCM wafer is ultrasonically treated in Piranha solution and then subjected to a first wash and a first dry. Then, the QCM wafer is immersed in a mixed solution of (3-aminopropyl)triethoxysilane and ethanol and then subjected to a second wash and a second dry. Next, the QCM wafer is immersed in maleic anhydride solution and then subjected to a third wash and a third dry.

3. The preparation method according to claim 1, characterized in that, In the prepolymer solution, the mass fraction of 3-acrylamidophenylboronic acid is 10-30%, the mass fraction of N-N'-methylenebisacrylamide is 0.5-5%, the mass fraction of acrylamide is 61-89%, and the mass fraction of 2,2-dimethoxy-phenylethyl ketone is 0.5-4%.

4. The preparation method according to claim 1, 2 or 3, characterized in that, The method for preparing the boric acid hydrogel coating is a UV-assisted polymerization method; the UV-assisted polymerization method includes the following steps: the prepolymerization solution is dropped onto the surface of a quartz plate, the QCM wafer is placed face down on the prepolymerization solution, the back of the QCM wafer is pressed, and the QCM wafer is irradiated with ultraviolet light, and then the QCM wafer with the boric acid hydrogel coating is separated from the quartz plate.

5. The preparation method according to claim 1, characterized in that, The coating method is spin coating; the spin coating includes low-speed spin coating and high-speed spin coating performed sequentially; the low-speed spin coating has a rotation speed of 200~1000 rpm and a time of 3~10 s, and the high-speed spin coating has a rotation speed of 2500~4000 rpm and a time of 10~40 s.

6. The preparation method according to claim 1, characterized in that, The oxygen flow rate for plasma treatment is 10-30 sccm, the time is 3-10 min, and the power is 100-300 W.

7. The preparation method according to claim 1, characterized in that, The mass ratio of tannic acid, polyethyleneimine, and polyvinylpyrrolidone in the coprecipitation solution is 2~6:1:5~30.

8. The antifouling superhydrophilic gel QCM sensor prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the antifouling superhydrophilic gel QCM sensor according to claim 8 in saliva sugar monitoring.

Images