A multi-channel electrochemical biosensor based on CRISPR / Cas12a and a breast cancer miRNA detection method

Abstract

The application discloses a CRISPR / Cas12a-based multi-channel electrochemical biosensor and a breast cancer miRNA detection method, wherein the CRISPR / Cas12a-based multi-channel electrochemical biosensor comprises a portable multi-channel electrochemical detection host and a functionalized modified sensing electrode; the application has high detection sensitivity; through a two-stage signal amplification strategy of rolling circle amplification pre-amplification and CRISPR / Cas12a cutting, low-abundance miRNA is converted into a large amount of detectable signals, the detection sensitivity is significantly improved, the extremely low-abundance target molecules in early-stage patient serum can be captured, and the early-stage tumor diagnosis demand is met; the application has excellent detection specificity; the sequence complementary pairing of a padlock probe and the precise target recognition of CRISPR / Cas12a form double specificity guarantee, homologous miRNA sequences can be effectively distinguished, the false positive rate is significantly reduced, and the diagnosis accuracy is improved.

Description

Technical Field

[0001] This invention relates to the field of biosensing and detection technology, specifically to a multi-channel electrochemical biosensor based on CRISPR / Cas12a and a method for detecting breast cancer miRNA. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women, and early diagnosis is crucial for improving patient survival rates. Liquid biopsy technology based on circulating microRNAs (miRNAs) in the blood is considered a highly promising direction for non-invasive early diagnosis.

[0003] However, existing clinical testing technologies have significant limitations, making it difficult to meet the needs of early screening and accurate diagnosis. Specifically, existing technologies have the following shortcomings: First, it is highly invasive and has poor compliance. Clinical diagnosis relies on tissue biopsy, which carries risks of bleeding and infection, making it unsuitable for large-scale early screening and long-term follow-up.

[0004] Second, traditional biomarkers are inadequate. Commonly used serum biomarkers such as carcinoembryonic antigen and carbohydrate antigen 15-3 have low sensitivity and specificity, which cannot meet the needs of early diagnosis.

[0005] Third, nucleic acid testing relies on large-scale equipment. Quantitative polymerase chain reaction is currently the mainstream method for miRNA detection, but it requires precise temperature control instruments, has a complicated operation process, and a long testing cycle, making it difficult to adapt to primary healthcare and point-of-care testing scenarios.

[0006] Fourth, existing CRISPR detection methods have limitations. Most CRISPR nucleic acid detection technologies rely on fluorescence signal readout, which results in high equipment costs and makes it difficult to achieve simultaneous detection of multiple targets.

[0007] Fifth, low-abundance targets are difficult to detect. The abundance of miRNA in serum is extremely low, and a single technology cannot simultaneously achieve both sensitivity and specificity. There is a lack of a systematically optimized integrated portable detection solution that combines amplification, recognition, and readout.

[0008] Therefore, developing a non-invasive, ultrasensitive, highly specific, and portable multi-target simultaneous detection technology is of great clinical value and social significance for early breast cancer screening.

[0009] Based on this, a multi-channel electrochemical biosensor based on CRISPR / Cas12a and a method for detecting breast cancer miRNA were designed. Summary of the Invention

[0010] The purpose of this invention is to provide a multi-channel electrochemical biosensor based on CRISPR / Cas12a and a method for detecting breast cancer miRNA, so as to solve the problems mentioned in the background art.

[0011] To achieve the above objectives, the present invention provides the following technical solution: A CRISPR / Cas12a-based multichannel electrochemical biosensor includes a portable multichannel electrochemical detection host and functionalized sensing electrodes; The portable multi-channel electrochemical detection host adopts a time-division multiplexing circuit design, integrating multiple independent three-electrode systems. Each three-electrode system includes a working electrode, a reference electrode, and a counter electrode, forming a closed-loop programmable electrochemical measurement platform. It has a built-in microcontroller unit that supports various electrochemical detection technologies such as square wave voltammetry, cyclic voltammetry, and electrochemical impedance spectroscopy. It is equipped with a self-calibration algorithm to eliminate interference from temperature drift and electrolyte fluctuations in real time. In the functionalized sensing electrode, the working electrode is a pure gold electrode, on which a methylene blue-labeled single-stranded DNA reporter probe is immobilized by thiol-gold self-assembly, and non-specific adsorption sites are blocked by 6-mercapto-1-hexanol to form a stable and uniform sensing interface.

[0012] Preferably, the method for preparing the sensing electrode includes the following steps: S1. Probe pretreatment: Mix 100mM tris(2-carboxyethyl)phosphine with 2μM methylene blue labeled single-stranded DNA reporter probe at a volume ratio of 2:3 and incubate at room temperature in the dark for 1h to inhibit the self-crosslinking and aggregation of thiol DNA. S2. Probe fixation: Take 6 μL of the above mixture and drop it onto the clean pure gold electrode surface. Incubate at room temperature in the dark for 1 hour to fix the probe to the electrode surface through thiol-gold covalent bonds. S3. Site blocking: Rinse the electrode with 20mM Tris-HCl buffer, dry it with inert gas, add 6μL of 1mM 6-mercapto-1-hexanol solution, and incubate at room temperature in the dark for 60min to block the unbound active sites on the electrode surface. S4. Post-processing: Rinse the electrode again with the above Tris-HCl buffer solution, dry it with inert gas, and set it aside for later use.

[0013] Preferably, the portable multi-channel electrochemical detection host detects the oxidation peak current of methylene blue in the potential range of -0.7V to 0V using square wave voltammetry. The peak current of the target-free system is used as the reference current, and the peak current of the target-containing system is used as the detection current. The current difference is calculated, and the current difference is linearly related to the target miRNA concentration within the detection range.

[0014] A CRISPR / Cas12a-based method for detecting breast cancer miRNAs includes the following steps: S1. Sample pretreatment: Collect 2 mL of peripheral venous blood from the subject, centrifuge at 1000 r / min for 15 min to separate the serum, extract total miRNA from the serum using a miRNA extraction kit, and store at -80℃ for later use. S2. Rolling Circulation Amplification Reaction: Locking probes were designed for the target miRNAs. The target miRNAs, locking probes, and 10×T4 DNA ligase buffer were mixed and heated at 95℃ for 5 min, then slowly cooled to room temperature. T4 DNA ligase was added, and the mixture was incubated at 23℃ for 2 h to ligate the ends of the locking probes and form a complete circular template. The circularized product was taken, and Phi29 DNA polymerase, deoxyribonucleoside triphosphate, 10× reaction buffer, and bovine serum albumin were added. The mixture was incubated at 30℃ for 120 min to generate a long DNA product containing a large number of repetitive target sequences. S3, CRISPR / Cas12a complex incubation: The rolling circle amplification product was mixed with LbaCas12a protein, corresponding crRNA, and 10×NEBuffer r2.1 buffer. The molar ratio of Cas12a to crRNA was 1:2. The mixture was incubated at 37°C in the dark for 120 min. When the target sequence is present, crRNA guides Cas12a to specifically recognize the target sequence and activate its trans-cleavage activity. S4. Electrochemical signal readout: 10 μL of the incubated reaction solution was dropped onto the surface of the multi-channel working electrode. After reacting at 37°C in the dark, the methylene blue oxidation peak current was recorded using square wave voltammetry within a potential range of -0.7V to 0V. The peak current of the target-free system was used as the reference current, and the peak current of the target-containing system was used as the detection current. The current difference was calculated. The current difference showed a linear relationship with the target miRNA concentration within the detection range, thereby achieving quantitative detection of the target nucleic acid.

[0015] Preferably, the target miRNA includes three breast cancer-related biomarkers: miRNA-21, miRNA-195, and miRNA-let-7a, and the simultaneous detection of the three target miRNAs is achieved through a multi-channel design.

[0016] Preferably, the method further includes a detection condition optimization step, in which the key parameters of the system are systematically optimized using a single-factor variable method to determine the optimal reaction conditions. These key parameters include: for the rolling circle amplification system, the reaction temperature is 30℃, the reaction time is 120 min, the Phi29 DNA polymerase concentration is 1 U / μL, and the deoxyribonucleoside triphosphate concentration is 2.0 mM; for the CRISPR system, the Cas12a to crRNA molar ratio is 1:2, the Cas12a reaction concentration is 100 nM, the reaction time is 120 min, and the reaction temperature is 37℃; for the electrode-modified methylene blue-labeled single-stranded DNA probe, the concentration is 2.0 μM, the 6-mercapto-1-hexanol concentration is 1.0 mM, and the 6-mercapto-1-hexanol incubation time is 60 min; and for the electrochemical detection, the square wave voltammetry has an amplitude of 50 mV, a frequency of 50 Hz, and an increment potential of 8 mV.

[0017] Preferably, the method further includes a selective verification step, in which a blank group and non-target miRNA sequences are selected as interfering substances for selective experiments. The concentration of all interfering substances is 20 times higher than the concentration of the target miRNA to verify the selective recognition ability of the detection system for the target miRNA.

[0018] Preferably, the method further includes a spiked recovery experiment step, in which different concentrations of standards are added to real serum samples, the concentrations are measured using corresponding sensors, the spiked recovery rate is calculated according to a formula, and the quantitative accuracy of the detection system in actual biological samples is verified.

[0019] Preferably, the method further includes a clinical sample testing step, in which pathologically confirmed breast cancer patients and healthy controls are selected, the clinical information of the patient group is complete, and those with other tumors, organ dysfunction or who have not signed informed consent are excluded; 2 mL of peripheral blood of the subjects is collected and the expression levels of miRNA-195, miRNA-21 and miRNA-let-7a are simultaneously detected by CRISPR electrochemical detection of serum samples, and the ability of the detection system to distinguish positive and negative samples in clinical samples is statistically analyzed to determine the detection efficacy.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention has high detection sensitivity. Through a two-stage signal amplification strategy of rolling circle amplification pre-amplification and CRISPR / Cas12a cleavage, low-abundance miRNAs are converted into a large number of detectable signals, which significantly improves the detection sensitivity. It can capture target molecules with extremely low abundance in the serum of early-stage patients, which is suitable for the needs of early tumor diagnosis. 2. This invention has excellent detection specificity. The sequence complementary pairing of the lock probe and the precise target recognition of CRISPR / Cas12a form a dual guarantee of specificity, which can effectively distinguish homologous miRNA sequences, significantly reduce the false positive rate, and improve diagnostic accuracy. 3. This invention has strong multi-target simultaneous detection capability. The multi-channel electrochemical design can simultaneously detect multiple breast cancer-related miRNA biomarkers. The combined diagnostic model greatly improves diagnostic accuracy and effectively solves the problem of high single-target missed diagnosis rate. 4. This invention is highly portable and versatile. The detection device is small and portable, requiring no large laboratory instruments or precision temperature control equipment, and can be adapted to scenarios such as primary hospitals and community health centers, supporting large-scale non-invasive early screening for breast cancer; 5. This invention is non-invasive and has high compliance, requiring only a small amount of peripheral blood to complete the test, avoiding the invasive risks and pain of tissue biopsy, improving screening compliance in high-risk groups, and is suitable for long-term follow-up monitoring; 6. The present invention has a lower detection cost, and the reagent and equipment costs are significantly lower than those of traditional quantitative polymerase chain reaction and fluorescence CRISPR detection, which is conducive to clinical promotion and large-scale population screening applications. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0022] In the attached diagram: Figure 1 This is a schematic diagram of the structure of the multi-channel electrochemical workstation of the present invention; Figure 2 Electrochemical impedance spectroscopy characterization diagrams of different modified electrodes of the present invention; Figure 3 The graph shows the optimization curves for the electrochemical detection of miRNA in this invention, where (a) is the optimized concentration of dNTPs, (b) is the optimized concentration of Phi29 DNA polymerase, (c) is the optimized incubation time of MCH, (d) is the optimized concentration of MCH, and (e) is the optimized optimal concentration of DNA probe. Figure 4 This is a graph showing the effect of different reaction times on the Cas12a trans-cleavage efficiency of miRNA-21, miRNA-195, and miRNA-let-7a according to the present invention. Figure 5 This is a curve showing the optimized Cas12a reaction concentration of the present invention; Figure 6 This is a standard curve for the electrochemical detection of miRNA in this invention; Figure 7 This is a figure showing the experimental results of electrochemical detection of miRNA interference in this invention; Figure 8 This is a comparison of the expression levels of miRNA-21, miRNA-195, and miRNA-let-7a in the serum of healthy individuals and breast cancer patients detected by the electrochemical method of this invention. Figure 9 This is a heatmap showing the expression of three miRNAs in the peripheral blood of breast cancer patients and healthy controls according to the present invention. Figure 10 This is a schematic diagram illustrating the amplification principle of the RCA system used for RNA detection in this invention; Figure 11 This is a schematic diagram of the RCA and CRISPR / Cas12a-based system for miRNA detection according to the present invention; Figure 12 This is a schematic diagram illustrating the electrode cutting principle of the present invention; Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1: Construction of a multichannel electrochemical biosensor This embodiment constructs a CRISPR / Cas12a-based multichannel electrochemical biosensor, comprising a portable multichannel electrochemical detection host and functionalized sensing electrodes.

[0025] The portable multi-channel electrochemical detection host adopts a time-division multiplexing circuit design, integrating multiple independent three-electrode systems. Each three-electrode system includes a working electrode, a reference electrode, and a counter electrode, forming a closed-loop programmable electrochemical measurement platform. The host has a built-in microcontroller unit that supports various electrochemical detection technologies such as square wave voltammetry, cyclic voltammetry, and electrochemical impedance spectroscopy. The host is equipped with a self-calibration algorithm that can eliminate environmental interferences such as temperature drift and electrolyte fluctuations in real time. The device is compact and can operate independently without large laboratory instruments, making it suitable for bedside and field testing scenarios.

[0026] In the functionalized sensing electrode, the working electrode is a pure gold electrode, on which a methylene blue-labeled single-stranded DNA reporter probe is immobilized by thiol-gold self-assembly, and non-specific adsorption sites are blocked by 6-mercapto-1-hexanol to form a stable and uniform sensing interface.

[0027] The method for preparing the sensing electrode is as follows: S1. Probe pretreatment: Mix 100mM tris(2-carboxyethyl)phosphine with 2μM methylene blue labeled single-stranded DNA reporter probe at a volume ratio of 2:3 and incubate at room temperature in the dark for 1h to inhibit the self-crosslinking and aggregation of thiol DNA. S2. Probe fixation: Take 6 μL of the above mixture and drop it onto the clean pure gold electrode surface. Incubate at room temperature in the dark for 1 hour to fix the probe to the electrode surface through thiol-gold covalent bonds. S3. Site blocking: Rinse the electrode with 20mM Tris-HCl buffer (containing 100mM sodium chloride, pH 7.4), dry it with argon gas, add 6μL of 1mM 6-mercapto-1-hexanol solution, and incubate at room temperature in the dark for 60min to block the unbound active sites on the electrode surface. S4. Post-processing: Rinse the electrode again with the above Tris-HCl buffer solution, dry it with argon gas, and set it aside for later use.

[0028] The modification process was verified using electrochemical impedance spectroscopy: the bare gold electrode exhibited the lowest electron transfer resistance and the smallest impedance spectrum semicircle diameter; after immobilizing methylene blue-labeled single-stranded DNA, the negatively charged nucleic acid strands formed an electrostatic repulsion layer on the electrode surface, significantly increasing the electron transfer resistance and the semicircle diameter; upon binding the Cas12a-crRNA complex with the target sequence, a protein-nucleic acid complex formed on the electrode surface, resulting in a moderate increase in impedance, confirming successful construction of the sensing interface and normal biorecognition response; these characterization results are as follows: Figure 2 As shown.

[0029] Example 2: Detection method for breast cancer miRNA This embodiment provides a CRISPR / Cas12a-based method for detecting breast cancer miRNAs. It utilizes a multi-channel electrochemical biosensor constructed in Example 1, with miRNA-21, miRNA-195, and miRNA-let-7a as joint detection targets. The specific steps include: S1. Sample pretreatment: Collect 2 mL of peripheral venous blood from the subject, centrifuge at 1000 r / min for 15 min to separate the serum, extract total miRNA from the serum using a miRNA extraction kit, and store at -80℃ for later use. S2. Rolling circle amplification reaction: Lock-on probes were designed for the three target miRNAs, and amplification was completed according to the following procedure: (1) Probe circularization: Mix the target miRNA, lock probe and 10×T4 DNA ligase buffer, heat at 95℃ for 5 min and then slowly cool to room temperature; add T4 DNA ligase and incubate at 23℃ for 2 h to ligate the two ends of the lock probe to form a complete circular template. (2) Rolling circle extension: Take the circularized product, add Phi29 DNA polymerase, deoxyribonucleoside triphosphate, 10× reaction buffer and bovine serum albumin, and incubate at 30℃ for 120 min to generate a long-chain DNA product containing a large number of repetitive target sequences. S3, CRISPR / Cas12a complex incubation: The rolling circle amplification product was mixed with LbaCas12a protein, corresponding crRNA, and 10×NEBuffer r2.1 buffer. The molar ratio of Cas12a to crRNA was 1:2. The mixture was incubated at 37°C in the dark for 120 min. When the target sequence is present, crRNA guides Cas12a to specifically recognize the target sequence and activate its trans-cleavage activity. S4. Electrochemical signal readout: Take 10 μL of the incubated reaction solution and drop it onto the surface of the multi-channel working electrode. After reacting at 37°C in the dark, use square wave voltammetry to detect the oxidation peak current of methylene blue in the potential range of -0.7V to 0V.

[0030] Using the peak current of the target-free system as the reference current I0 and the peak current of the target-containing system as the detection current I1, the current difference ΔI = I0 - I1 is calculated. The current difference ΔI is linearly related to the target miRNA concentration within the detection range, thereby enabling the quantitative detection of target nucleic acid.

[0031] The amplification principle of the RCA system in this detection method is as follows: Figure 10 As shown, the overall detection principle based on the RCA and CRISPR / Cas12a systems is as follows: Figure 11 As shown, the electrode cutting principle is as follows: Figure 12 As shown.

[0032] Example 3: Optimization of Detection Conditions The key parameters of the system were systematically optimized using the single-factor variable method to determine the optimal reaction conditions. Rolling circle amplification system: reaction temperature 30℃, reaction time 120 min, Phi29 DNA polymerase concentration 1 U / μL, deoxyribonucleoside triphosphate concentration 2.0 mM.

[0033] CRISPR system: Cas12a to crRNA molar ratio 1:2, Cas12a concentration 100 nM, reaction time 120 min, reaction temperature 37℃.

[0034] Electrode modification: Methylene blue labeled single-stranded DNA probe concentration 2.0 μM, 6-mercapto-1-hexanol concentration 1.0 mM, 6-mercapto-1-hexanol incubation time 60 min.

[0035] Electrochemical detection: square wave voltammetry with an amplitude of 50mV, a frequency of 50Hz, and an incremental potential of 8mV.

[0036] The optimization results of each parameter are as follows: Figure 3 , Figure 4 , Figure 5 As shown.

[0037] Example 4: Establishment of Standard Curve Under optimal detection conditions, different concentrations of miRNA-21, miRNA-195, and miRNA-let-7a were detected to determine the linear range and limit of detection for electrochemical detection; the standard curve for electrochemical detection of miRNA is shown below. Figure 6 As shown.

[0038] Example 5: Selective Validation To examine the selectivity of the detection system for target miRNAs, blank control and non-target miRNA sequences were selected as interfering substances for selectivity experiments. The concentrations of all interfering substances were 20 times higher than the target miRNA concentration during the selectivity experiments, verifying the selective recognition ability of the detection system for target miRNAs. Experimental results show that the detection system of this invention has excellent selectivity for target miRNAs and can effectively distinguish non-target sequences. The interference experiment results for electrochemical detection of miRNAs are as follows: Figure 7 As shown.

[0039] Example 6: Spiked Recovery Experiment To verify the accuracy of the detection system in quantifying target analytes in actual samples, spiked recovery experiments were conducted on miRNA-21, miRNA-195, and miRNA-let-7a. The experiments involved adding different concentrations of standards to real serum samples, measuring the concentrations using corresponding sensors, and calculating the spiked recovery rate according to the formula.

[0040] The specific experimental design is as follows: For miRNA-21, standards at three concentration levels of 0.1 fM, 1 fM, and 10 fM were added to real serum samples, and each concentration was measured in triplicate. For miRNA-195, standards at three concentration levels of 100 fM, 500 fM, and 1000 fM were added to real serum samples, and each concentration was measured in triplicate. For miRNA-let-7a, standards at three concentration levels of 10 fM, 100 fM, and 500 fM were added to real serum samples, and each concentration was measured in triplicate.

[0041] The experimental results are shown in Table 1; Table 1: Spike recovery experiments of three target miRNAs

[0042] Experimental results show that the spiked recoveries of the target analytes are all within a reasonable range, with small relative standard deviations. This fully demonstrates that the designed CRISPR electrochemical detection system has excellent detection accuracy for the three target miRNAs in the serum matrix. The method can effectively overcome interference from complex matrices and meet the requirements for quantitative analysis of actual biological samples.

[0043] Example 7: Clinical Sample Testing Patients with pathologically confirmed breast cancer and healthy controls were selected. Patients with complete clinical information were excluded, and those with other tumors, organ dysfunction, or who did not sign informed consent forms were excluded. The expression levels of miRNA-195, miRNA-21, and miRNA-let-7a were simultaneously detected in 2 mL peripheral blood samples from all subjects using CRISPR electrochemical detection. The CRISPR electrochemical detection was applied to the above-mentioned breast cancer patients and healthy controls to determine the ability of the detection system to distinguish between positive and negative samples in clinical samples. Finally, the detection results were statistically analyzed to determine the detection efficacy of the detection system, including sensitivity, selectivity, and Youden index.

[0044] The results of electrochemical detection of the expression levels of miRNA-21, miRNA-195, and miRNA-let-7a in the serum of healthy individuals and breast cancer patients are as follows: Figure 8 As shown; heatmaps of expression of three miRNAs in peripheral blood of breast cancer patients and healthy controls are shown below. Figure 9 As shown.

[0045] Example 8: Alternative Solution The target miRNA type can be replaced according to different target detectable substances, such as other miRNA markers of different cancers, pathogen nucleic acids, etc. The crRNA sequence should also be adjusted accordingly, so that this device can be applied to nucleic acid detection of other diseases.

[0046] The electrode types and report probe systems of the electrochemical detection module can adopt other mature electrochemical sensing strategies, such as using different electroactive labels or impedance detection methods.

[0047] The target and probe replacement: The target nucleic acid can be replaced according to the detection requirements, and the lock-type probe and crRNA sequence can be adjusted accordingly, so that this system can be extended to nucleic acid detection of other diseases.

[0048] The electrochemical system can be replaced by replacing the electroactive markers, such as ferrocene or ruthenium complexes, or by using other electrochemical detection modes such as impedance or amperometric methods to replace the square wave voltammetry.

[0049] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0050] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A CRISPR / Cas12a-based multi-channel electrochemical biosensor, characterized in that, Includes a portable multi-channel electrochemical detection unit and functionalized sensing electrodes; The portable multi-channel electrochemical detection host adopts a time-division multiplexing circuit design and integrates multiple independent three-electrode systems. Each three-electrode system includes a working electrode, a reference electrode, and a counter electrode, forming a closed-loop programmable electrochemical measurement platform. It has a built-in microcontroller unit that supports multiple electrochemical detection techniques, including square wave voltammetry, cyclic voltammetry, and electrochemical impedance spectroscopy; it is equipped with a self-calibrating algorithm to eliminate interference from temperature drift and electrolyte fluctuations in real time. In the functionalized sensing electrode, the working electrode is a pure gold electrode, on which a methylene blue-labeled single-stranded DNA reporter probe is immobilized by thiol-gold self-assembly, and non-specific adsorption sites are blocked by 6-mercapto-1-hexanol to form a stable and uniform sensing interface.

2. The multichannel electrochemical biosensor based on CRISPR / Cas12a according to claim 1, characterized in that, The method for preparing the sensing electrode includes the following steps: S1. Probe pretreatment: Mix 100mM tris(2-carboxyethyl)phosphine with 2μM methylene blue labeled single-stranded DNA reporter probe at a volume ratio of 2:3 and incubate at room temperature in the dark for 1h to inhibit the self-crosslinking and aggregation of thiol DNA. S2. Probe fixation: Take 6 μL of the above mixture and drop it onto the clean pure gold electrode surface. Incubate at room temperature in the dark for 1 hour to fix the probe to the electrode surface through thiol-gold covalent bonds. S3. Site blocking: Rinse the electrode with 20mM Tris-HCl buffer, dry it with inert gas, and then add 6μL of 1mM 6-mercapto-1-hexanol solution. Incubate at room temperature in the dark for 60min to block the unbound active sites on the electrode surface. S4. Post-processing: Rinse the electrode again with the above Tris-HCl buffer solution, dry it with inert gas, and set it aside for later use.

3. The multichannel electrochemical biosensor based on CRISPR / Cas12a according to claim 1, characterized in that, The portable multi-channel electrochemical detection host detects the oxidation peak current of methylene blue in the potential range of -0.7V to 0V using square wave voltammetry. The peak current of the target-free system is used as the reference current, and the peak current of the target-containing system is used as the detection current. The current difference is calculated, and the current difference is linearly related to the target miRNA concentration within the detection range.

4. A method for detecting breast cancer miRNAs based on CRISPR / Cas12a, characterized in that, Includes the following steps: S1. Sample pretreatment: Collect 2 mL of peripheral venous blood from the subject, centrifuge at 1000 r / min for 15 min to separate the serum, extract total miRNA from the serum using a miRNA extraction kit, and store at -80℃ for later use. S2. Rolling Circulation Amplification Reaction: Locking probes were designed for the target miRNAs. The target miRNAs, locking probes, and 10×T4 DNA ligase buffer were mixed and heated at 95℃ for 5 min, then slowly cooled to room temperature. T4 DNA ligase was added, and the mixture was incubated at 23℃ for 2 h to ligate the ends of the locking probes and form a complete circular template. The circularized product was taken, and Phi29 DNA polymerase, deoxyribonucleoside triphosphate, 10× reaction buffer, and bovine serum albumin were added. The mixture was incubated at 30℃ for 120 min to generate a long DNA product containing a large number of repetitive target sequences. S3, CRISPR / Cas12a complex incubation: The rolling circle amplification product was mixed with LbaCas12a protein, corresponding crRNA, and 10×NEBuffer r2.1 buffer. The molar ratio of Cas12a to crRNA was 1:

2. The mixture was incubated at 37°C in the dark for 120 min. When the target sequence is present, crRNA guides Cas12a to specifically recognize the target sequence and activate its trans-cleavage activity. S4. Electrochemical signal readout: 10 μL of the incubated reaction solution was dropped onto the surface of the multi-channel working electrode. After reacting at 37°C in the dark, the methylene blue oxidation peak current was recorded using square wave voltammetry within a potential range of -0.7V to 0V. The peak current of the target-free system was used as the reference current, and the peak current of the target-containing system was used as the detection current. The current difference was calculated. The current difference showed a linear relationship with the target miRNA concentration within the detection range, thereby achieving quantitative detection of the target nucleic acid.

5. The method for detecting breast cancer miRNA based on CRISPR / Cas12a according to claim 4, characterized in that, The target miRNAs include three breast cancer-related biomarkers: miRNA-21, miRNA-195, and miRNA-let-7a. The simultaneous detection of the three target miRNAs is achieved through a multi-channel design.

6. The method for detecting breast cancer miRNA based on CRISPR / Cas12a according to claim 4, characterized in that, The method also includes a detection condition optimization step, in which key parameters of the system are systematically optimized using a single-factor variable method to determine the optimal reaction conditions. These key parameters include: for the rolling circle amplification system, the reaction temperature is 30℃, the reaction time is 120 min, the Phi29 DNA polymerase concentration is 1 U / μL, and the deoxyribonucleoside triphosphate concentration is 2.0 mM; for the CRISPR system, the Cas12a to crRNA molar ratio is 1:2, the Cas12a concentration is 100 nM, the reaction time is 120 min, and the reaction temperature is 37℃; for the electrode-modified methylene blue-labeled single-stranded DNA probe, the concentration is 2.0 μM, the 6-mercapto-1-hexanol concentration is 1.0 mM, and the 6-mercapto-1-hexanol incubation time is 60 min; and for electrochemical detection, the square wave voltammetry has an amplitude of 50 mV, a frequency of 50 Hz, and an increment potential of 8 mV.

7. The method for detecting breast cancer miRNA based on CRISPR / Cas12a according to claim 4, characterized in that, The method also includes a selective verification step, in which blank groups and non-target miRNA sequences are selected as interfering substances for selective experiments. The concentration of all interfering substances is 20 times higher than the concentration of the target miRNA to verify the selective recognition ability of the detection system for the target miRNA.

8. The method for detecting breast cancer miRNA based on CRISPR / Cas12a according to claim 4, characterized in that, The method also includes a spiked recovery experiment step, in which different concentrations of standards are added to real serum samples, the concentrations are measured using corresponding sensors, and the spiked recovery rate is calculated according to a formula to verify the quantitative accuracy of the detection system in actual biological samples.

9. The method for detecting breast cancer miRNA based on CRISPR / Cas12a according to claim 4, characterized in that, The method also includes a clinical sample testing step, in which pathologically confirmed breast cancer patients and healthy controls are selected. The clinical information of the patient group is complete, and those with other tumors, organ dysfunction or who have not signed informed consent are excluded. 2 mL of peripheral blood is collected from the subjects, and the expression levels of miRNA-195, miRNA-21 and miRNA-let-7a are detected simultaneously by CRISPR electrochemical detection. The ability of the detection system to distinguish positive and negative samples in clinical samples is statistically analyzed to determine the detection efficacy.

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