CN114324506A

CN114324506A - Electrochemical biosensing composition, working solution, sensor, device and application thereof

Info

Publication number: CN114324506A
Application number: CN202111677287.8A
Authority: CN
Inventors: 万家余; 卜胜君; 刘文森; 李忠义; 郝镯
Original assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Current assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-04-12

Abstract

The invention relates to the field of electrochemical biosensors, in particular to an electrochemical biosensor composition, a working solution, a sensor, a device and application thereof. The invention introduces CRISPR/Cas12a combined with click chemistry driven exponential amplification reaction (EXPAR) into an electrochemical biosensor to detect miRNA-21. When miRNA-21 exists, a click chemistry-exponential amplification reaction can be triggered in the electrochemical biosensor, a large number of nucleic acid fragments are generated, the trans-cleavage capability of CRISPR-Cas12a is stimulated, and hairpin DNA immobilized on the surface of an electrode is cleaved. Under the optimal condition, the lowest detection limit of the electrochemical biosensor can reach 1 fM. Therefore, the proposed electrochemical biosensor can sensitively and efficiently detect miRNAs and may become a potential analytical tool for POC testing and on-site molecular diagnostics.

Description

Electrochemical biosensing composition, working solution, sensor, device and application thereof

Technical Field

The invention relates to the field of electrochemical biosensors, in particular to an electrochemical biosensor composition, a working solution, a sensor, a device and application thereof.

Background

The nucleic acid detection is a common, high-efficiency and specific detection method, and plays an important role in the aspects of rapid disease diagnosis, food safety, environmental pollution detection and the like. Currently, real-time fluorescent quantitative PCR (qRT-PCR) is considered as a gold standard for detecting nucleic acids. However, the qRT-PCR method is long in time consumption, low in cost, complex in procedure, low in sensitivity and too much in equipment requirement, and thus cannot meet the requirements of practical application. Therefore, establishing a sensitive nucleic acid detection method has important significance for environmental pollution monitoring, food safety and disease diagnosis.

MicroRNAs (miRNAs) are small single-stranded RNAs consisting of 22 nucleotides, which play important regulatory roles in various biological processes including cell development, differentiation, immune response and tumorigenesis by participating in the control of post-transcriptional gene expression, and have become biomarkers for the diagnosis of many tumor molecules, including neurodegenerative diseases, chronic cardiovascular diseases and cancer. Sensitive point-of-care (POC) methods are crucial for detecting mirnas.

Currently, several Cas12 a-based miRNA detection methods have been designed. For example, Wang et al describe a Cas12 a-derived miRNA sensing technology based on rolling circle amplification; peng et al designed a sensitive method 13 with high cost performance and detection of miRNAs by assembling Cas12a with a catalytic hairpin; chen et al established a POC method based on Cas12a sensor to detect miRNA31-5p, and combined with a superimposed primer amplification reaction; sun et al developed a cas12 a-mediated signaling platform for sensitive and specific miRNA detection by coupling ligase-assisted probe ligation, DNAzyme and RNA polymerase-assisted amplification; gong et al developed a method for detecting miRNA16 with a bispecific nuclease-assisted CRISPR-Cas12 a; chen et al established an ultra-sensitive miR-21 detection strategy based on CRISPR/Cpf17-19 multistage displacement amplification and trans-cleavage activity. All of these methods require several nucleic acid amplification techniques, such as rolling circle amplification, catalytic hairpin assembly, etc., to improve the platform for Cas12a to detect mirnas.

The exponential amplification reaction (EXPAR) has catalytic activities of DNA polymerase and endonuclease, can realize rapid cyclic amplification of a large number of short nucleic acid fragments, and has received much attention because of its simplicity and sensitivity. In standard EXPAR, a short DNA primer hybridizes to the template strand, replicates along the template strand with the aid of a DNA polymerase, and undergoes a sequential displacement event with the aid of a nicking endonuclease. Nicking endonucleases recognize specific sequences in the extended duplex, causing the nucleic acid strand to break and replace the newly replicated strand. In previous studies, a fluorescent biosensor was constructed by modifying the EXPAR for miRNA detection, improving the target-induced strand amplification reaction. Yang et al successfully established an EXPAR-driven fluorescence biosensor based on a three-dimensional biped DNA walker to detect miRNA. In addition, a sensitive method for analyzing miRNA by fluorescence is developed based on EXPAR combined with DNA template silver nanocluster.

Traditional miRNAs detection methods such as northern blotting, microarray and qRT-PCR remain widely used methods, and in particular qRT-PCR is more considered as the "gold standard" for the detection of miRNAs. However, these techniques suffer from drawbacks such as differences in kit sensitivity, inevitable false positive results, time-consuming, high requirements for instrumentation and equipment, etc. that do not allow for more intensive studies of miRNAs.

Poor sensitivity: since some miRNAs are present in very low amounts in tissues or cells, they need to be amplified for subsequent detection.

Disclosure of Invention

In view of the above, the invention provides an electrochemical detection method and a sensor for micro rna by using a click chemistry binding index amplification reaction to assist CRISPR-Cas12 a.

In order to achieve the above object, the present invention provides the following technical solutions:

in a first aspect, the present invention provides an electrochemical biosensor composition comprising: azide (N3) modified first ODNs probe, Aza-dibenzocyclooctane (Aza-DBCO) modified second ODNs probe, primers, dNTPs, Klenow fragment, nt.bbvci, LbCas12a, and CRISPR RNA.

The invention provides a high-sensitivity electrochemical microRNA detection platform based on click chemistry driven EXPAR Cas12a (the principle is shown in figure 1). microRNA mediates the linkage of two oligonucleotide single-stranded DNA fragments for use as an EXPAR template. Enzyme-free click chemistry between azide (N3) and Aza-dibenzocyclooctane (Aza-DBCO) modified ODNs achieves high ligation efficiencies depending on miRNA. The resulting binding template consists of two nick sites between the target miRNA binding site, the primer binding site and the Cas12a activator binding site, extended to give the final single-stranded DNA product (FP). The translocated target miRNA elicits a ligation reaction of two ODNs probes. The nicking endonuclease nt. One of the DNA fragments is identical to the target miRNA sequence, and the other activates Cas12 a. The displacement of the same DNA segment of the target miRNA is used as a bridge to trigger the click reaction of the two ODNs probes, and the exponential generation of FPs is further promoted. These extension, cleavage, extension, and substitution reactions are repeated to generate a large number of short fragments of DNA that can activate Cas12 a. Activating the DNA fragment of Cas12a can open the trans-cleavage activity of Cas12a immobilized on the surface of the electrode to cleave hairpin DNA electrochemically, thereby altering the electrochemical signal. The method is based on a click chemistry EXPAR and Cas12a system, is coupled with an electrochemical biosensor, realizes qualitative and quantitative detection of microRNA, and provides a potential application value for a novel nucleic acid analysis method.

In a second aspect, the invention also provides the application of the electrochemical biosensor composition in preparing a working solution of an electrochemical biosensor or an electrochemical biosensor.

In a third aspect, the invention also provides a working solution of the electrochemical biosensor, which comprises the electrochemical biosensor composition and acceptable auxiliary materials or auxiliary agents.

In a fourth aspect, the invention also provides application of the working solution of the electrochemical biosensor in preparing the electrochemical biosensor.

In a fifth aspect, and more importantly, the present invention also provides an electrochemical biosensor, a working fluid comprising the electrochemical biosensor composition or the electrochemical biosensor, and an electrode system.

In some embodiments of the invention, the electrode system comprises a reference electrode, an auxiliary electrode, and a working electrode;

the reference electrode comprises an Ag/AgCl electrode; and/or

The auxiliary electrode comprises a platinum wire; and/or

The working electrode comprises a gold electrode.

In a sixth aspect, the invention also provides the application of the electrochemical biosensor in the preparation of devices for nucleic acid detection, environmental pollution monitoring, food safety detection or disease diagnosis.

In some embodiments of the invention, the nucleic acids include, but are not limited to, microRNAs.

In some embodiments of the invention, the nucleic acid includes, but is not limited to, miRNA-21.

In some embodiments of the invention, the disease includes, but is not limited to: one or more of a neurodegenerative disease, a chronic cardiovascular disease, or cancer.

In a seventh aspect, the present invention further provides a method for detecting nucleic acid, wherein nucleic acid to be detected is mixed with the electrochemical biosensor composition or the working solution of the electrochemical biosensor, and the mixture is placed on a working electrode of the electrochemical biosensor for detection.

In some embodiments of the invention, the method for detecting nucleic acids specifically comprises:

MicroRNAs and ODNs (containing Probe A and Probe B) at various concentrations, Probe A and Probe B (each 1. mu.M), Probe A modified Aza-DBCO. Probe B modified N3 and maintained at 37 ℃ for 60 minutes. In the presence of miRNA21, Probe A and Probe B are joined into one nucleic acid strand. Then, 6. mu.L of the ligation product and 1. mu.L of 10. mu.M primer were in 4. mu.L of 10. mu.L of XKlenow buffer and 10 XCutSmart, at 95 ℃ for 5 minutes. Then slowly cooled to 25 deg.C for more than 20min, and adding 4. mu.L 25mM dNTPs, 0.5. mu.L 5U/. mu.L Klenow fragment, 0.5. mu.L 10U/. mu.L. To the mixture was added nt. Addition of ddH₂O brought the final volume of the mixture to 20. mu.L. The reaction system was incubated at 37 ℃ for 80min and then at 80 ℃ for 20min to inactivate the Klenow fragment and nt. Then 7. mu.L of the reaction product, 4. mu.L of LbCas12a (0.3. mu.M), 1. mu.L crRNA (4. mu.M), 1. mu.L RNase (10U) were mixed well and dropped onto the gold electrode.

Compared with the traditional PCR method, the detection method based on the nucleic acid amplification technology has higher sensitivity, particularly the isothermal nucleic acid amplification technology which is developed rapidly does not need precise temperature conditions, gets rid of the dependence on precise equipment, and has simpler and more convenient operation. According to the invention, signals are amplified by combining click chemistry with EXPAR, and then the signals are output by a CRISPR system. The sensitivity detection of miRNA is realized. Poor specificity: the experiment realizes the detection of specific RNA by designing the sequence specificity of click chemistry and combining with microRNA. Time consumption: the experiment can be used for rapidly detecting the microRNA in a short time by exponentially amplifying signals. The invention can stably and rapidly measure the gold electrode signal through a small electrochemical workstation, and the electrochemical signal is particularly sensitive. The signal is not required to be measured by a large instrument, and the result can be visually observed.

In conclusion, the invention provides a CRISPR/Cas12a electrochemical biosensor based on click chemistry-EXPAR, which is used for sensitive detection of miRNA. The biosensor combines the self-signal amplification of the CRISPR-Cas12a system with the click chemistry-EXPAR mediated signal conversion, realizes the multi-cycle miRNA amplification detection, and has high efficiency and high sensitivity. In consideration of the immobilization capability and expandability of click chemistry-EXPAR, the developed biosensor can also be applied to different miRNA targets, without changing the CRISPR-Cas12a component, and only a part of nucleic acid sequences of two miRNA nucleic acid probes is required to be modified. The versatility of the developed electrochemical biosensor was confirmed by using real serum samples with different concentrations of miRNA21 added. Therefore, the invention expands the application of the biosensor in the aspect of miRNA detection, and the developed electrochemical biosensor can become a valuable tool for clinical prediction and molecular diagnosis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

Fig. 1 shows a schematic diagram of electrochemical detection of miRNA mediated by criprpr-cas 12 a;

FIG. 2 shows cyclic voltammetry for the process of modifying a gold electrode, and; wherein, a: bare electrode, b: DNA-MB-MCH-cas12a modified electrode, c: DNA-MB-MCH modified electrode, d: a DNA-MB-MCH modified electrode;

FIG. 2B shows an impedance method of a process of modifying a gold electrode; wherein, a: bare electrode, b: DNA-MB-MCH-cas12a modified electrode, c: DNA-MB modified electrode, d: a DNA-MB-MCH modified electrode;

FIG. 2C shows differential pulse voltammetry for the process of modifying gold electrodes; wherein, a: blank negative control; b: crRNA-free DNA-MB-MCH-Cas12 a; c: DNA-MB-MCH; d: DNA-MB-MCH-Cas12 a;

FIG. 3 shows click chemistry of gel electrophoresis to determine ligation products; from left to right (Lanes 1-6): probe a, Probe B, miRNA21, template, Probe a + Probe B + miRNA21, Probe a + Probe B with miRNA 21; the first lane is Marker;

FIG. 4 shows the optimization of experimental conditions; wherein, fig. 4A shows Cas12a concentration; FIG. 4B shows click segment doses; fig. 4C shows nt.bbvci concentrations; FIG. 4D shows EXPAR reaction times; FIG. 4E shows crRNA concentrations; FIG. 4F shows click chemistry reaction times;

figure 5 shows the quantitative analysis of the target miRNA21 using this method; wherein, FIG. 5A shows the sensitivity of the developed electrochemical sensor for detecting miRNA 21; FIG. 5B shows a calibration graph of DPV versus peak current intensity for lg c; each miRNA21 concentration was determined three times;

FIG. 6 shows the specificity of the developed electrochemical biosensor (miRNA concentration: 10)⁹fM); error bars are the standard deviation of at least three measurements.

Detailed Description

The invention discloses an electrochemical biological sensing composition, a working solution, a sensor, a device and application thereof, and a person skilled in the art can appropriately improve process parameters for realization by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

MicroRNAs (miRNAs) play a very important role in biological processes and are used as detection markers for a variety of diseases, including neurodegenerative diseases, chronic cardiovascular diseases and cancer. Sensitive point-of-care (POC) methods are crucial for detecting mirnas. The invention introduces CRISPR/Cas12a combined with click chemistry driven exponential amplification reaction (EXPAR) into an electrochemical biosensor to detect miRNA-21. The target miRNA-21 triggers a click chemistry-exponential amplification reaction in the electrochemical biosensor to generate a large number of nucleic acid fragments, stimulates the trans-cutting capability of CRISPR-Cas12a, and cuts hairpin DNA fixed on the surface of an electrode. Under the optimal condition, the lowest detection limit of the electrochemical biosensor can reach 1 fM. Therefore, the proposed electrochemical biosensor can sensitively and efficiently detect miRNAs and may become a potential analytical tool for POC testing and on-site molecular diagnostics.

Chemical and instrument:

klenow fragment (3'-5' exo-) (5U/. mu.L), enzyme stock (100mM KPO)₄1mM DTT,0.1mM EDTA, 50% glycerol; 10 XKlenow buffer (500mM NaCl,100mM Tris-HCl,100mM MgCl)₂10mM DTT; pH 7.9(25 ℃), from Biotechnology Ltd, Shanghai, China. dNTPs (25mM) and 10 XTA (TBE) premix were purchased from Biotechnology Ltd. BbvCI (10U/. mu.L), 10 × CutSmart (100mM Mg (Ac)₂,200mM Tris-Ac,1000μg/mL BSA,500mM KAc；Hiscribe^TMT7 kit for rapid and high-yield RNA synthesis,

RNA purification kit, LbaCas12a 10 XNEB buffer 2.1(100mM MgCl)₂100mM Tris-HCl, 1000. mu.g/mL BSA,500mM NaCl; pH 7.9 at 25 ℃) obtained from New England Biotechnology co., Ltd. (MA, USA). 6-mercapto-1-hexanol and tris- (2-carboxy)Base) -phosphate hydrochloride (TCEP) was purchased from Sigma-Aldrich (st. louis, USA).

The DNA fragments used in the present invention were all synthesized by Sangon Biotech co. (table 1). Specific sequence information for all ODNs is shown in table 1. Ultrapure water (18.2 M.OMEGA.. multidot.cm) was supplied by milliq systems (Bedford, MA, USA), and all nucleic acid strands can be diluted with ultrapure water.

All electrochemical signals were measured by the CHI 660E electrochemical workstation (chenhua instrument, shanghai, china). The electrode system comprises an Ag/AgCl electrode, a platinum wire and a gold electrode as a reference electrode, an auxiliary electrode and a working electrode. Gel electrophoresis was performed using a Bio-Rad electrophoresis apparatus (USA) and visualization (Shenhua science and technology, Inc., Hangzhou, China).

TABLE 1 DNA fragments used in this study

In the electrochemical biosensing composition, the working solution, the sensor, the device and the application thereof, all the raw materials, the reagents and the device can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 synthesis of CRISPR guide RNA

A total of 2. mu. L CRISPR RNA (crRNA) template and 2. mu.L of T7 promoter (100. mu.M) were mixed with 14. mu.L of LRNase-free water and the mixture was heated to 95 ℃ for 5 minutes. Then, the temperature of the mixture was slowly lowered to 20 minutes and 25 ℃, and 10 μ L (100mm nucleotide buffer and 2 μ L T7 were mixed and added to the mixture, single-stranded DNA was degraded with DNase I, kept at 37 ℃ for 15min, and 45 μ L RNase-free water was added, then, the mixture was incubated at 37 ℃ for 16 hours to obtain a target crRNA of sufficient quality, the crRNA transcript was purified with miRcute miRNA Isolation Kit, centrifuged at 13000rpm for 1min, washed 3 times with washing buffer, and stored in an enzyme-free tube at-20 ℃.

Example 2 electrode treatment and functionalization of gold electrode sensing surfaces

First, the gold electrode was polished with 0.05% aluminum powder to achieve a mirror-like surface smoothness of the electrode surface. Next, the dust on the surface of the gold electrode was removed by ultrasonic treatment with deionized water. The gold electrode was then placed in a freshly prepared solution of piranha water (volume ratio: 3:1 sulfuric acid/hydrogen peroxide) for 30 minutes to corrode impurities, thoroughly rinsed with deionized water, and electrochemically evaluated and CV 0.5H₂SO₄Ranging from 0.2 to 1.4V and a scan rate of 50mV/s up to a stable full CV peak. The amplitude of the electrochemical impedance spectrum is 5mV, and the scanning frequency range is 0.1-10⁵Hz. In 20mM PBS (2.5mM MgCl2,50mM NaCl; pH 7.4). DNA-MB was immobilized on a gold electrode and dissolved in 10mM Tris-HCl buffer solution (containing 5mM MgCl) with 10mM TCEP₂0.5M NaCl; pH 7.4). Subsequently, 10. mu.L of DNA-MB was added to the pretreated electrode surface and incubated in the dark at room temperature for 10 h. Then, the electrode was rinsed with ultrapure water, 5. mu.L of 1mM MCH solution was added to the electrode, and the electrode was soaked for 60min, and finally, the remaining portion of the electrode surface was completely removed with ultrapure water for subsequent operations.

EXAMPLE 3 preparation of Polyacrylamide gel electrophoresis

A 15% native polyacrylamide gel was prepared and the ligation product of click chemistry was determined. Electrophoresis was performed using 1 XTBE buffer (89mM triboric acid, 2mM EDTA; subsequently, the gel was stained with 10 XSSYBR Green II and visualized using a gel imaging system.

Example 4 detection of MicroRNA

Different concentrations of microRNAs and ODNs (containing Probe A and Probe B), Probe A and Probe B (each 1. mu.M), Probe A modified Aza-DBCO. Probe B modified N3 and maintained at 37 ℃ for 60 minutes. In the presence of miRNA21, Probe A and Probe B are joined into one nucleic acid strand. Then, 6. mu.L of the ligation product and 1. mu.L of 10. mu.M primer were incubated in 4. mu.L of buffer at 95 ℃ for 5 minutes. Then slowly cooled to 25 degrees C, for more than 20min, and continue to add 4U L25 mM dNTPs, 0.5L 5U/L Klenow fragment. 0.5 μ L of 10U/. mu.L Nt. BbvCI enzyme was added to the mixture. ddH2O was added to bring the final volume of the mixture to 20. mu.L. The reaction system was incubated at 37 ℃ for 80min and then at 80 ℃ for 20min to inactivate the Klenow fragment and nt. Finally, the reaction product was mixed well with 7. mu.L of LbCas12a (0.3. mu.M), 4. mu.L of crRNA (4. mu.M) and 1. mu.L of RNase (10U), and the mixture was dropped onto a gold electrode.

Effect example 1

As shown in fig. 1, the detailed workflow of CRISPR/Cas sensor was demonstrated with miRNA21 as the target template. miRNA21 can link and facilitate click chemistry ligation reactions between ODNs (including Probe a and Probe B) by complementary hybridization. Each miRNA21 serves as a bridge to connect ODNs (containing Probe a and Probe B) to create a 5'-3' external replication template. When the template strand is bound to the primer, DNA polymerization is initiated in the presence of dNTPs. Then, two cleavage sites recognized by the added nt. One is used for starting the CRISPR system, and the other can continue bridging the click chemistry reaction and then enter the next group of circulation to achieve the amplification purpose.

Effect example 2 electrochemical characterization of the biosensor according to the present invention

Due to the high-efficiency electron transfer capacity of the surface of the electrochemical biosensor, a pair of clear redox peaks can be observed on the pretreated bare gold electrode. And after the DNA-MB is fixed on the surface of the gold electrode, the electron transfer is obviously inhibited, and the oxidation reduction peak is obviously reduced. In addition, addition of MCH blocks the remaining active sites, further reducing CV signal. In contrast, after CRISPR/Cas12A cleavage, the modified MB-DNA separated from the electrode and the electroactive site was again exposed, resulting in an increase in the redox peak (fig. 2A). FIG. 2 shows a reaction solution in [ Fe (CN)6]^3-/4Results of EIS in solution. The semi-circular diameter increases with stepwise modification of the DNA-MB resistance compared to a bare electrode. After MCH surface blocking, the resistance of the DNA-MB-MCH modified electrode is further increased due to the increase of electron transfer resistance. As expected, the resistance response curve of the DNA-MB-MCH modified electrode is reduced after shearing by CRISPR/Cas12 a. These phenomena indicate the success of CRISPR/Cas12a biosensor preparation (fig. 2B), and show the feasibility of using this sensor under different conditions. MB acts as a signal output tag, causing a strong DPV response, resulting in a strong peak current, which is caused byIn the case where a crRNA binding initiation system is required; in contrast, in the absence of crRNA, there was essentially no change in signal. Furthermore, the absence of Cas12a has a negligible effect on DPV signals (fig. 2C).

Effect example 3 Polyacrylamide gel electrophoresis

Whether miRNA could link Probe a and Probe B was detected using 15% polyacrylamide gel electrophoresis (fig. 3). As shown in fig. 3, columns 1-3 represent probe a, probe B, and miRNA21, respectively; track 4 as template (base sequence of Probe A and Probe B); lane 5 is the connection of Probe A, Probe B and miRNA 21; lane 6 shows Probe a and Probe B are free of miRNA 21. As can be seen from the figure, the bands are in line with the template, confirming that Probe A and Probe B are linked together in the presence of miRNA.

Effect example 4 optimization of experiment conditions

In order to improve the detection performance of the electrochemical biosensor, 6 parameters influencing the detection reaction are optimized to different degrees: (A) CRISPR/Cas12a concentration, (B) Klenow fragment (3'-5' exo-) concentration; (C) BbvCI concentration; (D) EXPAR reaction time; (E) the crRNA concentration; (F) click chemistry reaction time. As shown in fig. 4, the signal intensity change (Δ I%) is calculated by the formula of Δ I% — background signal-target signal)/background signal) × 100%. As can be seen from fig. 4A, the cleavage efficiency of CRISPR/Cas12a increases with increasing concentration of CRISPR/Cas12 a. Therefore, to save costs, we chose 0.2 μ M as the optimal concentration for CRISPR/Cas12 a. Klenow fragment and nt. The CRISPR/Cas12a is sheared by 4U/. mu.L Klenow fragment, and the shearing efficiency is highest. With respect to the nt.bbvci concentration, Δ I% gradually increased with increasing concentration of nt.bbvci, peaking at 4U/μ L of nt.bbvci (fig. 4B). Therefore, 5U/μ lnt. bbvci was selected as the optimal concentration for this study (fig. 4C). When the EXPAR reaction time is increased from 20min to 40min, the shearing efficiency of CRISPR/Cas12a is rapidly improved; but the cleavage efficiency remained relatively stable with further increase in reaction time. Therefore, 40min was chosen as the optimal EXPAR reaction time (fig. 4D). Studies of the effect of 6 different crRNA concentrations (50, 100, 150, 200, and 250nM) showed that the cleavage efficiency of the CRISPR/Cas12a system remained stable with increasing crRNA concentration (fig. 4E). miRNA, Probe a and Probe B also influence Δ I% by click chemistry reaction time, Δ I% increasing with increasing click chemistry reaction time (fig. 4F).

Effect example 5 quantification of miRNA21 target

To test the ability of this method to detect miRNA21 under optimal experimental conditions, a series of solutions containing different concentrations of miRNA21 were investigated. The peak current induced by MB varied with increasing miRNA21 concentration. Decreasing Δ I% concentration progressively decreased miRNA21 (10)⁰，10¹，10³，10⁵，10⁷，10⁹And blank control), indicating a correlation between the concentrations of one sensitivity-responsive miRNA21 and the first signal (fig. 5b) and the linear equation can be given as follows: Δ I% ═ 7.888 × 30.24X + (R2 ═ 0.9869) detection limit 1fM (fig. 5 b). These results indicate that the developed electrochemical sensor has good detection sensitivity to miRNA21, compared to some previously reported methods.

TABLE 2 FIG. 5 corresponding tables

10⁹ fM	10⁷ fM	10⁵ fM	10³ fM	10¹fM
					99.7	86	67.7	53	43.2
99.6	86.7	68.8	54	41.9
					99.7	89.2	70.3	55	43.1

Effect example 6 specificity

To evaluate the selectivity and specificity of electrochemical biosensors, mirnas of different degrees of mutation, including miRNA21-1 (10), were used under the same reaction conditions⁹fM)、miRNA21-2(10⁹fM)、miRNA21-3(10⁹fM)、miRNA21-4(10⁹fM) and miRNA141 (10)⁹fM). As shown in fig. 6, miRNA21 showed a strong response throughout the experiment with a decrease in DPV signal and an increase in Δ I%. Whereas the DPV signals of miRNA141, miRNA21-1, miRNA21-2, miRNA21-3 and miRNA21-4 were not significantly changed, Cas12a did not have any reaction and was almost the same as the blank control. These results indicate that the developed electrochemical biosensor has high specificity and effectiveness.

TABLE 3 specificity FIG. 6 corresponding table

MiRNA141	MiRNA21-1	MiRNA21-2	MiRNA21-3	MiRNA21-4	control	MiRNA21
							28.4	26.6	24.3	24.3	20.8	22.9	99.7
26.6	26.3	25.7	24.3	23.1	19.2	99.6
							29.5	22.9	27.1	22.9	25.7	19.3	99.7

Effect example 7 recovery experiment

The developed biosensor platform was verified by a recovery testAnd its application potential and reliability in nucleic acid detection. Briefly, different concentrations of miRNA21 (10) were used⁷、10⁵And 10³fM) was added to a 10-fold dilution of the real biological serum sample and detected using the developed electrochemical biosensor. As shown in Table 4, 10 was added⁷,10⁵And 10³The sample labeling recovery rates of fM miRNA21 are respectively 98.2%, 97.3% and 100.9%, which indicates that the developed biosensor can effectively detect miRNA21 in complex environment and different biological samples.

TABLE 4 detection of miRNA in real serum samples by developed biosensors

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Claims

1. An electrochemical biosensor composition, comprising: azide-modified ODNs probes, DBCO-modified ODNs probes, primers, dNTPs, Klenow fragment, nt.bbvci, LbCas12a, and CRISPRRNA.

2. Use of the electrochemical biosensor composition according to claim 1 for preparing a working fluid for electrochemical biosensors or electrochemical biosensors.

3. The working solution of an electrochemical biosensor, comprising the electrochemical biosensor composition according to claim 1 and an acceptable adjuvant or auxiliary agent.

4. Use of the working fluid of an electrochemical biosensor according to claim 3 for the preparation of an electrochemical biosensor.

5. An electrochemical biosensor comprising the electrochemical biosensor composition according to claim 1 or the working solution of the electrochemical biosensor according to claim 3, and an electrode system.

6. The electrochemical biosensor of claim 5, wherein the electrode system comprises a reference electrode, an auxiliary electrode, and a working electrode;

the reference electrode comprises an Ag/AgCl electrode; and/or

The auxiliary electrode comprises a platinum wire; and/or

The working electrode comprises a gold electrode.

7. Use of the electrochemical biosensor according to claim 5 or 6 for the preparation of a device for nucleic acid detection, environmental pollution monitoring, food safety detection or disease diagnosis.

8. The use of claim 7, wherein said nucleic acids include, but are not limited to, microRNAs.

9. The use of claim 7, wherein the disease includes, but is not limited to: one or more of a neurodegenerative disease, a chronic cardiovascular disease, or cancer.

10. A method for detecting a nucleic acid, which comprises mixing a nucleic acid to be detected with the electrochemical biosensor composition according to claim 1 or the working solution of the electrochemical biosensor according to claim 3, and placing the mixture on the working electrode of the electrochemical biosensor according to claim 5 or 6 for detection.