Nanometer enzyme electrode array chip and multi-element detection application thereof

Abstract

The invention discloses a nano enzyme electrode array chip, which is characterized in that three nano enzymes of CuO.Fe2O3, CuO at Fc and NiO at Co are firstly synthesized to respectively realize high-sensitivity and high-stability detection of glucose, fructosamine and lactic acid, and the three nano enzymes can be used as an effective alternative scheme of a traditional enzyme electrode, and then the nano enzyme electrode array chip is prepared based on the three nano enzymes to realize high-sensitivity and high-stability detection of glucose, fructosamine and lactic acid. And in combination with a portable multi-channel electrochemical workstation, synchronous and real-time detection of three diabetes related biomarkers such as glucose, fructosamine and lactic acid in saliva can be realized. The invention also discloses application of the nano-enzyme electrode array chip in multi-element detection of diabetes related biomarkers in saliva.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and more specifically, relates to a nanozyme electrode array chip and its multivariate detection application. Background Art

[0002] The glucose concentration in saliva is about 1% of the blood sugar concentration, and electrochemical sensors can very sensitively detect glucose concentrations as low as 1.5 μM. There have been many studies on the instant detection of glucose in saliva. However, glucose in saliva may be decomposed by bacteria and enzymes in the mouth, thus affecting the stability of its content. Therefore, people try to introduce more markers for multivariate detection to enhance the accuracy of diabetes monitoring. Summary of the invention

[0003] The present invention aims to solve the above technical problems. To this end, the present invention proposes a nanozyme electrode array chip and its multivariate detection application for the simultaneous real-time detection of three diabetes-related markers: glucose, fructosamine and lactic acid in saliva.

[0004] Before describing the scheme of the present invention, the following English abbreviations are explained: PB: Prussian blue acid; APTES: silane coupling agent acid; Fc: ferrocene; H 2 C 2 O 4 :oxalic acid.

[0005] The technical solution of the present invention is as follows:

[0006] In one aspect, the present invention provides a method for preparing a nanozyme electrode array chip, comprising:

[0007] S1, CuO·Fe 2 O 3 Synthesis: First, CuO, Fe 2 O 3 It is synthesized with PB as a precursor, and then CuO and Fe 2 O 3 The nanoparticles were mixed in Nafion (0.50 wt%) solution at a ratio of 1:4 to obtain CuO·Fe 2 O 3 .

[0008] S2. Synthesis of CuO@Fc: First, CuO was synthesized by precipitation followed by annealing, and APTES was used to functionalize CuO with amine groups to obtain CuO-NH 2 With the help of EDC / NHS coupling agent, Fc was covalently linked to CuO-NH 2 On top, CuO@Fc was obtained.

[0009] S3. Synthesis of NiO@Co: Flower-like structured NiO@Co nanomaterials were synthesized by hydrothermal method.

[0010] S4, the electrode array was made by screen printing, gold nanoparticles were modified on the electrode surface by electrodeposition, and the working electrode was modified by drop casting to prepare CuO·Fe 2 O 3 / AuNPs / SPCE, CuO@Fc / AuNPs / SPCE and NiO@Co / AuNPs / SPCE electrodes.

[0011] In some embodiments, step S1 specifically comprises: firstly, 0.50 g of CuCl 2 Dissolve in 5.0 mL of water and shake to fully dissolve, then add 1.0 mL of 0.80 M oxalic acid solution. There will be CuC in the solution. 2 O 4 Precipitation appears and the solution color changes from dark blue to green-blue. Filter out CuC 2 O 4 The remaining solution was then transferred to a muffle furnace and calcined at 450°C for 4 hours. After the calcination, the temperature was cooled to room temperature, and the black CuO powder was collected and fully ground, washed three times with pure water, and dried in an oven at 50°C. 2 O 3 It was synthesized using PB as a precursor. First, K 3 [Fe(CN) 6 ] and FeCl 3 The solutions were mixed in equal volumes, and the resulting precipitate was aged at room temperature for two days, filtered, and washed with distilled water. The resulting PB solid was dried in an oven at 60 °C, ground, and used as a precursor for the synthesis of α-Fe 2 O 3 The PB powder was placed in a muffle furnace and calcined at 550°C for 4 hours to obtain a compact α-Fe 2 O 3 Finally, CuO and Fe 2 O 3 The nanoparticles were mixed in Nafion (0.50 wt%) solution at a ratio of 1:4 to obtain CuO·Fe 2 O 3 .

[0012] In some embodiments, step S2 specifically comprises: first, 0.68 g of CuCl 2 -2H 2 O and 0.15 g of citric acid were mixed in 60 ml of distilled water and stirred vigorously at room temperature, and then 4.0 g of NaOH was added to the mixed solution to form Cu(OH)2 A blue precipitate was obtained. After 10 minutes, the blue precipitate was filtered and washed three times with distilled water and ethanol. After drying, it was dried in an oven at 60°C for 6 hours. The powder was annealed in a muffle furnace at 400°C for 4 hours to obtain black CuO. CuO was amine-functionalized using APTES. 60 mg of CuO was dispersed in 60 mL of ethanol by ultrasonic treatment, and then 1.0 mL of APTES was added to the suspension under vigorous stirring. After stirring at room temperature for 12 hours, the resulting mixture was washed three times with distilled water and ethanol. It was dried at 60°C to obtain CuO-NH 2 With the help of EDC / NHS coupling agent, Fc was covalently linked to CuO-NH 2 In a 10mL solution containing 2.0×10 2 Add 10 ml of a solution containing 0.40 M EDC and 0.10 M NHS to the beaker containing the 10 μg / L Fc solution and stir for 2 hours to activate the carboxyl groups on Fc. Then add 0.10 g CuO-NH 2 After stirring overnight, the mixture was centrifuged to obtain a precipitate, which was washed three times with pure water to remove unbound Fc. Finally, the precipitate was dried in an oven at 60 °C for 5 hours to obtain CuO@Fc solid.

[0013] In some embodiments, step S3 specifically comprises: adding 1.6 mmol Ni(NO 3 )·6H 2 O and 3.5 mmol urea were added to 100 mL pure water, and 0.050 g Co(NO 3) 6H 2 O was doped with Co element, and the mixture was stirred at room temperature for 30 minutes and then transferred to a Teflon-lined autoclave. The autoclave was then placed in an oven and kept at 150°C for 24 hours. The container was then allowed to cool naturally to room temperature. The precipitate generated in the container was collected by centrifugation. The collected precipitate was thoroughly rinsed with pure water and ethanol, dried at 60°C for 12 hours, and calcined at 450°C in ambient air for 4 hours to obtain NiO@Co powder.

[0014] In some embodiments, the specific steps of making an electrode array by screen printing in step S4 are as follows: First, the PET substrate is ultrasonically washed with ethanol for 10 minutes. After that, silver paste is deposited on the PET substrate to construct a conductive circuit, and after it is cured, the first layer of carbon paste is deposited on the PET substrate to manufacture three working electrodes and one counter electrode. Next, the printed electrode is annealed at a temperature of 120°C for 15 minutes. Then, a second layer of printing is performed using Ag / AgCl paste as a reference electrode. Curing is performed again at 120°C for 15 minutes. Subsequently, silver paste is deposited by screen printing to achieve connection between the conductive parts. Finally, the covering layer is printed using an insulating polymer slurry. The prepared screen-printed electrodes are stored at room temperature.

[0015] In some embodiments, step S4 further comprises: first, ultrasonically cleaning the screen-printed electrode with ethanol and distilled water for 2 minutes, and then drying it for later use. Next, in order to further enhance the conductivity of the electrode, gold nanoparticles are modified on the surface of the electrode by electrodeposition. 100 μL of 3.0 mM HAuCl 4 The solution was dropped onto the screen-printed electrode surface and then electrodeposited at a constant potential of −0.4 V for 50 s. Afterwards, it was rinsed with ultrapure water to remove excess HAuCl 4 , and the electrode surface modified with gold nanoparticles (AuNPs / SPCE) was obtained. Next, the working electrode was modified by drop casting. 2 O 3 The three nanozyme materials, CuO@Fc, and NiO@Co, were dissolved in a mixed solution of deionized water and 0.50wt% Nafion (5:1, v / v) to a concentration of 1.0 mg / mL. Then, the 1.0 mg / mL nanozyme suspension was evenly dripped onto the surfaces of the three working electrodes to prepare CuO·Fe 2 O 3 / AuNPs / SPCE, CuO@Fc / AuNPs / SPCE and NiO@Co / AuNPs / SPCE electrodes. Finally, the electrodes were naturally dried at room temperature.

[0016] On the other hand, the present invention also provides a nanozyme electrode array chip prepared according to the above preparation method.

[0017] On the other hand, the present invention also provides the application of the above-mentioned nanozyme electrode array chip in the multivariate detection of diabetes-related biomarkers in saliva.

[0018] In the present invention, CuO·Fe 2 O 3The three types of nanozymes, CuO@Fc, and NiO@Co, respectively achieved high-sensitivity and high-stability detection of glucose, fructosamine, and lactic acid, and can be used as an effective alternative to traditional enzyme electrodes. Nanozyme electrode array chips were prepared based on these three nanozymes. Combined with a portable multi-channel electrochemical workstation, they can achieve simultaneous and instant detection of three diabetes-related biomarkers, including glucose, fructosamine, and lactic acid, in saliva. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the reaction mechanism of nanozyme electrode array multivariate detection;

[0020] Figure 2 It is CuO·Fe 2 O 3 SEM image (A) and corresponding EDS spectrum (B); Fe 2 O 3 XRD patterns of CuO (C) and CuO (D); Fe 2 O 3 (E) and XPS spectra of CuO (F);

[0021] Figure 3 It is (A)CuO·Fe 2 O 3 CV responses of CuO·Fe / AuNPs / SPCE to different glucose concentrations; (B) When 0.005-2.2 mM glucose was continuously added to 0.1 M NaOH solution, 2 O 3 / AuNPs / SPCE glucose oxidation amperometric response; (C)CuO·Fe 2 O 3 Linear calibration results of / AuNPs / SPCE current response to glucose;

[0022] Figure 4 SEM image (A) of CuO@Fc, corresponding EDS spectrum (B), X-ray diffraction pattern (C) and XPS spectrum (D);

[0023] Figure 5 (A) CV response of CuO@Fc / AuNPs / SPCE to different concentrations of fructosamine; (B) amperometric response of CuO@Fc / AuNPs / SPCE to fructosamine when 50-350 μM fructosamine was added continuously to 0.1 M NaOH; (C) Linear calibration result of CuO@Fc / AuNPs / SPCE to fructosamine current response;

[0024] Figure 6The SEM image (A) of NiO@Co, the corresponding EDS spectrum (B), X-ray diffraction pattern (C) and XPS spectrum (D);

[0025] Figure 7 (A) CV response of NiO@Co / AuNPs / SPCE to different concentrations of lactic acid; (B) Amperometric response of lactic acid oxidation of NiO@Co / AuNPs / SPCE when 100-2700 μM lactic acid was continuously added to 0.1 M NaOH; (C) Linear calibration result of NiO@Co / AuNPs / SPCE to fructosamine current response;

[0026] Figure 8 Consistency analysis of the results of glucose (A), fructosamine (B) and lactic acid (C) in clinical saliva samples determined by a multi-channel portable electrochemical workstation based on nanozyme electrode array and the standard colorimetric method (number of samples N = 10). DETAILED DESCRIPTION

[0027] The technical scheme of the present invention is further described in detail below in conjunction with specific embodiments, but does not constitute any limitation to the present invention. Unless otherwise specified, the reagents, equipment and methods used in the present invention are conventional commercially available reagents, equipment and conventional methods in the art.

[0028] Example 1 Preparation of Nanozyme Electrode Array Chip

[0029] The three working electrodes of the electrode array are modified with CuO.Fe 2 O 3 , CuO@Fc and NiO@Co three nanozyme materials to achieve simultaneous instant detection of glucose, fructosamine and lactic acid. The reaction mechanism of nanozyme electrode array multivariate detection is as follows Figure 1 shown.

[0030] The preparation process of the nanozyme electrode array chip is as follows:

[0031] S1, CuO·Fe 2 O 3 Synthesis: CuO was synthesized by precipitation and annealing. 0.50 g CuCl 2 Dissolve in 5.0 mL of water and shake to fully dissolve, then add 1.0 mL of 0.80 M oxalic acid solution. There will be CuC in the solution. 2 O 4 Precipitation appears and the solution color changes from dark blue to green-blue. Filter out CuC 2 O 4The remaining solution was then transferred to a muffle furnace and calcined at 450°C for 4 hours. After the calcination, the temperature was cooled to room temperature, the black CuO powder was collected and fully ground, washed three times with pure water, dried in an oven at 50°C overnight, and stored for subsequent characterization and use. 2 O 3 It was synthesized using PB as a precursor. First, K 3 [Fe(CN) 6 ] and FeCl 3 The solutions were mixed in equal volumes, and the resulting precipitate was aged at room temperature for two days, filtered, and washed with distilled water. The resulting PB solid was dried in an oven at 60 °C, ground, and used as a precursor for the synthesis of α-Fe 2 O 3 The PB powder was placed in a muffle furnace and calcined at 550°C for 4 hours to obtain a compact α-Fe 2 O 3 . CuO and Fe 2 O 3 The nanoparticles were mixed and stored in Nafion (0.50 wt %) solution at a ratio of 1:4.

[0032] S2. Synthesis of CuO@Fc: First, 0.68 g CuCl 2 -2H 2 O and 0.15 g of citric acid were mixed in 60 ml of distilled water and stirred vigorously at room temperature, and then 4.0 g of NaOH was added to the mixed solution to form Cu(OH) 2 A blue precipitate was obtained. After 10 minutes, the blue precipitate was filtered and washed three times with distilled water and ethanol. After drying, it was dried in an oven at 60°C for 6 hours. The powder was annealed in a muffle furnace at 400°C for 4 hours to obtain black CuO. CuO was amine-functionalized using APTES. 60 mg of CuO was dispersed in 60 mL of ethanol by ultrasonic treatment, and then 1.0 mL of APTES was added to the suspension under vigorous stirring. After stirring at room temperature for 12 hours, the resulting mixture was washed three times with distilled water and ethanol. It was dried at 60°C to obtain CuO-NH 2 With the help of EDC / NHS coupling agent, Fc was covalently linked to CuO-NH 2 In a 10mL solution containing 2.0×10 2 Add 10 ml of a solution containing 0.40 M EDC and 0.10 M NHS to the beaker containing 1 μg / L Fc solution and stir for 2 hours to activate the carboxyl groups on Fc. Then add 0.10 g CuO-NH 2After stirring overnight, the mixture was centrifuged to obtain a precipitate, which was washed three times with pure water to remove unbound Fc. Finally, the precipitate was dried in an oven at 60 °C for 5 hours to obtain CuO@Fc solid.

[0033] S3. Synthesis of NiO@Co: Flower-like NiO@Co nanomaterials were synthesized by hydrothermal method. 1.6mmol Ni(NO 3 )·6H 2 O and 3.5mmol urea were added to 100mL pure water, and 0.050g Co(NO 3) 6H 2 O was doped with Co element, and the mixture was stirred at room temperature for 30 minutes and then transferred to a Teflon-lined autoclave. The autoclave was then placed in an oven and kept at 150°C for 24 hours. The container was then allowed to cool naturally to room temperature. The precipitate generated in the container was collected by centrifugation. The collected precipitate was thoroughly rinsed with pure water and ethanol, dried at 60°C for 12 hours, and calcined at 450°C in ambient air for 4 hours to obtain NiO@Co powder.

[0034] S4. Make an electrode array by screen printing. The specific steps are as follows: First, the PET substrate is ultrasonically washed with ethanol for 10 minutes. After that, silver paste is deposited on the PET substrate to construct a conductive circuit. After it is cured, the first layer of carbon paste is deposited on the PET substrate to make three working electrodes and one counter electrode. Then, the printed electrode is annealed at 120°C for 15 minutes. Then, a second layer of Ag / AgCl paste is used for printing as a reference electrode. Curing is performed again at 120°C for 15 minutes. Subsequently, silver paste is deposited by screen printing to achieve connection between the conductive parts. Finally, the covering layer is printed using an insulating polymer paste. The prepared screen-printed electrodes are stored at room temperature.

[0035] Before using the screen-printed electrode, a series of cleaning and modification steps are required to optimize its performance. The specific steps are as follows: First, the screen-printed electrode is ultrasonically cleaned with ethanol and distilled water for 2 minutes, and then dried for use. Next, in order to further enhance the conductivity of the electrode, gold nanoparticles are modified on the electrode surface by electrodeposition. 100.0 μL of 3.0 mM HAuCl 4 The solution was dropped onto the screen-printed electrode surface and then electrodeposited at a constant potential of −0.4 V for 50 s. Afterwards, it was rinsed with ultrapure water to remove excess HAuCl 4, and the electrode surface modified with gold nanoparticles (AuNPs / SPCE) was obtained. Next, the working electrode was modified by drop casting. The three nanozyme materials were dissolved in a mixed solution of deionized water and 0.50wt% Nafion (5:1, v / v) to a concentration of 1.0mg / mL. Then, 1.0mg / mL of the nanozyme suspension was evenly dripped on the surfaces of the three working electrodes to prepare CuO·Fe 2 O 3 / AuNPs / SPCE, CuO@Fc / AuNPs / SPCE and NiO@Co / AuNPs / SPCE electrodes. Finally, the electrodes were naturally dried at room temperature.

[0036] Figure 2 (A) is CuO and Fe 2 O 3 Scanning electron microscope image of nanozyme material, irregular CuO particles and stacked two-dimensional Fe 2 O 3 CuO·Fe 2 O 3 Elemental mapping of composite materials ( Figure 2 (B)) The presence of Cu, O, and Fe elements can be seen, confirming that CuO·Fe 2 O 3 The composite nanozyme material was successfully synthesized. 2 O 3 The powder and CuO powder were characterized by X-ray diffraction (XRD) and compared with standard cards. Figure 2 (C) Display Fe 2 O 3 The XRD spectrum of the standard card is in good correspondence. Obvious diffraction peaks can be observed at angles of 24.2°, 33.2°, 35.6°, 40.9°, 49.5°, 54.1°, 57.6°, 62.4° and 64.0°. These peaks correspond to Fe 2 O 3 The (012), (104), (110), (113), (024), (116), (018), (214) and (300) crystal planes reflect. Figure 2 As shown in (D), the diffraction peaks of the synthesized CuO powder centered at 43.3°, 50.4° and 74.1° on the (111), (200) and (220) crystal planes correspond to the typical characteristic peaks of copper and are in good agreement with the standard CuO nanosheets, verifying the successful formation of the two-dimensional CuO crystal structure. X-ray photoelectron spectroscopy (XPS) technology was further used to analyze the composition and binding state of the elements. The results are shown in Figure 2 (E) and Figure 2(F) 2 O 3 The material has two peaks at 711.2 and 724.5 eV, which are attributed to Fe 2p3 / 2 and Fe 2p1 / 2, respectively. There is also a satellite peak at 718.9 eV, which is the characteristic of Fe(Ⅲ). The two strong peaks at 953.4 eV and 933.4 eV in the XPS of the CuO material are attributed to Cu 2p1 / 2 and Cu 2p3 / 2, respectively. At the same time, the appearance of satellite peaks at 940 eV and 962.2 eV further confirms that the valence state of Cu is Cu(Ⅱ). The valence states of Fe and Cu in the two materials enable the hybrid material to have corresponding electrocatalytic properties.

[0037] like Figure 3 As shown in (A), after adding 100 μM glucose to 0.10 M NaOH buffer, CuO·Fe 2 O 3 The / AuNPs / SPCE electrode showed an obvious current response near 0.40 V. Therefore, the present invention selects 0.40 V as the excitation voltage for the chronoamperometric response. Figure 3 (B) shows the CuO·Fe under the excitation voltage of 0.40 V when glucose is continuously added 2 O 3 Current response of / AuNPs / SPCE. When the current reaches a stable state in the buffer, high-concentration glucose mother solution is continuously added, and its concentration is gradually increased to 5μM, 10μM, 50μM, 100μM, 250μM, until it increases to 2.2mM. With the increase of glucose concentration, the response current increases steadily. Figure 3 (C) Yes Figure 3 (B) The corresponding calibration curve shows that CuO·Fe 2 O 3 The stable current of AuNPs / SPCE changes with the glucose concentration and shows a good linear relationship in the range of 0.005–2.2 mM (y=0.0022x+0.11). 2 =0.99. The detection limit of this method is 0.12 μM, which can meet the detection sensitivity requirement of glucose concentration in saliva ranging from 0.008–1.77 mM.

[0038] Similarly, the CuO@Fc material was characterized using SEM. Figure 4 As shown in (A), CuO@Fc exhibits a two-dimensional irregular shape of CuO nanosheets, and the introduction of ferrocene has no effect on its morphology. Figure 4 (B) is the corresponding elemental spectrum, where the presence of Cu, O, and Fe can be observed, further verifying that ferrocene is successfully connected to the surface of CuO through the coupling of carboxyl and amino groups. Figure 4 (C) is the XRD spectrum of CuO@Fc nanoparticles. The position of the diffraction peaks basically corresponds to the CuO standard card, and its basic structure of CuO can be seen. Figure 4 (D) XPS spectrum shows Cu 2p1 / 2 and Cu 2p3 / 2 at around 935 eV and 955 eV respectively, which belong to CuO. CuO is still the main component of the nanomaterial.

[0039] Figure 5 (A) is the CV spectrum of the CuO@Fc / AuNPs / SPCE electrode in 0.1M NaOH solution in the presence and absence of 100μM fructosamine. It can be found that in the presence of 100μM fructosamine, the peak current density of the electrode near 0.45V increased significantly, representing the oxidation reaction of enaminol here. The CuO@Fc / AuNPs / SPCE modified electrode detected fructosamine by chronoamperometry, such as Figure 5 As shown in (B), at an excitation voltage of 0.45 V, high-concentration mother solution was added continuously to increase the fructosamine concentration in the buffer by 50 μM each time. It can be observed that the steady-state current increases with the increase in fructosamine concentration. Figure 5 (C) is the linear calibration result of CuO@Fc / AuNPs / SPCE to the fructosamine current response, which shows a logarithmic relationship in the range of 50μM–350μM, with a detection limit of 29.21μM, which can meet the detection requirements of fructosamine in the range of 70–300μM in saliva.

[0040] In order to identify that NiO@Co has been successfully synthesized, its morphology was characterized by scanning electron microscopy, such as Figure 6 As shown in (A), it can be observed that the NiO@Co particles have a classic nanoflower-like structure consisting of curled and wrinkled 2D nanosheets (thickness ∼10 nm). The composition of NiO@Co was evaluated by EDS, as shown in Figure 6 As shown in (B), the synthesized NiO@Co contains characteristic peaks of both Ni and Co, confirming the successful introduction of Co. Similarly, the XRD spectrum of NiO@Co shows diffraction peaks at 37.6°, 43.2°, 62.8°, 75.3° and 79.1°, which are respectively attributed to the (111), (200), (220), (311) and (322) planes, which are basically corresponding to the diffraction peaks of the NiO standard card. The appearance of the diffraction peak around 30° may be due to the influence of the doping of Co element on its crystal structure. Figure 6 (C)). The valence state conversion of Ni element is the key to its ability to sense lactic acid, so XPS is used to verify its chemical composition and valence state. Figure 6As shown in (D), the Ni 2p3 / 2 peak of NiO@Co can be decomposed into two fitting curves, and their binding energies are located at 855.2 eV and 856.5 eV, corresponding to Ni 2+ and Ni 3+ This indicates that the NiO@Co nanozyme has electrocatalytic active sites and can be used for the electrocatalytic reaction of lactic acid.

[0041] The present invention studies the electrocatalytic performance of NiO@Co to lactic acid by cyclic voltammetry and chronoamperometry. Figure 7 (A) shows the cyclic voltammograms of NiO@Co / AuNPs / SPCE in the presence and absence of 100 μM lactic acid in buffer. A clear oxidation peak can be observed around 0.50 V, which is due to the redox reaction of nickel (Ni 3+ / Ni 2+ ), lactic acid is electrochemically oxidized to pyruvic acid on the electrode surface, while Ni 3+ Reduction to Ni 2+ Afterwards, the amperometric response of NiO@Co / AuNPs / SPCE to different concentrations of lactic acid under 0.5 V voltage excitation was measured, such as Figure 7 (B) Figure 7 The calibration curve (C) shows that NiO@Co / AuNPs / SPCE has a good linear response to lactic acid in the range of 0.05–2.7 mM (y=0.00010x+0.14(R 2 =0.99)), with a detection limit of 16.81 μM, indicating that NiO@Co has excellent electrocatalytic ability for lactic acid and can meet the quantitative detection of lactic acid in the range of 0.1–2.5 mM in saliva.

[0042] Example 2 Application of Nanozyme Electrode Array Chip in Multiplex Detection of Diabetes-Related Biomarkers in Saliva

[0043] In this example, 10 clinical saliva samples were collected with the assistance of clinical hospital collaborators.

[0044] This embodiment combines the nanozyme electrode array chip with the multi-channel portable workstation previously developed by the inventors to construct a multiplex detection system.

[0045] After obtaining the saliva sample and performing a simple filtration treatment, the concentrations of the three substances were first detected using a kit and an ultraviolet spectrophotometer. Then, 25 μL of saliva was mixed with an equal volume of NaOH (0.2 M) solution and dripped onto the surface of the nanozyme electrode array. The three channels of the multi-channel portable electrochemical workstation were set to it detection mode, and the excitation voltages were 0.4 V, 0.45 V and 0.5 V, respectively. The excitation time was set to 100 s. After exporting the data, the current value was substituted into the formula for calculation to obtain the concentrations of the three substances in the corresponding samples. The detection results of the three substances obtained using the colorimetric kit and the method of this embodiment in 10 samples were subjected to Bland-Altman to evaluate the consistency of the two detection methods ( Figure 8 ), it can be found that the error of the analyte concentration detected by the nanozyme electrode array and the colorimetric kit is basically distributed within the range of ±1.96SD, and almost all points are within the 95% confidence interval. Therefore, Bland-Altman analysis shows that there is no significant difference in the detection accuracy between the multivariate detection system and the standard colorimetric kit.

Claims

1. A method for preparing a nanozyme electrode array chip, characterized in that: include: S1. Synthesis of CuO·Fe2O3: First, CuO was synthesized by precipitation followed by annealing. Fe2O3 was synthesized using PB as a precursor. Then, CuO and Fe2O3 nanoparticles were mixed in a Nafion (0.50 wt%) solution at a ratio of 1:4 to obtain CuO·Fe2O3. S2. Synthesis of CuO@Fc: First, CuO was synthesized by precipitation followed by annealing. APTES was used to functionalize CuO with amine groups to obtain CuO-NH2. Fc was covalently linked to CuO-NH2 with the help of EDC / NHS coupling agents to obtain CuO@Fc. S3. Synthesis of NiO@Co: Synthesis of flower-like structured NiO@Co nanomaterials by hydrothermal method; S4. Electrode arrays were fabricated by screen printing, gold nanoparticles were modified on the electrode surface by electrodeposition, and the working electrode was modified by drop casting to prepare CuO·Fe2O3 / AuNPs / SPCE, CuO@Fc / AuNPs / SPCE, and NiO@Co / AuNPs / SPCE electrodes, respectively.

2. The preparation method according to claim 1, characterized in that: Step S1 includes: S11. First, dissolve 0.50 g of CuCl2 in 5.0 mL of water and shake to fully dissolve it. Then add 1.0 mL of 0.80 M oxalic acid solution. CuC2O4 precipitates will appear in the solution. S12, after filtering out CuC2O4, transfer the remaining solution to a muffle furnace, calcine at 450°C, cool to room temperature, collect the black CuO powder, grind it thoroughly, wash it with pure water, and dry it in an oven at 50°C; S13, mixing equal volumes of K3[Fe(CN)6] and FeCl3 solutions with a concentration of 0.020M, aging the resulting precipitate at room temperature, filtering and washing with distilled water; drying the resulting PB solid in an oven at 60°C, placing the PB powder in a muffle furnace, and calcining at 550°C to obtain α-Fe2O3; S14. CuO and Fe2O3 nanoparticles are mixed in a Nafion (0.50 wt%) solution at a ratio of 1:4 to obtain CuO·Fe2O3.

3. The preparation method according to claim 1, characterized in that: Step S2 includes: S21, 0.68 g of CuCl2-2H2O and 0.15 g of citric acid were mixed in 60 ml of distilled water, and stirred vigorously at room temperature, and then 4.0 g of NaOH was added to the mixed solution to form a blue precipitate of Cu(OH)2; S22, filtering the blue precipitate, washing it with distilled water and ethanol, drying it, and drying it in an oven at 60°C, and annealing the powder in a muffle furnace at 400°C to obtain black CuO; S23, using APTES to functionalize CuO with amine groups, dispersing 60 mg of CuO in 60 mL of ethanol by ultrasonic treatment, and then adding 1.0 mL of APTES to the suspension under vigorous stirring, stirring at room temperature, and washing the resulting mixture with distilled water and ethanol; drying at 60 ° C to obtain CuO-NH2; S24, in a solution containing 10 mL of 2.0×10 2 To the beaker of the μg / L Fc solution, 10 ml of a solution containing 0.40 M EDC and 0.10 M NHS was added and stirred to activate the carboxyl groups on the Fc; then 0.10 g CuO-NH2 was added and stirred overnight, and the mixture was centrifuged to obtain a precipitate, which was washed with pure water to remove unbound Fc; finally, the precipitate was dried in an oven at 60°C to obtain a CuO@Fc solid.

4. The preparation method according to claim 1, characterized in that: Step S3 includes: S31, add 1.6mmol Ni(NO3)·6H2O and 3.5mmol urea to 100mL pure water, and add 0.050g Co(NO 3) 6H2O to introduce the doping of Co element, and the mixture was stirred at room temperature; S32, transfer to a Teflon-lined autoclave, then place the autoclave in an oven at 150°C, and then allow the container to cool naturally to room temperature; S33. The precipitate generated in the container is collected by centrifugation, and the collected precipitate is thoroughly rinsed with pure water and ethanol, dried at 60° C., and calcined at 450° C. in ambient air to obtain NiO@Co powder.

5. The preparation method according to claim 1, characterized in that: The preparation process of the screen-printed electrode in step S4 includes: S41, ultrasonically washing the PET substrate with ethanol, then depositing silver paste on the PET substrate to construct a conductive circuit, and after the silver paste is cured, depositing a first layer of carbon paste on the PET substrate to manufacture three working electrodes and one counter electrode; S42, annealing the printed electrode at a temperature of 120° C., then printing a second layer using Ag / AgCl paste as a reference electrode, and curing it again at 120° C.; S43, depositing silver paste by screen printing to achieve connection between conductive parts; S44. Printing a cover layer using an insulating polymer paste.

6. The preparation method according to claim 1, characterized in that: The steps of preparing CuO·Fe2O3 / AuNPs / SPCE, CuO@Fc / AuNPs / SPCE and NiO@Co / AuNPs / SPCE electrodes in step S4 include: 100 μL of 3.0 mM HAuCl4 solution was dropped onto the surface of the screen-printed electrode and then electrodeposited at a constant potential of -0.4 V. Afterwards, it was rinsed with ultrapure water to remove excess HAuCl4 to obtain an electrode surface modified with gold nanoparticles (AuNPs / SPCE). Next, the three nanozyme materials, CuO·Fe2O3, CuO@Fc, and NiO@Co, were dissolved in a mixed solution of deionized water and 0.50 wt% Nafion (5:1, v / v) to a concentration of 1.0 mg / mL. Then, 1.0 mg / mL of the nanozyme suspension was evenly dropped onto the surfaces of the three working electrodes to prepare CuO·Fe2O3 / AuNPs / SPCE, CuO@Fc / AuNPs / SPCE, and NiO@Co / AuNPs / SPCE electrodes, respectively.

7. A nanozyme electrode array chip prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the nanozyme electrode array chip according to claim 7 in the multivariate detection of diabetes-related biomarkers in saliva.

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