A temperature-responsive yeast cell-imprinted modified electrode, its preparation method, and its application.

Abstract

This invention belongs to the field of imprinted sensor fabrication and application technology, and discloses a temperature-responsive yeast cell imprinted modified electrode, its fabrication method, and its application. This invention utilizes chronoamperometry and drop casting to modify gold electrodes, obtaining Au / AuNPs / Ti3C2T. x Au / AuNPs modified electrode. Using fluorescently labeled yeast cells as catalyst and template molecules, N-isopropylacrylamide as the temperature-responsive functional monomer and α-methacrylic acid as the auxiliary functional monomer, the electrode surface was modified using the MVL ATRP method. After removing the yeast cells, Au / AuNPs / Ti3C2T electrode was obtained. x / AuNPs / CIPs modified electrode. This modified electrode can be used as an electrochemical sensor for detecting yeast cells. This sensor has advantages such as fast detection speed and high sensitivity, with a linear response range of 1.0 × 10⁻⁶ for detecting yeast cells. 2 ~1.0×10 9 cells / mL.

Description

Technical Field

[0001] This invention belongs to the field of imprint sensor fabrication and application technology. This invention relates to a temperature-responsive yeast cell imprint modified electrode, its preparation method, and its application. Background Technology

[0002] The vital life activities and functions of organisms are controlled by the interactions between specific macromolecules within cells. At the cellular level, dysfunctional molecular interactions often lead to cellular malfunctions and disease. Biomimetic tools for exploring molecular interactions have been used in cell imaging, improving drug delivery, tissue engineering, and diagnostics. Rapid and accurate cell analysis is crucial in clinical diagnosis, efficacy evaluation, and personalized treatment. Currently, methods for quantitative cell detection mainly include enzyme-linked immunosorbent assays (ELISA), polymerase chain reaction (PCR), and flow cytometry. However, these established methods have significant drawbacks, such as complex equipment and cumbersome procedures. Furthermore, the antibody activity in immunoassays is difficult to maintain, and natural antibodies are difficult to purify. Therefore, developing portable and low-cost methods for selectively detecting cells without antibodies is of great significance.

[0003] Cell-imprinted polymers (CIPs) mimic the specific recognition properties of natural receptors, exhibiting similar specificity, high selectivity, and high strength. They can be used as recognition elements to construct sensor platforms for cell recognition. CIPs are polymer materials synthesized by capturing the structural and chemical information of cells through template-assisted assembly of functional groups. Typically, the polymer is cured around a template cell, which is then removed, leaving pores whose size, shape, and function are complementary to the target cell. CIPs have attracted increasing attention due to their unique advantages such as low cost, reusability, and ease of handling.

[0004] MVL ATRP is an environmentally friendly method for preparing CIPs. Fluorescein is a commonly used photocatalyst in MVL ATRP, possessing visible light absorption, good chemical stability, long excited-state lifetime, and good redox potential. However, fluorescein is usually free and can remain in the imprinted polymer, which will affect subsequent analysis of the imprinted polymer. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a temperature-responsive yeast cell-imprinted modified electrode, its preparation method, and its application. Based on the MVL ATRP method in Ti3C2T… xCell-imprinted polymers were prepared on the surface of a gold disk electrode modified with AuNPs composite material, and yeast cells were detected with high sensitivity using an electrochemical method.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] A temperature-responsive yeast cell-imprinted modified electrode includes a gold disk electrode, chloroauric acid, and Ti3C2T. x Temperature-responsive functional monomer N-isopropylacrylamide (NIPAM), auxiliary functional monomer α-methacrylic acid (MAA), and crosslinking agent N,N'-methylene-Bis-acrylamide (MBA).

[0008] The gold disc electrode is not specifically limited, but the commercially available model CHI101 (Φ=2mm, purchased from Shanghai Chenhua Instrument Co., Ltd.) is preferred.

[0009] The specific steps of the above-mentioned method for preparing temperature-responsive yeast cell-imprinted modified electrodes are as follows:

[0010] a. A clean gold disk electrode was inserted into a 0.25–4% chloroauric acid solution (using ultrapure water as the solvent). Electrodeposition was performed for 400 s at a constant potential of -0.9 V using a chronoamperometry method. The electrode was then rinsed with ultrapure water and dried with nitrogen to obtain an Au / AuNPs-modified electrode. 5 μL of Ti3C2T4, which had been ultrasonically treated for 20 min, was then drop-cast onto the surface of the Au / AuNPs-modified electrode. x A solution of (0.25–4 mg / mL) (using ultrapure water as solvent) was dried in a dark room for 4 hours to obtain Au / AuNP / Ti3C2T. x The modified electrode was immersed in a chloroauric acid solution (0.25–4% by mass, with ultrapure water as the solvent) and electrodeposited for 400 s at a constant potential of -0.9 V using a chronoamperometry method. Afterward, it was rinsed with ultrapure water and dried with nitrogen to obtain Au / AuNPs / Ti3C2T. x / AuNPs modified electrode.

[0011] b. In Au / AuNPs / Ti3C2T x The Au / AuNPs electrode surface is self-assembled with an initiator (4-mercaptophenyl-2-bromo-2-methylpropanoate, 4-HTP-Br) to obtain Au / AuNPs / Ti3C2T. x / AuNPs / Br modified electrode.

[0012] c. Using 0.025–0.4 mol / L MBA as a crosslinking agent, 0.05–0.8 mol / L NIPAM as a temperature-responsive functional monomer, 0.05–0.8 mol / L MAA as an auxiliary functional monomer, and 0.25–4 mg / mL fluorescein isothiocyanate-labeled yeast cells (FITC-Yeast) as both an imprinting template and a photocatalyst, the mixture was sonicated for 5 min. 0.05–0.8 mL of triethylamine (TEA) was added, and nitrogen gas was purged for 10 min to deoxygenate the reaction system, yielding 10 mL of polymerization solution. At room temperature, the initiator-modified Au / AuNPs / Ti3C2T... x The Au / AuNPs / Br electrode was inserted into 2.5 mL of polymerization solution and irradiated with light for 20 h to obtain polymer-modified Au / AuNPs / Ti3C2T. x / AuNPs / Polymer electrodes.

[0013] The prepared Au / AuNPs / Ti3C2T x The AuNPs / Polymer electrode was placed in a 0.1 mol / L CH3COOH solution containing 2.5–40% SDS. An electric field of -0.4 V was applied for 1800 s using an impedance-time method to remove the yeast cells used as the template for the imprinted material. After washing the electrode three times with PBS and drying it with N2, the yeast cell imprinted modified electrode Au / AuNPs / Ti3C2T was obtained. x / AuNPs / CIPs.

[0014] This invention also claims protection for the application of the yeast cell-imprinted modified electrode Au / AuNPs / Ti3C2Tx / AuNPs / CIPs prepared by the above preparation method in the rapid and highly sensitive detection of yeast cells. Specifically:

[0015] Examining Au / AuNPs / Ti3C2T using the DPV method x The electrochemical recognition performance of the Au / AuNPs / CIPs electrode on yeast cells was investigated. First, the electrode was placed in a characterization solution for DPV scanning, and the oxidation peak current was recorded as I1. Then, the Au / AuNPs / Ti3C2T electrode was subjected to electrochemical scanning at room temperature. x Au / AuNPs / CIPs were incubated in yeast cell solutions of different concentrations for 10 min, and then rinsed three times with PBS. Using the Au / AuNPs / CIPs after yeast cell incubation as the working electrode, DPV scanning was performed in the characterization solution, and the oxidation peak current was recorded as I2. A linear regression equation was obtained by quantitative analysis of the relationship between the signal response (ΔI = I1 - I2) and the yeast cell concentration.

[0016] To investigate the prepared Au / AuNPs / Ti3C2T x The practical application prospects of / AuNPs / CIPs electrodes were explored, and a spiked recovery experiment was used to detect the actual sample, namely, commercially available yeast extract with known cell concentrations. First, a yeast extract with a concentration of 1.0 × 10⁻⁶ was prepared. 4 10 mL of sample solution containing 1.0 × 10⁻⁶ cells / mL was prepared, and then 10, 20, and 30 μL of a 1.0 × 10⁻⁶ cell / mL solution were added to the sample solution, respectively. 7 Yeast cell standard solution at cells / mL. Utilizing Au / AuNPs / Ti3C2T x The AuNPs / CIPs electrode was used to perform DPV detection on actual samples containing yeast cells, obtaining the signal response value of the spiked sample. Based on the linear regression equation ΔI(μA)=8.44logC(cells / mL)+7.62, the corresponding yeast cell concentration, spike recovery rate, RSD, and other data could be calculated. The final experimental results showed that the spike recovery rates were all between 98.5% and 108.0%, and the RSD values ​​were all less than 4%, indicating that the Au / AuNPs / Ti3C2T electrode was effective. x / AuNPs / CIPs electrodes can be used as sensors for detecting yeast cells in Saccharomyces boulardii.

[0017] The specific method for preparing FITC-Yeast labeled yeast cells is as follows:

[0018] 0.02 g of yeast extract was dispersed in 10 mL of CBS (0.05 mol / L, pH 9.3), then centrifuged at 2000 rpm for 5 min. After washing and centrifuging three times, the resulting yeast cells were uniformly dispersed in 10 mL of CBS and magnetically stirred for 30 min. 1.2 mg of fluorescein isothiocyanate (FITC) was dissolved in 6 mL of dimethyl sulfoxide (DMSO), and then the FITC solution was slowly added to the continuously stirred yeast cell solution. After stirring and reacting at 4 °C for 12 h, the mixture was centrifuged at 2000 rpm for 5 min, and the washing and centrifugation were repeated three times. The collected solid was dissolved in 10 mL of PBS (0.1 mol / L, pH 7.0) and subjected to fluorescence spectroscopy analysis. FITC-Yeast was obtained.

[0019] This invention provides a mild, accurate, efficient, and metal-catalyst-free method for preparing yeast cell-imprinted modified electrodes. The method involves preparing Ti3C2T-based electrodes. x A temperature-responsive fluorescently labeled yeast cell imprint sensor was constructed using an imprinted electrode made of AuNPs composite material. This sensor has advantages such as simple fabrication, high sensitivity, and fast detection speed.

[0020] The advantages of this invention compared to the prior art are:

[0021] The Au / AuNPs / Ti3C2T of the present invention x The / AuNPs / CIPs modified electrode can effectively identify yeast cells, meaning it can be used as an electrochemical sensor for detecting yeast cells. Experimental results show that this sensor can detect yeast cells rapidly and with high sensitivity, thus providing an efficient, inexpensive, and convenient analytical sensor for the field of yeast cell detection.

[0022] This invention belongs to the field of imprinted sensor fabrication and application technology, and discloses a temperature-responsive yeast cell imprinted modified electrode, its fabrication method, and its application. This invention utilizes chronoamperometry and drop casting to modify gold electrodes, obtaining Au / AuNPs / Ti3C2T. x Au / AuNPs modified electrode. Using fluorescently labeled yeast cells as both catalyst and template molecule, NIPAM as the temperature-responsive functional monomer and MAA as the auxiliary functional monomer, the electrode surface was modified using the MVL ATRP method. After removing the yeast cells, Au / AuNPs / Ti3C2T electrode was obtained. x / AuNPs / CIPs modified electrode. This modified electrode can be used as an electrochemical sensor for detecting yeast cells. This sensor has advantages such as fast detection speed and high sensitivity, with a linear response range of 1.0 × 10⁻⁶ for detecting yeast cells. 2 ~1.0×10 9 cells / mL.

[0023] Based on the high specific surface area, high catalytic activity, and ability to promote electron transfer reactions of gold nanoparticles, as well as the excellent properties of MXene materials, this invention combines the advantages of both materials, selecting one type of MXene material (Ti3C2T). x Ti3C2T with exceptional electrocatalytic activity and high electrical conductivity was synthesized using gold nanoparticles. x Composite electrode modified with AuNPs.

[0024] This invention utilizes the temperature-responsive monomer N-isopropylacrylamide (NIPAM) to prepare the temperature-responsive imprinted polymer PNIPAM. PNIPAM exhibits a significant chain contraction-extension conformational transition in response to changes in external temperature. It is hydrophilic below its lowest critical solution temperature (LCST = 32°C) and hydrophobic above its LCST. Temperature-responsive imprinted polymers have wide applications in biomedical materials, drug release, and substance separation.

[0025] To eliminate the negative effects of photocatalysts, this invention employs fluorescent labeling techniques to bind the photocatalyst fluorescein isothiocyanate (FITC) to template cells via physical adsorption or covalent bonding to form a complex. The fluorescence properties of the labeled substance then reflect the biomolecular performance information. After template removal, no photocatalyst residue remains. Attached Figure Description

[0026] Figure 1 In the electrode preparation process of Embodiment 1 of the present invention, different modified electrodes are prepared in the presence of 5 mM [FeCN)6. 3- / 4- Cyclic voltammogram in +0.1M KCl (pH 7.0 PBS) solution.

[0027] Figure 2 The bare gold disk electrode (A), Au / AuNPs modified electrode (B), and Au / AuNPs / Ti3C2T electrode are from Embodiment 1 of this invention. x Modified electrode (C), Au / AuNPs / Ti3C2T x / AuNPs(D) and Au / AuNPs / Ti3C2T x SEM image of / AuNPs / Polymer(E).

[0028] Figure 3 This is the Au / AuNPs / Ti3C2T of Embodiment 1 of the present invention. x Differential pulse voltammetry curve (A) and working curve (B) of / AuNPs / CIPs for yeast cell detection.

[0029] Figure 4 This is the imprinted modified electrode (Au / AuNPs / Ti3C2T) of Embodiment 1 of the present invention. x Selectivity plot of / AuNPs / CIPs).

[0030] Figure 5 The images show fluorescence spectra (A), linear regression equation (B), and fluorescence spectra (C) of a series of FITC solutions with different concentrations in Example 1 of this invention.

[0031] Figure 6 This is the Au / AuNPs / Ti3C2T of Embodiment 1 of the present invention. x Temperature response of / AuNPs / CIPs at pH=7. Detailed Implementation

[0032] The present invention is described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0033] Example 1:

[0034] a. A clean gold disk electrode was inserted into a 1% (w / w) chloroauric acid solution (using ultrapure water as the solvent). Electrodeposition was performed for 400 s at a constant potential of -0.9 V using a chronoamperometry method. The electrode was then rinsed with ultrapure water and dried with nitrogen to obtain an Au / AuNPs-modified electrode. 5 μL of Ti3C2T4, which had been ultrasonically treated for 20 min, was then drop-cast onto the surface of the Au / AuNPs-modified electrode. x A 1 mg / mL solution (using ultrapure water as solvent) was dried in a dark room for 4 hours. The modified electrode was then inserted into a 1% chloroauric acid solution (using ultrapure water as solvent) and electrodeposited for 400 seconds at a constant potential of -0.9 V using a chronoamperometry method. After rinsing with ultrapure water and drying with nitrogen, Au / AuNPs / Ti3C2T was obtained. x / AuNPs modified electrode.

[0035] b. In Au / AuNPs / Ti3C2T x The Au / AuNPs electrode surface is self-assembled with an initiator (4-mercaptophenyl-2-bromo-2-methylpropanoate, 4-HTP-Br) to obtain Au / AuNPs / Ti3C2T. x / AuNPs / Br modified electrode.

[0036] c. Using 0.1 mol / L MBA as a crosslinking agent, 0.2 mol / L NIPAM as a temperature-responsive functional monomer, 0.2 mol / L MAA as an auxiliary functional monomer, and 1 mg / mL FITC-Yeast as both an imprinting template and a photocatalyst, the mixture was ultrasonically vibrated for 5 min. 0.2 mL of triethylamine (TEA) was added, and nitrogen gas was purged through the reaction system for 10 min to remove oxygen, yielding 10 mL of polymerization solution. At room temperature, the initiator-modified Au / AuNPs / Ti3C2T... x The Au / AuNPs / Br electrode was inserted into 2.5 mL of polymerization solution and irradiated with light for 20 h to obtain polymer-modified Au / AuNPs / Ti3C2T. x / AuNPs / Polymer electrodes.

[0037] The prepared Au / AuNPs / Ti3C2T xThe / AuNPs / Polymer electrode was placed in a 0.1 mol / L CH3COOH solution containing 10% SDS, and an electric field of -0.4 V was applied for 1800 s using an impedance-time method to remove the imprinted template yeast cells. After washing the electrode three times with PBS and drying it with N2, a yeast cell imprinted polymer-modified electrode (Au / AuNPs / Ti3C2T) was obtained. x / AuNPs / CIPs).

[0038] Examining Au / AuNPs / Ti3C2T using the DPV method x The electrochemical recognition performance of the Au / AuNPs / CIPs electrode on yeast cells was investigated. First, the electrode was placed in a characterization solution for DPV scanning, and the oxidation peak current was recorded as I1. Then, the Au / AuNPs / Ti3C2T electrode was subjected to electrochemical scanning at room temperature. x Au / AuNPs / CIPs were incubated in yeast cell solutions of different concentrations for 10 min, and then rinsed three times with PBS. Using the Au / AuNPs / CIPs after yeast cell incubation as the working electrode, DPV scanning was performed in the characterization solution, and the oxidation peak current was recorded as I2. Quantitative analysis was performed by analyzing the relationship between the signal response (ΔI = I1 - I2) and the yeast cell concentration.

[0039] Figure 1 Modified electrodes at different stages were tested using 0.1 mol / L KCl + 5 mmol / L [Fe(CN)6] 3- / 4- CV curves in an electrolyte solution (0.1 mol / L PBS, pH 7.0). Curve 1 is the bare gold disk electrode before polymer modification. Curve 2 is the CV curve of Au / AuNPs, with a significantly higher peak current than Curve 1. This is because AuNPs increase the electrode surface area and electron transport capacity, leading to a significant increase in peak current. Curve 3 is the Au / AuNPs electrode after Ti3C2Tx modification, with a slightly lower current than Curve 2. This may be due to the lower conductivity of MXene material compared to AuNPs. Curve 4 is the Au / AuNPs / Ti3C2Tx electrode after AuNPs modification. x The current values ​​of the oxidation and reduction peaks of the electrode are significantly higher than those in curve 3. This is likely because AuNPs increase the surface area of ​​the electrode, and AuNPs have a high electron transport rate. Curve 5 shows the Au / AuNPs / Ti3C2T electrode modified with 4-HTP-Br. x The current at the / AuNPs electrode is lower than that in curve 4. This may be because 4-HTP-Br is an electrochemically inert substance, and its binding on the electrode would hinder [Fe(CN)6]. 3- / 4- The probe's poor electron transfer ability from solution to the electrode surface film leads to a decrease in the electrode's electron transfer rate. Further modification of the electrode surface with a polymer to prepare Au / AuNPs / Ti3C2T...x The / AuNPs / Polymer curve (curve 6) shows a sharp decrease in peak current compared to curve 3. This is likely due to the formation of a polymer film encapsulating yeast cells on the electrode surface. This polymer film is an electrochemically inert material that hinders the movement of the probe ions [Fe(CN)6]. 3- / 4- Reaching the electrode surface leads to a significant decrease in peak current. Curve 7 shows the Au / AuNPs / Ti3C2T curve after eluting the template yeast cells. x The peak current of the CV curve of / AuNPs / CIPs is significantly higher than that of curve 6. This may be because after eluting the template yeast cells, imprint pores appear on the electrode surface, making it easier for probe ions to diffuse to the electrode surface, resulting in an increase in peak current.

[0040] Figure 2 A through E are SEM images of the modified electrode surface. Figure 2 A is a SEM image of bare Au. As can be seen from the image, the surface of the unmodified electrode has scratches but is relatively smooth, and it has a relatively limited actual surface area. Figure 2 Image B is an SEM image of the Au / AuNPs modified electrode. It can be clearly seen that the surface of the gold disk electrode is modified with a large number of gold nanoparticles (AuNPs), indicating that the Au / AuNPs modified electrode was successfully prepared. Figure 2 C represents Au / AuNPs / Ti3C2T x The SEM image of the electrode surface shows that Ti3C2T x It has a unique two-dimensional layered structure resembling an accordion and a large surface area. Figure 2 D represents Au / AuNPs / Ti3C2T x The SEM image of the AuNPs electrode surface shows that AuNPs are uniformly deposited on Ti3C2T. x Modify the electrode surface. Figure 2 E represents Au / AuNPs / Ti3C2T x The SEM image of / AuNPs / Polymer shows that AuNPs / Ti3C2T x / AuNPs are covered with polymer, and the polymer partially encapsulates the yeast cells.

[0041] Figure 3 A represents Au / AuNPs / Ti3C2T x / AuNPs / CIPs modified electrodes were used as electrochemical biosensors to detect DPV curves of a series of yeast cells at different concentrations (yeast cell concentrations were 0, 9, and 9 × 10⁻⁶). 1 9×10 2 9×10 3 9×104 9×10 5 9×10 6 9×10 7 9×10 8 The figure shows that the DPV peak current decreases with increasing yeast cell concentration. This is because after the imprinted polymer recognizes yeast cells, the yeast cells occupy the imprinted cavities, blocking the diffusion of probe ions to the electrode surface, thus leading to a decrease in peak current. A plot of the DPV peak current difference (response signal, ΔI) against the logarithm of yeast cell concentration yields the working curve for yeast cell detection using this modified electrode. Figure 3 (As shown in B). The figure shows Au / AuNPs / Ti3C2T. x The linear regression equation for detecting yeast cells using an AuNPs / CIPs modified electrode was ΔI(μA) = 8.44logC(cells / mL) + 7.62, with a correlation coefficient of 0.993 and a linear response range of 1.0 × 10⁻⁶. 2 ~1.0×10 9 cells / mL.

[0042] To investigate Au / AuNPs / Ti3C2T x The selectivity of the / AuNPs / CIPs modified electrode was compared with that of autolyzed yeast (AY), dead yeast (DY), Escherichia coli (EC), human mammary epithelial cells (MCF-10A), and human breast cancer cells (MCF-7), as well as interference agents used in the experiment. Au / AuNPs / Ti3C2T x / AuNPs / CIPs and Au / AuNPs / Ti3C2T x / AuNPs / NIPs were detected using the DPV method at a concentration of 1.0 × 10⁻⁶. 5 Interference cell solution and yeast cell solution at cells / mL, the results are as follows Figure 4 As shown in the figure. The results in the figure indicate that Au / AuNPs / Ti3C2T x The signal response ΔI of the Au / AuNPs / CIPs electrode in yeast cells was 49.27 μA, which was 9.10, 8.87, 18.72, 31.05, and 24.23 times that of the corresponding signals detected by AY, DY, EC, MCF-10A, and MCF-7, respectively. This indicates that the Au / AuNPs / Ti3C2T electrode... x / AuNPs / CIPs electrodes have good recognition ability for yeast cells.

[0043] Electrode selectivity can also be evaluated using the imprinting factor (IF), which is calculated using the following formula:

[0044] IF = ΔI (CIPs) / ΔI (NIPs)

[0045] In the formula ΔI (CIPs) Au / AuNPs / Ti3C2T x / AuNPs / CIPs electrode signal response in yeast cells; ΔI (NIPs) Au / AuNPs / Ti3C2T x Signal response of / AuNPs / NIPs electrodes. From Figure 3 The data shows that the IF values ​​for electrodes Y, AY, DY, EC, MCF-10A, and MCF-7 are 5.73, 2.09, 2.11, 2.73, 1.28, and 1.42, respectively. The highest IF value indicates that Au / AuNPs / Ti3C2T x / AuNPs / CIPs electrodes have better selectivity for yeast cells.

[0046] To perform quantitative analysis of FITC-yeast, we measured the fluorescence spectra of a series of FITC solutions with different concentrations (the FITC concentrations in curves 1-10 are 2.50, 2.00, 1.50, 1.25, 1.00, 0.75, 0.625, 0.50, 0.3125, and 0.25 μmol / L, respectively) and plotted the fluorescence intensity against the concentration in the obtained fluorescence spectra. Figure 5 A shows the fluorescence spectra of a series of FITC solutions with different concentrations. As can be seen from the figure, the fluorescence intensity increases with increasing FITC solution concentration. (By: I) f =2.3φ f I0klc: where I f It is the fluorescence intensity, φ f Let I0 be the fluorescence quantum yield, I0 be the incident light intensity, k be the molar absorption coefficient of the fluorescent substance, l be the optical path length, and c be the concentration of the fluorescent substance. When I0 and l are constant, the fluorescence intensity is directly proportional to the concentration of the fluorescent substance. For example... Figure 3 As shown in B, the curve is I. f The relationship curve between c and the linear regression equation I f =959.637c(μmol / L)+128.187, R 2 =0.992.

[0047] like Figure 5As shown in Figure C, curve 1 is the fluorescence spectrum of yeast cells, curve 2 is the fluorescence spectrum of fluorescein isothiocyanate, and curve 3 is the fluorescence spectrum of FITC-Yeast, indicating successful preparation of FITC-Yeast. Substituting the fluorescence intensity of curve 2 into the linear regression equation I... f From 959.637c(μmol / L) + 128.187, the FITC content in fluorescently labeled cells (2 mg / ml) can be calculated to be 0.9616 μmol / L, meaning that one yeast cell corresponds to 1.16 × 10⁻⁶ μmol / L. 7 One FITC molecule.

[0048] To evaluate the temperature response of the polymer at pH=7, Au / AuNPs / Ti3C2T were subjected to temperature variations at 10, 20, 30, 40 °C and 50 °C, respectively. x / AuNPs / CIPs electrodes were immersed in a solution containing yeast cells (1.0 × 10⁻⁶). 3 The cells / mL were immersed in PBS (pH=7.0) for 15 min. After washing three times with PBS, the peak current value was measured using DPV. The results are as follows. Figure 6 As shown, Au / AuNPs / Ti3C2T can be observed. x / AuNPs / CIPs exhibited different peak current values ​​after incubation with yeast cell solutions at different temperatures, indicating that the polymer exhibits temperature responsiveness. Thermosensitive poly(N-isopropylacrylamide) (PNIPAM) is hydrophilic below its lowest critical solution temperature (LCST) of 32°C and hydrophobic above the LCST. For example... Figure 6 As shown in Figure B, a higher signal response is observed at a temperature of 30℃. Considering the convenience of the experiment, 30℃ was chosen as the optimal condition for subsequent experiments.

[0049] Example 2:

[0050] a. A clean gold disk electrode was inserted into a 0.5% chloroauric acid solution (using ultrapure water as the solvent). Electrodeposition was performed for 400 s at a constant potential of -0.9 V using a chronoamperometry method. The electrode was then rinsed with ultrapure water and dried with nitrogen to obtain an Au / AuNPs-modified electrode. 5 μL of Ti3C2T4, which had been ultrasonically treated for 20 min, was then drop-cast onto the surface of the Au / AuNPs-modified electrode. x A 1 mg / mL solution (using ultrapure water) was dried in a dark room for 4 hours. The modified electrode was then inserted into a 0.5% chloroauric acid solution (using ultrapure water) and electrodeposited for 400 seconds at a constant potential of -0.9 V using a chronoamperometry method. After rinsing with ultrapure water and drying with nitrogen, Au / AuNPs / Ti3C2T was obtained. x / AuNPs modified electrode.

[0051] b. In Au / AuNPs / Ti3C2T x The Au / AuNPs electrode surface is self-assembled with an initiator (4-mercaptophenyl-2-bromo-2-methylpropanoate, 4-HTP-Br) to obtain Au / AuNPs / Ti3C2T. x / AuNPs / Br modified electrode.

[0052] c. Using 0.05 mol / L MBA as a crosslinking agent, 0.1 mol / L NIPAM as a temperature-responsive functional monomer, 0.1 mol / L MAA as an auxiliary functional monomer, and 0.5 mg / mL FITC-Yeast as both an imprinting template and a photocatalyst, the mixture was ultrasonically vibrated for 5 min. 0.1 mL of triethylamine (TEA) was added, and nitrogen gas was purged through the reaction system for 10 min to remove oxygen, yielding 10 mL of polymerization solution. At room temperature, the initiator-modified Au / AuNPs / Ti3C2T... x The Au / AuNPs / Br electrode was inserted into 2.5 mL of polymerization solution and irradiated with light for 20 h to obtain polymer-modified Au / AuNPs / Ti3C2T. x / AuNPs / Polymer electrodes.

[0053] The prepared Au / AuNPs / Ti3C2T x The / AuNPs / Polymer electrode was placed in a 0.05 mol / L CH3COOH solution containing 5% SDS, and an electric field of -0.4 V was applied for 1800 s using an impedance-time method to remove the imprinted template yeast cells. After washing the electrode three times with PBS and drying it with N2, a yeast cell imprinted polymer-modified electrode (Au / AuNPs / Ti3C2T) was obtained. x / AuNPs / CIPs).

[0054] Examining Au / AuNPs / Ti3C2T using the DPV method x The electrochemical recognition performance of the Au / AuNPs / CIPs electrode on yeast cells was investigated. First, the electrode was placed in a characterization solution for DPV scanning, and the oxidation peak current was recorded as I1. Then, the Au / AuNPs / Ti3C2T electrode was subjected to electrochemical scanning at room temperature. xAu / AuNPs / CIPs were incubated in yeast cell solutions of different concentrations for 10 min, and then rinsed three times with PBS. Using the Au / AuNPs / CIPs after yeast cell incubation as the working electrode, DPV scanning was performed in the characterization solution, and the oxidation peak current was recorded as I2. Quantitative analysis was performed by analyzing the relationship between the signal response (ΔI = I1 - I2) and the yeast cell concentration.

[0055] Example 3:

[0056] a. A clean gold disk electrode was inserted into a 0.25% chloroauric acid solution (using ultrapure water as the solvent). Electrodeposition was performed for 400 s at a constant potential of -0.9 V using a chronoamperometry method. The electrode was then rinsed with ultrapure water and dried with nitrogen to obtain an Au / AuNPs-modified electrode. 5 μL of Ti3C2T4, which had been ultrasonically treated for 20 min, was then drop-cast onto the surface of the Au / AuNPs-modified electrode. x A 1 mg / mL solution (using ultrapure water) was prepared and dried in a dark room for 4 hours. The modified electrode was then inserted into a 0.25% chloroauric acid solution (using ultrapure water) and electrodeposited for 400 seconds at a constant potential of -0.9 V using a chronoamperometry method. After rinsing with ultrapure water and drying with nitrogen, Au / AuNPs / Ti3C2T was obtained. x / AuNPs modified electrode.

[0057] b. In Au / AuNPs / Ti3C2T x The Au / AuNPs electrode surface is self-assembled with an initiator (4-mercaptophenyl-2-bromo-2-methylpropanoate, 4-HTP-Br) to obtain Au / AuNPs / Ti3C2T. x / AuNPs / Br modified electrode.

[0058] c. Using 0.025 mol / L MBA as a crosslinking agent, 0.05 mol / L NIPAM as a temperature-responsive functional monomer, 0.05 mol / L MAA as an auxiliary functional monomer, and 0.025 mg / mL FITC-Yeast as both an imprinting template and a photocatalyst, the mixture was ultrasonically vibrated for 5 min. 0.05 mL of triethylamine (TEA) was added, and nitrogen gas was purged through the reaction system for 10 min to remove oxygen, yielding 10 mL of polymerization solution. At room temperature, the initiator-modified Au / AuNPs / Ti3C2T... x The Au / AuNPs / Br electrode was inserted into 2.5 mL of polymerization solution and irradiated with light for 20 h to obtain polymer-modified Au / AuNPs / Ti3C2T. x / AuNPs / Polymer electrodes.

[0059] The prepared Au / AuNPs / Ti3C2T x The / AuNPs / Polymer electrode was placed in a 0.025 mol / L CH3COOH solution containing 2.5% SDS, and an electric field of -0.4 V was applied for 1800 s using an impedance-time method to remove the imprinted template yeast cells. After washing the electrode three times with PBS and drying it with N2, a yeast cell imprinted polymer-modified electrode (Au / AuNPs / Ti3C2T) was obtained. x / AuNPs / CIPs).

[0060] Examining Au / AuNPs / Ti3C2T using the DPV method x The electrochemical recognition performance of the Au / AuNPs / CIPs electrode on yeast cells was investigated. First, the electrode was placed in a characterization solution for DPV scanning, and the oxidation peak current was recorded as I1. Then, the Au / AuNPs / Ti3C2T electrode was subjected to electrochemical scanning at room temperature. x Au / AuNPs / CIPs were incubated in yeast cell solutions of different concentrations for 10 min, and then rinsed three times with PBS. Using the Au / AuNPs / CIPs after yeast cell incubation as the working electrode, DPV scanning was performed in the characterization solution, and the oxidation peak current was recorded as I2. Quantitative analysis was performed by analyzing the relationship between the signal response (ΔI = I1 - I2) and the yeast cell concentration.

[0061] Example 4:

[0062] a. A clean gold disk electrode was inserted into a 2% (w / w) chloroauric acid solution (using ultrapure water as the solvent). Electrodeposition was performed for 400 s at a constant potential of -0.9 V using a chronoamperometry method. The electrode was then rinsed with ultrapure water and dried with nitrogen to obtain an Au / AuNPs-modified electrode. 5 μL of Ti3C2T4, which had been ultrasonically treated for 20 min, was then drop-cast onto the surface of the Au / AuNPs-modified electrode. x A 1 mg / mL solution (using ultrapure water) was prepared and dried in a dark room for 4 hours. The modified electrode was then inserted into a 2% chloroauric acid solution (using ultrapure water) and electrodeposited for 400 seconds at a constant potential of -0.9 V using a chronoamperometry method. After rinsing with ultrapure water and drying with nitrogen, Au / AuNPs / Ti3C2T was obtained. x / AuNPs modified electrode.

[0063] b. In Au / AuNPs / Ti3C2T xThe Au / AuNPs electrode surface is self-assembled with an initiator (4-mercaptophenyl-2-bromo-2-methylpropanoate, 4-HTP-Br) to obtain Au / AuNPs / Ti3C2T. x / AuNPs / Br modified electrode.

[0064] c. Using 0.2 mol / L MBA as a crosslinking agent, 0.4 mol / L NIPAM as a temperature-responsive functional monomer, 0.4 mol / L MAA as an auxiliary functional monomer, and 0.2 mg / mL FITC-Yeast as both an imprinting template and a photocatalyst, the mixture was ultrasonically vibrated for 5 min. 0.4 mL of triethylamine (TEA) was added, and nitrogen gas was purged through the reaction system for 10 min to remove oxygen, yielding 10 mL of polymerization solution. At room temperature, the initiator-modified Au / AuNPs / Ti3C2T... x The Au / AuNPs / Br electrode was inserted into 2.5 mL of polymerization solution and irradiated with light for 20 h to obtain polymer-modified Au / AuNPs / Ti3C2T. x / AuNPs / Polymer electrodes.

[0065] The prepared Au / AuNPs / Ti3C2T x The / AuNPs / Polymer electrode was placed in a 0.2 mol / L CH3COOH solution containing 20% ​​SDS, and an electric field of -0.4 V was applied for 1800 s using an impedance-time method to remove the imprinted template yeast cells. After washing the electrode three times with PBS and drying it with N2, a yeast cell imprinted polymer-modified electrode (Au / AuNPs / Ti3C2T) was obtained. x / AuNPs / CIPs).

[0066] Examining Au / AuNPs / Ti3C2T using the DPV method x The electrochemical recognition performance of the Au / AuNPs / CIPs electrode on yeast cells was investigated. First, the electrode was placed in a characterization solution for DPV scanning, and the oxidation peak current was recorded as I1. Then, the Au / AuNPs / Ti3C2T electrode was subjected to electrochemical scanning at room temperature. x Au / AuNPs / CIPs were incubated in yeast cell solutions of different concentrations for 10 min, and then rinsed three times with PBS. Using the Au / AuNPs / CIPs after yeast cell incubation as the working electrode, DPV scanning was performed in the characterization solution, and the oxidation peak current was recorded as I2. Quantitative analysis was performed by analyzing the relationship between the signal response (ΔI = I1 - I2) and the yeast cell concentration.

[0067] Example 5:

[0068] a. A clean gold disk electrode was inserted into a 4% (w / w) chloroauric acid solution (using ultrapure water as the solvent). Electrodeposition was performed for 400 s at a constant potential of -0.9 V using a chronoamperometry method. The electrode was then rinsed with ultrapure water and dried with nitrogen to obtain an Au / AuNPs-modified electrode. 5 μL of Ti3C2T4, which had been ultrasonically treated for 20 min, was then drop-cast onto the surface of the Au / AuNPs-modified electrode. x A 1 mg / mL solution (using ultrapure water) was dried in a dark room for 4 hours. The modified electrode was then inserted into a 4% chloroauric acid solution (using ultrapure water) and electrodeposited for 400 seconds at a constant potential of -0.9 V using a chronoamperometry method. After rinsing with ultrapure water and drying with nitrogen, Au / AuNPs / Ti3C2T was obtained. x / AuNPs modified electrode.

[0069] b. In Au / AuNPs / Ti3C2T x The Au / AuNPs electrode surface is self-assembled with an initiator (4-mercaptophenyl-2-bromo-2-methylpropanoate, 4-HTP-Br) to obtain Au / AuNPs / Ti3C2T. x / AuNPs / Br modified electrode.

[0070] c. Using 0.4 mol / L MBA as a crosslinking agent, 0.8 mol / L NIPAM as a temperature-responsive functional monomer, 0.8 mol / L MAA as an auxiliary functional monomer, and 4 mg / mL FITC-Yeast as both an imprinting template and a photocatalyst, the mixture was ultrasonically vibrated for 5 min. 0.8 mL of triethylamine (TEA) was added, and nitrogen gas was purged through the reaction system for 10 min to remove oxygen, yielding 10 mL of polymerization solution. At room temperature, the initiator-modified Au / AuNPs / Ti3C2T... x The Au / AuNPs / Br electrode was inserted into 2.5 mL of polymerization solution and irradiated with light for 20 h to obtain polymer-modified Au / AuNPs / Ti3C2T. x / AuNPs / Polymer electrodes.

[0071] The prepared Au / AuNPs / Ti3C2T xThe / AuNPs / Polymer electrode was placed in a 0.4 mol / L CH3COOH solution containing 40% SDS, and an electric field of -0.4 V was applied for 1800 s using an impedance-time method to remove the imprinted template yeast cells. After washing the electrode three times with PBS and drying it with N2, a yeast cell imprinted polymer-modified electrode (Au / AuNPs / Ti3C2T) was obtained. x / AuNPs / CIPs).

[0072] Examining Au / AuNPs / Ti3C2T using the DPV method x The electrochemical recognition performance of the Au / AuNPs / CIPs electrode on yeast cells was investigated. First, the electrode was placed in a characterization solution for DPV scanning, and the oxidation peak current was recorded as I1. Then, the Au / AuNPs / Ti3C2T electrode was subjected to electrochemical scanning at room temperature. x Au / AuNPs / CIPs were incubated in yeast cell solutions of different concentrations for 10 min, and then rinsed three times with PBS. Using the Au / AuNPs / CIPs after yeast cell incubation as the working electrode, DPV scanning was performed in the characterization solution, and the oxidation peak current was recorded as I2. Quantitative analysis was performed by analyzing the relationship between the signal response (ΔI = I1 - I2) and the yeast cell concentration.

[0073] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a temperature-responsive yeast cell-imprinted modified electrode, characterized in that, The specific steps are as follows: a. A clean gold disk electrode was inserted into a chloroauric acid solution with a mass concentration of 0.25–4%, and electrodeposition was performed for 400 s at a constant potential of -0.9 V using a chronoamperometry method. The electrode was then rinsed with ultrapure water and dried with nitrogen to obtain an Au / AuNPs-modified electrode. 5 μL of Ti3C2T4, which had been ultrasonically treated for 20 min, was then drop-cast onto the surface of the Au / AuNPs-modified electrode. x The solution was dried in a dark room for 4 hours to obtain Au / AuNP / Ti3C2T. x The modified electrode was immersed in a chloroauric acid solution with a mass concentration of 0.25–4%, and electrodeposition was performed for 400 s at a constant potential of -0.9 V using a chronoamperometry method. The electrode was then washed with ultrapure water and dried with nitrogen to obtain Au / AuNPs / Ti3C2T. x / AuNPs modified electrode; b. In Au / AuNPs / Ti3C2T x An initiator is self-assembled onto the surface of the AuNPs electrode to obtain Au / AuNPs / Ti3C2T. x / AuNPs / Br modified electrode; c. Using 0.025–0.4 mol / L N,N'-methylenebisacrylamide solution as a crosslinking agent, 0.05–0.8 mol / L NIPAM as a temperature-responsive functional monomer, 0.05–0.8 mol / L MAA as an auxiliary functional monomer, and 0.25–4 mg / mL FITC-Yeast as both an imprinting template and a photocatalyst, the mixture was ultrasonically vibrated for 5 min. Then, 0.05–0.8 mL of triethylamine was added, and nitrogen gas was purged for 10 min to deoxygenate the reaction system, yielding 10 mL of polymerization solution. At room temperature, the initiator-modified Au / AuNPs / Ti3C2T... x The Au / AuNPs / Br electrode was inserted into 2.5 mL of polymerization solution and irradiated with light for 20 h to obtain polymer-modified Au / AuNPs / Ti3C2T. x / AuNPs / Polymer electrodes; The prepared Au / AuNPs / Ti3C2T x The / AuNPs / Polymer electrode was placed in a 0.1 mol / L CH3COOH solution containing 2.5~40% SDS, and an electric field of -0.4 V was applied for 1800 s using the impedance-time method to remove the imprinted template yeast cells; After washing the electrode three times with PBS and drying it with N2, the yeast cell imprinted modified electrode Au / AuNPs / Ti3C2T was obtained. x / AuNPs / CIPs; NIPAM is N-isopropylacrylamide; MAA is α-methacrylic acid; and FITC-Yeast is yeast cells labeled with fluorescein isothiocyanate.

2. The application of the yeast cell imprinted modified electrode prepared by the preparation method described in claim 1 in the rapid and highly sensitive detection of yeast cells.

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