Photoelectrochemical analysis method based on pyroelectric effect and strand displacement reaction and application

Abstract

The application belongs to the technical field of photoelectrochemical analysis methods, and particularly relates to a photoelectrochemical analysis method based on pyroelectric effect and strand displacement reaction and application.The photoelectrochemical analysis method based on pyroelectric effect and strand displacement reaction has the characteristics that the method comprises the following steps: preparing an electrode interface based on a CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite; preparing a DNA beacon probe labeled with Ag2S NPs; preparing a magnetic bead DNA amplification carrier based on a strand displacement reaction; adding a detection object into the magnetic bead DNA amplification carrier, and performing photoelectric quantitative detection through an electrochemical workstation and a near-infrared lamp.The photoelectrochemical analysis method based on pyroelectric effect and strand displacement reaction has high amplification efficiency, good sensitivity and strong signal coupling effect.The method can be used for detecting miRNA-21.

Description

Technical Field

[0001] This invention belongs to the field of photoelectrochemical analysis methods, specifically relating to photoelectrochemical analysis methods and applications based on pyroelectric effect and chain displacement reaction. Background Technology

[0002] Photoelectrochemical analysis methods offer advantages such as high sensitivity and low background, with different excitation sources and energy forms of detection signals. Traditional photoelectrochemical analysis methods based on ultraviolet light excitation can generate photocurrents for analyte analysis by exciting photoelectric nanomaterials labeled with beacon probes at the electrode interface. However, this method is prone to damaging the biomolecules (such as DNA) constituting the beacon probes. In near-infrared light-driven photoelectrochemical analysis methods, the excitation of photoelectric nanomaterials can reduce damage to the beacon probes. Excited electrons in near-infrared light-responsive nanomaterials transfer from the valence band to the conduction band, generating electron-hole pairs and outputting a photocurrent, enabling quantitative analysis of the analyte. However, the high recombination rate of photogenerated electrons and holes limits the sensitivity of this method.

[0003] Isothermal amplification strategies based on DNA nanotechnology have become a major tool for signal enhancement, widely used in photoelectric analysis, electrochemiluminescence analysis, and fluorescence bioanalysis. Currently, DNA amplification strategies applied to near-infrared photoelectric analysis mainly include hybridization chain reaction (HCR) and rolling circle amplification (RBAC), which enhance photoelectric signals by amplifying DNA fragments. For example, CN116790722A discloses a method for photoelectrochemical detection of miRNA-21, which improves detection sensitivity through a biomolecule-based amplification strategy. The DNA Walker signal amplification reaction is triggered by the target, and the released walking chain can undergo cyclic amplification, resulting in a large number of substrate chains hybridizing with the walking chain, releasing a large amount of HRP enzyme. The addition of Na2S2O3 and H2O2 catalyzes the generation of a large amount of H2S, forming a CuS / CuO-Au semiconductor heterojunction, thereby increasing the photocurrent intensity. This is a photoelectrochemical sensing strategy for miRNA-21 signal enhancement. However, the steps of preparing composite semiconductor materials using ion exchange technology to enhance the photocurrent are cumbersome and time-consuming.

[0004] CN111638255A discloses a photoelectrochemical detection method for miRNA-21 content based on bismuth vanadate. This method combines miRNA-21-mediated rolling circle amplification, endonuclease-specific recognition hydrolysis reaction, and photoelectric signal excitation of BiVO4-modified electrodes caused by the specific intercalation of double-stranded DNA by methylene blue or Nell blue to construct a signal-enhanced detection. This method uses a combination of rolling circle amplification technology and endonuclease-specific recognition, resulting in a complex reaction system, requiring a large number of enzymes, and easily leading to false positives.

[0005] CN111220666A discloses a highly efficient miRNA detection method based on a chloro-heme-induced biocatalytic photosensitive interface. After modifying a paper chip with gold nanoparticles and cadmium sulfide quantum dots, hairpin-structured capture DNA is modified onto the electrode surface. Subsequently, the double-stranded structure formed by the hybridization of microRNA and the capture DNA is cleaved by DSN, releasing microRNA. The released microRNA then hybridizes with the capture DNA and is cleaved by DSN, forming a DSN-assisted microRNA cycle. Subsequently, numerous short DNA fragments on the electrode surface hybridize with subsequently added chloro-heme-labeled DNA to form a new double-stranded structure. In the presence of hydrogen peroxide, chloro-heme catalyzes 4-chloro-1-naphthol, forming an insoluble precipitate on the electrode surface, effectively inhibiting the transfer of ascorbic acid to the electrode surface, leading to a decrease in photocurrent. This method exhibits a low detection limit and a wide detection range, enabling the detection of trace amounts of RNA sequences. The hairpin substrate on the electrode surface is prone to spontaneous opening, resulting in false positives. Furthermore, the spatial steric hindrance between the hairpins reduces the loading efficiency of the electrode surface, thereby decreasing sensitivity. Summary of the Invention

[0006] The technical problem this invention aims to solve is to overcome the aforementioned deficiencies in existing technologies and provide a photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction, which has high amplification efficiency, good sensitivity, and strong signal coupling effect. This method can be used to detect miRNA-21.

[0007] The photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction described in this invention includes the following steps:

[0008] (1) Preparation of electrode interface based on CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite;

[0009] (2) Prepare DNA beacon probes labeled with Ag2S NPs;

[0010] (3) Preparation of magnetic bead DNA amplification vectors based on strand displacement reaction;

[0011] (4) The analyte was added to the magnetic bead DNA amplification carrier and photoelectric quantitative detection was performed using an electrochemical workstation and a near-infrared lamp to obtain a standard curve and detect the analyte.

[0012] Step (1) specifically includes the following steps:

[0013] a. Add cadmium nitrate and thiourea to ethylenediamine and stir. React at 135-145℃ for 10-14 h. Centrifuge, wash, and dry to obtain CdS nanowires.

[0014] b. Mix 2-mercaptobenzimidazole with ethanol, adjust the pH, add CdS nanowires, reflux at 65-75℃ for 22-26 h, cool, wash and dry to obtain CdS / 2-mercaptobenzimidazole complex.

[0015] c. Dissolve the CdS / 2-mercaptobenzimidazole complex in water, drop-coat it onto the surface of an FTO electrode, and dry it to obtain a CdS-2MBI modified photoelectrode;

[0016] d. Add DNA capture probes to the surface of the photoelectrode, incubate for 6-10 h, wash, then add 6-mercapto-1-hexanol and incubate for 30-50 min, then wash to obtain the electrode interface based on CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite.

[0017] The mass ratio of cadmium nitrate, thiourea, and 2-mercaptobenzimidazole is 1:(0.5-0.75):(1-1.24).

[0018] Step (2) specifically includes the following steps: silver nitrate is added to a mixture of 3-mercaptopropionic acid and ethylene glycol, nitrogen gas is introduced, and the mixture is heated at 140-150℃ until a black solution appears. After washing and drying, Ag2S NPs (silver sulfide nanoparticles) are obtained. Ag2S NPs are dispersed in water, 1-5 μmol / L of aminoated DNA is added, and the mixture is reacted for 10-16 h. Then, it is dispersed in PBS buffer to obtain DNA beacon probes labeled with Ag2S NPs at a concentration of 1-2 μmol / L.

[0019] The ratio of silver nitrate, 3-mercaptopropionic acid, and aminated DNA is (0.05-0.15)g:(1-2)mL:(2-10)μmol.

[0020] Step (3) specifically includes the following steps: adding carboxylated magnetic beads to an aqueous solution containing carbodiimide (EDC) and N-hydroxythiosuccinimide (NHS), incubating for 25-35 min, dispersing in PBS buffer, then adding aminoated F-DNA and R-DNA and co-incubating for 10-14 h, washing, adding the DNA beacon probe labeled with Ag2S NPs prepared in step (2) to react, and washing to obtain the magnetic bead DNA amplification vector based on strand displacement reaction.

[0021] The volume ratio of the carboxylated magnetic beads, aminolated F-DNA, R-DNA, and DNA beacon probe labeled with Ag2S NPs is (100-200):(15-25):(15-25):(15-25).

[0022] Step (4) specifically includes the following steps: adding different concentrations of the analyte to the magnetic bead DNA amplification vector based on strand displacement reaction prepared in step (3), and NEBuffer. TM 2 buffer, rCutsmart buffer TM The buffer, dNTPs, Klenow fragment polymerase, Nb.BtsI endonuclease and water were mixed and incubated for 1-1.5 h, and then reacted at 80-85 °C for 20-25 min to inactivate the enzyme. The released DNA beacon probe labeled with Ag2S NPs was collected by magnetic separation and dropped into the electrode interface of the CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite prepared in step (1) and incubated for 30-60 min. After washing, photoelectric testing was performed to quantify the analyte.

[0023] The magnetic bead DNA amplification vector based on strand displacement reaction, NEBuffer TM 2 buffer, rCutsmartbuffer TM The volume ratio of buffer, dNTPs, Klenow fragment polymerase, Nb.BtsI endonuclease and water was (10-30):(4-8):(4-8):(1-3):(1-5):(1-3):(15-20).

[0024] The application of the photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction is to quantitatively detect miRNA-21.

[0025] Specifically, the photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction includes the following steps:

[0026] (1) Preparation of electrode interface based on CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite:

[0027] a. Add 2.01g of cadmium nitrate and 1g-1.5g of thiourea to 50mL-60mL of ethylenediamine and stir. React at 135-145℃ for 10-14h in a solvothermal manner. After centrifugation, washing, and drying, CdS nanowires are obtained.

[0028] b. Mix 2g-2.5g of 2-mercaptobenzimidazole with 40mL of anhydrous ethanol, adjust the pH to 10-10.5, add CdS nanowires, reflux at 65-75℃ for 22-26h, cool, wash and dry to obtain CdS / 2-mercaptobenzimidazole complex.

[0029] c. Dissolve the CdS / 2-mercaptobenzimidazole complex in water to a concentration of 5.0 mg / mL, and drop 30-50 μL onto the surface of the FTO electrode. The effective area of ​​the drop coating is 28.26 mm². 2A CdS-2MBI modified photoelectrode with a diameter of 6 mm was obtained after drying.

[0030] d. Add 15 μL-30 μL of DNA capture probe at a concentration of 5 μmol / L to the photoelectrode surface, incubate for 6-10 h, wash several times with ultrapure water, then add 10 μL-20 μL of 6-mercapto-1-hexanol (MCH) at a concentration of 500 nmol / L, incubate for 30-50 min, and wash with ultrapure water to obtain the electrode interface based on CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite.

[0031] (2) Preparation of DNA beacon probes labeled with Ag2S NPs: Add 0.05g-0.15g of silver nitrate to a mixture of 1mL-2mL of 3-mercaptopropionic acid and 8mL-10mL of ethylene glycol, purge with nitrogen to remove oxygen, heat at 140-150℃ for 25min until a black solution appears, centrifuge, wash with acetone, ethanol and ultrapure water, freeze dry to obtain Ag2S NPs (silver sulfide nanoparticles); disperse Ag2S NPs in water at a concentration of 3mg / mL-8mg / mL, take 20μL and add 20μL of 1μmol / L-5μmol / L aminated DNA, react for 10-16h, purify by centrifugation multiple times, redisperse in 20μL-50μL of 0.01mol / L PBS buffer to obtain DNA beacon probes labeled with Ag2S NPs at a concentration of 1μmol / L-2μmol / L.

[0032] (3) Preparation of magnetic bead DNA amplification vector based on strand displacement reaction: 100 μL-200 μL of carboxylated magnetic beads were added to 100 μL-200 μL of aqueous solution containing 0.1-0.3 mol / L carbodiimide (EDC) and 0.01-0.03 mol / L N-hydroxythiosuccinimide (NHS). After incubation for 25-35 min, the mixture was dispersed in 100-200 μL of 0.01 mol / L PBS buffer. Then, 20 μL of aminoated F-DNA and 20 μL of L-DNA were added and co-incubated for 10-14 h. The mixture was washed with 0.01 mol / L PBS buffer and 20 μL of DNA beacon probe labeled with Ag2S NPs prepared in step (2) were added for reaction. The mixture was washed with 0.01 mol / L PBS buffer to obtain the magnetic bead DNA amplification vector based on strand displacement reaction.

[0033] (4) Disperse the DNA amplification vector from step (3) into 0.01 mol / L PBS buffer. Add 10-30 μL of the 10 μmol / L magnetic bead DNA amplification vector based on strand displacement reaction prepared in step (3) to different concentrations of the detection agent miRNA-21, and 4-8 μL of NE Buffer.TM 2 buffer, 4-8 μL rCutsmart buffer TM A mixture of buffer, 1-3 μL dNTPs (500 μmol / L), 1-5 μL Klenow fragment polymerase (4-8 U), 1 μL Nb.BtsI endonuclease (3-8 U), and 17 μL water was incubated for 1-1.5 h, followed by reaction at 80-85 °C for 20-25 min. The released DNA beacon probes labeled with Ag2S NPs were collected by magnetic separation and incubated at the electrode interface of the CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite prepared in step (1) for 30-60 min. After washing with ultrapure water, photoelectric testing was performed under the following conditions: excitation light 808 nm, power: 2.5 W cm⁻¹. -2 The analyte is quantified using an electric current to obtain a standard curve and then the analyte is detected.

[0034] DNA polymerase / endonuclease-assisted strand displacement amplification has become a unique isothermal amplification strategy, with amplification efficiency reaching hundreds of times within minutes. To further improve the detection sensitivity of near-infrared photoelectric analysis methods, isothermal amplification technology based on strand displacement reaction is integrated into near-infrared photoelectric analysis methods, which not only has high sensitivity but also good application prospects.

[0035] This invention proposes a near-infrared driven photoelectrochemical analysis method by integrating photoelectric and pyroelectric effects. After the addition of the analyte, a strand displacement reaction is triggered in a DNA amplification carrier based on magnetic beads, releasing numerous DNA beacon probes labeled with Ag2S NPs. These probes hybridize with capture probes at the electrode interface, immobilizing the Ag2S NPs on the electrode surface. Under near-infrared light irradiation, the Ag2S NPs generate a photocurrent and simultaneously act as a heat source for CdS / 2-mercaptobenzimidazole. Due to the temperature change, the spontaneous polarization of CdS / 2-mercaptobenzimidazole changes, generating a pyroelectric current, which, in conjunction with the photocurrent, achieves signal enhancement.

[0036] Compared with the prior art, the beneficial effects of the present invention are:

[0037] (1) The near-infrared photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction of the present invention integrates pyroelectric effect and photoelectric effect to achieve better signal coupling enhancement effect.

[0038] (2) The near-infrared photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction of the present invention achieves signal amplification by means of DNA chain displacement reaction, and exhibits the characteristics of high amplification efficiency and good sensitivity.

[0039] (3) The near-infrared photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction of the present invention uses the DNA beacon probe labeled with Ag2S as a photoelectric nanomaterial to generate photocurrent on the one hand, and as a heat source of pyroelectric composite material to assist it in generating thermocurrent on the other hand, which has the characteristics of multifunctionality and high utilization rate.

[0040] (4) The near-infrared photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction of the present invention is applied to the detection of miRNA-21, with high sensitivity, accurate detection results and high detection efficiency. Attached Figure Description

[0041] Figure 1 The image shows a transmission electron microscope (TEM) image of the CdS nanowires prepared in step (1)a of Example 1 at 100 nm.

[0042] Figure 2 The image is a transmission electron microscope (TEM) image at 100 nm of the CdS / 2-mercaptobenzimidazole complex prepared in step (1)b of Example 1.

[0043] Figure 3 The UV-Vis absorption spectra of the aminoated DNA, Ag2S NPs, and DNA beacon probe labeled with Ag2S NPs in step (2) of Example 1 are shown.

[0044] Figure 3 In the middle: a) Aminated DNA; b) DNA beacon probe labeled with Ag2S NPs; c) Ag2S NPs.

[0045] Figure 4 This is a graph showing the detection current of different concentrations of the detectant miRNA-21 in Example 1, where a→g is a curve of concentration from low to high.

[0046] Figure 5 The standard curve prepared in Example 1.

[0047] Figure 6 This is a graph showing the effect of Klenow fragment polymerase concentration on the current intensity of photoelectric signals in this invention.

[0048] Figure 7 This is a graph showing the effect of Nb.BtsI endonuclease concentration on the current intensity of photoelectric signals in this invention.

[0049] Figure 8 This is a graph showing the effect of reaction time on the current intensity of the photoelectric signal in this invention.

[0050] Figure 9 This is a comparison graph showing the effect of different electrode interfaces on the current intensity of photoelectric signals in Example 1 and Comparative Example 1.

[0051] Figure 10 This is a graph showing the results of a repeatability test of the near-infrared photoelectrochemical analysis method based on the pyroelectric effect and chain displacement reaction of the present invention. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments.

[0053] The reagents used in the examples are sourced and specified as shown in Table 1. All others are commercially available products.

[0054] Table 1 Reagent Specifications

[0055]

[0056]

[0057] Example 1

[0058] The photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction includes the following steps:

[0059] (1) Preparation of electrode interface based on CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite:

[0060] a. Add 2.01 g of cadmium nitrate and 1.2 g of thiourea to 55 mL of ethylenediamine and stir. React solvothermically at 140 °C for 12 h. After centrifugation, washing, and drying, CdS nanowires are obtained. Figure 1 As shown;

[0061] b. Mix 2g of 2-mercaptobenzimidazole with 40mL of anhydrous ethanol, adjust the pH to 10, add CdS nanowires, reflux at 70℃ for 24h, cool, wash, and dry to obtain the CdS / 2-mercaptobenzimidazole complex, as follows: Figure 2 As shown;

[0062] c. Dissolve the CdS / 2-mercaptobenzimidazole complex in water to a concentration of 5.0 mg / mL, and drop 40 μL onto the surface of the FTO electrode. The effective area of ​​the drop coating is 28.26 mm². 2 A CdS-2MBI modified photoelectrode with a diameter of 6 mm was obtained after drying.

[0063] d. Add 20 μL of DNA capture probe with a concentration of 5 μmol / L to the surface of the photoelectrode, incubate for 8 h, wash several times with ultrapure water, add 15 μL of MCH with a concentration of 500 nmol / L, incubate for 40 min, and wash with ultrapure water to obtain the electrode interface based on CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite.

[0064] (2) Preparation of DNA beacon probes labeled with Ag2S NPs: 0.1 g of silver nitrate was added to a mixture of 2 mL of 3-mercaptopropionic acid and 8 mL of ethylene glycol. Nitrogen gas was purged to remove oxygen, and the mixture was heated at 145 °C for 25 min until a black solution appeared. After centrifugation, the solution was washed with acetone, ethanol, and ultrapure water, and then freeze-dried to obtain Ag2S NPs. Ag2S NPs were dispersed in water at a concentration of 6 mg / mL. 20 μL of this solution was added to 20 μL of 4 μmol / L aminated DNA and reacted for 12 h. After multiple centrifugations for purification, the solution was redispersed in 30 μL of 0.01 mol / L PBS buffer to obtain a DNA beacon probe labeled with Ag2S NPs at a concentration of 1.5 μmol / L. Figure 3 As shown, the prepared beacon probe (1.5 μM) exhibits a specific absorption peak of DNA at 260 nm and a strong absorption peak at 808 nm.

[0065] (3) Preparation of magnetic bead DNA amplification vector based on strand displacement reaction: 150 μL of carboxylated magnetic beads were added to 150 μL of aqueous solution containing 0.2 mol / L EDC and 0.02 mol / L NHS. After incubation for 30 min, the mixture was dispersed in 150 μL of 0.01 mol / L PBS buffer. Then, 20 μL of amino-modified F-DNA and 20 μL of L-DNA were added and co-incubated for 12 h. The mixture was washed with 0.01 mol / L PBS buffer and 20 μL of DNA beacon probe labeled with Ag2S NPs prepared in step (2) were added for reaction. The mixture was washed with 0.01 mol / L PBS buffer to obtain the magnetic bead DNA amplification vector based on strand displacement reaction.

[0066] (4) Disperse the DNA amplification vector from step (3) into 0.01 mol / L PBS buffer. Add 20 μL of the detection agent miRNA-21 at concentrations of 0 pmol / L, 1 pmol / L, 10 pmol / L, 100 pmol / L, 1 nmol / L, 10 nmol / L, and 100 nmol / L to 10 μL of the magnetic bead DNA amplification vector based on strand displacement reaction prepared in step (3) and 6 μL of NE Buffer. TM 2 buffer, 6 μL rCutsmart buffer TMA mixture of buffer, 2 μL dNTP (500 μmol / L), 3 μL Klenow fragment polymerase (6 U), 1 μL Nb.BtsI endonuclease (6 U), and 17 μL water was incubated for 1 h, followed by reaction at 80 °C for 20 min. The released DNA beacon probe labeled with Ag2S NPs was collected by magnetic separation and incubated at the electrode interface of the CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite prepared in step (1) for 40 min. After washing with ultrapure water, photoelectric testing was performed under the following conditions: excitation light 808 nm, power: 2.5 W cm⁻¹. -2 The analyte is quantified using an electric current, and the detection results for different concentrations are as follows: Figure 4 As shown, the current value gradually increased and then stabilized as the miRNA-21 concentration increased from 1 pmol / L to 100 nmol / L. The current C was recorded for each concentration. miRNA-21 The current value, expressed as lgC miRNA-21 Using the x-axis as the horizontal axis and the current value I as the vertical axis, a standard curve was constructed. The standard curve is I = 24.883lgC. miRNA-21 +375.68, where R 2 =0.993, such as Figure 5 As shown, its detection limit was calculated to be 54 fM (3 S / N).

[0067] Based on embodiment 1, this invention has prepared a standard curve for a photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction. Furthermore, experiments were conducted to explore the optimal performance of this analytical method from the following three aspects.

[0068] (1) The effect of Klenow fragment polymerase concentration on the chain displacement reaction was investigated. The signal was detected within a final concentration range of 0.05–0.25 U / μL. The optimal signal value was found at a concentration of 0.20 U / μL. Figure 6 As shown.

[0069] (2) The effect of Nb.BtsI endonuclease concentration on the chain displacement reaction was investigated. The signal was detected within a final concentration range of 0.1–0.5 U / μL. The optimal signal value was found at a concentration of 0.4 U / μL. Figure 7 As shown.

[0070] (3) The effect of reaction time on the chain displacement reaction was investigated. The signal was detected within the range of 30 min to 180 min. The experiment showed that the signal value reached its optimal value at a concentration of 120 min. Figure 8 As shown.

[0071] Comparative Example 1

[0072] This comparative example follows the same steps as Example 1, except that it uses an electrode interface based on pure CdS pyroelectricity. Under the same concentration (10 nmol / L) of the target analyte, the current response value of the electrode interface using pure CdS pyroelectricity is lower than that of the electrode interface based on CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite. This indicates that pure CdS pyroelectricity has poor performance, resulting in poor detection and low sensitivity. Figure 9 As shown, a represents the result of Example 1, and b represents the result of Comparative Example 1.

[0073] The present invention provides a near-infrared photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction, and verifies its recovery rate and reproducibility.

[0074] (1) The photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction of the present invention was verified by addition recovery method. The verification parameters and steps were the same as in Example 1. The practical application of the proposed biosensor was evaluated by standard addition method. miRNA-21 at concentrations of 0 pmol / L, 2 pmol / L, 20 pmol / L and 200 pmol / L were added to serum diluted 100 times and detected. The recovery results are shown in Table 2.

[0075] Table 2 Results of Spiked Recovery Experiment

[0076]

[0077] (2) A reproducibility experiment was conducted on the photoelectrochemical analysis method based on the pyroelectric effect and chain displacement reaction of the present invention. The experimental parameters and steps were the same as in Example 1. In addition, five parallel photoelectrodes based on the electrode interface of CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite were prepared, and miRNA-21 at the same concentration (10 nmol / L) was detected to evaluate the reproducibility of the detection method. Figure 10 As shown, with a relative standard deviation (RSD) of 2.5%, the currents of the five photoelectrodes are basically consistent, indicating that the detection method has good reproducibility.

[0078] Of course, the above description is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the embodiments of the present invention. The present invention is also not limited to the above examples, and all equivalent changes and improvements made by those skilled in the art within the scope of the present invention should fall within the patent coverage of the present invention.

Claims

1. A photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction, characterized in that, Includes the following steps: (1) Preparation of electrode interface based on CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite: a. Add cadmium nitrate and thiourea to ethylenediamine and stir. React at 135-145℃ for 10-14h, centrifuge, wash and dry to obtain CdS nanowires; b. Mix 2-mercaptobenzimidazole with ethanol, adjust the pH, add CdS nanowires, reflux at 65-75℃ for 22-26 h, cool, wash and dry to obtain CdS / 2-mercaptobenzimidazole complex. c. Dissolve the CdS / 2-mercaptobenzimidazole complex in water, drop-coat it onto the surface of an FTO electrode, and dry it to obtain a CdS-2MBI modified photoelectrode; d. Add DNA capture probe to the surface of photoelectrode, incubate for 6-10 h and wash, then add 6-mercapto-1-hexanol and incubate for 30-50 min and wash again to obtain the electrode interface based on CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite. (2) Preparation of DNA beacon probes labeled with Ag2S NPs: Add silver nitrate to a mixture of 3-mercaptopropionic acid and ethylene glycol, purge with nitrogen, heat at 140-150℃ until a black solution appears, wash and dry to obtain Ag2S NPs; disperse Ag2S NPs in water, add 1-5 μmol / L of aminated DNA and react for 10-16 h, then disperse in PBS buffer to obtain DNA beacon probes labeled with Ag2S NPs at a concentration of 1-2 μmol / L; (3) Preparation of magnetic bead DNA amplification vector based on strand displacement reaction: Carboxylated magnetic beads were added to an aqueous solution containing carbodiimide and N-hydroxythiosuccinimide, incubated for 25-35 min, dispersed in PBS buffer, and then aminated F-DNA and R-DNA were added and co-incubated for 10-14 h. After washing, the DNA beacon probe labeled with Ag2S NPs prepared in step (2) was added to react, and the mixture was washed to obtain the magnetic bead DNA amplification vector based on strand displacement reaction. (4) The analyte was added to the magnetic bead DNA amplification carrier and photoelectric quantitative detection was performed using an electrochemical workstation and a near-infrared lamp to obtain a standard curve and detect the analyte.

2. The near-infrared photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction according to claim 1, characterized in that: The mass ratio of cadmium nitrate, thiourea, and 2-mercaptobenzimidazole is 1:(0.5-0.75):(1-1.24).

3. The photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction according to claim 1, characterized in that: The ratio of silver nitrate, 3-mercaptopropionic acid, and aminoated DNA is (0.05-0.15) g: (1-2) mL: (2-10) μmol.

4. The photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction according to claim 1, characterized in that: The volume ratio of the reaction of carboxylated magnetic beads, aminolated F-DNA, R-DNA, and DNA beacon probe labeled with Ag2S NPs was (100-200):(15-25):(15-25):(15-25).

5. The photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction according to claim 1, characterized in that, Step (4) specifically includes the following steps: adding different concentrations of the analyte to the magnetic bead DNA amplification vector based on strand displacement reaction prepared in step (3), and NEBuffer. TM 2 buffer, rCutsmart buffer TM The buffer, dNTPs, Klenow fragment polymerase, Nb.BtsI endonuclease and water were mixed and incubated for 1-1.5 h, and then reacted at 80-85 °C for 20-25 min. The released DNA beacon probe labeled with Ag2S NPs was collected by magnetic separation and dropped into the electrode interface of the CdS / 2-mercaptobenzimidazole pyroelectric nanocomposite prepared in step (1) and incubated for 30-60 min. After washing, photoelectric testing was performed to quantify the analyte.

6. The photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction according to claim 5, characterized in that, NEBuffer, a magnetic bead DNA amplification vector based on strand displacement reaction. TM 2 buffer, rCutsmart buffer TM The volume ratio of buffer, dNTP, Klenow fragment polymerase, Nb.BtsI endonuclease and water was (10-30):(4-8):(4-8):(1-3):(1-5):(1-3):(15-20).

7. An application of the photoelectrochemical analysis method based on pyroelectric effect and chain displacement reaction as described in any one of claims 1-6, characterized in that, Used for quantitative detection of miRNA-21.

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