Electrochemical biosensor based on the interaction between copper ions and DNA and use thereof

Abstract

本发明属于电化学检测领域，具体涉及基于铜离子与DNA相互作用制备的电化学生物电极及其用途。本发明提供了一种简单的新型电化学生物电极，通过肾上腺素与Cu2+、DNA之间的相互作用，所得到的电极Cu2+ / dsDNA / AuE对肾上腺素的氧化表现出优异的电催化活性。该电极具有制备简单、检测限低、灵敏度高、线性范围广等优点，可用于实际样品中肾上腺素的测定。

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection, specifically relating to an electrochemical bioelectrode prepared based on the interaction between copper ions and DNA and its applications. Background Technology

[0002] Epinephrine (EP) is a hormone secreted by the adrenal medulla and is a very important catecholamine neurotransmitter in the mammalian central nervous system. It exists in nerve tissue and body fluids in the form of large organic cations and plays an important role in regulating the physiological processes of living systems. [1] Metabolic abnormalities can also lead to certain diseases, such as Parkinson's and Alzheimer's. [2] Adrenaline is also commonly used as a drug in humans to treat neurological disorders, asthma, bronchial asthma, acute allergies, bradycardia, glaucoma, and as a hemostatic agent. Therefore, the determination of adrenaline concentration is of great significance for the diagnosis of certain diseases, pharmacological research, and the development of life sciences.

[0003] Currently, electrochemical technology has been widely applied and has become one of the means and methods for analyzing and detecting adrenaline. Electrochemical biosensors are developed by integrating electronic transduction elements and biorecognition elements. On the one hand, electronic components provide a high-performance electrochemical biosensing platform for detecting target molecules with high sensitivity and rapid response. On the other hand, based on the specific recognition of biomolecules, biorecognition elements contribute to the high selectivity of biosensors.

[0004] Deoxyribonucleic acid (DNA), as an excellent biomaterial, has become a cornerstone for constructing novel devices in biosensor technology. Due to its ordered nanostructure and highly programmable properties, DNA can interact with small molecules, metal ions, or nanoparticles. DNA-based electrochemical biosensors offer advantages such as simple operation, fast response, and low cost, and have been widely used in biochemical analysis due to their high sensitivity and selectivity. The phosphate backbone, the deoxyribose containing O atoms, and the bases containing N and O atoms in the DNA structure make it a natural target for metal ion binding. Transition metal ions tend to form internal spherical complexes with DNA bases. It is generally believed that metal ions bind to purine bases G and A through the N7 atom. The O6 atom of G and the N1 atom of A are also metal ion binding sites. Pyrimidine bases T and C bind to metal ions through O2 and N3 atoms, respectively.

[0005] Copper ions, due to their important biological roles, are among the most extensively and thoroughly studied metal ions in their interaction with DNA. It has been reported that Cu... 2+It has a higher binding affinity to GC-rich DNA than to AT-containing DNA. [1] Furthermore, the main binding mechanism of copper ions to DNA differs depending on the concentration of copper ions or the ratio of copper ions to bases, and their affinity also varies. Specifically, when the concentration of copper ions or the ratio of copper ions to bases is high, copper ions primarily bind to the N7 and O6 atoms of guanine (G) and the N3 and O2 atoms of cytosine (C). When the DNA strand contains consecutive guanine ions, copper ions will bind to the N7 and O6 atoms of two adjacent guanine ions on the same strand, forming a "G-Cu(II)-G sandwich structure." This binding mechanism enhances the base stacking between adjacent guanine ions and the hydrogen bonding in the guanine-cytosine base pair. [2,3] Materazzi and others [4] Kaur et al. investigated the thermal analytical properties of coordination compounds formed by adrenaline with certain divalent transition metal ions. [5] An electrochemical sensor based on metal ion-PANI-Nano-ZSM-5 was constructed and developed for the simultaneous determination of adrenaline, acetaminophen, and folic acid, while Cu... 2+ The glassy carbon electrode modified with PANI-Nano-ZSM-5 exhibited the highest electrocatalytic activity, but the preparation process of this electrode was complex and time-consuming. Summary of the Invention

[0006] To address the problems of complex and time-consuming existing electrode preparation processes, this invention provides an electrochemical bioelectrode prepared based on the interaction between copper ions and DNA.

[0007] The electrochemical bioelectrode prepared based on the interaction between copper ions and DNA provided by this invention is prepared by the following method:

[0008] a. Polish the gold electrode sequentially with 0.3μm and 0.05μm Al2O3 powders on chamois cloth, then ultrasonically clean it sequentially in acetone, anhydrous ethanol, and ultrapure water, and dry it with N2; then immerse the gold electrode in 0.5M H2SO4 solution for electrochemical pretreatment until a relatively stable cyclic voltammetry curve is obtained, with a potential range of 0–1.5V; finally, thoroughly clean the gold electrode with ultrapure water and dry it with N2 to obtain the pretreated gold electrode;

[0009] b. At room temperature, drop 10 μL of PBS buffer containing 1 μM ssDNA (single-stranded DNA) onto the pretreated gold electrode in the dark for 12 hours. Then rinse the electrode with PBS buffer and ultrapure water and dry it under N2 flow to obtain the modified electrode ssDNA / AuE.

[0010] c. Then, the ssDNA / AuE electrode was incubated in 10 μL Tris-HCl buffer containing 0.9 mM MCH (6-mercapto-1-hexanol) for 60 minutes to obtain the electrode MCH / ssDNA / AuE.

[0011] d. Spread 10 μL of SSC buffer solution containing 1 μM dsDNA (complementary strand DNA) onto the MCH / ssDNA / AuE electrode for 1 h to complete hybridization and obtain electrode dsDNA / AuE;

[0012] e. Place the electrode dsDNA / AuE in a Cu-containing environment. 2+ The electrode was stirred continuously in the buffer solution for 30–70 min in the dark, then rinsed with PBS buffer and ultrapure water, and dried with N2 to obtain the Cu electrode. 2+ / dsDNA / AuE.

[0013] In the above method for preparing an electrochemical bioelectrode based on the interaction between copper ions and DNA, the scan rate during the electrochemical pretreatment in step a is 100 mV / s.

[0014] In the above method for preparing an electrochemical bioelectrode based on the interaction between copper ions and DNA, the PBS buffer in step b is composed of 10 mM Na2HPO4, 10 mM NaH2PO4 and 0.1 M KCl, with a pH of 7.0.

[0015] In the above method for preparing an electrochemical bioelectrode based on the interaction between copper ions and DNA, the SSC buffer in step c is composed of 0.15M sodium chloride and 15mM sodium citrate, with a pH of 7.0.

[0016] In the above method for preparing an electrochemical bioelectrode based on the interaction between copper ions and DNA, step e involves Cu... 2+ The buffer solution is Cu 2+ PBS buffer with a concentration range of 1–12 mM.

[0017] The present invention also provides the use of the electrochemical bioelectrode prepared based on the interaction between copper ions and DNA in the construction of an electrochemical biosensor for detecting adrenaline.

[0018] The operating method for the above-mentioned applications is as follows: Place the Cu electrode... 2+ / dsDNA / AuE was inserted into PBS buffer containing EP (adrenaline), and electrochemical detection was performed using the DPV method (differential pulse voltammetry), with a scan potential of -0.1 to 0.6 V and a pulse amplitude of 50 mVs. –1 The pulse width is 0.05s.

[0019] In the above-mentioned operating methods, the PBS buffer containing EP needs to be deoxygenated by purging with nitrogen before preparation.

[0020] This invention provides a simple novel electrochemical bioelectrode that utilizes adrenaline and Cu 2+ The interaction between DNA and the resulting electrode Cu 2+ / dsDNA / AuE exhibits excellent electrocatalytic activity for the oxidation of adrenaline. This electrode has advantages such as simple preparation, low detection limit, high sensitivity, and wide linear range, and can be used for the determination of adrenaline in real samples. Attached Figure Description

[0021] Figure 1 A schematic diagram of the electrochemical bioelectrode prepared based on the interaction between copper ions and DNA, provided by this invention, in the detection of adrenaline.

[0022] Figure 2 A. Electrochemical impedance characterization of electrodes with different modifications: (a) bare AuE; (b) ssDNA / AuE; (c) MCH / ssDNA / AuE; (d) dsDNA / AuE; (e) Cu 2+ / dsDNA / AuE; B. DPV curves of different modified electrodes in epinephrine buffer.

[0023] Figure 3 The responsiveness of different DNA sequences to the adrenaline oxidation peak current.

[0024] Figure 4 A. In Cu 2+ Cyclic voltammograms of adrenaline were measured on / dsDNA / AuE at different scan rates (40–160 mV / s); B. Linear relationship between the square root of the scan rate and the peak current.

[0025] Figure 5 The responsiveness of DNA concentration to the peak current of adrenaline oxidation.

[0026] Figure 6 Cu 2+ The response of concentration to the peak current of adrenaline oxidation.

[0027] Figure 7 Cu 2+ The responsiveness of immobilization time to the peak current of adrenaline oxidation.

[0028] Figure 8 Cu 2+ The responsiveness of the buffer solution to the peak current of adrenaline oxidation.

[0029] Figure 9A. The responsiveness of the pH of the adrenaline buffer solution to the peak current of adrenaline oxidation; B. Linear relationship between pH and peak potential.

[0030] Figure 10 The responsiveness of adrenaline enrichment time to the peak current of adrenaline oxidation.

[0031] Figure 11 A. Based on Cu 2+ DPV curves of / dsDNA / AuE in buffer solutions containing different concentrations of adrenaline (1–1000 μM); B. Linear relationship between adrenaline concentration and peak current.

[0032] Figure 12 Adrenaline in Cu 2+ Interference performance test results on / dsDNA / AuE.

[0033] Figure 13 Based on Cu 2+ Stability study of / dsDNA / AuE. Detailed Implementation

[0034] The application of electrochemical bioelectrodes prepared based on the interaction between copper ions and DNA in the construction of electrochemical biosensors for detecting adrenaline is as follows: the electrode Cu... 2+ / dsDNA / AuE was inserted into PBS buffer containing EP (adrenaline), and electrochemical detection was performed using the DPV method (differential pulse voltammetry), with a scan potential of -0.1 to 0.6 V and a pulse amplitude of 50 mVs. –1 The pulse width is 0.05s.

[0035] In the above operating method, the electrode Cu 2+ The preparation method of / dsDNA / AuE is as follows:

[0036] a. Polish the gold electrode sequentially with 0.3μm and 0.05μm Al2O3 powders on chamois cloth, then ultrasonically clean it sequentially in acetone, anhydrous ethanol, and ultrapure water, and dry it with N2; then immerse the gold electrode in 0.5M H2SO4 solution for electrochemical pretreatment until a relatively stable cyclic voltammetry curve is obtained, with a potential range of 0–1.5V; finally, thoroughly clean the gold electrode with ultrapure water and dry it with N2 to obtain the pretreated gold electrode;

[0037] b. At room temperature, drop 10 μL of PBS buffer containing 1 μM ssDNA onto a pretreated gold electrode in the dark for 12 hours. ssDNA self-assembles onto the gold electrode surface using S-Au (which utilizes the bonding between sulfur and gold). Then, rinse the electrode with PBS buffer and ultrapure water to remove DNA that has not bound to the electrode, and dry it under N2 flow to obtain the modified electrode ssDNA / AuE.

[0038] c. Then, the ssDNA / AuE electrode was incubated in 10 μL Tris-HCl buffer containing 0.9 mM MCH for 60 minutes to block the active site of the electrode and obtain the electrode MCH / ssDNA / AuE.

[0039] d. Drop 10 μL of SSC buffer solution containing 1 μM dsDNA onto the MCH / ssDNA / AuE electrode and hybridize for 1 h to obtain electrode dsDNA / AuE;

[0040] e. Place the electrode dsDNA / AuE in a Cu-containing environment. 2+ In the buffer solution, stir continuously for 30–70 minutes in the dark. Rinse the electrode with PBS buffer and ultrapure water, and dry with N2 to allow Cu to mature. 2+ It binds to the bases in the DNA molecule through interaction, resulting in the electrode Cu. 2+ / dsDNA / AuE.

[0041] The above electrode Cu 2+ In the preparation method of / dsDNA / AuE, the scan rate during the electrochemical pretreatment in step a is 100mV / s.

[0042] The above electrode Cu 2+ In the preparation method of / dsDNA / AuE, the PBS buffer in step b is composed of 10mM Na2HPO4, 10mM NaH2PO4 and 0.1M KCl, with a pH of 7.0.

[0043] The above electrode Cu 2+ In the preparation method of / dsDNA / AuE, the SSC buffer in step c consists of 0.15M sodium chloride and 15mM sodium citrate, with a pH of 7.0. The MCH is diluted to the required concentration in Tris-HCl buffer (10mM, pH 7.0).

[0044] The above electrode Cu 2+ In the preparation method of / dsDNA / AuE, step e describes Cu 2+ The buffer solution is Cu 2+ PBS buffer with a concentration range of 1–12 mM.

[0045] The ss DNA used in this invention was purchased from Shanghai Sangon Biotech Co., Ltd., and its base sequence is as follows:

[0046] P1: 5'—SH—(CH2)6—GGGGCCGGGG—3' (The G at the 5' end has an SH-(CH2)6-substituent group);

[0047] P2:5'—CCCCGGCCCC—3';

[0048] P3:5'—SH—(CH2)6—GCGCGCGCGC—3' (The G at the 5' end has an SH-(CH2)6-substituent group);

[0049] P4:5'—GCGCGCGCGC—3'.

[0050] The ssDNA that is self-assembled onto the gold electrode in this invention is P1 or P3. ssDNA-P1 and ssDNA-P2 are complementary to obtain dsDNA-P1&P2; ssDNA-P3 and ssDNA-P4 are complementary to obtain dsDNA-P3&P4.

[0051] The above electrode Cu 2+ In the preparation method of / dsDNA / AuE, the specific operation steps of dsDNA / AuE in step d are as follows: the ssDNA-P1 / AuE (or ssDNA-P3 / AuE) electrode is incubated in 10 μL Tris-HCl buffer containing 0.9 mM MCH for 60 minutes to block the active site of the electrode and obtain electrode MCH / ssDNA-P1 / AuE (or MCH / ssDNA-P3 / AuE); 10 μL SSC buffer solution containing 1 μM dsDNA-P2 (or dsDNA-P4) is dropped onto the MCH / ssDNA-P1 / AuE (or MCH / ssDNA-P3 / AuE) electrode for 1 hour to complete hybridization, and electrode dsDNA-P1&P2 / AuE (or dsDNA-P3&P4 / AuE) is obtained by base complementary pairing.

[0052] EP (epinephrine) was purchased from Shanghai Maclean's Biochemical Technology Co., Ltd. Epinephrine hydrochloride injection was purchased from Grand Pharmaceutical (China) Co., Ltd. MCH (6-mercapto-1-hexanol) was purchased from Sigma-Aldrich (Gillingham, UK). MCH was diluted to the required concentration in Tris-HCl buffer (10 mM, pH 7.0). All experimental water was ultrapure water.

[0053] Male SD rats were purchased from the Animal Experiment Center of Ningxia Medical University. Blood samples were taken from the rats' abdomens, incubated at 4°C for 1 hour, centrifuged at 4°C to obtain the supernatant serum, and then acetonitrile was added to remove the protein, resulting in the processed rat serum.

[0054] The equipment used included an oven, magnetic stirrer, centrifuge, and CNC ultrasonic cleaner. All electrochemical measurements were performed on a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., China). A conventional three-electrode system was used, with bare gold electrodes or modified gold electrodes (AuE, 3 mm) as the working electrodes. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) were performed, with a platinum wire as the counter electrode and Ag / AgCl as the reference electrode.

[0055] Example 1: Electrochemical characterization of electrodes with different modifications

[0056] Electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) were used to characterize gold electrodes with different modifications. The impedance spectra included a semicircle in the high-frequency region and a linear region in the low-frequency region. The diameter of the semicircle in the high-frequency region was equal to the interfacial electron transfer resistance (Ret). Ret can reflect the electron transfer information at the electrode interface and can characterize the stepwise construction process of the sensor. Figure 2 A shows five differently modified gold electrodes in 5 mM [Fe(CN)6] containing 0.1 M KCl. 3- / 4- The Nyquist plots obtained are shown in the figure. It is clear that the bare gold electrode (AuE) exhibits a small semicircle, indicating a low electron transfer resistance (Ret) at the electrode interface for the redox probe (line a). After immobilizing ssDNA onto the bare gold electrode, the diameter of the semicircle increases (line b), indicating that the interfacial charge transfer between the bare gold electrode and ferricyanide ions is significantly blocked, as the DNA membrane acts as a barrier. After sequentially immobilizing MCH and dsDNA-P1 & P2 onto the electrode, the diameter of the semicircle gradually increases (lines c, d). Since MCH is non-conductive, its electrode conductivity decreases, the repulsive force between the redox probe and double-stranded DNA is delayed, and the interfacial electron transfer resistance gradually increases. When Cu... 2+ When immobilized on the electrode (e-line), the diameter of the semicircle was found to decrease significantly, and the electron transfer resistance decreased. This is due to Cu 2+ It is a positive charge, which will promote the interaction between the electrode and [Fe(CN)6]. 3- / 4- Charge transfer.

[0057] Figure 2 B showed naked AuE, dsDNA-P1&P2 / AuE, and Cu. 2+The DPV response of dsDNA-P1&P2 / AuE in deoxygenated 10 μM EP. Compared to naked AuE, dsDNA-P1&P2 / AuE shows a decrease in EP peak current because dsDNA is electronegative, reducing electron transfer of EP molecules at the electrode surface. When Cu is immobilized... 2+ After that, Cu 2+ / dsDNA-P1&P2 / AuE significantly increased the EP peak current, which means that Cu 2+ It increases the conductivity of the electrode interface, which has a good catalytic effect on EP and can better recognize EP molecules.

[0058] Example 2: Selection of DNA Sequence

[0059] To improve the responsiveness to the adrenaline peak current, the DPV method was used to investigate the effect of DNA immobilized with different sequences (ssDNA-P1, ssDNA-P3, dsDNA-P1&P2, dsDNA-P3&P4) on the EP peak current. Figure 3 As shown, when guanine in the immobilized DNA strand serves as a spacer, its response to the EP peak current is relatively small, and the response is smaller for single-stranded DNA than for double-stranded DNA. This indicates that Cu... 2+ The interaction between Cu and bases is weak, resulting in a weak catalytic ability for EP. When the guanine in the immobilized DNA strand is continuous (dsDNA-P1&P2), a significant increase in the peak current of EP is observed, further illustrating the effect of Cu. 2+ The strong interaction with continuous guanine also verifies that Cu 2+ A stable “G-Cu(II)-G sandwich structure” is formed with guanine.

[0060] Example 3: Effect of scan rate on the electrochemical behavior of adrenaline

[0061] In PBS buffer (pH 7.0) containing 10 μM EP, the effect of scan rate (v) on peak current (Ip) was investigated by cyclic voltammetry (CV) within a scan rate range of 40–160 mV / s, further exploring the redox reaction mechanism of EP on the electrode dsDNA-P1&P2 surface. Figure 4 As shown in Figure A, only the oxidation peak appeared in the CV curve of EP, indicating that the reaction of EP on the electrode surface is irreversible. Simultaneously, as the square root of the scan rate increases, the oxidation peak current also gradually increases, exhibiting a good linear positive correlation. Figure 4 B), the linear equation is Ip(μA)=0.57v 1 / 2 -2.1(R 2 =0.9825), which means that EP at electrode Cu 2+The / dsDNA / AuE surface belongs to a diffusion-controlled process.

[0062] Example 4: Optimization of Experimental Conditions

[0063] To improve the electrode Cu 2+ The analytical performance of the / dsDNA / AuE electrochemical sensor was investigated in detail, focusing on the influence of experimental conditions on the peak current of the electrochemical electrode (EP). The Cu electrode used in this series of experiments... 2+ Both dsDNA and AuE use Cu electrodes. 2+ / dsDNA-P1&P2 / AuE.

[0064] 1) Figure 5 The effect of DNA concentration on the peak current of EP was shown. When the DNA concentration reached 1.0 μM, the peak current of EP reached its maximum value, and the peak current tended to stabilize as the DNA concentration increased. Therefore, the concentration of immobilized DNA was chosen to be 1.0 μM.

[0065] 2) Figure 6 Cu was shown 2+ The effect of Cu concentration on the peak current of EP, when Cu 2+ When the concentration of Cu reaches 10 mM, the peak current of EP reaches its maximum value, and with the increase of Cu... 2+ As the concentration increases, the peak current tends to stabilize; therefore, immobilized Cu is selected. 2+ The concentration is 10 mM.

[0066] 3) Figure 7 Cu was shown 2+ The effect of Cu enrichment time on the peak current of EP can be seen from the figure. 2+ When the enrichment time was 60 min, the peak current reached its maximum value, and as the enrichment time increased, the peak current no longer increased, reaching a saturation state. Therefore, Cu was selected for subsequent experiments. 2+ The enrichment time was 60 min.

[0067] 4) Figure 8 Cu was shown 2+ The effect of buffer pH on the peak current of EP: The peak current of EP increased from pH 5.0 to 7.0 and then decreased from pH 7.0 to 9.0. The maximum peak current was obtained at pH 7.0. Therefore, pH 7.0 was selected as the optimal condition.

[0068] 5) This invention also optimized the pH of the EP buffer solution and investigated the effects of different pH values ​​on the peak potential and peak current of the EP. The results showed that... Figure 9A) Within the pH range of 5–9, the peak potential of EP shifts significantly and gradually becomes negative with increasing pH, and the oxidation peak current first increases and then decreases with increasing pH, reaching its maximum value at pH = 7.0. Considering the physiological pH value of human body fluids, subsequent experiments were conducted at pH 7.0. Figure 9 B) Furthermore, the oxidation peak potential of EP (E) was further discovered. pa The equation shows a good linear relationship with pH, ​​and the linear equation is: E pa (V)=-0.020pH+

[0069] 0.38(R 2 =0.9922). This indicates that adrenaline at electrode Cu 2+ The reaction on / dsDNA / AuE involves electrons.

[0070] 6) This invention also explored the effect of adrenaline accumulation time on peak current, such as... Figure 10 As shown, the peak current of EP reaches its maximum value when the enrichment time is 180s, and the peak current tends to stabilize as the enrichment time increases. Therefore, the enrichment time of adrenaline is chosen to be 180s.

[0071] Example 5 Based on electrode Cu 2+ Determination of adrenaline by / dsDNA / AuE

[0072] Under the experimental conditions selected in this invention, a sensor was constructed using the electrodes provided in this invention, and different concentrations of adrenaline were detected using the DPV method: the electrode Cu... 2+ / dsDNA-P1&P2 / AuE was inserted into PBS buffer containing EP (the PBS buffer containing EP needs to be purged with nitrogen before preparation). Electrochemical detection was performed using the DPV method, with a scan potential of -0.1 to 0.6 V and a pulse amplitude of 50 mVs. –1 The pulse width is 0.05s.

[0073] Depend on Figure 11 It can be seen that within the concentration range of 1–12.5 μM, the oxidation peak current of adrenaline exhibits a good linear relationship with its concentration, with the linear equation being Ipa(μA) = 0.14C. EP (μM)-0.042(R 2 =0.9949), and furthermore, in the range of 12.5–1000 μM, the peak current of adrenaline also showed a linear relationship with its concentration, with the linear equation being Ipa(μA) = 0.0090C(μM) + 1.6(R). 2 =0.9971), detection limit is 4.7×10 -8 M(S / N=3).

[0074] Table 1 Cu 2+ Comparison of / dsDNA / AuE with other electrodes for detecting adrenaline

[0075] working electrode method Linear range (μM) Detection limit (μM) References OMC-NiO / GCE DPV 0.8～50.0 0.085 6 Pt / POM / HCS–GCE DPV 0.16～1195 0.057 7 CoMnZIF-CNF DPV 5～1000 0.22 8 PDMS@cZIF / GCE DPV 1～60 0.13 9 MWCNT-PANI-TiO2 / Au DPV 4.9～76.9 0.16 10 MGR-Ox-Au@MIP / CPE DPV 0.08～1 0.053 11 EDDPT / GO / CPE DPV 1.5～600 0.65 12 <![CDATA[Cu 2+ / dsDNA-P1&P2 / AuE]]> DPV 1～1000 0.047 This invention

[0076] Table 1 lists and compares the performance of modified electrodes for adrenaline detection disclosed in other references. The results show that the Cu electrode provided by this invention... 2+ Compared with other modified electrodes, / dsDNA-P1&P2 / AuE has a wider linear range and a lower detection limit.

[0077] Example 6: Reproducibility, Selectivity, and Stability

[0078] The reproducibility of the sensor constructed using the electrodes of this invention was evaluated by DPV analysis: Five electrodes immobilized under identical conditions were used to detect 5 μM epinephrine under optimal conditions, with an RSD (relative standard deviation) of 5.6%. The effects of adding some common coexisting ions to a 5 μM epinephrine buffer solution on Ip were investigated. Figure 12 The results showed that 200 times the amount of glycine (Gly), phenylalanine (Phe), alanine (Ala), and citric acid (CA), 100 times the amount of NaCl, and 2 times the amount of ascorbic acid (AA) had virtually no interference with the peak current of adrenaline, with an RSD of 2.7%, indicating that the modified electrode has strong anti-interference ability.

[0079] like Figure 13 As shown, electrodes modified under the same conditions were placed at 4°C, and adrenaline levels were measured on days 1, 3, and 5. The results showed that the peak current was stable with an RSD of 3.5%. These results indicate that the Cu electrode... 2+ / dsDNA-P1&P2 / AuE exhibits good reproducibility, selectivity, and stability.

[0080] Example 7: Determination of adrenaline in rat serum samples

[0081] To further verify the accuracy of the electrode of the present invention, Cu electrode was used. 2+ The sensor constructed using / dsDNA-P1&P2 / AuE was used to measure EP in rat serum samples added using the standard addition method.

[0082] Blood samples were collected from the abdominal abdomen of rats and processed to obtain rat serum. The serum was diluted 200-fold with PBS buffer (pH 7.0) before use. A known amount of epinephrine was added to the diluted rat serum samples to obtain serum samples containing different concentrations of epinephrine, as shown in Table 2. The recoveries of different samples ranged from 97.9% to 106.0%, and the relative standard deviations (RSDs) ranged from 3.6% to 4.7%.

[0083] Table 2. Determination of adrenaline in rat serum samples

[0084]

[0085] The results in Table 2 demonstrate that this sensor can be applied to the detection of adrenaline in biological samples with good accuracy.

[0086] Example 8: Determination of actual samples

[0087] Epinephrine hydrochloride injection (1 mg / mL) was diluted to different concentrations with deoxygenated PBS buffer (pH 7.0) and tested using Cu electrodes. 2+ / dsDNA-P1&P2 / AuE was measured under optimal conditions for epinephrine hydrochloride injection.

[0088] Table 3. Determination of epinephrine in epinephrine hydrochloride injection

[0089]

[0090] As shown in Table 3, the results indicate that the electrode provided by the present invention can detect adrenaline in actual samples.

[0091] This invention provides a series of Cu electrodes for the electrochemical detection of adrenaline in buffer solutions and real samples. 2 + / dsDNA / AuE. We utilize dsDNA, Cu... 2+ The interaction between Cu and adrenaline led to the development of a simple and sensitive electrode. 2+ / dsDNA / AuE can be used to construct electrochemical biosensors, and the successful construction of the sensor was demonstrated using DPV and EIS. Under optimal conditions, the electrode Cu 2+ / dsDNA-P1&P2 / AuE was used to detect EP and achieved a low detection limit (47 nM) and a wide linear range (1–1000 μM), enabling selective detection of EP even in the presence of various interfering substances. Furthermore, the series of Cu electrodes provided by this invention... 2+The sensor constructed using / dsDNA / AuE exhibited good reproducibility and stability. Satisfactory results were obtained by measuring EP in epinephrine hydrochloride injection and serum samples using the constructed sensor.

[0092] References:

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[0104] 12、JOSEPH T,THOMAS T,THOMAS N.Graphene oxide modified carbon pasteelectrodefor handy and ultra-sensitive determination of Epinephrine in thepresence of uric and ascorbicacids[J].Electroanalysis,2020,32(11):2463-2473。

Claims

1. An electrochemical bioelectrode based on the interaction between copper ions and DNA is prepared by the following method: a. Polish the gold electrode sequentially with 0.3μm and 0.05μm Al2O3 powders on chamois cloth, then ultrasonically clean it sequentially in acetone, anhydrous ethanol, and ultrapure water, and dry it with N2; then immerse the gold electrode in 0.5M H2SO4 solution for electrochemical pretreatment until a relatively stable cyclic voltammetry curve is obtained, with a potential range of 0–1.5V; finally, thoroughly clean the gold electrode with ultrapure water and dry it with N2 to obtain the pretreated gold electrode; b. At room temperature, drop 10 μL of PBS buffer containing 1 μM single-stranded DNA onto the pretreated gold electrode and protect it from light for 12 hours. Then rinse the electrode with PBS buffer and ultrapure water and dry it under N2 flow to obtain the modified electrode ssDNA / AuE. c. Then, the ssDNA / AuE electrode was incubated in 10 μL of Tris-HCl buffer containing 0.9 mM 6-mercapto-1-hexanol for 60 minutes to obtain electrode MCH / ssDNA / AuE. d. Spread 10 μL of SSC buffer solution containing 1 μM complementary DNA onto the MCH / ssDNA / AuE electrode for 1 h to complete hybridization and obtain electrode dsDNA / AuE; e. Place the electrode dsDNA / AuE in a Cu-containing environment. 2+ The electrode was stirred continuously in the buffer solution for 30-70 minutes in the dark, then rinsed with PBS buffer and ultrapure water, and dried with N2 to obtain the Cu electrode. 2+ / dsDNA / AuE; The base sequence of the single-stranded DNA described in step b is as follows: P1: 5'—SH—(CH2)6—GGGGCCGGGG—3' (The G at the 5' end has an SH-(CH2)6-substituent group); P2: 5'—CCCCGGCCCC—3'; P3: 5'—SH—(CH2)6—GCGCGCGCGC—3' (The G at the 5' end has an SH-(CH2)6-substituent group); P4: 5'—GCGCGCGCGC—3'; The complementary DNA mentioned in step d refers to: ssDNA self-assembled onto the gold electrode as P1 or P3, ssDNA-P1 and ssDNA-P2 being complementary to obtain dsDNA-P1&P2; ssDNA-P3 and ssDNA-P4 being complementary to obtain dsDNA-P3&P4.

2. The electrochemical bioelectrode prepared based on the interaction between copper ions and DNA according to claim 1, characterized in that: During the electrochemical pretreatment described in step a, the scan rate is 100 mV / s.

3. The electrochemical bioelectrode prepared based on the interaction between copper ions and DNA according to claim 1, characterized in that: The PBS buffer in step b consists of 10 mM Na2HPO4, 10 mM NaH2PO4 and 0.1 M KCl, with a pH of 7.

0.

4. The electrochemical bioelectrode prepared based on the interaction between copper ions and DNA according to claim 1, characterized in that: The SSC buffer in step d consists of 0.15M sodium chloride and 15mM sodium citrate, with a pH of 7.

0.

5. The electrochemical bioelectrode prepared based on the interaction between copper ions and DNA according to claim 1, characterized in that: Step e describes Cu 2+ The buffer solution is Cu 2+ PBS buffer with a concentration range of 1~12mM.

6. The use of the electrochemical bioelectrode prepared based on the interaction between copper ions and DNA as described in any one of claims 1 to 5 in the construction of an electrochemical biosensor for detecting adrenaline.

7. The use according to claim 6, characterized in that: The operation method is to place the electrode Cu 2+ / dsDNA / AuE was inserted into PBS buffer containing epinephrine, and electrochemical detection was performed using differential pulse voltammetry, with a scan potential of -0.1 to 0.6 V and a pulse amplitude of 50 mVs. –1 The pulse width is 0.05 s.

8. The use according to claim 7, characterized in that: The PBS buffer containing adrenaline needs to be purged with nitrogen to remove oxygen before preparation.

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