A method for improving the detection performance of electrochemical biosensors and its application in ratiometric electrochemical biosensors

Abstract

The present invention relates to a method for improving the detection performance of an electrochemical biosensor and its application in a ratiometric electrochemical biosensor. Methylene blue and cholesterol-modified double-stranded DNA probes are immobilized on a gold electrode modified with a 1-hexanethiol assembly layer via hydrophobic interaction, thereby improving the stability of the methylene blue at the gold electrode interface and the immobilization efficiency of the double-stranded DNA probe at the gold electrode interface. In a potassium ferricyanide-mediated electrocatalytic system, the methylene blue on the gold electrode serves as a signal unit, and ferrocene is introduced as another signal unit. The 1-hexanethiol assembly layer on the gold electrode can shield [Fe(CN)6] 3‒ Electron transfer between the electrode interface and methylene blue enables simultaneous signal amplification of both methylene blue and ferrocene. Methylene blue, acting as an electron mediator, enhances the signal response of ferrocene, achieving secondary signal amplification of ferrocene, thereby improving sensor detection sensitivity. The ratiometric electrochemical biosensor constructed using this method exhibits excellent sensitivity, accuracy, and selectivity.

Description

Technical Field

[0001] The present invention relates to the fields of electrochemical analysis, sensors and medical diagnosis, and in particular to a method for improving the detection performance of an electrochemical biosensor and its application in a ratiometric electrochemical biosensor. Background Art

[0002] Electrochemical methods offer advantages such as simplicity, selectivity, low cost, rapid analysis, ease of integration, and miniaturization. They are widely used in disease diagnosis, environmental monitoring, and food quality monitoring. However, electrochemical DNA biosensors suffer from heterogeneous DNA assembly at the electrode interface and variability in the effective area of ​​different electrode interfaces, leading to inconsistent initial background currents across electrodes and causing background signal drift, severely impacting detection reproducibility and stability. To improve the accuracy of electrochemical DNA biosensors, researchers have proposed the concept of "ratiometric electrochemical sensors." These sensors incorporate an additional redox indicator as a reference and utilize the signal ratio of two different electrochemical indicators to address background signal drift. Currently, methylene blue (MB) and ferrocene (Fc) are widely used in the construction of ratiometric electrochemical sensors. MB and Fc offer unique advantages, such as: 1) their reversible redox properties and relatively independent electrochemical response potentials, enabling simultaneous detection; and 2) their covalent attachment to DNA. However, the number of MB and Fc modified on DNA is limited, so its response at the electrode interface is limited, which usually cannot meet the needs of sensitive detection of low-concentration targets.

[0003] To improve the sensitivity of ratiometric electrochemical DNA biosensors, researchers have introduced nucleic acid amplification reactions into electrochemical sensors to enhance their sensitivity. However, these amplification methods still suffer from complex procedures, long reaction times, and enzyme inactivation. Although some studies have achieved sensitive amplification-free detection by introducing nanoparticles, the complex and expensive preparation process of nanoparticles has hindered their widespread application in real-world sample testing.

[0004] Potassium ferrocyanide ([Fe(CN)6] 3- )-mediated electrocatalytic technology provides a new approach to solve the above reactions. [Fe(CN)6] 3- MB-mediated signal amplification reaction is a classic electrocatalytic reaction and is widely used in mutant genes, DNA damage, single nucleotide polymorphisms, Hg 2+ 、melamine and other biological molecules. This reaction mainly uses the high concentration of [Fe(CN)6] 3-The redox reaction of [Fe(CN)6] provides a large amount of electrons for MB, promoting the redox and cyclic regeneration of MB and generating a significant catalytic current. 3- The mediated electrocatalytic reaction can also achieve Fc signal amplification. 3- The electron transfer between the electrodes will affect the normal progress of the electrocatalytic reaction. Therefore, it is usually necessary to assemble high-density DNA probes or long alkyl chains such as dodecyl mercaptan and 12-azidodecane-1-thiol at the electrode interface to shield [Fe(CN)6] 3- However, the high-density DNA probe assembly layer will hinder the hybridization or conformational change of DNA at the electrode interface, and the long alkyl chain may affect the conductivity of the electrode, thereby inhibiting the signal response of the electrode interface.

[0005] It is worth noting that the 1-hexanethiol (HT) assembly layer has good hydrophobicity and a carbon chain of appropriate length, which can form a tight assembly layer at the electrode interface and inhibit the [Fe(CN)6] 3- Electron transfer between the electrode interface and [Fe(CN)6] 3- Mediated electrocatalytic reaction. However, there are still certain problems with the electrocatalytic reaction based on the HT assembly layer: 1) Although HT has good stability, it is easy to adsorb MB, which also has a hydrophobic structure, through hydrophobic interaction, resulting in a higher background signal; 2) DNA probes are usually terminally modified with cholesterol, which is embedded in the HT assembly layer through hydrophobic interactions to achieve its fixation at the electrode interface. Compared with the traditional method of relying on Au-S bonds to fix the probe, the cholesterol fixation method can avoid the DNA probe competing with the thiol assembly layer (such as MCH) for gold sites, resulting in low fixation efficiency of the DNA probe. However, this non-covalent binding method that relies on hydrophobic interactions still has problems such as weak probe fixation and easy detachment during repeated elution, resulting in low fixation efficiency of the probe at the electrode interface.

[0006] In addition, the sensor can achieve rapid detection of targets in a relatively short period of time, which is of great significance for instant detection. The hybridization efficiency at the electrode interface is an important factor affecting the detection time of DNA electrochemical sensors. Shorter DNA has a faster hybridization efficiency at the electrode interface, but the melting temperature of short DNA fragments is low, making it difficult to form a thermodynamically stable hybrid double-stranded structure at room temperature. Although longer DNA chains can form thermodynamically stable double-stranded structures, long single-stranded DNA is very prone to aggregation or entanglement at the interface, which greatly affects the hybridization efficiency. Therefore, how to improve the hybridization efficiency of DNA at the electrode interface remains a challenge. The base stacking force is the π orbital stacking force between adjacent bases in the longitudinal direction of the DNA molecule, as well as the longitudinal hydrophobic force and van der Waals force between bases. By utilizing the base stacking force, the thermodynamic stability of short-chain DNA hybridization can be effectively improved, so that short-chain DNA can also achieve rapid and effective hybridization at room temperature. Summary of the Invention

[0007] In view of this, an object of the present invention is to provide a method for improving the detection performance of an electrochemical biosensor and application of the method in a ratiometric electrochemical biosensor.

[0008] The object of the present invention is achieved through the following technical solutions:

[0009] A method for improving the detection performance of an electrochemical biosensor comprises utilizing hydrophobic interactions to immobilize a double-stranded DNA probe whose terminal is modified with cholesterol and MB on a gold electrode with a surface modified with a HT assembly layer. The hydrophobic interaction between MB and the HT assembly layer can improve the stability of MB at the terminal of the DNA double-stranded probe in the sensor at the gold electrode interface. The hydrophobic interactions between cholesterol and the HT assembly layer, and between MB and the HT assembly layer, can produce a synergistic effect to improve the immobilization efficiency of the DNA double-stranded probe in the sensor at the gold electrode interface. In [Fe(CN)6] 3- In the mediated electrocatalytic system, the MB modified at the end of the double-stranded DNA probe fixed on the gold electrode serves as a signal unit, and Fc is introduced as another signal unit. The HT assembly layer modified on the gold electrode surface can shield [Fe(CN)6] 3- The electron transfer between the MB and the gold electrode interface realizes the simultaneous signal amplification of MB and Fc, improving the sensitivity of sensor detection. At the same time, MB as an electron mediator can further enhance the signal response of Fc, realize the secondary signal amplification of Fc, and further improve the sensitivity of sensor detection.

[0010] The above method is used in constructing an electrochemical biosensor, wherein the electrochemical biosensor is a ratiometric electrochemical biosensor that uses MB as a reference signal and Fc as a target signal.

[0011] A ratiometric electrochemical biosensor constructed using the above method comprises a gold electrode, a capture probe Cp, an auxiliary probe Ap, a signal probe Sp, and an electrochemical detection solution; the surface of the gold electrode is modified with an HT assembly layer, the 5' end of the capture probe Cp is modified with cholesterol, the 3' end of the auxiliary probe Ap is modified with MB, the 3' end of the signal probe Sp is modified with Fc, and the electrochemical detection solution contains [Fe(CN)6] 3- PBS buffer; the sequences of the capture probe Cp, auxiliary probe Ap, and signal probe Sp are designed based on the principle of base complementary pairing according to the sequence of the detection target;

[0012] The sequence of the capture probe Cp modified with cholesterol at the 5' end is: 5'-Cholesteryl-TEG-GGAGTCTTGGACGACGGATTGCGGG-3'; the sequence of the auxiliary probe Ap modified with MB at the 3' end is: 5'-GTCGTCCAAGACTCC-MB-3'; the sequence of the signal probe Sp modified with Fc at the 3' end is: 5'-TGCCAATGTGATCTT-Fc-3'; [Fe(CN)6] 3- The concentration is 10 mM.

[0013] The preparation method of the ratiometric electrochemical biosensor comprises the following steps:

[0014] 1) polishing a bare gold electrode with alumina powder, then ultrasonically cleaning it in anhydrous ethanol and deionized water, drying it with nitrogen, and then placing the electrode in a 0.5 M sulfuric acid solution and performing a cyclic voltammetry scan at a scan rate of 0.1 V / s between -0.35 and +1.5 V to clean the electrode until the cyclic voltammetry curve is stable. Then, the electrode is removed and rinsed with ultrapure water, and dried with nitrogen to obtain a treated gold electrode;

[0015] 2) Immerse the treated gold electrode in HT solution, incubate at room temperature for 1 hour, then rinse with ultrapure water and blow dry with nitrogen to obtain a GE / HT electrode;

[0016] 3) preparing a mixture of a capture probe Cp modified with cholesterol at the 5' end and an auxiliary probe Ap modified with MB at the 3' end using a DNA fixation buffer, and annealing and hybridizing to obtain a Cp-Ap solution;

[0017] 4) Add Cp-Ap solution dropwise onto the GE / HT electrode, incubate at room temperature for 1 h, then rinse with ultrapure water and blow dry with nitrogen to obtain a GE / HT / Cp-Ap electrode;

[0018] 5) Prepare a mixture of the test solution containing the target T and the signal probe Sp modified with Fc at the 3' end using DNA fixation buffer, which is recorded as T-Sp solution;

[0019] 6) Add T-Sp solution dropwise onto the GE / HT / Cp-Ap electrode, incubate at room temperature for 30 min, then rinse with ultrapure water and blow dry with nitrogen to obtain a GE / HT / Cp-Ap-T-Sp electrode;

[0020] 7) Place the GE / HT / Cp-Ap-T-Sp electrode in the electrochemical detection solution and scan using square wave voltammetry;

[0021] The annealing and hybridization conditions are as follows: first incubating in a 95°C metal bath for 5 minutes, then cooling to 25°C at a rate of 1°C / min;

[0022] The formula of the DNA fixation buffer is: 10mM Tris, 1mM EDTA·2Na·2H2O, 50mM NaCl, 1mM MgCl2; pH 8.0;

[0023] The target T sequence is 5'-AAGAUCACAUUGGCACCCGCAAUCC-3'.

[0024] The application of the above-mentioned ratiometric electrochemical biosensor in the preparation of new coronavirus detection products.

[0025] The mechanism of the present invention is as follows:

[0026] The detection mechanism of the electrochemical biosensor of the present invention is as follows Figure 6 As shown. First, HT is fixed to the gold electrode surface through Au-S bonds to form a compact HT assembly layer. Then, the capture probe Cp modified with cholesterol at the 5' end and the auxiliary probe Ap modified with MB at the 3' end are annealed and hybridized to form a Cp-Ap fragment. The Cp-Ap fragment is fixed to the HT assembly layer through the synergistic cooperation of the terminal cholesterol and MB ( Figure 6 A). Then, based on the principle of base stacking, a signal probe Sp with an Fc modified at the 3' end was designed to specifically recognize the novel coronavirus target fragment T. When the target fragment T is present, T hybridizes with Sp to form Sp-T with a short overhang. Driven by the base stacking force, Sp-T is quickly captured by the Cp-Ap on the interface, forming a stable hybrid complex, which allows the Fc modified on Sp to be attracted to the electrode interface. After voltage is applied, a large amount of MB and Fc react with [Fe(CN)6] 3- In contrast, when T is absent, Sp cannot be captured by Cp-Ap on the electrode interface, and thus the Fc signal cannot be detected ( Figure 6B). Ultimately, by recording the changes in Fc catalytic current induced by different concentrations of the target fragment T and calculating the current ratio of the Fc signal to the Cp-Ap-terminated MB signal, accurate, sensitive, and rapid detection of T was achieved.

[0027] The beneficial effects of the present invention are:

[0028] (1) The synergistic immobilization method of cholesterol and MB not only effectively improves the immobilization efficiency of the probe at the electrode interface, but also cleverly avoids the problem of nonspecific adsorption of MB on the HT assembly layer.

[0029] (2) The hydrophobic HT can form a compact assembly layer at the electrode interface, thereby shielding [Fe(CN)6] 3- Electron transfer reaction with the electrode interface, thereby achieving [Fe(CN)6] 3- The mediated MB and Fc simultaneously amplify the signals and improve the sensitivity of sensor detection.

[0030] (3) MB in the electrocatalytic system can act as an electron mediator to achieve secondary signal amplification of Fc, thereby further improving the sensitivity of detection. This method has the advantages of high amplification efficiency, simple operation, fast reaction speed, and low cost.

[0031] (4) By introducing base stacking force, the thermodynamic stability of the short overhang fragment at the end of the double-stranded capture probe is improved, the stability of the hybridization product is improved, and thus the hybridization efficiency of DNA is improved.

[0032] (5) The ratiometric electrochemical detection method using MB as a reference is similar to the internal standard comparison method in chromatographic analysis. It greatly reduces the background signal drift caused by factors such as different electrode interfaces, different probe assembly concentrations, and probe degradation and dissociation caused by non-targets, thereby improving the sensitivity and stability of the detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : The binding between HT and MB and Fc in Example 1. A: Schematic diagram of the binding structure of HT and MB and the corresponding E ad B: Schematic diagram of the structure of HT binding to Fc and the corresponding E ad Gray balls represent carbon atoms, white balls represent hydrogen atoms, yellow balls represent sulfur atoms, blue balls represent nitrogen atoms, and purple balls represent iron atoms.

[0034] Figure 2In Example 2, the EIS method was used to compare the immobilization efficiencies of different immobilization methods. Model a corresponds to curve a, showing the EIS plot for MB immobilization (GE / HT / DNA1). Model b corresponds to curve b, showing the EIS plot for cholesterol immobilization (GE / HT / DNA2). Model c corresponds to curve c, showing the EIS plot for the coordinated immobilization of cholesterol and MB (GE / HT / DNA3).

[0035] Figure 3 :[Fe(CN)6] in Example 3 3- The feasibility of simultaneous signal amplification of MB and Fc mediated by β-catenin. Model a corresponds to curve a, which means that when [Fe(CN)6] 3- SWV diagram of the P1-P2 double chain fixed to the HT assembled electrode (GE / HT / DNA1) when [Fe(CN)6] 3- SWV diagram of the P1-P2 double chain modified with MB and Fc fixed on the HT assembled electrode (GE / HT / DNA2) when it is not present. Model c corresponds to curve c, which shows that when [Fe(CN)6] 3- SWV plot of the P1-P2 duplex modified with MB and Fc immobilized on the HT assembled electrode (GE / HT / DNA2) in the presence of .

[0036] Figure 4 SWV characterization of Fc secondary signal amplification using MB as an electron mediator in Example 4. Model a corresponds to curve a, showing the SWV plot when only MB is modified on the P1-P2 duplex (GE / HT / DNA1). Model b corresponds to curve b, showing the SWV plot when only Fc is modified on the P1-P2 duplex (GE / HT / DNA2). Model c corresponds to curve c, showing the SWV plot when both MB and Fc are modified on the P1-P2 duplex (GE / HT / DNA3).

[0037] Figure 5 In Example 6, the SWV method was used to compare the current responses of DNAs with different structures. Model a corresponds to curve a, which shows the SWV plot of Cp-T (GE / HT / Cp-T). Model b corresponds to curve b, which shows the SWV plot of Cp-Ap-T (GE / HT / Cp-Ap-T). Model c corresponds to curve c, which shows the SWV plot of Cp-T-Sp (GE / HT / Cp-T-Sp). Model d corresponds to curve d, which shows the SWV plot of Cp-Sp-T-Sp (GE / HT / Cp-Sp-T-Sp).

[0038] Figure 6 : Detection mechanism of the ratiometric electrochemical biosensor of the present invention.

[0039] Figure 7: In Example 7, the sensitivity of the ratiometric electrochemical biosensor was investigated using the SWV method. A: SWV plots in the presence of different concentrations of target T. B: Changes in the ratio of Fc to MB peak current (ΔI Fc / I MB ) and the logarithm of T concentration (logC RNA ) linear analysis diagram.

[0040] Figure 8 In Example 8, the selectivity of the ratiometric electrochemical biosensor was investigated using the SWV method. DETAILED DESCRIPTION

[0041] To illustrate the technical content, characterization and performance analysis methods of the present invention in detail, the following embodiments of the present invention will be described in detail with reference to the accompanying drawings. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used can be purchased commercially.

[0042] The DNA fixation buffer has the following formula: 10 mM Tris, 1 mM EDTA·2Na·2H2O, 50 mM NaCl, 1 mM MgCl2; pH 8.0.

[0043] The following contains 10mM [Fe(CN)6] 3- The formula of PBS buffer is: 10mM K3[Fe(CN)6], 8mMNa2HPO4, 136mM NaCl, 2mM KH2PO4, 2.6mM KCl.

[0044] The sequences of the following probes and targets are:

[0045] Capture probe Cp: 5'-GGAGTCTTGGACGACGGATTGCGGG-3',

[0046] Capture probe Cp modified with cholesterol at the 5' end:

[0047] 5'-Cholesteryl-TEG-GGAGTCTTGGACGACGGATTGCGGG-3';

[0048] Auxiliary probe Ap: 5'-GTCGTCCAAGACTCC-3',

[0049] Auxiliary probe Ap modified with MB at the 3′ end: 5′-GTCGTCCAAGACTCC-MB-3′;

[0050] Signal probe Sp: 5'-TGCCAATGTGATCTT-3',

[0051] The signal probe Sp with Fc modified at the 3' end: 5'-GGAGTCTTGGACGACTGTACTGCTGA-Fc-3';

[0052] Probe P1: 5'-GGAGTCTTGGACGACTGTACTGCTGA-3',

[0053] Probe P1 modified with cholesterol at the 5' end and Fc at the 3' end:

[0054] 5'-Cholesteryl-TEG-GGAGTCTTGGACGACTGTACTGCTGA-Fc-3',

[0055] Probe P1 modified with cholesterol at the 5' end:

[0056] 5'-Cholesteryl-TEG-GGAGTCTTGGACGACTGTACTGCTGA-3';

[0057] Probe P2: 5'-TCAGCAGTACAGTCGTCCAAGACTCC-3',

[0058] Probe P2 modified with MB at the 3′ end: 5′-TCAGCAGTACAGTCGTCCAAGACTCC-MB-3′;

[0059] Target T: 5'-AAGAUCACAUUGGCACCCGCAAUCC-3',

[0060] Target T modified with MB at the 5' end: 5'-MB-AAGATCACATTGGCACCCGCAATCC-3'.

[0061] The method for obtaining the gold electrode after the following treatment is:

[0062] A bare gold electrode with a diameter of 2 mm was polished with 0.3 μm and 0.05 μm alumina powder in sequence, and then ultrasonically cleaned in anhydrous ethanol and deionized water for 3 minutes, dried with nitrogen, and then placed in a 0.5 M sulfuric acid solution and cyclic voltammetry was performed at a scan rate of 0.1 V / s between -0.35 and +1.5 V to clean the electrode until the cyclic voltammetry curve was stable. The electrode was then taken out and rinsed with ultrapure water, dried with nitrogen to obtain the treated gold electrode.

[0063] Example 1:

[0064] Density functional theory was used to calculate the binding forces between HT and MB, and between HT and Fc. The experimental steps are as follows:

[0065] (1) All structures involved in the calculation were subjected to geometry optimization and frequency calculation at the B3LYP / 6-31G(d)&LANL2DZ level to ensure that these structures are the minima on their respective potential energy surfaces.

[0066] (2) The adsorption energy (E) of the HT-X (X = MB or Fc) complex was calculated at the B3LYP / 6-311++G(d,p)&LANL2DZ level. ad ).

[0067] In both steps (1) and (2), a density-based solvation model was used and the effect of aqueous solution was considered. In addition, DFT-D3 (BJ) dispersion correction was used to consider long-range interactions.

[0068] The calculation formula of adsorption energy in step (2) is: E ad =E (HT-X) -E X -E HT Among them, E HT 、E X 、E (X-HT) are the zero-point correction energies of HT, X, and HT-X, respectively (all of which are the sum of the electron energy at the B3LYP / 6-311++G(d,p)&LANL2DZ calculation level and the zero-point correction value obtained by optimization at the B3LYP / 6-31G(d)&LANL2DZ level).

[0069] Schematic diagram of the structure of HT and MB binding and the corresponding E ad Value Figure 1 A shows the structural diagram of HT binding to Fc and the corresponding E ad Value Figure 1 B. Among them, the gray balls represent carbon atoms, the white balls represent hydrogen atoms, the yellow balls represent sulfur atoms, the blue balls represent nitrogen atoms, and the purple balls represent iron atoms. ad The more negative it is, the stronger the binding ability between substances.

[0070] Example 2:

[0071] The EIS method was used to compare the fixation efficiency of different fixation methods to verify that the synergistic fixation of MB and cholesterol improves the fixation efficiency of the probe at the electrode interface. The experimental steps are as follows:

[0072] (1) The treated gold electrode was immersed in a 5 mM HT solution and incubated at room temperature for 1 h to cover the electrode surface with a tight alkyl assembly layer. The electrode surface was then rinsed with ultrapure water and dried with nitrogen to obtain a gold electrode with a surface modified with a HT assembly layer (denoted as GE / HT).

[0073] (2) Three probe mixtures were prepared using DNA fixation buffer. Mixture 1 contained 1 μM capture probe Cp and 1 μM auxiliary probe Ap modified with MB at the 3' end. Mixture 2 contained 1 μM capture probe Cp modified with cholesterol at the 5' end and 1 μM auxiliary probe Ap. Mixture 3 contained 1 μM capture probe Cp modified with cholesterol at the 5' end and 1 μM auxiliary probe Ap modified with MB at the 3' end. Mixture 1 was incubated in a 95°C metal bath for 5 min, then cooled to 25°C at a rate of 1°C / min to complete annealing. The resulting hybridization product solution was recorded as double-stranded DNA 1 and stored in a 4°C refrigerator for later use. Mixtures 2 and 3 were annealed in the same manner, and the resulting hybridization product solutions were recorded as double-stranded DNA 2 and double-stranded DNA 3, respectively.

[0074] (3) 4 μL of double-stranded DNA 1 solution, double-stranded DNA 2 solution, or double-stranded DNA 3 solution was added to the GE / HT electrode and incubated at room temperature for 1 h. The electrode surface was then rinsed with ultrapure water and dried with nitrogen gas to obtain a double-stranded DNA 1-modified electrode (denoted as GE / HT / DNA1), a double-stranded DNA 2-modified electrode (denoted as GE / HT / DNA2), and a double-stranded DNA 3-modified electrode (denoted as GE / HT / DNA3).

[0075] (4) The GE / HT / DNA1, GE / HT / DNA2, and GE / HT / DNA3 electrodes were immersed in an impedance detection buffer (the impedance detection buffer formula is: 10 mM potassium ferrocyanide, 10 mM potassium ferrocyanide, 0.1 M potassium chloride) for electrochemical impedance spectroscopy (EIS); wherein the parameters of the electrochemical impedance spectroscopy are: running time 2 s, initial potential 0.21 V, amplitude 0.005 V, and scanning frequency range 1 to 100,000 Hz.

[0076] Characterization of the fixation efficiency of different fixation methods is as follows Figure 2 As shown in the figure, model a corresponds to curve a and represents the GE / HT / DNA1 electrode, model b corresponds to curve b and represents the GE / HT / DNA2 electrode, and model c corresponds to curve c and represents the GE / HT / DNA3 electrode. As can be seen from the figure, the hydrophobic structure of MB can interact hydrophobically with HT, and cholesterol can also hydrophobically bind to HT. The synergistic effect of MB, cholesterol, and HT can achieve efficient immobilization of MB- and cholesterol-modified double-stranded DNA probes at the gold electrode interface.

[0077] Example 3:

[0078] Verification of [Fe(CN)6] by SWV method 3- The feasibility of simultaneous signal amplification of MB and Fc mediated by ELISA. The experimental steps are as follows:

[0079] (1) The treated gold electrode was immersed in a 5 mM HT solution and incubated at room temperature for 1 h to cover the electrode surface with a tight alkyl assembly layer. The electrode surface was then rinsed with ultrapure water and dried with nitrogen to obtain a gold electrode with a surface modified with a HT assembly layer (denoted as GE / HT).

[0080] (2) Two probe mixtures were prepared using DNA fixation buffer. Mixture 1 contained 2.5 μM probe P1 and 2.5 μM probe P2, and mixture 2 contained 2.5 μM probe P1 modified with cholesterol at the 5' end and Fc at the 3' end, and 2.5 μM probe P2 modified with MB at the 3' end. Mixtures 1 and 2 were incubated in a 95°C metal bath for 5 min, then cooled to 25°C at a rate of 1°C / min to complete annealing. The resulting hybridization product solutions were designated as double-stranded DNA 1 and double-stranded DNA 2, respectively, and stored in a 4°C refrigerator for later use.

[0081] (3) 4 μL of 2.5 μM double-stranded DNA 1 solution or double-stranded DNA 2 solution was added to the GE / HT electrode and incubated at room temperature for 1 h. The electrode surface was then rinsed with ultrapure water and dried with nitrogen gas to obtain a double-stranded DNA 1-modified electrode (denoted as GE / HT / DNA1) and a double-stranded DNA 2-modified electrode (denoted as GE / HT / DNA2), respectively.

[0082] (4) A three-electrode system was formed with GE / HT / DNA1 electrode or GE / HT / DNA2 electrode as the working electrode, Ag / AgCl electrode as the reference electrode, and platinum wire electrode as the counter electrode. The reaction was carried out in PBS buffer or a solution containing 10 mM [Fe(CN)6] 3- PBS buffer was used as the electrochemical detection solution, and a CHI 660E electrochemical workstation was used to perform square wave voltammetry (SWV), wherein the parameters of the square wave voltammetry scan were: scanning potential range -0.5 to +0.7 V, standing time 2 s, and frequency 50 Hz.

[0083] [Fe(CN)6] 3- The feasibility of simultaneous signal amplification of MB and Fc mediated by Figure 3 As shown, model a corresponds to curve a, which means that when [Fe(CN)6] 3- SWV diagram of GE / HT / DNA1 electrode when [Fe(CN)6] 3- SWV diagram of GE / HT / DNA2 electrode when [Fe(CN)6] 3- SWV diagram of GE / HT / DNA2 electrode when HT is present. As shown in the figure, hydrophobic HT can form a compact assembly layer at the electrode interface, thereby shielding [Fe(CN)6] 3-Electron transfer reaction with the electrode interface, thereby realizing [Fe(CN)6] 3- Mediated simultaneous signal amplification of MB and Fc.

[0084] Example 4:

[0085] The SWV method was used to verify the feasibility of using MB as an electron mediator to achieve secondary Fc signal amplification. The experimental steps are as follows:

[0086] (1) The treated gold electrode was immersed in a 5 mM HT solution and incubated at room temperature for 1 h to cover the electrode surface with a tight alkyl assembly layer. The electrode surface was then rinsed with ultrapure water and dried with nitrogen to obtain a gold electrode with a surface modified with a HT assembly layer (denoted as GE / HT).

[0087] (2) Three probe mixtures were prepared using DNA fixation buffer. Mixture 1 contained 5 μM probe P1 modified with cholesterol at the 5' end and Fc at the 3' end, and 5 μM probe P2. Mixture 2 contained 5 μM probe P1 modified with cholesterol at the 5' end and 5 μM probe P2 modified with MB at the 3' end. Mixture 3 contained 5 μM probe P1 modified with cholesterol at the 5' end and Fc at the 3' end, and 5 μM probe P2 modified with MB at the 3' end. Mixture 1, mixture 2, and mixture 3 were incubated in a 95°C metal bath for 5 min, and then cooled to 25°C at a rate of 1°C / min to complete annealing. The resulting hybridization product solutions were recorded as double-stranded DNA 1, double-stranded DNA 2, and double-stranded DNA 3, respectively, and stored in a 4°C refrigerator for later use.

[0088] (3) 4 μL of double-stranded DNA 1 solution, double-stranded DNA 2 solution, or double-stranded DNA 3 solution was added to the GE / HT electrode and incubated at room temperature for 1 h. The electrode surface was then rinsed with ultrapure water and dried with nitrogen gas to obtain a double-stranded DNA 1-modified electrode (denoted as GE / HT / DNA1), a double-stranded DNA 2-modified electrode (denoted as GE / HT / DNA2), and a double-stranded DNA 3-modified electrode (denoted as GE / HT / DNA3).

[0089] (4) A three-electrode system was formed with GE / HT / DNA1 electrode, GE / HT / DNA2 electrode, or GE / HT / DNA3 electrode as the working electrode, Ag / AgCl electrode as the reference electrode, and platinum wire electrode as the counter electrode. The three-electrode system was placed in a solution containing 10 mM [Fe(CN)6] 3- The samples were placed in PBS buffer and square wave voltammetry (SWV) was performed using a CHI 660E electrochemical workstation. The SWV scan parameters were as follows: scanning potential range -0.5 to +0.7 V, standing time 2 s, and frequency 50 Hz.

[0090] MB as an electron mediator to achieve Fc secondary signal amplification SWV characterization diagram Figure 4 As shown in the figure, model a corresponds to curve a, which represents the SWV plot of the GE / HT / DNA1 electrode; model b corresponds to curve b, which represents the SWV plot of the GE / HT / DNA2 electrode; and model c corresponds to curve c, which represents the SWV plot of the GE / HT / DNA3 electrode. As can be seen from the figure, MB in the electrocatalytic system can act as an electron mediator, further enhancing the current response of Fc.

[0091] Example 5:

[0092] The secondary structures of different DNA double strands were fitted using Nupack software, and the corresponding secondary structure free energies were compared. The DNA mode was selected for the nucleic acid type, the fitting temperature was room temperature (25°C), the initial concentration of each nucleic acid sequence was set to 1 μM, and Na + Mg 2+ The ion concentrations are 0.05 M and 0.0125 M. The more negative the secondary structure free energy is, the more stable the structure is.

[0093] As shown in Table 1, the free energy of the secondary structure of Cp-T is -18.98 kcal / mol, the free energy of the secondary structure of Cp-T-Ap is -42.08 kcal / mol, the free energy of the secondary structure of Cp-T-Sp is -41.43 kcal / mol, and the free energy of Cp-T-Ap-Sp is -64.53 kcal / mol. As can be seen from the table, the free energy of Cp-T-Ap-Sp is the most negative, indicating that it is the most stable structure compared to the other structures.

[0094] Table 1 Free energy of different DNA secondary structures

[0095]

[0096] Example 6:

[0097] The SWV method was used to compare the current response of DNA with different structures to verify that the introduction of base stacking force improved the stability of the hybridization product and thus improved the hybridization efficiency. The experimental steps are as follows:

[0098] (1) The treated gold electrode was immersed in a 5 mM HT solution and incubated at room temperature for 1 h to cover the electrode surface with a tight alkyl assembly layer. The electrode surface was then rinsed with ultrapure water and dried with nitrogen to obtain a gold electrode with a surface modified with a HT assembly layer (denoted as GE / HT).

[0099] (2) Four probe-target mixtures were prepared using DNA fixation buffer. Mixture 1 contained 1 μM capture probe Cp and 1 μM target T modified with MB at its 5' end. Mixture 2 contained 1 μM capture probe Cp, 1 μM target T modified with MB at its 5' end, and 1 μM auxiliary probe Ap. Mixture 3 contained 1 μM capture probe Cp, 1 μM target T modified with MB at its 5' end, and 1 μM signal probe Sp. Mixture 4 contained 1 μM capture probe Cp, 1 μM target T modified with MB at its 5' end, 1 μM auxiliary probe Ap, and 1 μM signal probe Sp. Mixtures 1, 2, 3, and 4 were incubated in a 95°C metal bath for 5 min, then cooled to 25°C at a rate of 1°C / min to complete annealing. The resulting hybridization product solutions were designated as hybridization solution 1, hybridization solution 2, hybridization solution 3, and hybridization solution 4, respectively.

[0100] (3) 4 μL of hybridization solution 1, hybridization solution 2, hybridization solution 3, or hybridization solution 4 was added to the GE / HT electrode and incubated at room temperature for 1 h. The electrode surface was then rinsed with ultrapure water and dried with nitrogen gas to obtain an electrode modified with hybridization solution 1 (denoted as GE / HT / Cp-T), an electrode modified with hybridization solution 2 (denoted as GE / HT / Cp-Ap-T), an electrode modified with hybridization solution 3 (denoted as GE / HT / Cp-T-Sp), and an electrode modified with hybridization solution 4 (denoted as GE / HT / Cp-Sp-T-Sp).

[0101] (4) A three-electrode system was formed with a GE / HT / Cp-T electrode or a GE / HT / Cp-Ap-T electrode or a GE / HT / Cp-T-Sp electrode or a GE / HT / Cp-Sp-T-Sp electrode as a working electrode, an Ag / AgCl electrode as a reference electrode, and a platinum wire electrode as a counter electrode. The three-electrode system was placed in a solution containing 10 mM [Fe(CN)6] 3- The samples were placed in PBS buffer and subjected to square wave voltammetry (SWV) scanning using a CHI 660E electrochemical workstation. The parameters of the square wave voltammetry scanning were as follows: scanning potential range -0.5 to +0.1 V, voltage increment 0.004 V, amplitude 0.025 V, frequency 50 Hz, and dwell time 2 s.

[0102] SWV with different DNA structures Figure 5 As shown, model a corresponds to curve a, which represents the SWV diagram of the GE / HT / Cp-T electrode; model b corresponds to curve b, which represents the SWV diagram of the GE / HT / Cp-Ap-T electrode; model c corresponds to curve c, which represents the SWV diagram of the GE / HT / Cp-T-Sp electrode; model d corresponds to curve d, which represents the SWV diagram of the GE / HT / Cp-Sp-T-Sp electrode.

[0103] Example 7:

[0104] The sensitivity of the sensor is investigated using the SWV method. The experimental steps are as follows:

[0105] (1) The pretreated gold electrode was immersed in a 5 mM HT solution and incubated at room temperature for 1 h to cover the electrode surface with a tight alkyl assembly layer. The electrode surface was then rinsed with ultrapure water and dried with nitrogen to obtain a gold electrode with a surface modified with a HT assembly layer (denoted as GE / HT).

[0106] (2) Prepare a probe mixture using DNA fixation buffer. The mixture contains 0.5 μM capture probe Cp modified with cholesterol at the 5' end and 0.5 μM auxiliary probe Ap modified with MB at the 3' end. Incubate the mixture in a 95°C metal bath for 5 min and then cool to 25°C at a rate of 1°C / min to complete annealing. The resulting hybridization product solution is designated Cp-Ap.

[0107] (3) 4 μL of 0.5 μM Cp-Ap solution was added to the GE / HT electrode and incubated at room temperature for 1 h. The electrode surface was then rinsed with ultrapure water and dried with nitrogen. The resulting electrode was designated as GE / HT / Cp-Ap.

[0108] (4) Dissolve the target T in DEPC water to prepare a concentration of 10 0 fM, 10 2 fM, 10 3 fM, 10 5 fM, 10 6 fM, 10 7 fM, 10 8 fM target T solution; the signal probe Sp modified with Fc at the 3' end was dissolved in DNA fixation buffer to prepare a Sp solution with a concentration of 10 μM; 2 μL of target T solutions of different concentrations and 8 μL of Sp solution were mixed in an EP tube containing 10 μL DNA fixation buffer to prepare a mixed solution with a total volume of 20 μL (denoted as T-Sp).

[0109] (5) 4 μL of the T-Sp mixture was added to the GE / HT / Cp-Ap electrode and incubated at room temperature for 30 min. The electrode surface was then rinsed with ultrapure water and dried with nitrogen gas. The resulting electrode was designated GE / HT / Cp-Ap-T-Sp.

[0110] (6) A three-electrode system was formed with a GE / HT / Cp-Ap-T-Sp electrode as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum wire electrode as the counter electrode. The three-electrode system was placed in a solution containing 10 mM [Fe(CN)6] 3-The cells were placed in a PBS buffer solution and subjected to square wave voltammetry (SWV) scanning using a CHI660E electrochemical workstation. The parameters of the square wave voltammetry scanning were as follows: scanning potential range -0.5 to +0.1 V, voltage increment 0.004 V, amplitude 0.025 V, frequency 50 Hz, and standing time 2 s.

[0111] SWV diagrams in the presence of different concentrations of target T are shown in Figure 2. Figure 7 As shown in A, the concentrations of target T in the detection system are 0 fM, 10 1 fM, 10 2 fM, 10 4 fM, 10 5 fM, 10 6 fM, 10 7 fM. The logarithm of the concentration of T (logC RNA ) and the ratio of current change (ΔI Fc / I MB ) standard curve as shown in Figure 7 As shown in B.

[0112] Example 8:

[0113] The SWV method was used to investigate the selectivity of the sensor. The experimental steps are as follows:

[0114] (1) The pretreated gold electrode was immersed in a 5 mM HT solution and incubated at room temperature for 1 h to cover the electrode surface with a tight alkyl assembly layer. The electrode surface was then rinsed with ultrapure water and dried with nitrogen to obtain a gold electrode with a surface modified with a HT assembly layer (denoted as GE / HT).

[0115] (2) Prepare a probe mixture using DNA fixation buffer. The mixture contains 10 μM capture probe Cp modified with cholesterol at the 5' end and 10 μM auxiliary probe Ap modified with MB at the 3' end. Incubate the mixture in a 95°C metal bath for 5 min and then cool to 25°C at a rate of 1°C / min to complete annealing. The resulting hybridization product solution is designated Cp-Ap. Next, dilute the Cp-Ap solution to 0.5 μM using DNA fixation buffer.

[0116] (3) 5 μL of 0.5 μM Cp-Ap solution was added to the GE / HT electrode and incubated at room temperature for 1 h. The electrode surface was then rinsed with ultrapure water and dried with nitrogen. The resulting electrode was designated as GE / HT / Cp-Ap.

[0117] (4) The target T and the interfering sequence (selected from V. vulnificus, E. coli O157: H7, MERS-CoV, and Influenza A related genomic fragments) were dissolved in DEPC water to prepare a test solution with a concentration of 1 pM; 2 μL of the test solution and 8 μL of a 10 μM signal probe Sp modified with Fc at the 3' end were mixed, and DNA fixation buffer was added to a volume of 20 μL to obtain a mixed solution of the test substance signal probe.

[0118] (5) 5 μL of the sample signal probe mixture obtained in step (4) was added to the GE / HT / Cp-Ap electrode and incubated at room temperature for 30 min. The electrode surface was then rinsed with ultrapure water and dried with nitrogen. The electrode obtained was used as the working electrode, and a three-electrode system was formed with an Ag / AgCl electrode as the reference electrode and a platinum wire electrode as the counter electrode. The three-electrode system was placed in a solution containing 10 mM [Fe(CN)6] 3- The samples were placed in PBS buffer and subjected to square wave voltammetry (SWV) scanning using a CHI 660E electrochemical workstation. The parameters of the square wave voltammetry scanning were as follows: scanning potential range -0.5 to +0.1 V, voltage increment 0.004 V, amplitude 0.025 V, frequency 50 Hz, and dwell time 2 s.

[0119] like Figure 8 As shown, the detection signal of target T is significantly greater than the detection signals of other interference sequences.

[0120] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A ratiometric electrochemical biosensor, characterized in that: The invention comprises a gold electrode, a capture probe Cp, an auxiliary probe Ap, a signal probe Sp and an electrochemical detection solution; the surface of the gold electrode is modified with a 1-hexanethiol assembly layer, the 5' end of the capture probe Cp is modified with cholesterol, the 3' end of the auxiliary probe Ap is modified with methylene blue, the 3' end of the signal probe Sp is modified with ferrocene, and the electrochemical detection solution contains [Fe(CN)6] 3- PBS buffer; the sequences of the capture probe Cp, auxiliary probe Ap, and signal probe Sp are designed based on the principle of base complementary pairing according to the sequence of the detection target; The sequence of the capture probe Cp modified with cholesterol at the 5' end is: 5'-Cholesteryl-TEG-GGAGTCTTGGACGACGGATTGCGGG-3'; the sequence of the auxiliary probe Ap modified with methylene blue at the 3' end is: 5'-GTCGTCCAAGACTCC-MB-3'; the sequence of the signal probe Sp modified with ferrocene at the 3' end is: 5'-TGCCAATGTGATCTT-Fc-3'; [Fe(CN)6] 3- The concentration is 10 mM.

2. The ratiometric electrochemical biosensor according to claim 1, wherein: The working principle of the ratiometric electrochemical biosensor is as follows: a DNA double-stranded probe with terminal modifications of cholesterol and methylene blue is fixed on a gold electrode with a surface modified with a 1-hexanethiol assembly layer by utilizing hydrophobic interaction; the hydrophobic interaction between methylene blue and the 1-hexanethiol assembly layer can improve the stability of the methylene blue at the terminal of the DNA double-stranded probe in the sensor at the gold electrode interface; the hydrophobic interaction between cholesterol and the 1-hexanethiol assembly layer, and the hydrophobic interaction between methylene blue and the 1-hexanethiol assembly layer can produce a synergistic effect to improve the fixation efficiency of the DNA double-stranded probe in the sensor at the gold electrode interface; in [Fe(CN)6] 3- In the mediated electrocatalytic system, the methylene blue modified at the end of the double-stranded DNA probe fixed on the gold electrode serves as a signal unit, and ferrocene is introduced as another signal unit. The 1-hexanethiol assembly layer modified on the gold electrode surface can shield [Fe(CN)6] 3- The electron transfer between the gold electrode interface realizes the simultaneous signal amplification of methylene blue and ferrocene, thereby improving the sensitivity of sensor detection. At the same time, methylene blue as an electron mediator can further enhance the signal response of ferrocene, realize the secondary signal amplification of ferrocene, and further improve the sensitivity of sensor detection.

3. The method for preparing a ratiometric electrochemical biosensor according to claim 1, wherein: The steps include: 1) Polish a bare gold electrode with alumina powder, then ultrasonically clean it in anhydrous ethanol and deionized water, blow dry it with nitrogen, and then place the electrode in 0.5M sulfuric acid solution and perform cyclic voltammetry scanning at a scan rate of 0.1V / s between -0.35 and +1.5V to clean the electrode until the cyclic voltammetry curve is stable. Then remove the electrode, rinse it with ultrapure water, and blow dry it with nitrogen to obtain the treated gold electrode; 2) Immerse the treated gold electrode in 1-hexanethiol solution, incubate at room temperature for 1 hour, then rinse with ultrapure water and blow dry with nitrogen to obtain a GE / HT electrode; 3) Prepare a mixture of a capture probe Cp modified with cholesterol at the 5' end and an auxiliary probe Ap modified with methylene blue at the 3' end using DNA fixation buffer, and perform annealing and hybridization to obtain a Cp-Ap solution; 4) Add Cp-Ap solution to the GE / HT electrode, incubate at room temperature for 1 hour, then rinse with ultrapure water and blow dry with nitrogen to obtain a GE / HT / Cp-Ap electrode; 5) Prepare a mixture of the target T and the signal probe Sp modified with ferrocene at its 3' end using DNA fixation buffer, which is referred to as T-Sp solution. 6) Add T-Sp solution to the GE / HT / Cp-Ap electrode, incubate at room temperature for 30 minutes, then rinse with ultrapure water and blow dry with nitrogen to obtain a GE / HT / Cp-Ap-T-Sp electrode; 7) Place the GE / HT / Cp-Ap-T-Sp electrode in the electrochemical detection solution and scan using square wave voltammetry.

4. The preparation method according to claim 3, wherein: The annealing and hybridization conditions are as follows: first incubating in a 95° C. metal bath for 5 minutes, and then cooling to 25° C. at a rate of 1° C. / min.

5. The preparation method according to claim 3, wherein: The formula of the DNA fixation buffer is: 10 mM Tris, 1 mM EDTA·2Na·2H2O, 50 mM NaCl, 1 mM MgCl2; pH 8.

0.

6. The preparation method according to claim 3, wherein: The target T sequence is 5'-AAGAUCACAUUGGCACCCGCAAUCC-3'.

7. Use of the ratiometric electrochemical biosensor according to claim 1, characterized in that: Used to prepare new coronavirus testing products.

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