Multi-miRNA detection electrochemical biosensor, preparation method, product and application

Abstract

The invention provides a multiple miRNA detection electrochemical biosensor, a preparation method, a product and application, and belongs to the technical field of electrochemical detection. The multiple miRNA detection electrochemical biosensor is developed on the basis of an amino-functionalized nano material or a modified avidin nano material, and a specific capture probe and a signal probe marked with different metal ions or biotin are designed; by combining electrochemical detection methods such as two-step sandwich type hybridization reaction and differential pulse voltammetry or square wave voltammetry, quantitative simultaneous detection of multiple miRNAs is realized, the detection process is simplified, the detection efficiency is improved, and the problem that multiple markers cannot be simultaneously detected by a traditional method is solved. In addition, by optimizing hybridization reaction conditions and probe design, the specificity of the sensor is improved, cross reaction is reduced, meanwhile, the stability and reproducibility of detection results are guaranteed, and finally an efficient and accurate tool is provided for clinical detection of miRNA.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of electrochemical detection, and particularly relates to a multiplex miRNA detection electrochemical biosensor, a preparation method, a product and an application. BACKGROUND

[0002] As a key endogenous non-coding RNA molecule, microRNA (miRNA) plays an irreplaceable regulatory role in core biological processes such as cell proliferation, differentiation and apoptosis, and its abnormal expression is closely related to the occurrence and development of various diseases. Therefore, the detection of miRNA is of great significance for early accurate clinical diagnosis.

[0003] However, the current miRNA detection technology has many problems and cannot meet the needs of clinical accuracy, efficiency and multi-index joint detection. The mainstream detection methods include real-time fluorescent quantitative PCR, Northern blotting, chip technology, etc.: real-time fluorescent quantitative PCR has a certain sensitivity, but it needs multiple operations such as RNA extraction, reverse transcription and PCR amplification, takes several hours, and has high requirements for personnel skills and experimental environment, which cannot be quickly detected; Northern blotting is complex to operate, requires a large amount of samples, has limited detection ability for low-abundance miRNA, and is prone to false negatives; although chip technology can theoretically detect multiple indexes, it has high cost, poor specificity, is easily interfered by cross-reaction, and its repeatability is difficult to guarantee, which limits its clinical routine application. In order to overcome the shortcomings of traditional methods, electrochemical biosensors have attracted much attention in the field of biomarker detection due to their advantages of simple operation, rapid response (completed within tens of minutes), low cost and easy miniaturization. However, the existing electrochemical biosensors still have shortcomings in detecting multiple miRNAs: first, the multi-detection capability is insufficient, which cannot simultaneously detect multiple miRNAs in a single system, and cannot meet the needs of clinical multi-marker joint analysis to improve the comprehensiveness of diagnosis; second, the sensitivity and specificity are poor, the detection lower limit of low-abundance miRNA in clinical samples is high, it is difficult to capture trace expression changes, and it is easy to cross-react with non-target molecules in complex matrix, which leads to a decrease in the accuracy of the results and cannot provide reliable basis for disease diagnosis. In terms of material application, gold nanoparticles (AuNPs) are widely used in the construction of biosensors due to their good electrical conductivity, surface plasmon resonance effect and excellent biocompatibility; polyethyleneimine (PEI) is a water-soluble polymer rich in amino groups, which can stabilize AuNPs through metal-amino complexation to avoid aggregation, and also provides abundant amino groups for probe fixation and metal ion chelation, laying an ideal material foundation for constructing a high-performance multi-detection platform. Therefore, PEI-AuNP nanocomposites show dual-function potential, which can simplify the sensor manufacturing process and realize the stable construction of aptamer conjugates and efficient chelation of metal ions through structural characteristics.

[0004] While existing technologies attempt to use nanomaterials to construct sensors for detecting biomarkers, problems still exist: First, there is a lack of targeted probe design strategies; second, the results are not standardized, making it difficult to avoid errors in sample processing and operation, thus reducing the reliability of the results; third, the sensitivity, selectivity, stability, and reproducibility are poor, failing to meet the clinical requirements for accurate detection of low-abundance miRNAs and making it difficult to adapt to the need for simultaneous detection of multiple miRNAs in disease diagnosis. In summary, existing miRNA detection technologies have four major shortcomings: First, they are complex and time-consuming, with traditional methods being cumbersome and inefficient, failing to meet the needs of rapid clinical diagnosis; second, they cannot simultaneously detect multiple biomarkers, making it difficult to achieve combined detection of multiple miRNAs and limiting the comprehensiveness of diagnostic information; third, they lack sensitivity, with limited ability to detect low-abundance miRNAs, easily leading to missed diagnoses and misdiagnoses due to missed detection of trace expression changes; and fourth, their specificity and multiplex detection capabilities need improvement, as existing electrochemical biosensors are prone to cross-reactivity and have poor specific recognition capabilities, making it difficult to achieve accurate simultaneous detection of multiple indicators. Therefore, overcoming the shortcomings of existing miRNA detection methods, such as complex operation, long time consumption, inability to simultaneously detect multiple biomarkers, and insufficient sensitivity and specificity, and designing a technical solution that can simultaneously, accurately, and with high sensitivity detect multiple miRNAs is crucial for clinical disease diagnosis. Summary of the Invention

[0005] The purpose of this invention is to provide a multiplex miRNA detection electrochemical biosensor, its preparation method, product, and application, which can realize the simultaneous detection of multiple miRNAs. The method is simple, highly sensitive, accurate, and specific, providing an efficient and accurate tool for the clinical detection of miRNAs.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a multiplex miRNA detection electrochemical biosensor, wherein the PEI-AuNPs-based multiplex miRNA detection electrochemical biosensor comprises the following structure: Substrate electrode, modification layer, and capture probe layer; The substrate electrode includes a carbon-based electrode; The modified layer includes amino-functionalized nanomaterials or nanomaterials modified with avidin; The capture probe layer includes capture probes.

[0007] Preferably, the carbon-based electrode includes one of screen-printed carbon electrode, glassy carbon electrode, and carbon paste electrode; The amino-functionalized nanomaterials include one of the following: polyethyleneimine gold nanoparticles, amino-modified silver nanoparticles, and amino-functionalized carbon nanotubes.

[0008] The present invention also provides a miRNA detection product, which includes the multiplex miRNA detection electrochemical biosensor and signal probe.

[0009] Preferably, the signal probe comprises a signal probe modified with a metal ion having a characteristic redox potential or a signal probe labeled with biotin; the metal ion includes Pb. 2+ Ag + Cd 2+ Cu 2+ One or more of them.

[0010] This invention also provides a method for preparing a multiplex miRNA detection electrochemical biosensor, comprising the following steps: After pretreating the substrate electrode, modify it with amino-functionalized nanomaterials or avidin-modified nanomaterials, then fix the capture probe to obtain the detection electrode. Integrate the detection electrode into the same sensor platform to obtain a multiplex miRNA detection electrochemical biosensor.

[0011] Preferably, the fixation conditions for the fixed capture probe are 20~30℃ for 0.5~2h.

[0012] This invention also provides a method for detecting multiplex miRNAs, comprising the following steps: (1) Design capture probes and labeled signal probes according to multiple target miRNAs; (2) The signal probe to be labeled is functionalized with carboxyl groups and labeled with metal ions with different characteristic redox potentials to obtain the signal probe; or biotin is labeled on the signal probe to be labeled to obtain the signal probe. (3) Using the above-mentioned method for preparing a multiplex miRNA detection electrochemical biosensor, the capture probe prepared in step (1) is used to design detection electrodes for multiple target miRNAs, and the detection electrodes are integrated into the same sensor platform to obtain a multiplex miRNA detection electrochemical biosensor. (4) Mix the signal probe with the sample to be tested and incubate to obtain a hybridization complex solution; (5) Drop the hybridization complex solution onto the surface of the multiplex miRNA detection electrochemical biosensor prepared in step (3), so that the target miRNA in the hybridization complex specifically binds to the corresponding capture probe in the capture probe layer, forming a sandwich structure of 'capture probe-target miRNA-signal probe', and then perform electrochemical detection. Record the peak current according to the characteristic redox potential of the metal ions to obtain the detection result.

[0013] Preferably, in step (1), the capture probe is complementary to the miRNA sequence; the signal probe to be labeled is complementary to the miRNA sequence.

[0014] Preferably, in step (4), the incubation time is 40 to 80 minutes.

[0015] Preferably, in step (5), the electrochemical detection method includes differential pulse voltammetry or square wave voltammetry.

[0016] This invention also provides the application of the above-mentioned multiplex miRNA detection electrochemical biosensor or miRNA detection product in multiplex miRNA detection.

[0017] The substrate electrode of this invention is a carbon-based electrode. Using a carbon-based electrode as the conductive substrate of the sensor has the advantages of low cost, easy mass production, and stable conductivity, providing physical support for subsequent modification layers and probe fixation.

[0018] This invention modifies the surface of a substrate electrode with amino-functionalized nanomaterials, which can obtain stable nanoparticles through metal-amino complexation. These nanoparticles have excellent conductivity and biocompatibility, and can enhance the electrochemical signal response of the electrode.

[0019] The capture probe of the present invention can be covalently fixed to the surface of the modified layer and also bind to miRNA. Through complementary base pairing, it can achieve specific recognition and preliminary fixation of target miRNA.

[0020] The signal probe of this invention is a carboxyl-functionalized signal probe, which can covalently bind to the amino group in the modified layer through the carboxyl group; at the same time, it labels different metal ions, and provides quantifiable electrochemical signals through metal ion labeling to achieve indirect detection of target miRNA.

[0021] The signal probe of the present invention is also complementary to the miRNA sequence, which can form a sandwich structure of capture probe-miRNA-signal probe.

[0022] The beneficial effects of this invention compared to the prior art are as follows: This invention develops a multiplex miRNA detection electrochemical biosensor based on amino-functionalized nanomaterials or avidin-modified nanomaterials. By designing specific capture probes and signal probes labeled with different metal ions or biotin, and combining a two-step sandwich hybridization reaction with electrochemical detection methods such as differential pulse voltammetry or square wave voltammetry, it achieves simultaneous quantitative detection of multiple miRNAs, simplifying the detection process, improving detection efficiency, and solving the problem that traditional methods cannot simultaneously detect multiple biomarkers. This invention also improves the sensor's specificity and reduces cross-reactivity by optimizing hybridization reaction conditions and probe design, while ensuring the stability and reproducibility of detection results, ultimately providing an efficient and accurate tool for the clinical detection of miRNAs.

[0023] The present invention utilizes amino-functionalized nanomaterials or avidin-modified nanomaterials, along with the signal amplification effect of metal ions and biotin labeling, to improve detection sensitivity and meet the detection requirements for low-abundance miRNAs. Simultaneously, the design of specific capture probes and optimized hybridization conditions reduce cross-reactivity, ensuring high selectivity for detection. Furthermore, the sensor exhibits good stability and reproducibility, providing an accurate and reliable tool for the clinical diagnosis of diseases such as Candida albicans infection. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The image shows the characterization results of PEI-AuNPs in Example 1 of this invention, where A is the UV-Vis spectrum of PEI, B is the UV-Vis spectrum of PEI-AuNPs, and C is the observation result of transmission electron microscopy. Figure 2 The images shown are the scanning electron microscope (SEM) analysis results in Example 1 of the present invention, where A is the SEM analysis image of the bare electrode surface and B is the SEM analysis image of the electrode surface after PEI-AuNPs deposition. Figure 3 This invention relates to the detection principle and results of the multiplex miRNA detection electrochemical biosensor based on PEI-AuNPs in Example 2 of this invention. Figure 4 The results of four repeated measurements of the sensor for miR-155 detection in Experiment Example 1 of the present invention are shown. A is a line graph of the four repeated measurements of the sensor for miR-155 detection, and B is a bar graph of the four repeated measurements of the sensor for miR-155 detection. Detailed Implementation Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] 1. Polyethyleneimine-Gold Nanoparticles (PEI-AuNPs): A composite material formed by combining polyethyleneimine (PEI) and gold nanoparticles (AuNPs). PEI is a highly branched, water-soluble polymer containing primary, secondary, and tertiary amine groups; AuNPs are gold nanoparticles with a diameter of approximately 20 nm. The two are bonded together through metal-amine complexation, exhibiting good conductivity, biocompatibility, and abundant reactive groups, making it suitable for constructing sensor modification layers.

[0031] 2. MicroRNAs (miRNAs): A class of endogenous non-coding RNA molecules that play important regulatory roles in biological processes such as cell proliferation, differentiation, and apoptosis. Abnormal expression of these microRNAs is associated with various diseases. The expression levels of miR-146a, miR-146b, and miR-155 detected in this invention may be abnormal during Candida albicans infection, and they can serve as potential biomarkers.

[0032] 3. Screen-printed carbon electrode (SPCE): A substrate electrode made of carbon as the electrode material through screen printing technology. It has the characteristics of low cost, easy mass production and stable conductivity, and is the substrate of the sensor of this invention.

[0033] 4. Capture probe (CP): A nucleic acid probe used to specifically capture target miRNAs. The CP-146a / b in this invention can simultaneously capture miR-146a and miR-146b, while CP-155 is used to capture miR-155. Target recognition is achieved through complementary base pairing.

[0034] 5. Signal probe (SP): A labeled nucleic acid probe that generates a detectable signal upon binding to the target miRNA. The SP-146b in this invention is labeled with Pb. 2+ SP-155 is marked with Ag + It is covalently fixed to the modified layer to provide a signal for electrochemical detection.

[0035] 6. Differential pulse voltammetry (DPV): An electrochemical detection method that detects the redox signal of metal ions by applying a pulse voltage and recording the change in current with potential. In this invention, it is used to detect Pb. 2+ and Ag + The peak current is used to achieve quantitative analysis of the target miRNA.

[0036] 7. Acetic acid buffer solution: The buffer solution used for electrochemical detection in this invention has a pH value of 4.5, providing a suitable chemical environment for the redox reaction of metal ions.

[0037] 8. Hybridization reaction: The process by which nucleic acid molecules form double strands through complementary base pairing. This invention uses a two-step hybridization reaction to enable the target miRNA to bind to the signal probe and the capture probe, thereby achieving target immobilization on the electrode surface.

[0038] This invention provides a method for detecting multiplex miRNAs, comprising the following steps: (1) Design capture probes and labeled signal probes according to multiple target miRNAs; (2) The signal probe to be labeled is functionalized with carboxyl groups and labeled with metal ions with different characteristic redox potentials to obtain the signal probe; or biotin is labeled on the signal probe to be labeled to obtain the signal probe. (3) Prepare “PEI-AuNPs / SPCE” modified electrodes, and select the number of modified electrodes according to the number of target miRNAs. Mix the capture probes corresponding to multiple target miRNAs that are detected on the same modified electrode in the same volume ratio (if two miRNAs are detected, the volume ratio of capture probes is 1:1, if three miRNAs are detected, it is 1:1:1, and so on), and fix them on the modified electrode. Integrate the prepared multiple modified electrodes into the same sensor platform to obtain a multiplex miRNA detection electrochemical biosensor. For example, capture probes of n miRNAs can be fixed on one modified electrode and m electrodes can be set. Then the sensor can realize the detection of m×n miRNAs.

[0039] (4) Mix the signal probe with the sample to be tested and incubate to obtain a hybridization complex solution; (5) Drop the hybridization complex solution onto the surface of the multiplex miRNA detection electrochemical biosensor prepared in step (3) for electrochemical detection, and record the peak current based on the characteristic redox potential of the metal ions to obtain the detection results.

[0040] Example 1 Example 1 of this invention provides a method for preparing a multiplex miRNA detection electrochemical biosensor based on PEI-AuNPs, the specific steps of which are as follows: (1) Modification of the substrate electrode 1) Preparation of PEI-AuNPs Soak the glassware and magnetic stir bar overnight in a mixture of nitric acid and hydrochloric acid (1:3). A 50 mL aqueous solution of HAuCl4·3H2O with a concentration of 2.5 mmol / L was prepared, and a 5 mL solution of polyethyleneimine (PEI 25kB) with a mass fraction of 10% was prepared. The two solutions were then mixed and heated to 80°C with continuous stirring for 1.5 hours. The reaction of the amino group of PEI with Au... 3+ Metal-amino complexation was used to form PEI-encapsulated AuNPs (PEI-AuNPs). During this process, the solution color changed from light yellow to deep red, indicating that PEI-stabilized gold nanoparticles (PEI-AuNPs) had been formed. The synthesized nanoparticles were then cooled to room temperature and stored at 4°C for later use. PEI-AuNPs were characterized using UV-Vis spectroscopy, and the results are as follows: Figure 1 As shown.

[0041] Figure 1The visible spectrum of PEI showed low absorbance, while PEI-AuNPs exhibited a characteristic absorption peak at 524 nm, confirming the successful synthesis of PEI-AuNPs. Further observation by transmission electron microscopy (TEM) revealed that PEI-AuNPs were spherical, uniformly dispersed in solution, with an average diameter of 20 nm. This uniform dispersion provides a stable basis for subsequent sensor construction, and the amino groups of PEI can provide abundant reaction sites. The excellent conductivity of AuNPs helps to enhance the electrochemical signal.

[0042] 2) Modification of the substrate electrode Take two screen-printed carbon electrodes (SPCE, the number of basic electrodes increases with the number of miRNAs to be detected), treat surface impurities with ethanol, rinse with ultrapure water, and dry with nitrogen (to avoid residual moisture affecting the modification).

[0043] Take 10 μL of the PEI-AuNPs solution prepared in step 1), drop it onto the pretreated SPCE surface, and let it stand and dry at 25°C for 1 hour to allow PEI-AuNPs to bind to SPCE through physical adsorption, thus obtaining the “PEI-AuNPs / SPCE” modified electrode.

[0044] (2) Immobilization of the capture probe layer Take the “PEI-AuNPs / SPCE” modified electrode prepared in step (1), add 10 μL of a 1 μM capture probe mixture dropwise to the electrode surface, with the volume ratio of different capture probes being 1:1, and fix the capture probe mixture onto the two electrodes respectively.

[0045] The interaction between gold nanoparticles and β-thiol groups was utilized to promote the coupling modification of the probe with PEI-AuNPs. After the modified electrode was thoroughly rinsed with ultrapure water, 10 μL of MCH solution was added and incubated at 25°C for 30 minutes to block non-specific binding sites. After rinsing the electrode three times with ultrapure water, it was air-dried again at 25°C to obtain a PEI-AuNPs-based electrochemical biosensor for multiplex miRNA detection, which could be stored at 4°C or used directly in subsequent experiments.

[0046] Images of the sensor fabrication process were observed using a scanning electron microscope, and the results are as follows: Figure 2 As shown.

[0047] Figure 2 The SPCE bare electrode surface is a clean and uniform granular carbon surface. After the deposition of PEI-AuNPs, the SEM images show that the SPCE electrode surface is mainly covered by dispersed PEI-AuNPs.

[0048] Example 2 Example 2 of this invention uses Candida albicans as an example, according to Figure 3 The principle shown is to detect three microRNAs: miR-146a, miR-146b, and miR-155. The specific steps are as follows: (1) Sequence design The capture probe (SEQ ID NO.4, SEQ ID NO.5) and signal probe (SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8) were designed based on the sequences shown in SEQ ID NO.1~3. The signal probe is a carboxyl-functionalized probe and is labeled with Pb using SP-146a / b. 2+ SP-155 marked Ag + (See Table 1).

[0049] Table 1 Gene and probe sequences

[0050] (2) Fabrication of a multiplex miRNA detection electrochemical biosensor based on PEI-AuNPs Using the method of Example 1, in step (2) the capture probe layer fixation step, the capture probe is fixed on the surface of the modified electrode to obtain a multiplex miRNA detection electrochemical biosensor based on PEI-AuNPs.

[0051] (3) Detection of microRNAs (miR-146a and miR-155) 1) Hybridization reaction Samples containing target microRNAs (miR-146a and miR-155) were mixed with signal probes (SP-146a and SP-155) in a buffer solution and incubated at 37°C for 60 minutes to form hybridization complexes. During this process, miR-146a and SP-146b (Pb) were... 2+ The markers form the "SP-146b / miR-146a complex" through complementary base pairing; miR-155 and SP-155 (Ag) + (The marker) forms the “SP-155 / miR-155 complex” through complementary base pairing.

[0052] The hybridization complex was then dropped onto the electrode surface of a PEI-AuNPs-based multiplex miRNA detection electrochemical biosensor and incubated at 37°C for 60 minutes to allow the complex to bind to the capture probe. The reaction was followed by rinsing with PBS buffer. During this process, the "SP-146b / miR-146a complex" bound to CP-146a / b via complementary base pairing; the "SP-155 / miR-155 complex" bound to CP-155 via complementary base pairing. Ultimately, the target miRNA was immobilized on the electrode surface through a sandwich structure of "capture probe-target miRNA-signal probe"; after the reaction, PBS was rinsed to remove unbound complexes.

[0053] DPV, by applying a pulsed voltage (potential range -0.7 to 0.2V), causes metal ions (Pb) on the electrode surface to... 2 + Ag + A redox reaction occurs, generating a characteristic peak current. Pb 2+ A peak current is generated at -0.6V, and its intensity is linearly related to the concentrations of miR-146a and miR-146b. Ag + A peak current is generated at 0.08V, and its intensity is linearly related to the concentration of miR-155.

[0054] Quantitative analysis: Based on the calibration curve of peak current and known concentration of miRNA, the levels of miR-146a, miR-146b and miR-155 in the sample were calculated, and miR-155 was used as an internal reference for standardization to improve the accuracy and reliability of detection.

[0055] 2) Electrochemical detection: The electrode immobilized with the target miRNA was placed in an acetate buffer solution at pH 4.5 (containing 10 mMPb). 2+ and 10mM Ag + The differential pulse voltammetry (DPV) method was used for detection, with a potential range of -0.7 to 0.2 V, and a value of -0.6 V (Pb) was recorded. 2 + ) and 0.08V (Ag) + The peak current at the target microRNA concentration was used to calculate the level of miR-146a or miR-146b in the sample based on the linear relationship between the peak current and the target microRNA concentration, and miR-155 was used as an internal reference for standardization.

[0056] (4) The detection of microRNAs (miR-146b and miR-155) was performed using the method in step (3), employing the sequences listed in Table 1. Among these, miR-146b and SP-146b (Pb2+ The markers form the "SP-146b / miR-146b complex" through complementary base pairing; the "SP-146b / miR-146b complex" binds to CP-146a / b through complementary base pairing.

[0057] (5) Detection principle The detection principle of miR-146a and miR-146b is as follows: Specific recognition: Because miR-146a and miR-146b have the same seed sequence and their mature sequences differ by only one base, the complementary sequence of CP-146a / b covers the common part of both, and can bind to both miRNAs simultaneously; at the same time, the sequence of SP-146b is complementary to both (SP-146b is completely complementary to miR-146b; SP-146b is complementary to miR-146a, matching by one base), forming a ternary complex of "CP-146a / b / miR-146a / SP-146b" and "CP-146a / b / miR-146b / SP-146b".

[0058] Signal generation: Pb labeled SP-146b 2+ In DPV assays, a specific current is generated at -0.6V, which indirectly reflects the levels of the two miRNAs. Because the CP-146a / b / miR-146b / SP-146b ternary complex binds more stably due to its perfect match, while CP-146a / b / miR-146a / SP-146b, having the same seed sequence but with mature sequences differing by only one base, binds slightly less strongly. This difference is reflected in the DPV data plot as a difference in the peak value at -0.6V.

[0059] The detection principle of miR-155: Specific recognition: The sequence of CP-155 is completely complementary to that of miR-155, and it specifically binds to miR-155 only; the sequence of SP-155 is complementary to that of miR-155, forming a “CP-155 / miR-155 / SP-155” complex.

[0060] Signal generation: SP-155 marked Ag + A specific current is generated at 0.08V in DPV detection, and this current quantitatively reflects the level of miR-155, such as... Figure 3 As shown.

[0061] This embodiment uses a screen-printed carbon electrode (SPCE) modified with polyethyleneimine gold nanoparticles (PEI-AuNPs) as a substrate. A two-step sandwich hybridization reaction of "specific capture probe - miRNA - signal probe" is employed to immobilize the target miRNA on the electrode surface. Differential pulse voltammetry (DPV) is then used to detect Pb at a specific potential. 2+ and Ag + The peak current was used to achieve quantitative analysis of "miR-146a and miR-155" and "miR-146b and miR-155" respectively, and miR-155 was used as an internal reference for standardization, which effectively improved the accuracy and reliability of the detection.

[0062] Advantages of substrate modification: PEI-AuNPs modified on the surface of SPCE substrates provide abundant reaction sites due to the amino-rich nature of PEI, while AuNPs enhance detection performance by leveraging their excellent conductivity and biocompatibility, thus laying a key material foundation for multiplex detection.

[0063] Application value: This sensor can accurately detect the levels of miR-146a, miR-146b and miR-155 in human serum, with high sensitivity and high selectivity. It can provide an effective tool for the clinical diagnosis of related diseases and has clear clinical application value.

[0064] Experimental Example 1 Experimental Example 1 of this invention tested the performance of the PEI-AuNPs-based multiplex miRNA detection electrochemical biosensor prepared in Example 2. The specific steps are as follows: Stability test: The sensor was stored at 4°C for a certain period of time, and tested every three days to calculate the relative standard deviation (RSD).

[0065] Reproducibility test: Repeated tests were performed on samples of the same concentration, and the RSD was calculated. Results are shown in the graph. Figure 4 As shown.

[0066] Effects: Test results show that the calibration curve has a good linear relationship, indicating that the sensor has high sensitivity; the DPV signal of the target microRNA is significantly higher than that of the interfering microRNA, indicating good selectivity; the RSD of the stability and reproducibility tests is low, confirming that the sensor has stable performance and reliable detection results, and can meet the actual detection needs.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multiplex miRNA detection electrochemical biosensor, characterized in that, The multiplex miRNA detection electrochemical biosensor includes the following structure: Substrate electrode, modification layer, and capture probe layer; The substrate electrode includes a carbon-based electrode; The modified layer includes amino-functionalized nanomaterials or nanomaterials modified with avidin; The capture probe layer includes capture probes.

2. The multiplex miRNA detection electrochemical biosensor according to claim 1, characterized in that: The carbon-based electrode includes one of screen-printed carbon electrode, glassy carbon electrode, and carbon paste electrode; The amino-functionalized nanomaterials include one of the following: polyethyleneimine gold nanoparticles, amino-modified silver nanoparticles, and amino-functionalized carbon nanotubes.

3. A miRNA detection product, characterized in that, The miRNA detection product includes the multiplex miRNA detection electrochemical biosensor and signal probe described in claim 1.

4. The miRNA detection product according to claim 3, characterized in that, The signal probe includes a signal probe modified with a metal ion having a characteristic redox potential or a signal probe labeled with biotin; the metal ion includes Pb. 2+ Ag + Cd 2+ Cu 2+ One or more of them.

5. A method for preparing the multiplex miRNA detection electrochemical biosensor according to claim 1, characterized in that, Includes the following steps: After pretreating the substrate electrode, modify it with amino-functionalized nanomaterials or avidin-modified nanomaterials, then fix the capture probe to obtain the detection electrode. Integrate the detection electrode into the same sensor platform to obtain a multiplex miRNA detection electrochemical biosensor.

6. The method for preparing the multiplex miRNA detection electrochemical biosensor according to claim 5, characterized in that, The fixation conditions for the fixed capture probe are 20~30℃ for 0.5~2h.

7. A method for detecting multiplex miRNAs, characterized in that, Includes the following steps: (1) Design capture probes and labeled signal probes according to multiple target miRNAs; (2) The signal probe to be labeled is functionalized with carboxyl groups and labeled with metal ions with different characteristic redox potentials to obtain the signal probe; Alternatively, biotin can be labeled onto the signal probe to be labeled to obtain the signal probe; (3) Using the preparation method of the multiple miRNA detection electrochemical biosensor described in claim 5, the capture probe prepared in step (1) is used to design detection electrodes for multiple target miRNAs respectively, and the detection electrodes are integrated into the same sensor platform to obtain the multiple miRNA detection electrochemical biosensor; (4) Mix the signal probe from step (2) with the sample to be tested and incubate to obtain a hybridization complex solution; (5) Drop the hybridization complex solution onto the surface of the multiplex miRNA detection electrochemical biosensor prepared in step (3), so that the target miRNA in the hybridization complex specifically binds to the corresponding capture probe in the capture probe layer, forming a sandwich structure of 'capture probe-target miRNA-signal probe', and then perform electrochemical detection. Record the peak current according to the characteristic redox potential of the metal ions to obtain the detection result.

8. The method for detecting multiple miRNAs according to claim 7, characterized in that, In step (1), the capture probe is complementary to the miRNA sequence; the signal probe to be labeled is complementary to the miRNA sequence.

9. The method for detecting multiple miRNAs according to claim 7, characterized in that, In step (4), the incubation time is 40-80 min; in step (5), the electrochemical detection method includes differential pulse voltammetry or square wave voltammetry.

10. The application of the electrochemical biosensor for multiplex miRNA detection according to any one of claims 1 to 2 or the miRNA detection product according to any one of claims 3 to 4 in multiplex miRNA detection.

Images