Electrochemical biosensing electrode constructed based on click chemistry reaction and having multiple detection capability, preparation method and application

Abstract

The invention provides an electrochemical biosensing electrode which is constructed based on click chemistry reaction and has multiple detection capabilities, a preparation method and application, the electrode comprises two or more than two independent working electrodes, a reference electrode and a counter electrode, the working electrodes are screen-printed carbon electrodes, the working electrodes are arranged in an array, and the reference electrode is arranged in the array. And different probes are modified on the working electrode. According to the invention, multiple biomarkers including but not limited to p53 gene, thrombin and vascular endothelial growth factor 165 (VEGF165) can be detected at the same time, and the detection capability can reach the skin molar concentration level; even in complex biological matrixes such as serum and diluted serum, the method still shows excellent performance and high repeatability; according to the invention, a one-time multi-channel design is adopted, the manufacturing process is simplified, the cost is reduced, and the device is suitable for on-site rapid detection application.

Description

Technical Field

[0001] The present invention relates to the field of biosensor technology, and in particular to an electrochemical biosensor electrode with multiple detection capabilities constructed based on click chemistry reactions, a preparation method, and applications. Background Art

[0002] With the growing demand for faster, more sensitive, and more portable biomarker detection systems, the development of highly sensitive and selective detection methods for the simultaneous detection of DNA, RNA, and proteins has become increasingly important. Multiplex detection technology can quantitatively analyze multiple target substances in a single test, and compared with traditional detection methods, it has significant advantages in improving overall diagnostic accuracy, reducing detection costs, and shortening analysis time. These advantages make multiplex detection technology an obvious goal in promoting the development of on-site molecular diagnostic equipment. In addition, especially when biological samples are scarce, it is becoming increasingly important to obtain key information about DNA and protein markers from the same sample.

[0003] Traditional immunosensors rely on antibody / antigen recognition strategies and have been shown to be effective in protein detection, especially for single protein detection. However, cross-reactivity of antibodies may hinder their application in multiplex detection. In contrast, aptamers (short, antibody-like DNA oligonucleotides) have shown great potential for application in protein detection. Aptamers, which can bind to their targets with high affinity after folding into specific structures, have attracted widespread attention and have been used in multiplex detection based on various signal readout strategies (such as fluorescence, surface plasmon resonance, Raman spectroscopy, and field-effect transistors).

[0004] To meet the requirements of portable detection platforms in terms of sensitivity, accuracy, rapidity, miniaturization, cost-effectiveness, and ease of use, electrochemical biosensors have great potential as a development platform for multiplexed detection portable devices, mainly due to their low manufacturing cost, simple operation, and high feasibility. In addition, the multiplexed detection modes of electrochemical analysis can be divided into single-electrode mode and electrode array mode. For electrode arrays, complex manufacturing processes and multi-channel electrochemical analysis equipment are usually required. However, the need to distinguish signal labels on a single electrode substrate to achieve multiplexed detection has posed new technical challenges. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention proposes an electrochemical biosensor electrode with multiple detection capabilities constructed based on click chemistry reaction, a preparation method and application.

[0006] An electrochemical biosensor electrode with multiple detection capabilities constructed based on click chemistry reaction has two or more independent working electrodes, a reference electrode and a counter electrode. The working electrode is a screen-printed carbon electrode arranged in an array and modified with different probes.

[0007] Furthermore, the reference electrode is a silver chloride electrode, and the counter electrode is a platinum electrode.

[0008] Furthermore, the probe is an oligonucleotide probe with one end modified with an alkynyl group or dibenzocyclooctyne (DBCO) and the other end modified with methylene blue.

[0009] A method for preparing an electrochemical biosensor electrode with multiple detection capabilities based on the above-mentioned click chemistry reaction comprises the following steps:

[0010] A gold film was formed on the surface of a screen-printed carbon electrode by electrochemical deposition;

[0011] Modifying the gold film surface with a thiol containing an azide group according to claim 1;

[0012] Oligonucleotide probes modified with alkynyl or dibenzocyclooctyne (DBCO) are selectively immobilized on the electrode surface via electrochemically triggered or ring strain-triggered click chemistry reactions.

[0013] Furthermore, the thiol containing an azide group is 1-azidoundecethiol.

[0014] Furthermore, the electrochemically triggered click chemistry reaction selectively generates an active monovalent copper ion catalyst by controlling the electrode potential, thereby performing an alkynyl-azide cycloaddition reaction on a desired electrode.

[0015] An application of the electrochemical biosensor electrode with multiple detection capabilities constructed based on the above-mentioned click chemistry reaction comprises the following steps:

[0016] The electrochemical biosensor electrode with multiple detection capabilities constructed based on click chemistry reaction is connected to the electrochemical workstation through a wire; the electrochemical biosensor electrode is immersed in the solution to be detected, and the electrochemical workstation is turned on to detect the detection target.

[0017] Furthermore, the detection target is a protein, a nucleic acid or a small molecule compound.

[0018] Beneficial effects:

[0019] 1. High sensitivity and high specificity: The present invention can simultaneously detect multiple biomarkers including but not limited to the p53 gene, thrombin, and vascular endothelial growth factor 165 (VEGF165), with a detection capability reaching picomolar concentration levels;

[0020] 2. Good reproducibility and stability: Even in complex biological matrices such as serum and diluted serum, the present invention still shows excellent performance and high reproducibility;

[0021] 3. High cost-effectiveness, fast manufacturing and easy operation: The present invention adopts a disposable multi-channel design, which simplifies the manufacturing process and reduces costs, and is suitable for on-site rapid detection applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the preparation process of an electrochemical biosensor electrode with multiple detection capabilities based on click chemistry reactions;

[0023] Figure 2 Schematic diagram of the detection of an electrochemical biosensor electrode with multiple detection capabilities based on click chemistry reactions.

[0024] Figure 3 The calibration curves of the sensor electrode constructed based on the alkyne-azide cycloaddition click reaction in Example 1 for different targets are shown in Figure 1. (a) shows the change in the detection signal when the sensor detects the p53 target; (b) shows the change in the detection signal when the sensor detects thrombin; (c) shows the change in the detection signal when the sensor detects VEGF. 165 Detection signal changes when

[0025] Figure 4 The repeatability and reproducibility curves of the sensor electrode constructed based on the ring tension-triggered click reaction in Example 1, (a) 200nM p53 DNA, 100nM thrombin and 20.0nM VEGF 165 ACV curves before protein binding (black line), after binding (red line), and regeneration (blue line); (b) kinetic scatter plots of hybridization / binding to targets and regeneration of the three working electrodes; (c) the first three binding-regeneration plots of the three sensor platforms;

[0026] Figure 5 The calibration curves of the sensor electrode constructed based on the ring tension-triggered click reaction for different targets are shown in Figure 2. (a) is the ACV curve for detecting p53; (b) is the dynamic relationship between the signal inhibition ratio and target concentration when detecting p53; (c) is the dynamic relationship between the signal inhibition ratio and target concentration when detecting VEGF. 165 ACV curve of protein; (d) for detecting VEGF 165 The dynamic relationship between the protein signal growth ratio and the target concentration;

[0027] Figure 6The long-term stability of the sensor electrode constructed based on the ring tension-triggered click reaction, (a) is the ACV curve of the sensor detecting the p53 target at 0 days; (b) is the ACV curve of the sensor detecting the p53 target at 7 days; (c) is the ACV curve of the sensor detecting the p53 target at 30 days; (d) is the signal inhibition ratio comparison diagram when the sensor detects the p53 target; (e) is the comparison diagram of the sensor detecting VEGF 165 ACV curve of the target on day 0; (f) VEGF detected by the sensor 165 ACV curve of the target at 7 days; (g) sensor detection of VEGF 165 ACV curve of the target at 30 days; (h) sensor detects VEGF 165 Comparison chart of target signal growth ratios. DETAILED DESCRIPTION

[0028] Click chemistry, particularly copper ion-catalyzed or dibenzocyclooctyne (DBCO) ring strain-triggered 1,2,3-triazole-forming reactions, has proven to be highly useful for immobilizing DNA or other molecules on solid surfaces. This reaction is quantitative and regioselective across a wide range of solvents, pH values, and temperatures. Electrochemically triggered click chemistry can selectively functionalize multiple electrodes, making it a viable strategy for multiplexed detection.

[0029] While traditional chemical click reactions offer an alternative sensor fabrication strategy, electrically triggered click reactions are more suitable for site-specific immobilization of biorecognition probes on multiplexed sensors because they can guide interfacial reactions through potential-controlled ligation. This paper proposes an electrochemical biosensor electrode with multiplexed detection capabilities constructed based on click chemistry, as well as its preparation method and application.

[0030] The following is a further explanation of the technical solution of the present invention in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. Figure 1 and Figure 2 .

[0031] Example 1:

[0032] 1. Electrode pretreatment

[0033] A three-channel screen-printed carbon electrode (SPCE) was selected as the substrate material; the SPCE was immersed in an electrolyte containing 1.2 mg / mL tetrachloroauric acid (HAuCl4); a voltage of -0.40 V (relative to the -Ag / AgCl reference electrode) was applied, and a platinum electrode was used as the counter electrode to form a closed circuit path. Electroplating was performed under stirring conditions for 20 minutes to form a gold film electrode.

[0034] 2. Azide group modification

[0035] The gold film electrode was immersed in a 2.0 mM 1-azidoundecanethiol solution (3:1 ethanol / water mixed solution) and reacted for 20 minutes; the electrode was immersed in a 2.0 mM 11-mercapto-1-undecanol solution (4:1 ethanol / water mixed solution) for passivation for 3 hours to remove nonspecific binding substances; after the reaction, the electrode was washed with deionized water and ethanol in sequence and blown dry with nitrogen gas.

[0036] 3. Fixation of the probe

[0037] The modified electrode was immersed in a click reaction solution containing 2.0 mM p53 DNA probe, 200 mM copper sulfate (CuSO4) and 900 mM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA); a -0.40 V potential was applied to the working electrode 1 (W1) and the reaction was carried out for 30 minutes so that the divalent copper ions in the solution were electrochemically reduced to monovalent copper ions; then, the electrode was removed from the solution and washed with deionized water, 5% Tween and ethanol in sequence, and dried for use; the above steps were repeated to fix the thrombin oligonucleotide probe on the working electrode 2 (W2) and the VEGF oligonucleotide probe on the working electrode 3 (W3). 165 Oligonucleotide probes.

[0038] 4. Electrochemical Detection

[0039] The modified multi-channel electrode array was connected to a multi-channel electrochemical workstation. Alternating current voltammetry (ACV) was used for detection, with the potential range set to -0.15 V to -0.42 V, the frequency to 10 Hz, and the amplitude to 25 mV. The electrodes were immersed in a physical buffer solution (phys2, pH 7.4) for 20 minutes for equilibrium. Different concentrations of target substances (p53 DNA, thrombin, and VEGF) were added to the electrochemical reaction cell in sequence. 165 ); start the electrochemical workstation to detect the detection target, collect AC voltammograms, and record the changes in peak current until a stable peak current is observed; analyze the changes in peak current before and after target binding, and calculate the signal enhancement or inhibition rate to achieve quantitative detection.

[0040] 5. Electrode regeneration process and method

[0041] For electrochemical biosensor electrodes with multiple detection capabilities constructed based on click chemistry reactions, there are two ways to achieve regeneration. When the detection target is a nucleic acid target, the electrode regeneration is achieved by destroying the double-stranded DNA structure through deionized water washing. This is because the hybridization of double-stranded DNA requires a certain concentration of metal cations, such as sodium ions and magnesium ions. When washed with deionized water, due to the lack of metal cations, the nucleic acid target is eluted due to electrostatic repulsion, and the electrode returns to its original state. When the detection target is a protein target, the electrode regeneration is achieved by soaking in 6.0M guanidine hydrochloride solution for 5 minutes and then rinsing with deionized water. Soaking in guanidine hydrochloride solution is to denature the protein, so that the binding force between the protein and the probe is reduced. After rinsing with deionized water, the electrode returns to its original state again.

[0042] 6. Performance Verification

[0043] Table 1 shows the detection results of the sensing electrode constructed based on the alkyne-azide cycloaddition click reaction in 50% fetal bovine serum in this example.

[0044] Figure 3 The calibration curves of the sensor electrode constructed based on the alkyne-azide cycloaddition click reaction in Example 1 for different targets are shown in Figure 1. (a) shows the change in the detection signal when the sensor detects the p53 target; (b) shows the change in the detection signal when the sensor detects thrombin; (c) shows the change in the detection signal when the sensor detects VEGF. 165 Detection signal changes when Figure 4 The repeatability and reproducibility curves of the sensing electrode constructed based on ring tension triggered click reaction for different targets in Example 1, (a) is 200nM p53 DNA, 100nM thrombin and 20.0nM VEGF 165 ACV curves before protein binding (black line), after binding (red line) and regeneration (blue line); (b) kinetic scatter plots of hybridization / binding to targets and regeneration of the three working electrodes; (c) the first three binding-regeneration graphs of the three sensor platforms.

[0045] Table 1

[0046]

[0047] Table 1 evaluates the detection ability of the sensing electrode prepared by alkyne-azide cycloaddition click reaction in 50% fetal bovine serum. In order to improve the detection accuracy, three target concentrations within the detectable range were selected, namely low, medium and high. The calculation of the average total detection amount of the target is based on the signal increase and decrease ratio before and after the addition of the target. The theoretical target concentration is calculated by this ratio, and the ratio of it to the actual value is calculated as the recovery rate. The calculation results show that the recovery rates of the three targets ranged from 87.3% to 118.0%, and their standard deviations were 2.1% (p53 target detection), 3.1% (thrombin target detection) and 1.6% (VEGF target detection). 165 These results indicate that the sensing electrode constructed based on the alkyne-azide cycloaddition click reaction has potential applications in biological sample analysis.

[0048] Figure 3 The detection performance of the sensor electrode constructed based on the alkyne-azide cycloaddition click reaction was demonstrated, and three calibration curves were obtained by comparing the relationship between the signal suppression ratio (SS) or signal enhancement ratio (SE) and the target concentration. Figure 3 As shown in Figure 2, when the electrode is immersed in a solution containing three target substances, the signal suppression ratio or signal enhancement ratio will change significantly, and the corresponding signal ratio will be obtained according to the concentration of the target substance. Under the optimal experimental conditions, such as Figure 3 As shown in a, with the increase of p53 DNA concentration, the signal suppression ratio of the electrode to the p53 target increases. In the concentration range of 1.0 nM to 128 nM, the signal suppression ratio has a good linear correlation with the concentration (inset, R 2 =0.98), its linear equation is SS(%)=0.55x+0.78, and the detection limit is 0.35nM. Figure 3 b shows that as the concentration of thrombin increases, the signal suppression ratio of the electrode for thrombin target detection also increases, and the target concentration shows a good linear correlation when it is between 0.8 nM and 40.0 nM (inset, R 2 =0.99), its equation is SS(%)=1.26x+1.75, and the detection limit reaches 0.22nM. Figure 3 c, along with VEGF 165 As the concentration increased, the signal growth ratio increased accordingly, showing an excellent linear correlation between the concentrations of 0.1 nM and 5.0 nM (inset, R 2 =0.99), its equation is SE(%)=22.97x+1.84, and the detection limit is 0.014nM. The above results show that the sensing electrode can achieve quantitative analysis of the target.

[0049] Figure 4The ACV peak current changes during the equilibrium-hybridization / binding-regeneration process of the sensing electrode prepared by the alkyne-azide cycloaddition click reaction are shown. In the phys2 buffer solution, the initial ACV peak current of the p53 and thrombin target electrodes is higher, while that of VEGF is higher. 165 The initial current of the target is low, which is related to the starting conformation of the probe. When the three targets are added, the hybridization / binding ACV peak current of channel 1 and channel 2 decreases, which is consistent with the probe conformational change of the signal attenuation sensor. The target induces the hairpin conformation to transform into a double-stranded structure and a G-quadruplex structure. The ACV peak current of channel 3 increases, which is consistent with the conformational change of the signal gain sensor, and the straight-chain structure transforms into a hairpin structure. After treatment with deionized water or guanidine hydrochloride, the ACV currents of the three channels can almost return to the initial state, indicating that the nucleic acid probe is fixed to the SPCE surface by covalent bonds, rather than physical or electrostatic adsorption. Dynamic characteristic studies have shown that by recording the changes in the ACV response current at the same time interval, the hybridization / binding kinetics of the three sensors can be understood. As Figure 4 As shown in b, the equilibrium time of the three sensors is similar, and the ACV current stabilizes after about 20 minutes. However, there are slight differences in the time required for each sensor to fully reach signal saturation: 40 minutes are required for detection of p53, 50 minutes for thrombin, and 50 minutes for VEGF. 165 The results show that the kinetics of the sensing electrode are similar to those of the traditional gold-thiol constructed sensor platform (40 minutes). In the electrode regeneration study, when detecting p53, the double-stranded structure was destroyed by elution with deionized water, and the recovery current was 99.2±2.3%. This is because DNA hybridization requires metal ions to maintain. When deionized water removes metal cations, electrostatic repulsion elutes the target DNA. Detection of thrombin and VEGF 165 When the electrode is regenerated, it needs to be soaked in 6.0M guanidine hydrochloride for 5 minutes and then rinsed with deionized water because the aptamer binds more strongly to protein than to double-stranded DNA. In phys2 buffer, the ACV current recovery rates are 98.6±1.9% and 95.3±1.1%, respectively. Regeneration is a key measurement criterion for this electrode. Figure 4 As shown in Figure c, the first three equilibrium-detection-regeneration studies showed that the relative standard errors of the hybridization / binding responses of the device were 1.11%, 1.49%, and 3.41%, respectively. This shows that the sensing electrode based on the alkynyl-azide cycloaddition reaction covalently immobilizes the nucleic acid probe on the SPCE surface, and even after multiple detection-regeneration, the detection ability remains stable. This shows that the electrochemical sensor based on the alkynyl-azide cycloaddition click reaction has comparable performance to that of electrochemical sensors constructed by traditional methods.

[0050] Example 2:

[0051] 1. Electrode pretreatment

[0052] A three-channel screen-printed carbon electrode (SPCE) was selected as the substrate material; the SPCE was immersed in an electrolyte containing 1.2 mg / mL tetrachloroauric acid (HAuCl4); a voltage of -0.40 V (relative to the -Ag / AgCl reference electrode) was applied, and a platinum electrode was used as the counter electrode to form a closed circuit path. Electroplating was performed under stirring conditions for 20 minutes to form a gold film electrode.

[0053] 2. Azide group modification

[0054] First, the SPCE was immersed in an aqueous solution of 1.2 mg / ml chloroauric acid (1.5% hydrochloric acid and 0.1 M sodium chloride) and stirred for 20 minutes while applying a voltage of -0.4 V to the working electrode surface of the SPCE. The SPCE surface was then rinsed with deionized water and ethanol and dried with nitrogen to obtain a gold-deposited SPCE (Au-SPCE). The resulting Au-SPCE required electrode activation by cyclic voltammetry in 0.5 M and 0.05 M sulfuric acid solutions to obtain an electrode surface with more active sites and a cleaner surface. Subsequently, the activated Au-SPCE was incubated for 20 minutes in a mixed solution containing 2.0 mM 1-azido-undecanethiol (ethanol / water, volume ratio 3:1). 1-azido-undecanethiol self-assembled and immobilized on the Au-SPCE surface via gold-thiol bonds. The azido-functionalized Au-SPCE was incubated overnight in a solution containing 2.0 mM 11-mercapto-1-undecanol (ethanol / water, volume ratio 4:1) to block the active sites on the Au-SPCE surface and remove nonspecific adsorption on the electrode surface. Finally, the electrode was rinsed multiple times with deionized water to remove the physically adsorbed compounds on the electrode surface, resulting in an azido / hydroxyl mixed self-assembled monolayer Au-SPCE (N3-Au-SPCE).

[0055] 3. Fixation of the probe

[0056] The azide group-modified electrode prepared in the previous step (named: N3-Au-SPCE) was immersed in a solution containing 2.0 μM p53 oligonucleotide probe (named: DBCO-p53-MB) modified with dibenzocyclooctyne at one end and methylene blue at the other end or 300 nM VEGF (named: DBCO-p53-MB) modified with dibenzocyclooctyne at one end and methylene blue at the other end. 165 Oligonucleotide probe (named: DBCO-VEGF 165The probe was covalently bonded to the Au-SPCE surface by a ring strain-driven cyclization reaction (SPAAC) between the azide group of N3-Au-SPCE and the DBCO modified end of the probe. Finally, the Au-SPCE surface was repeatedly rinsed three times with deionized water, 0.5% Tween, and ethanol to detect p53 targets and VEGF. 165 The target biosensor is prepared.

[0057] 4. Electrochemical Detection

[0058] The modified multi-channel electrode array was connected to a multi-channel electrochemical workstation. Alternating current voltammetry (ACV) was used for detection, with the potential range set to -0.15 V to -0.42 V, the frequency to 10 Hz, and the amplitude to 25 mV. The electrodes were immersed in a physiological buffer solution (phys2, pH 7.4) for 20 minutes for equilibrium. Different concentrations of target substances (p53 DNA, thrombin, and VEGF) were added to the electrochemical reaction cell in sequence. 165 protein); start the electrochemical workstation to detect the detection target, collect AC voltammograms, and record the changes in peak current until a stable peak current is observed; analyze the changes in peak current before and after target binding, and calculate the signal enhancement or inhibition rate to achieve quantitative detection.

[0059] 5. Electrode regeneration process and method

[0060] For an electrochemical biosensor electrode with multiple detection capabilities constructed based on a ring tension-triggered click chemistry reaction, the method for obtaining regenerative properties is as follows. When the detection target is a nucleic acid target, the electrode regenerative properties are obtained by destroying the double-stranded DNA structure by rinsing with deionized water. This is because the hybridization of double-stranded DNA requires a certain concentration of metal cations, such as sodium ions and magnesium ions. When rinsed with deionized water, due to the lack of metal cations, the nucleic acid target is eluted under electrostatic repulsion, and the electrode returns to its initial state. When the detection target is a protein target, the electrode regenerative properties are obtained by first soaking it in a 6.0 mol / L guanidine hydrochloride solution for 5 minutes, and then rinsing it with deionized water. Soaking in the guanidine hydrochloride solution is to denature the protein, thereby reducing the binding force between the protein and the probe. After rinsing with deionized water, the electrode returns to its initial state again.

[0061] 6. Performance Verification

[0062] Table 2 shows the detection results of the sensing electrode constructed based on the click reaction triggered by ring tension in 50% fetal bovine serum in this embodiment.

[0063] Table 2

[0064]

[0065] Figure 5 Calibration curves for different targets detected by the sensor electrode constructed based on ring tension-triggered click reaction. AC voltammograms of the sensor after adding different concentrations of target to phys2 buffer and the dynamic relationship between signal suppression ratio or signal increase ratio and target concentration. (a) is the ACV curve for detecting p53; (b) is the dynamic relationship between signal suppression ratio and target concentration when detecting p53; (c) is the dynamic relationship between signal suppression ratio and target concentration when detecting VEGF 165 ACV curve of protein; (d) detection of VEGF 165 The dynamic relationship between the protein signal growth ratio and the target concentration; Figure 6 The long-term stability of the sensor electrode constructed based on the ring tension-triggered click reaction, (a) is the ACV curve of the sensor detecting the p53 target at 0 days; (b) is the ACV curve of the sensor detecting the p53 target at 7 days; (c) is the ACV curve of the sensor detecting the p53 target at 30 days; (d) is the signal inhibition ratio comparison diagram when the sensor detects the p53 target; (e) is the comparison diagram of the sensor detecting VEGF 165 ACV curve of the target on day 0; (f) VEGF detected by the sensor 165 ACV curve of the target at 7 days; (g) sensor detection of VEGF 165 ACV curve of the target at 30 days; (h) sensor detects VEGF 165 Comparison chart of target signal growth ratios.

[0066] Table 2 evaluates the detection ability of the sensing electrode prepared based on the alkyne-azide ring addition click reaction in 50% fetal bovine serum. When detecting the p53 target, the electrode was incubated in 50% serum containing 5.0, 20.0, and 100.0nM p53 target chains for 1 hour, and its response ACV peak current was obtained. The recovery results were calculated by signal suppression ratio. By comparing the calculated value with the theoretical value, it was found that the spike recovery rate of the electrode in detecting the p53 target ranged from 98.0% to 105.0%, and the relative standard deviation was less than 2.2%. Detection of VEGF 165 The electrode was exposed to 0.5, 1.0, and 2.0 nM VEGF. 165The peak current of the ACV response was obtained by incubating the target in 50% serum for 1 hour, and the recovery rate was calculated by the signal suppression ratio. The calculated value was compared with the theoretical value to obtain the electrode's performance in detecting VEGF. 165 The target spike recoveries ranged from 80% to 105%, with relative standard deviations of less than 1.7%.

[0067] Figure 5 The calibration curves of the sensor electrode constructed by the ring tension-triggered click reaction for different targets are shown. Under the optimal conditions, such as Figure 5 As shown in a, when the concentration of p53 target chain increased from 0 nM to 300 nM, the ACV peak current of the sensor gradually decreased. This signal change is consistent with the electrochemical behavior of the signal attenuation sensor. Figure 5 b shows that the signal inhibition ratio exhibits a good linear relationship with the p53 target chain concentration in the range of 1.00 nM to 180 nM (inset), with the linear formula being Y=0.37x+0.71 and the detection limit being 0.76 nM. Figure 5 c The performance of the electrode in detecting VEGF165 was investigated. 165 When the protein concentration increased from 0 nM to 15.0 nM, the ACV peak current of the sensor also increased, which is consistent with the characteristics of a signal attenuation electrochemical sensor. Figure 5 d, signal growth ratio and VEGF 165 The concentrations exhibited two linear relationships: the linear equation from 0.15 nM to 1.00 nM was Y = 89.65x - 6.20, and the linear equation from 1.00 nM to 5.00 nM was Y = 38.25x + 50.26, with a detection limit of 8.20 pM. This demonstrates that the ring tension-triggered click reaction constructed on the SPCE surface can quantitatively detect the target.

[0068] Figure 6 The stability of the sensor electrode constructed based on the ring tension-triggered click reaction was evaluated. The sensor was stored at -20°C, and the effects of different storage times on its ACV curve and signal inhibition / increase ratio were studied. Figure 6 a shows that the ACV initial peak of the sensor was high when detecting the p53 target on day 0. After adding 300nM target, the ACV curve dropped significantly, and the signal inhibition ratio was 73.8±1.4%. After storage for 7 days, the ACV curve of the p53 E-DNA sensor was as follows Figure 6 b shows that the initial ACV peak current and the mixed ACV current after target binding are almost unchanged, and the signal suppression ratio is 73.2±1.8%. After storage for 30 days, the ACV curve is as follows Figure 6c shows that the initial ACV peak current and the mixed ACV current after target binding fluctuate slightly, and the signal inhibition ratio is 70.6±2.1%. Comparison of the changes in signal inhibition ratio at different times ( Figure 6 d), the changes were small, indicating the stability of p53 detection. Figure 6 e studied the effect of the sensor electrode on VEGF 165 VEGF was detected on day 0. 165 When the ACV initial peak value is low, after adding 10.0nM target, the ACV peak current increases significantly, and the signal growth ratio is 264.4±2.6%. The ACV curve after 7 days of storage is as follows Figure 6 f shows that the change is not significant, and the signal growth ratio is 259.7±3.4%. The ACV curve after storage for 30 days is as follows Figure 6 g shows a slight change, with a signal growth ratio of 254.4±2.7%. Figure 6 h Comparison of the signal growth ratio at different times shows that the change is very small, indicating that this sensor is suitable for detecting VEGF 165 These results show that the sensing electrode constructed by the click reaction triggered by ring tension has excellent stability.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An electrochemical biosensor electrode with multiple detection capabilities constructed based on click chemistry reactions, comprising two or more independent working electrodes, a reference electrode, and a counter electrode. The working electrodes are screen-printed carbon electrodes arranged in an array and modified with different probes.

2. The electrochemical biosensor electrode with multiple detection capabilities constructed based on click chemistry reaction according to claim 1, characterized in that: The reference electrode was a silver chloride electrode, and the counter electrode was a platinum electrode.

3. The electrochemical biosensor electrode with multiple detection capabilities constructed based on click chemistry reaction according to claim 1, characterized in that: The probe is an oligonucleotide probe with one end modified with an alkynyl group or a dibenzocyclooctyne and the other end modified with methylene blue.

4. A method for preparing an electrochemical biosensor electrode with multiple detection capabilities based on click chemistry according to any one of claims 1 to 3, comprising the following steps: A gold film was formed on the surface of a screen-printed carbon electrode by electrochemical deposition; Modifying the gold film surface with a thiol containing an azide group according to claim 1; Oligonucleotide probes modified with alkynyl or dibenzocyclooctyne are selectively immobilized on the electrode surface through electrochemically triggered or ring strain-triggered click chemistry reactions.

5. The preparation method according to claim 4, characterized in that The thiol containing an azide group is 1-azidoundecethiol.

6. The preparation method according to claim 4, characterized in that The electrochemically triggered click chemistry reaction selectively generates an active monovalent copper ion catalyst by controlling the electrode potential, thereby performing an alkynyl-azide cycloaddition reaction on a desired electrode.

7. A use of an electrochemical biosensor electrode with multiple detection capabilities constructed based on click chemistry according to any one of claims 1 to 3, comprising the following steps: The electrochemical biosensor electrode with multiple detection capabilities constructed based on click chemistry reaction is connected to the electrochemical workstation through a wire; the electrochemical biosensor electrode is immersed in the solution to be detected, and the electrochemical workstation is turned on to detect the detection target.

8. The use according to claim 7, characterized in that The detection targets are proteins, nucleic acids or small molecules.

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