Construction method of biosensor based on NiCoO nano-enzyme and RBP4 detection application

Abstract

The invention discloses a construction method of a biosensor based on NiCoO nano-enzyme, which comprises the steps of synthesis of T-COF (at) CS, preparation of NiCoO nano-enzyme and preparation of NiCoO-Ab2 bioconjugate, a novel PEC biosensor is constructed based on spherical T-COF (at) CS and NiCoO nano-enzyme, is used for high-selectivity and ultra-sensitive detection of RBP4, has excellent anti-interference capability, and can be used for detecting RBP4. False positive or negative detection results can be eliminated, and good reproducibility and stability are achieved. The invention provides a good platform for ultra-sensitive detection of RBP4 and early monitoring and diagnosis of T2DM, also provides a good platform for analysis of other biomarkers, and has a wide application prospect in the aspect of biological analysis.

Description

Technical Field

[0001] The present invention relates to a construction method of a biosensor based on NiCoO nanozyme and its application in RBP4 detection, belonging to the fields of functional biomaterials and biosensing technology. Background Art

[0002] Retinol binding protein 4 (RBP4) is an important protein involved in vitamin A transport and metabolism in vivo. In recent years, a large number of studies have shown that RBP4 is closely related to various diseases. Diabetes is a global chronic metabolic disease, and type 2 diabetes (T2DM) accounts for the vast majority of diabetic patients. The development of T2DM is often relatively concealed, and in the early stage, it may only show mild metabolic abnormalities. If not detected and intervened in time, as the disease progresses, it will cause a series of serious complications, such as cardiovascular diseases, kidney diseases, retinopathy, and neuropathy, which will cause great damage to the health and quality of life of patients, and at the same time bring a heavy burden to social medical resources. Therefore, developing a highly efficient, sensitive, and specific RBP4 detection method is of great significance for the early screening, diagnosis, evaluation of treatment effects, and research on the pathogenesis of these diseases.

[0003] As an emerging biodietection technology, photoelectrochemical (PEC) biosensors use light to excite photoactive materials to generate electron-hole pairs, and realize the quantitative detection of target biomolecules by detecting the changes in photoelectric signals. They have the advantages of low background signals, simple operation, and easy miniaturization. As a material with multiple oxidation states and excellent catalytic properties, NiCoO has great application potential in the field of nanozymes. Covalent organic framework materials (COFs), which connect organic building units through covalent bonds to form a porous framework with a periodic structure, have become excellent candidates for photoactive materials due to their high specific surface area, porous structure, and rich functional groups. In summary, developing a simple and highly sensitive NiCoO nanozyme-based biosensor for RBP4 detection has important scientific significance and practical application value, which is also the key problem that the present invention is committed to solving. Summary of the Invention The technical problem to be solved by the present invention is: to provide a construction method of a biosensor based on NiCoO nanozyme and its application in RBP4 detection. The present invention constructs a novel biosensor based on spherical T-COF@CS and NiCoO nanozyme for highly selective and ultrasensitive and accurate detection of RBP4.

[0005] The technical solutions adopted by the present invention to solve the technical problems are as follows: A construction method of a biosensor based on NiCoO nanozyme, comprising the following parts: (1)Synthesis of T-COF@CS Add 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (0.04 mmol, 14.2 mg), 1,3,5-benzenetricarbaldehyde (0.04 mmol, 6.5 mg), PVP (25 mg) and acetonitrile (5 mL) into a glass bottle. After ultrasonic treatment for 30 min, add 12M CH₃COOH₃ (0.4 mL), seal the glass bottle, and let it stand at room temperature for 96 h. Wash the separated product with N,N-dimethylformamide, and then place it in a vacuum oven at 70 °C for 12 h to obtain yellow solid T-COF; further mix 250 µL of T-COF (2 mg / mL) with 200 µL of 0.025 %wt CS solution dissolved in 1 % acetic acid, react in a metal bath for 3 h and then centrifuge to obtain T-COF@CS, and then add deionized water to make a 350 µL T-COF@CS solution; (2)Preparation of NiCoO nanozyme First dissolve 1,3,5-benzenetricarboxylic acid (2 mmol, 420.3 mg) in 90 mL of H₂O at 100 °C, and at the same time dissolve Co(CH₃COO)₂·4H₂O (2 mmol, 498.2 mg) and Ni(NO₃)₂·6H₂O (2 mmol, 581.6 mg) in another 10 mL of H₂O; then pour the latter solution into the former solution, stir magnetically for 15 min and then cool to room temperature. After centrifugation, obtain a pink precipitate. Wash the precipitate with water and ethanol and then centrifuge, and place it in an oven at 40 °C to dry; place the NiCo-BTC nanorods in an air environment and carbonize at 350 °C for 2 h to obtain NiCoO nanozyme; (3)Preparation of NiCoO-Ab2 conjugate Couple NiCoO nanozyme and Ab2 through electrostatic interaction: Add 2 mg of NiCoO nanozyme into 5 mL of PBS (0.01 M, pH 7.4) and mix well by ultrasonic treatment. Then add 600 µL of 10 µg mL⁻¹ Ab2 into the NiCoO nanozyme solution and mix. Shake well in a metal bath at 4 °C for 10 h; then add 400 µL of 1.0 wt% BSA solution and shake well in a metal bath at 4 °C for 1 h to prevent non-specific binding, and then centrifuge; Resuspend the prepared NiCoO-Ab2 conjugate in 1 mL of PBS (0.01M, pH 7.4) and store it at 4 °C for later use.

[0006] The application of the above constructed PEC biosensor in detecting RBP4 includes the following parts: (1) The ITO electrode was ultrasonically cleaned with 1 mol / L aqueous NaOH solution, 1 mol / L NaOH ethanol and water (1:1, v / v) solution, and Mili ultrapure water in sequence for 20 min. The conductive surface of the ITO electrode was determined by a multifunctional household electric meter, and then the electrode was placed at 60 °C and dried for 2 h; (2) 25 μL of T-COF@CS solution was dropped onto the surface of the ITO electrode and dried naturally to obtain the T-COF@CS / ITO electrode; (3) 20 μL of Ab1 (1 μg / mL-1) suspension was added and incubated at 37 °C for at least 1 h, and then washed with Mili ultrapure water to remove unbound Ab1 completely; (4) The surface of the electrode was covered with 20 μL of 0.5 wt% BSA solution, and non-specific binding was blocked for 30 min under air environmental conditions and then thoroughly rinsed with Mili ultrapure water; (5) 20 μL of RBP4 antigen solution with different concentrations was transferred onto the electrode surface and incubated at 37 °C for 90 min; (6) The above electrode was reacted with 20 μL of NiCoO-Ab2 conjugate at 37 °C for 90 min, and further washed with Mili water to obtain the NiCoO-Ab2 / RBP4 / BSA / Ab1 / T-COF@CS / ITO electrode; (7) 20 μL of 20 mmol / L 4-CN solution was dropped onto the electrode and incubated at room temperature for 20 min; (8) The obtained electrode was tested in Tris-HCl solution (0.1 mol / L, pH 7.4) containing 0.1 mol / L AA.

[0007] The T-COF@CS as the substrate material has excellent photocurrent signal, and the NiCoO nanorods as the signal probe have enhanced nanozyme activity.

[0008] The positive and beneficial effects that the present invention can achieve are as follows: The present invention constructs a novel PEC biosensor based on spherical T-COF@CS and NiCoO nanozyme for highly selective and ultrasensitive detection of RBP4. The large specific surface area, excellent optical properties and good electron transfer ability of the one-dimensional NiCoO nanozyme can effectively reduce the recombination of photogenerated electrons / holes and improve the detection sensitivity.

[0009] Under the optimal experimental conditions, the constructed PEC biosensor of the present invention shows a wide linear range (0.01–10000 pg / mL) and a low detection limit (8 fg / mL) for the detection of RBP4.

[0010] The present invention has excellent anti-interference ability, can eliminate false positive or negative detection results, and has good reproducibility and stability. It provides a good platform for ultrasensitive detection of RBP4 and early monitoring and diagnosis of T2DM, and also provides a good platform for the analysis of other biomarkers, showing broad application prospects in bioanalysis. Brief Description of the Drawings Figure 1 It is the schematic diagram of the NiCoO nanozyme PEC biosensor for detecting RBP4 in the present invention; Figure 2 It is the SEM image of T-COF; Figure 3 It is the SEM image of T-COF@CS; Figure 4 It is the SEM image of NiCo-BTC nanorods; Figure 5 It is the SEM image of NiCoO nanorods; Figure 6 It is the XPS image of NiCoO nanorods; Figure 7 It is the EIS response diagram of the RBP4 system in the present invention; Figure 8 It is the PEC response diagram of the RBP4 system in the present invention; In Figure 7 and Figure 8 : (a) T-COF@CS / ITO, (b) Ab1 / T-COF@CS / ITO, (c) BSA / Ab1 / T-COF@CS / ITO, (d) RBP4 / BSA / Ab1 / T-COF@CS / ITO (0.01 ng / mL RBP4), (e) NiCoO-Ab2 / RBP4 / BSA / Ab1 / T-COF@CS / ITO (1 μg / mL Ab1, 0.01 ng / mL sCD146), (f) after NiCoO-Ab2 / RBP4 / BSA / Ab1 / T-COF@CS / ITO is incubated with 4-CN; Figure 9 It is the test electrode bias optimization curve diagram in the present invention; Figure 10 It is the RBP4 incubation time optimization curve diagram in the present invention; Figure 11 It is the nanozyme-catalyzed 4-CN reaction time optimization curve diagram in the present invention; Figure 12 It is the photocurrent response diagram of the PEC biosensor for detecting RBP4 in the present invention. In the figure, the concentrations of RBP4 (a→h) are: 10, 10 2 and 103 , 10 4 , 10 5 , 10 6 , 5×10 6 and 10 7 fg / mL; Figure 13 is the standard curve graph; Figure 14 is the optoelectronic signal graph showing that the PEC biosensor is selective for RBP4. In the graph, the concentration of each interfering protein is 500 pg / mL, and the concentration of RBP4 in the mixed group is 10 pg / mL; Figure 15 is the photocurrent signal graph showing the stability of the PEC biosensor's response to the RBP4 antibody; Figure 16 is the optoelectronic signal graph showing the reproducibility of the PEC biosensor for detecting RBP4; Figure 17 is the optoelectronic signal graph of the PEC biosensor and ELISA kits for detecting RBP4 respectively. Detailed implementation manners The present invention will be further explained and described below with reference to the accompanying drawings and specific experimental examples: I. In this experimental example, the experimental materials and instruments used are as follows:

[0013] Retinol-binding protein 4 (RBP4), retinol-binding protein 4 monoclonal antibody (RBP4 Ab1), goat anti-rabbit IgG H&L (RBP4 Ab2) and RBP4 ELISA kit were all purchased from Henan Tianchi Biotechnology Co., Ltd.; bovine serum albumin (BSA), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), alpha-fetoprotein (AFP), neuron-specific enolase (NSE) were all from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0014] In addition, all aqueous solutions were prepared using ultrapure water from a Milli-Q filtration system (Millipore Corp., USA). X-ray photoelectron spectroscopy (XPS) was obtained on a multi-functional imaging electron spectrometer (Thermo ESCALAB250XI). A xenon lamp (PLS-SXE300) equipped with a 420 nm cut-off filter was used as the light source (λ>420 nm). PEC and electrochemical impedance spectroscopy (EIS) measurements were performed on a CHI660E electrochemical workstation, where an ITO electrode (diameter, 5.6 mm) was used as the working electrode, a saturated calomel electrode (SCE) was used as the reference electrode, and a platinum wire was used as the auxiliary electrode. EIS measurements were carried out in 5 mmol / L (1:1) [Fe(CN)6]3- / 4- It was carried out in a solution containing 0.1 mol / L KCl, with a frequency range of 0.1 Hz to 100 kHz and an amplitude of 5 mV. The PEC test was carried out in a Tris-HCl solution (0.1 mol / L, pH 7.4) containing 0.1 mol / L ascorbic acid (AA) at -0.2 V.

[0015] II. Experimental Contents A. A method for constructing a biosensor based on NiCoO nanozyme, including the following parts: (1) Synthesis of T-COF@CS 2,4,6-Tris(4-aminophenyl)-1,3,5-triazine (0.04 mmol, 14.2 mg), 1,3,5-benzenetricarbaldehyde (0.04 mmol, 6.5 mg), PVP (25 mg) and acetonitrile (5 mL) were added to a glass bottle. After ultrasonic treatment for 30 min, 12M CH3COOH3 (0.4 mL) was added, and the glass bottle was sealed and left standing at room temperature for 96 h. The separated product was washed with N,N-dimethylformamide and then placed in a vacuum oven at 70 °C for 12 h to obtain yellow solid T-COF; further, 250 µL of T-COF (2 mg / mL) was mixed with 200 µL of 0.025 %wt CS solution dissolved in 1 % acetic acid, reacted in a metal bath for 3 h and then centrifuged to obtain T-COF@CS, and then ionized water was added to make a 350 µL T-COF@CS solution; (2) Preparation of NiCoO nanozyme 1,3,5-Benzenetricarboxylic acid (2 mmol, 420.3 mg) was first dissolved in 90 mL of H2O at 100 °C, while Co(CH3COO)2·4H2O (2 mmol, 498.2 mg) and Ni(NO3)2·6H2O (2 mmol, 581.6 mg) were dissolved in another 10 mL of H2O; then the latter solution was poured into the former solution, magnetically stirred for 15 min and then cooled to room temperature. After centrifugation, a pink precipitate was obtained. The precipitate was washed with water and ethanol and then centrifuged, and placed in an oven at 40 °C to dry; the NiCo-BTC nanorods were placed in an air environment and carbonized at 350 °C for 2 h to obtain NiCoO nanozyme; (3) Preparation of NiCoO-Ab2 conjugate The NiCoO nanozyme was coupled with Ab2 by electrostatic interaction: 2 mg of NiCoO nanozyme was added to 5 mL of PBS (0.01 M, pH 7.4) and ultrasonically mixed, and then 600 µL of 10 µg mL -1Ab2 was added to the NiCoO nanozyme solution and mixed, and shaken evenly in a metal bath at 4 °C for 10 h; then 400 µL of 1.0 wt% BSA solution was added and shaken evenly in a metal bath at 4 °C for 1 h to prevent non-specific binding, and then centrifuged; the prepared NiCoO-Ab2 bioconjugate was resuspended in 1 mL of PBS (0.01 M, pH 7.4) and stored at 4 °C for later use.

[0016] B. Application of the PEC biosensor constructed in step A in detecting RBP4, including the following parts: (1) The ITO electrode was ultrasonically cleaned successively with 1 mol / L aqueous NaOH solution, 1 mol / L ethanol and water (1:1, v / v) solution of NaOH, and Mili ultrapure water for 20 min. The conductive surface of the ITO electrode was determined by a multifunctional household electric meter, and then the electrode was placed at 60 °C and dried for 2 h; (2) 25 µL of T-COF@CS solution was dropped onto the surface of the ITO electrode and dried naturally to obtain the T-COF@CS / ITO electrode; (3) 20 µL of Ab1 (1 µg / mL -1 ) suspension was incubated at 37 °C for at least 1 h, and then washed with Mili ultrapure water to remove unbound Ab1 completely; (4) The surface of the electrode was covered with 20 µL of 0.5 wt% BSA solution, and non-specific binding was blocked for 30 min under air environmental conditions and then thoroughly rinsed with Mili ultrapure water; (5) 20 µL of RBP4 antigen solution containing different concentrations was transferred to the surface of the electrode and incubated at 37 °C for 90 min; (6) The above electrode was reacted with 20 µL of NiCoO-Ab2 conjugate at 37 °C for 90 min and further washed with Mili water to obtain the NiCoO-Ab2 / RBP4 / BSA / Ab1 / T-COF@CS / ITO electrode; (7) 20 µL of 20 mmol / L 4-CN solution was dropped onto the electrode and incubated at room temperature for 20 min; (8) The obtained electrode was tested in a Tris-HCl solution (0.1 mol / L, pH 7.4) containing 0.1 mol / L AA.

[0017] III. Results and Discussion: A. Characterization of T-COF, T-COF@CS and NiCo-BTC, NiCoO In the SEM image of T-COF, it is clearly shown that T-COF has a regular spherical morphology (asFigure 2 ), after subsequent compounding of CS and T-COF, it can be clearly seen that T-COF is covered by CS, forming a protruding shell (as Figure 3 ).

[0018] The SEM image of NiCo-BTC shows that it has a regular nanorod shape (as Figure 4 ). Directly carbonizing NiCo-BTC in air, NiCoO still maintains the nanorod morphology, but there are some wrinkles on the surface (as Figure 5 ). The surface chemical composition and element valence state of NiCoO were studied by XPS method (as Figure 6 ). The XPS spectral study results of NiCoO show the presence of four elements: Ni, Co, and O. The XPS spectrum of Ni 2p can be divided into two parts, consisting of two spin-orbit doublets (Ni 2+ and Ni 3+ ) and two shake-up satellites. The peaks at 854.02 eV and 871.38 eV are labeled as Ni 2+ , while the peaks at 855.70 eV and 872.84 eV are assigned to Ni 3+ . In the Co 2p spectrum, the peaks at 779.31 and 794.26 eV belong to the Co 2p3 / 2 and Co 2p1 / 2 orbits of Co 3+ , and the peaks at 780.82 and 795.81 eV belong to the Co 2p3 / 2 and Co2p1 / 2 orbits of Co 2+ . The XPS spectrum of C 1s can be divided into two peaks (284.66 eV, 286.30 eV), which are attributed to C-C and C-O-C respectively. The peaks at 529.61 eV, 531.13 eV, and 532.80 eV in O 1s are attributed to O-Ni, C=O, and C-O-C respectively.

[0019] B. Feasibility analysis Electrochemical impedance spectroscopy (EIS) is an effective method to verify the preparation process of PEC biosensors. Figure 7 The EIS response curves showing the charge transfer resistance (R ct ) at the electrode interface at different assembly stages are presented. The T-COF@CS / ITO electrode shows a smaller electron transfer resistance (curve a). However, gradually introducing Ab1 and BSA onto the electrode surface leads to an increasing R ct value (curves b and c). When the target antibody is incubated with the biosensor, the R value increases significantly (curve d), which can be attributed to the obvious steric hindrance of the cells bound to the electrode. After introducing NiCoO-Ab2 into the electrode, R ctThe value further increases (curve e), which can be interpreted as the weak charge transfer of the NiCoO-Ab2 semiconductor. Subsequently, NiCoO-Ab2 nanozyme catalyzes 4-CN to produce insoluble 4-CD precipitate, and the insoluble precipitate hinders 3- / 4- the arrival at the electrode interface, resulting in an increase in R ct value (curve f). The changes observed in EIS confirm the successful construction of the PEC biosensor.

[0020] To further trace the assembly process of the PEC biosensor, the PEC performance at each step of electrode assembly was investigated (such as Figure 8 ). The initial T-COF@CS / ITO electrode had a large photocurrent (curve a). After assembling Ab1 and BSA, the photocurrent continuously decreased due to enhanced steric hindrance and deteriorated conductivity (curves b and c). After the RBP4 antibody was captured by the primary antibody on the electrode, the photoelectric response significantly decreased (curve d), which may be affected by the poor conductivity of the antibody, indicating that the biosensor successfully captured the antibody. The electron transport process of T-COF@CS nanoparticles was affected by the photocurrent quenching effect of NiCoO-Ab2 and the steric hindrance effect with poor conductivity, resulting in a further decrease in photocurrent (curve e). The further decrease in photocurrent indicates that NiCoO-Ab2 can act as a nanozyme with catalase-like activity and can effectively catalyze 4-CN to generate insoluble 4-CD precipitate, thereby preventing AA from providing electrons (curve f). These results demonstrate the feasibility of the NiCoO nanozyme-based PEC biosensor for RBP4 detection.

[0021] C. Optimization of experimental conditions To enable the designed PEC biosensor to detect RBP4 with ultrahigh sensitivity and accuracy, the present invention optimized parameters such as the test bias voltage, RBP4 incubation time, and nanozyme-catalyzed 4-CN time.

[0022] To select the optimal bias voltage for the test electrode, the degree of change in the PEC signal caused by different bias voltages of the test electrode was analyzed. As Figure 9 shown, as the bias voltage decreased, the photocurrent signal gradually increased and reached a plateau at a bias voltage of -0.2 V. Therefore, -0.2 V was selected as the optimal bias voltage for the test electrode.

[0023] Secondly, to obtain satisfactory detection efficiency, the specific immune binding time between the antibody and the antigen was investigated. As Figure 10As shown, it was observed that as the immunoreaction time extended from 0 to 90 min, the optoelectronic response value of the electrode gradually decreased, indicating that with the extension of time, the content of RBP4 captured on the electrode increased. When the binding time was further extended, the photocurrent response did not decrease significantly. Therefore, the optimal reaction time between Ab1 and RBP4 was selected as 90 min.

[0024] Subsequently, in order to further improve the detection efficiency, the time for the NiCoO nanozyme-catalyzed reaction to generate insoluble precipitate was studied. The results are as Figure 11 shown. As the NiCoO nanozyme-catalyzed time increased, the PEC signal weakened, and the downward trend weakened after 20 min. Therefore, 20 min was selected as the optimal time for the NiCoO nanozyme to catalyze 4-CN.

[0025] D. Standard curve Under the optimal experimental conditions, the developed PEC biosensing platform was applied to the determination of different concentrations of RBP4. As can be seen from Figure 12 , due to the consumption of AA, the photocurrent decreased with the increase of illumination time. In this invention, the maximum value of the response was used for the photocurrent value. By changing the concentration of RBP4 from 0.00001 to 10 ng mL -1 , the relationship between the PEC signal value and the logarithm of the RBP4 concentration was shown to be a linear relationship (as Figure 13 ), and the regression equation was I (μA)=1.189397 - 0.080987lgC RBP4 (fg / mL), R 2 = 0.9994, and the detection limit was 8 fg / mL (S / N = 3). It indicated that the established PEC cell biosensor had high sensitivity. The PEC cell sensing method proposed in this section showed a wider linear range and a lower detection limit, with relatively excellent detection performance.

[0026] E. Selectivity, reproducibility and stability investigation (1) Selectivity To further explore the selectivity of the designed PEC biosensor, some potential interferents such as AFP, PSA, NSE and CEA were selected as interfering substances. As can be seen from Figure 14 , in the absence of the target, there was no obvious change in the optoelectronic signal compared with the blank. When RBP4 was present, the optoelectronic signal decreased significantly, indicating that RBP4 could be specifically captured through antigen-antibody binding. When RBP4 or a mixture of RBP4 and other substances was present, the corresponding optoelectronic signal decreased significantly, and the signal after mixing was the same as that of pure RBP4. These results indicated that the prepared nanozyme biosensor had satisfactory selectivity and could be used for the detection of RBP4.

[0027] (2) Stability The present invention studied the stability of the developed biosensor to ensure its practical application. As Figure 15 shown, when stored at 4 °C for 7 days and 14 days respectively, the biosensor could maintain 93.8% and 91.1% of the initial photocurrent response for RBP4 antibody (0.01 ng / mL). These results indicate that the constructed PEC biosensor has good stability.

[0028] (3) Reproducibility The present invention investigated the reproducibility of the designed PEC biosensor. Five independent electrodes assembled in different batches were used to detect RBP4 (0.1 pg / mL), and the relative standard deviation (RSD) value of the PEC responses on the five independent electrodes was 2.2% (as Figure 16 ). These results indicate that the constructed PEC biosensor has good reproducibility and stability.

[0029] F, Standard addition recovery experiment In order to further evaluate the feasibility of the constructed PEC biosensing strategy in the detection of actual samples, the present invention conducted a standard addition recovery experiment. Different concentrations of RBP4 (10 pg / mL, 100 pg / mL, and 5000 pg / mL) were added to human serum samples diluted 10-fold. The experimental results show that the average standard addition recovery rates in high, medium, and low groups of human plasma samples were 98.2 - 103.4%, meeting the requirements for the detection of biological samples, indicating that the PEC biosensor constructed in the present invention may be used for the detection of actual samples. These results show that the PEC biosensor has potential application prospects in detecting biomarkers of T2DM in serum.

[0030] G, Methodology comparison The results of detecting RBP4 in human serum by the established PEC method and a traditional ELISA kit are as Figure 17 shown. The results of the PEC biosensor and the ELISA kit showed good correlation (P < 0.05), indicating that the results of the PEC biosensor were consistent with those of the commercially available ELISA kit. These results indicate that the developed PEC biosensor has good accuracy and great potential in clinical applications.

[0031] H, Conclusion The present invention constructs a novel PEC biosensor based on spherical T-COF@CS and NiCoO nanozymes for highly selective and ultrasensitive detection of RBP4. The large specific surface area, excellent optical properties and good electron transfer ability of one-dimensional NiCoO nanozymes can effectively reduce the recombination of photo-generated electrons / holes and improve the detection sensitivity. Under the optimal experimental conditions, the PEC biosensor proposed by the present invention shows a wide linear range (0.01–10,000 pg / mL) and a low detection limit (8 fg / mL) for the detection of RBP4.

[0032] The novel PEC biosensor constructed by the present invention has excellent anti-interference ability, can eliminate false positive or negative detection results, and has good reproducibility and stability.

[0033] The present invention provides a good platform for ultrasensitive detection of RBP4 for early monitoring and diagnosis of T2DM, and also provides a good platform for the analysis of other biomarkers, showing broad application prospects in bioanalysis.

[0034] Although specific experimental examples of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these examples without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A construction method of a NiCoO-based biosensor, characterized in that It includes the following parts: (1)Synthesis of T-COF@CS Add 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (0.04 mmol, 14.2 mg), 1,3,5-benzenetricarbaldehyde (0.04 mmol, 6.5 mg), polyvinylpyrrolidone (25 mg), and acetonitrile (5 mL) into a glass bottle. After ultrasonic treatment for 30 min, add 12M CH3COOH3 (0.4 mL), seal the glass bottle, and let it stand at room temperature for 96 h. Wash the separated product with N,N-dimethylformamide, and then place it in a vacuum oven at 70 °C for 12 h to obtain yellow solid T-COF; further mix 250 µL of T-COF (2 mg / mL) with 200 µL of 0.025 %wt CS solution dissolved in 1 % acetic acid, react in a metal bath for 3 h, then centrifuge to obtain T-COF@CS, and then add deionized water to make a 350 µL T-COF@CS solution; (2)Preparation of NiCoO nanozyme First dissolve 1,3,5-benzenetricarboxylic acid (2 mmol, 420.3 mg) in 90 mL of H2O at 100 °C, and at the same time dissolve Co(CH3COO)2·4H2O (2 mmol, 498.2 mg) and Ni(NO3)2·6H2O (2 mmol, 581.6 mg) in another 10 mL of H2O; then pour the latter solution into the former solution, stir magnetically for 15 min, cool to room temperature, centrifuge to obtain a pink precipitate, wash the precipitate with water and ethanol and then centrifuge, and place it in an oven at 40 °C to dry; place the NiCo-BTC nanorods in an air environment and carbonize at 350 °C for 2 h to obtain NiCoO nanozyme; (3)Preparation of NiCoO-Ab2 conjugate Couple NiCoO nanozyme with Ab2 by electrostatic interaction: Add 2 mg of NiCoO nanozyme into 5 mL of PBS (0.01 M, pH 7.4), mix well by ultrasonic treatment, and then add 600 µL of 10 µg mL -1 Ab2 into the NiCoO nanozyme solution and mix. Shake well in a metal bath at 4 °C for 10 h; then add 400 µL of 1.0 wt% BSA solution, shake well in a metal bath at 4 °C for 1 h to prevent non-specific binding, and then centrifuge; Resuspend the prepared NiCoO-Ab2 conjugate in 1 mL of PBS (0.01 M, pH 7.4) and store it at 4 °C for later use.

2. Use of the biosensor constructed according to claim 1 in detecting RBP4, characterized in that, It includes the following parts: (1)Ultrasonically clean the ITO electrode successively with 1 mol / L aqueous NaOH solution, 1 mol / L NaOH ethanol and water (1:1, v / v) solution, and Mili ultrapure water for 20 min. Determine the conductive surface of the ITO electrode with a multifunctional household electric meter, and then place the electrode in an oven at 60 °C to dry for 2 h; (2)Take 25 µL of T-COF@CS solution and drop it on the surface of the ITO electrode to dry naturally to obtain the T-COF@CS / ITO electrode; (3) Add 20 µL of Ab1 (1 µg / mL -1 ) suspension and incubate at 37 °C for 1 h, then wash with Mili ultrapure water to remove unbound Ab1 completely; (4)Cover the electrode surface with 20 µL of 0.5 wt% BSA solution, block non-specific binding for 30 min under air environment conditions, and then rinse thoroughly with Mili ultrapure water; (5)Transfer 20 µL of RBP4 antigen solution with different concentrations to the electrode surface and incubate at 37 °C for 90 min; (6) The above electrode was reacted with 20 µL of NiCoO-Ab2 conjugate at 37 °C for 90 min and further washed with Mili water to obtain the NiCoO-Ab2 / RBP4 / BSA / Ab1 / T-COF@CS / ITO electrode; (7) 20 µL of 20 mmol / L 4-CN solution was dropped onto the electrode and incubated at room temperature for 20 min; (8) The obtained electrode was tested in a Tris-HCl solution (0.1 mol / L, pH 7.4) containing 0.1 mol / L AA.

3. Use of the biosensor according to claim 2 for detecting RBP4, characterized in that, The described T-COF@CS as a substrate material has excellent photocurrent signals, and NiCoO nanorods as signal probes have enhanced nanozyme activity.

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