Programmable two-phase electrochemical biosensor and application thereof in microRNA molecular detection

Abstract

The invention provides a programmable dual-phase electrochemical biosensor and an application of the programmable dual-phase electrochemical biosensor in microRNA (Ribonucleic Acid) molecular detection. The programmable two-phase electrochemical biosensor comprises a CRISPR / Cas12a system, a double-stranded DNA amplicon for the system, and a gold electrode, the activated CRISPR / Cas12a system carries out trans-cutting on the thiolation reporter gene on the surface of the gold electrode and then extends Poly-G, and methylene blue is combined to generate a signal which can be in direct proportion to the concentration of miRNA molecules; according to the method, the signal amplification and transduction processes are separated by utilizing complementary advantages of homogeneous phase and interface reactions, the signal amplification is quickly and effectively carried out without being limited by dynamics and space of a surface system, the homogeneous phase system can realize quick molecular diffusion, unlimited enzyme-substrate interaction and a programmable reaction way, and the detection sensitivity is high. The complete amplification in the solution can effectively eliminate steric hindrance and simplify the detection work flow.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a programmable biphasic electrochemical biosensor and application thereof in microRNA detection. BACKGROUND

[0002] MicroRNA (miRNA) is a class of endogenous non-coding RNA molecules with a length of about 18-24 nucleotides, which can regulate gene expression at the post-transcriptional level. Mature miRNA binds to the 3' untranslated region (3'UTR) of the target mRNA, leading to mRNA degradation or translation inhibition. Studies have shown that the expression levels of certain miRNAs in cancer are significantly different from those in normal tissues, for example, miRNA-21 is overexpressed in prostate cancer, breast cancer and lung cancer, while let-7 miRNA family members are down-regulated in some cancers. These miRNAs may act as oncogenes or tumor suppressors in the occurrence and development of cancer, and are widely distributed in human body fluids such as blood, saliva and sweat, which can be used as important biomarkers for cancer diagnosis and prognosis.

[0003] Molecular diagnostics plays a key role in the early detection and monitoring of diseases through sensitive and specific analysis of biomolecular targets. Among various biomolecular targets, microRNA (miRNA) is a class of short non-coding RNA molecules involved in post-transcriptional gene regulation, which has become a powerful biomarker for a series of pathological conditions, including cancer, cardiovascular disease and neurodegenerative disease. Notably, miRNA-21 has attracted extensive attention due to its abnormal expression in multiple tumor types and its role in promoting tumorigenesis, making it a priority target for early cancer diagnosis.

[0004] To meet the analytical needs of miRNA detection, various strategies have been developed, including colorimetric, fluorescent and electrochemical modes. In particular, electrochemical biosensing has emerged as a leading technology due to its high sensitivity, low cost, ease of miniaturization and direct electrical signal output. This approach has significant advantages, including the ability to concentrate target recognition events or enzyme products at the electrode surface, resulting in high local signal density and enhanced detection sensitivity. In addition, the spatial confinement of the electrode interface maximizes the reduction of non-specific interactions, ensuring stable low background signals even in complex biological samples. These advantages highlight its potential for widespread application in portable diagnostics and on-site biological analysis.

[0005] In recent years, various nucleic acid amplification strategies have been integrated into electrochemical biosensing platforms, including strand displacement amplification (SDA), rolling circle amplification (RCA), hybridization chain reaction (HCR), catalytic hairpin assembly (CHA), and isothermal amplification coupled CRISPR / Cas system, greatly improving their sensitivity and target specificity. In the usual practice, these amplification reactions are usually carried out directly on the electrode surface by layer-by-layer or step-by-step decoration of multiple DNA recognition elements and amplification modules. For example, electrodes are sequentially modified with capture probes, signal sensors and amplification scaffolds to facilitate multi-step reactions in a narrow interfacial environment. This surface-limited cascade configuration can achieve ultra-sensitive detection of miRNA targets and allow precise control of molecular recognition and signal propagation, usually resulting in impressive detection limits. However, despite these advantages, amplification strategies limited to the electrode interface face several key challenges. First, the crowded and space-constrained environment of the electrode surface limits molecular accessibility and impairs enzyme kinetics, reducing overall reaction efficiency. Second, the multi-step manufacturing process usually involves repeated immobilization, hybridization and washing, which is labor-intensive, time-consuming and prone to batch-to-batch variability, affecting reproducibility. Third, limited control over probe orientation and packing density imposes limitations on amplification efficiency, and the prolonged incubation time required for surface-based reactions reduces their compatibility with time-sensitive diagnostic applications. Overall, these limitations reduce the scalability and practicality of such systems.

[0006] Based on the problems existing in the prior art, the present application provides a programmable biphasic electrochemical biosensor, a homogeneous system that can achieve rapid molecular diffusion, unrestricted enzyme-substrate interaction and programmable reaction pathways, and complete amplification in solution that can effectively eliminate steric hindrance and simplify the detection workflow, achieving ultra-sensitive detection of miRNA molecules, especially miRNA-21. SUMMARY

[0007] Therefore, in order to solve the above problems, the present application provides a programmable biphasic electrochemical biosensor and its application in microRNA molecule detection.

[0008] In order to achieve the above purpose, the present application provides a programmable biphasic electrochemical biosensor, which comprises a CRISPR / Cas12a system activated under homogeneous conditions and combined with an interface Poly-G signal transduction for detection of miRNA molecules.

[0009] Specifically, the programmable biphasic electrochemical biosensor at least comprises:

[0010] a double-stranded DNA amplicon; including a target-triggered hairpin polymerization reaction of a palindromic allosteric hairpin probe HP, to generate the double-stranded DNA amplicon;

[0011] - CRISPR / Cas12a system;

[0012] - gold electrode; a thiolated reporter gene is immobilized on the surface of the gold electrode;

[0013] Under homogeneous conditions, the double-stranded DNA amplicon-activated CRISPR / Cas12a system trans-cleaves the thiolated reporter gene, and then extends a G-rich poly-guanine Poly-G sequence on the interface of the gold electrode. The G-rich sequence binds methylene blue to generate a redox-active signal that is directly proportional to the concentration of miRNA molecules, thereby realizing the detection of miRNA molecules.

[0014] In some embodiments, the stem of the palindromic allosteric hairpin probe HP comprises 16 bp of palindromic base pairs. In the presence of miRNA-21, the two ends of the palindromic allosteric hairpin probe HP form a HP-miRNA-21 complex through base complementary pairing when forming a rigid structure.

[0015] In some embodiments, the preparation method of the palindromic allosteric hairpin probe comprises: dissolving the DNA single strand of the palindromic allosteric hairpin probe HP in TAE / Na + buffer to obtain a mixed solution; then heating the mixed solution at 95°C for 5 min, and then reducing to room temperature to form a hairpin structure, thereby obtaining the palindromic allosteric hairpin probe HP.

[0016] In some embodiments, the sequence of the DNA single strand of the palindromic allosteric hairpin probe HP is shown in SEQ. ID. NO. 1.

[0017] In some embodiments, the homogeneous interface comprises a thiolated reporter gene immobilized on a gold electrode.

[0018] In some embodiments, the immobilization method comprises: dropping the thiolated reporter gene probe diluted by TCEP buffer on the surface of the gold electrode, and assembling in the dark; after washing and air-drying, continuing to drop MCH, blocking non-specific adsorption sites in the dark, and then washing and air-drying to obtain the gold electrode with immobilized thiolated reporter gene.

[0019] Preferably, the TCEP buffer comprises: 0.1M NaCl, 1mM EDTA, 1M Tris-HCl with pH = 7.4.

[0020] In some embodiments, the hairpin polymerization reaction comprises: mixing the palindromic allosteric hairpin probe HP, 1x TE buffer, DEPC-treated water, Bsm DNAase, Bsm DNAase buffer, dNTPs, and miRNA-21, and incubating at 37°C for 1 h, followed by heating to 80°C for 10 min to terminate the polymerization reaction, to obtain reaction solution 1.

[0021] In some embodiments, the trans-cleavage comprises: mixing the reaction solution 1 and a CRISPR / Cas12a system, NE buffer TM r2.1, and DEPC water, and incubating at 37°C for 1 h to obtain reaction solution 2;

[0022] In some embodiments, the reaction solution 2 is dropped on a gold electrode on which a thiolated reporter gene is immobilized, and incubated at 30°C for 50 min for cleavage.

[0023] In some embodiments, the CRISPR / Cas12a system comprises: Cas12a, crRNA, and a ssDNA gene reporter probe.

[0024] In some embodiments, the CRISPR / Cas12a system comprises: 20 μL of reaction solution 1, 8 μL of NE buffer r2.1, 7 μL of DEPC-treated water, 1 μL of 1 μM Cas 12a, 1 μL of 1 μM crRNA, and 2 μL of 5 μM ssDNA gene reporter probe per 40 μL.

[0025] Preferably, the sequence of the crRNA is shown in SEQ. ID NO. 7.

[0026] Preferably, the sequence of the ssDNA gene reporter probe is shown in SEQ. ID NO. 4.

[0027] In some embodiments, 5 μL of the reaction solution 2 is incubated on a gold electrode.

[0028] In some embodiments, the method for immobilizing a reporter gene probe on the gold electrode comprises: dropping the thiolated reporter gene DNA diluted with TCEP buffer on the gold electrode to assemble in the dark for 30 min; after rinsing and air-drying, continue to drop MCH, block the non-specific adsorption sites in the dark for 1 h, and then clean and air-dry to obtain the gold electrode on which the reporter gene probe is immobilized.

[0029] In some embodiments, in the presence of dGTP, the gold electrode is dropped with a TdT extension solution, incubated, and a 3'-hydroxyl end initiates a terminal deoxynucleotidyl transferase (TdT)-mediated extension of poly-G to obtain a gold electrode with a G-rich sequence.

[0030] In some embodiments, the TdT extension solution comprises TdT buffer, CoCl2, dGTP, and TdT.

[0031] In some embodiments, the intercalation generates a redox-active signal comprising dropping methylene blue (MB) dilution on the gold electrode of the G-rich sequence, assembling for 10 min at room temperature in the dark, methylene blue selectively binds to the G-rich sequence, rinsing and air-drying.

[0032] As an aspect of the application, the application also provides a use of the programmable biphasic electrochemical biosensor as described above in detecting miRNA21.

[0033] As an aspect of the application, the application also provides a method for detecting miRNA21 based on the programmable biphasic electrochemical biosensor, comprising co-incubating the programmable biphasic electrochemical biosensor as described above with the target miRNA21, i.e., quantitatively detecting the miRNA21.

[0034] As an aspect of the application, the application also provides a diagnostic reagent comprising the programmable biphasic electrochemical biosensor provided in the above technical solution. Based on the precise and high-sensitivity detection of miRNA21 by the programmable biphasic electrochemical biosensor, the programmable biphasic electrochemical biosensor can be applied to a diagnostic reagent or a diagnostic drug for miRNA21-related diseases.

[0035] The application has the following beneficial technical effects:

[0036] (1) The programmable allosteric hairpin polymerization, CRISPR / Cas12a activation and TdT-mediated poly-G amplification are integrated to form an electrochemical biosensor system in the application, the signal transduction step is limited to the electrode interface, the biphasic architecture cooperatively combines the amplification efficiency of the liquid system with the sensitivity and low-noise reading realized by the electrochemical sensor, thereby ensuring spatial limitation, low background noise and high signal sensitivity, and an efficient cascade signal amplification strategy for ultra-trace molecular diagnosis is constructed, and quantitative detection of miRNA molecules is realized.

[0037] (2) The application separates the signal amplification and transduction processes by taking advantage of the complementary advantages of homogeneous (liquid phase) and interface (electrode combination) reactions, which can quickly and effectively amplify the signal without the kinetic and spatial limitations of surface systems. Unlike the surface-limited reactions in the prior art, the homogeneous system can realize rapid molecular diffusion, unrestricted enzyme-substrate interaction and programmable reaction pathways, and the amplification in solution completely eliminates spatial steric hindrance and simplifies the detection workflow.

[0038] (3) The miRNA detection method provided by the application is carried out under homogeneous conditions, all recognition and amplification processes are carried out under homogeneous conditions, so that signal amplification can be carried out quickly and effectively, and is not limited by the dynamics and space of the surface system, and only the generation of the final signal, including Cas12a-mediated thiolated DNA cleavage and poly-G synthesis, is limited to the electrode interface, and the electrode interface can ensure accurate and quantitative electrochemical reading. In addition to this spatial decoupling but functionally integrated strategy overcomes the limitations of traditional interface-level systems, while fully utilizing the advantages of liquid-phase biochemistry, the resulting biosensor exhibits excellent analytical performance, including atto-molar sensitivity, high sequence specificity and excellent reproducibility.

[0039] (4) The programmable biphasic electrochemical biosensor provided by the application provides a universal framework for precise molecular diagnosis by allowing extensive adaptability to other nucleic acid targets through the modularity of the detection platform and the programmable characteristics. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1A is a structure schematic diagram of the palindromic allosteric hairpin probe HP provided by an embodiment of the application.

[0041] Figure 1B is a principle diagram of the palindromic allosteric hairpin probe HP combined with homogeneous CRISPR / Cas12a activation in an embodiment of the application.

[0042] Figure 1C is a design principle diagram of the biphasic electrochemical biosensor for miRNA-21 detection in an embodiment of the application.

[0043] Figures 2A-2B are respectively DPV and gel electrophoresis PAGE feasibility verification diagrams of the biphasic electrochemical biosensor provided by an embodiment of the application.

[0044] Figure 3A is a CV feasibility verification diagram of the biphasic electrochemical biosensor provided by an embodiment of the application.

[0045] Figure 3B is an EIS curve feasibility verification diagram of the biphasic electrochemical biosensor provided by an embodiment of the application.

[0046] Figure 3C is a flow chart of the gradual assembly in the gold electrode surface modification process in the electrochemical biosensor in an embodiment of the application.

[0047] Figures 4A-4D are respectively four dNTP-induced DPV comparison diagrams provided by an embodiment of the application.

[0048] Figures 5A-5Dare respectively sensitivity determination graph and linear range and standard curve graph of the biphasic electrochemical biosensor provided by an embodiment of the present application.

[0049] Figures 6A-6C are respectively non-homologous miRNA specificity analysis determination graphs of the biphasic electrochemical biosensor provided by an embodiment of the present application.

[0050] Figures 6D-6F are respectively specificity analysis determination graphs of the biphasic electrochemical biosensor provided by an embodiment of the present application for base mutation miRNA-21 mutant.

[0051] Figure 7A is a schematic diagram of extraction and purification and detection of miRNA-21 in actual samples by RT-qPCR and the biphasic electrochemical biosensor provided by an embodiment of the present application.

[0052] Figures 7B-7C are respectively detection results graphs of miRNA-21 in actual samples by the biphasic electrochemical biosensor and RT-qPCR provided by an embodiment of the present application.

[0053] Figure 7D is a ROC curve graph of detection of miRNA-21 in actual samples by the biphasic electrochemical biosensor and RT-qPCR provided by an embodiment of the present application.

[0054] Figures 8A-8B are respectively fluorescence curves of trans-cleavage activity of CRISPR / Cas12a triggered by homogeneous dsDNA amplification product provided by embodiment 2 of the present application.

[0055] Figure 9A is a DPV analysis graph of the biphasic electrochemical biosensor under different Bsm DNA polymerase addition conditions provided by an embodiment of the present application.

[0056] Figure 9B is a DPV analysis graph of the biphasic electrochemical biosensor under different Bsm DNA polymerase reaction time conditions provided by an embodiment of the present application.

[0057] Figure 9C is a DPV current analysis graph of the biphasic electrochemical biosensor under different Cas12a-crRNA incubation time conditions on the electrode surface provided by an embodiment of the present application.

[0058] Figure 10 is an analysis graph of storage stability of the biphasic electrochemical biosensor evaluated for 7 consecutive days provided by an embodiment of the present application. DETAILED DESCRIPTION

[0059] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application.

[0060] The homogeneous allosteric hairpin polymerization reaction provided by the present application initiates the formation of tandem DNA scaffolds and chain extension when the target is recognized, the CRISPR / Cas12a-mediated transcleavage, the double-stranded DNA amplicon generated from the solution activates to cut the electrode-fixed reporter gene probe, the poly-G extended on the electrode surface catalyzed by terminal deoxynucleotidyl transferase (TdT), and the redox active signal generated by methylene blue (MB) intercalation.

[0061] The embodiments of the present application provide the aforementioned palindromic allosteric hairpin probe HP, and the stem of the palindromic allosteric hairpin probe HP comprises palindromic base pairs of 16 bp, and when a rigid structure is formed, the two ends of the HP can form an HP-miRNA-21 complex through base complementary pairing.

[0062] The embodiments of the present application provide a preparation method of the aforementioned palindromic allosteric hairpin probe (HP), comprising the following steps: adding a single strand of the HP and TAE / Na + buffer in a total reaction system; after denaturation at 95℃ for 5 min, naturally cooling to room temperature, and then long-term storage at-20℃.

[0063] The embodiments of the present application also provide a method for the aforementioned palindromic allosteric hairpin probe to undergo target-triggered polymerization and bidirectional chain extension to generate a double-stranded DNA (dsDNA) amplicon in a homogeneous phase, comprising the following steps:

[0064] The palindromic allosteric hairpin probe HP is provided.

[0065] The palindromic allosteric hairpin probe HP is mixed with 1x TE buffer, DEPC-treated water, Bsm DNA polymerase, Bsm DNA polymerase buffer, dNTPs and different concentrations of miRNA-21, and incubated at 37℃ for 1 h, and then heated to 80℃ for 10 min to terminate the polymerization reaction, to obtain reaction solution 1.

[0066] In some specific embodiments, the present application also provides a method for the aforementioned dsDNA to activate a CRISPR / Cas12a complex, comprising the following steps:

[0067] The reaction solution 1 and Cas 12a, crRNA, NE buffer TMr2.1, DEPC water, ssDNA-Reporter mixed, incubated at 37℃ for 1h; using F97Pro fluorescence spectrophotometer (Lengguang, Shanghai) to obtain fluorescence spectrum. The excitation wavelength is set to 490nm, and the emission spectrum is recorded in the range of 500-650nm. For performance evaluation, the peak fluorescence intensity at 520nm is used.

[0068] The embodiment of the present application also provides a method for fixing the thiolated reporter gene DNA (SH-PHO) on the gold electrode as described above, comprising:

[0069] The TCEP buffer-diluted SH-PHO is dropped on the gold electrode to assemble for 30min in the dark; after washing and drying, MCH is continuously dropped to block the non-specific adsorption sites in the dark for 1h, and then the electrode surface is washed with ddH2O and dried.

[0070] The embodiment of the present application also provides a method for cutting the thiolated reporter gene DNA (SH-PHO) fixed on the gold electrode as described above, comprising: adding Cas 12a, crRNA, NE buffer TM r2.1, DEPC water and reaction solution 1 are mixed to obtain reaction solution 2, which is dropped on the assembled gold electrode, and then incubated in a 37℃ constant temperature incubator for 50min; and then the electrode surface is washed with ddH2O and dried.

[0071] The embodiment of the present application also provides a method for exposing the 3'-hydroxyl end to initiate the deoxynucleotide transferase (TdT)-mediated poly-guanine (poly-G) extension in the presence of dGTP as described above, comprising:

[0072] The Cas 12a, crRNA, NE buffer TM r2.1, DEPC water and reaction solution 1 are mixed to obtain reaction solution 2, which is dropped on the assembled gold electrode, and then incubated in a 37℃ constant temperature incubator for 50min; and then the electrode surface is washed with ddH2O and dried.

[0073] The embodiment of the present application also provides a method for methylene blue to selectively bind to G-rich sequences to generate a strong voltammetry signal proportional to the original miRNA-21 concentration as described above, comprising:

[0074] The TdT extension solution (including: TdT buffer, CoCl2, dGTP and TdT) was added on the gold electrode, and placed in a 37℃ constant temperature oven for incubation for 30 min to extend poly-G, and then the electrode surface was rinsed with ddH2O and dried. Then, the MB diluent was added on the surface of the gold electrode, and assembled at room temperature for 10 min in the dark, and then the electrode surface was rinsed with 1*PBS (pH = 7.4) and dried.

[0075] The method for generating a strong voltammetric signal proportional to the original miRNA-21 concentration: the above-mentioned reaction completed gold electrode is placed in an electrolyte, the gold electrode is used as the working electrode, the platinum wire electrode is used as the counter electrode, and the Ag / AgCl2 electrode is used as the reference electrode, and the three-electrode system is immersed into the corresponding detection solution at room temperature for electrochemical detection. The characteristic is that the electrochemical detection differential pulse voltammetry curve (potential is 0.1 to-0.5, potential increment 0.004 V, amplitude 50 mV), the cyclic voltammetry curve (scanning parameter-0.2 to 0.6, scanning speed is 0.1 V / s) and the EIS curve.

[0076] The application provides an application of a method based on CRISPR / Cas12a driving poly-G extension in super-sensitive analysis of miRNA-21 in human whole blood RNA samples.

[0077] The miRNA extraction kit is used for miRNA extraction of fresh anticoagulant whole blood of breast cancer patients and healthy people. Reverse transcription-real-time fluorescent quantitative PCR (RT-qPCR) is carried out by using the extracted and purified miRNA, and the results show that the programmable biphasic electrochemical biosensor provided by the application has reliability in miRNA molecule detection and accuracy in trace detection.

[0078] In the homogeneous phase, the palindromic allosteric hairpin probe undergoes target-triggered polymerization and bidirectional strand extension to generate double-stranded DNA amplicon (dsDNA), and the dsDNA activates the CRISPR / Cas12a system to cut the thiolated reporter gene DNA (SH-PHO) fixed on the gold electrode; in the presence of dGTP, the exposed 3'-hydroxyl end initiates the terminal deoxynucleotidyl transferase (TdT)-mediated poly-G extension, and methylene blue selectively binds to the G-rich sequence to generate a strong voltammetric signal proportional to the original miRNA-21 concentration, and reliable detection of human blood samples is successfully realized.

[0079] In the following examples, the experimental materials used in the examples are commercially available from conventional biochemical reagent companies unless otherwise specified.

[0080] All HPLC purified DNA sequences (Table 1) are synthesized and quantified by Sangon Biotechnology Co., Ltd. (Shanghai, China).

[0081] Bsm DNA polymerase and 10x Bsm buffer (200 mM Tris-HCl (pH 8.8, 25 °C), 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% (v / v) Tween20) were purchased from Thermo Fisher Scientific (Shanghai, China); Terminal Transferase (TdT) and 10TdT buffer (50 mM Potassium Acetate, 20 mM Tris-acetate 10 mM Magnesium Acetate (pH 7.9 / 25 °C)), 2.5 mM CoCl2, NE buffer TM r2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 pg / mL Recombinant Albumin (pH 7.9 / 25 °C), LbaCas12a (Cpf1) and Lba Cas12a Diluent (500 mM NaCl, 20 mM NaOAc, 0.1 mM EDTA, 0.1 mM TCEP buffer, 50% Glycerol, pH 6 / 25 °C) were purchased from New England Biolabs (USA) Ltd. (Beijing, China); dNTPs (10 mM dATP, 10 mM dGTP, 10 mM dCTP, and 10 mM dTTP), 100 mM dGTP, N, N, N’, N’ Tetramethyl ethylene diamine (TEMED), 30% Acrylamide: Bisacrylamide (29: 1), 1x TBE (89 mM Tris-borate, 2 mM EDTA, 25 °C / pH = 8.2-8.4), 6x DNA loading buffer, 25-500 bp DNA Marker were provided by Sangon Biotech (Shanghai, China).

[0082] The gold disc electrode, counter electrode and reference electrode used in the experiment were purchased from Yuan Kang Tongfa Laboratory Equipment Trading Co., Ltd. (Tianjin, China).

[0083] The resistivity of the ultrapure water used throughout the experiment was 18.2 M Ω / cm, which was obtained from the Milli-A10 system (Mil-lipore).

[0084] Instruments

[0085] Fluorescence spectra were measured using a F97 Pro Fluorescence Spectrophotometer (Shanghai Lin-Guang Technology Co., Ltd.). Electrophoresis results were analyzed using a gel imaging system GelDoc Go and Image Lab analysis software (Bio-Rad Laboratories, Inc.). Electrochemical signals were measured using a CHI660F electrochemical workstation (Chen Hua Instruments Co., Ltd., Shanghai, China).

[0086] The technical solutions of the present application are further described in detail below through specific examples.

[0087] The sequences used in the present application are shown in Table 1.

[0088] Table 1 Sequence Listing

[0089]

[0090] Example 1

[0091] The present embodiment provides a programmable biphasic electrochemical biosensor for miRNA detection, which includes the combination of homogeneous CRISPR / Cas12a activation and interfacial Poly-G signal transduction for the ultra-sensitive analysis of miRNA.

[0092] In order to realize the programmable biphasic electrochemical detection of miRNA-21, the present application provides a layered signal amplification strategy, which integrates allosteric hairpin polymerization, CRISPR / Cas12a activation and TdT-mediated poly-G synthesis. Both hairpin polymerization and Cas12a activation occur completely in the homogeneous phase, thereby realizing efficient and rapid target amplification. In contrast, poly-G synthesis is limited to the electrode interface, where redox-active electrochemical signals are generated, completing the spatially decoupled amplification-transduction structure.

[0093] Specifically, first, the present application constructs a palindrome allosteric hairpin probe HP with a stem-loop configuration, Figure 1A The structural schematic diagram of the palindrome allosteric hairpin probe HP is shown, which has a palindrome sequence at both the 5' and 3' ends, and a loop region with a complementary sequence to the target miRNA-21, and a protospacer adjacent motif (PAM) sequence (TTTG) required for recognition by the Cas12a-crRNA complex is embedded. Upon introduction of the target miRNA-21, the hairpin probe HP is activated by the Cas12a-crRNA complex to form a complex HP*, Figure 1BAs shown, the hybridization between miRNA-21 and the loop region destroys the stem loop of HP, exposing the palindromic end, and this conformational change triggers the intermolecular base pairing between the exposed palindromic arms of the multiple HP / miRNA-21 complexes, spontaneously assembling into a long catenated DNA scaffold, which serves as a substrate for Bsm DNA polymerase to catalyze bidirectional primer extension, including (i) 5'→3' extension of the 3' end of the HP itself, and (ii) 5'→3' extension of the 3' end of the hybridized miRNA-21, forming an elongated miRNA-21 mimic strand; as the strand displacement progresses, the extended mimic is released and can reinitiate the unwinding and hybridization with a new HP molecule, thus establishing a target recycling amplification loop, this cooperative allosteric hairpin polymerization reaction generates long double-stranded DNA (dsDNA) amplicons containing two PAM flanking motifs, which can serve as binding sites for Cas12a-crRNA complex to activate the nuclease and induce its trans -cleavage activity. Further, refer to Figure 1C When the activated Cas12a is introduced into the AuE surface modified with SH-PHO and passivated with MCH, it cleaves the immobilized reporter probe, exposing multiple 3'-hydroxyl ends as the initiation sites for template-free nucleotide polymerization by TdT in the presence of deoxyguanosine triphosphate (dGTP), thereby generating poly-G strands. MB, as a redox-active dye, has a high affinity for G-rich sequences and can selectively bind to poly-G strands, generating an amplified electrochemical signal that can be detected by cyclic voltammetry. In the absence of miRNA-21, HP maintains its closed hairpin conformation, excluding loop hybridization, palindrome exposure, intermolecular assembly, and strand extension, and cannot form miRNA-21 mimics or catenated dsDNA, leaving Casl2a in an inactive state. Therefore, SH-PHO remains intact, TdT has no accessible 3'-OH end to act on, and MB cannot accumulate on the electrode, resulting in a negligible background signal. This contrast illustrates the powerful analytical capability of the biphasic electrochemical platform for molecular diagnostics, successfully enabling reliable detection of human blood samples.

[0094] Specifically, the specific steps of constructing the programmable biphasic electrochemical biosensor for detecting miRNA-21 include:

[0095] Step one, providing a palindromic allosteric hairpin probe HP.

[0096] The preparation method of the palindromic allosteric hairpin probe HP includes: adding HP single strand (the sequence is shown as SEQ. ID NO. 1) and TAE / Na+buffer buffer in the total reaction system, denaturing at 95℃ for 5min, and then naturally cooling to room temperature, and then storing at-20℃ for a long time; wherein, the TAE / Na +The buffer includes 10 mM Tris, 1 mM EDTA, 50 mM NaCl, pH = 8.0; the concentration of HP is 100 μM.

[0097] The total reaction system contains 45 μL TAE / Na + buffer and 5 μL HP single strand ((100 μM).

[0098] Step two, provide double-stranded DNA amplicon dsDNA

[0099] In a homogeneous system, the palindromic allosteric hairpin probe triggers polymerization and bidirectional chain extension reaction by the target to generate double-stranded DNA amplicon dsDNA, and the specific method comprises the following steps: mixing the palindromic allosteric hairpin probe HP, 1×TE buffer, DEPC treated water, Bsm DNA polymerase, Bsm DNA polymerase buffer, dNTPs and different concentrations of miRNA-21 to obtain a mixed system, and incubating at 37℃ for 1h, and then heating to 80℃ for 10min to terminate the polymerization reaction, to obtain reaction liquid 1.

[0100] The concentration of the palindromic allosteric hairpin probe HP is 1 μM; the amount of Bsm DNA polymerase is 2.5 U; the Bsm DNA polymerase buffer is 10×; and the concentration of dNTPs is 25 mM.

[0101] The mixed system contains 9 μL 1×TE buffer, 2 μL DEPC treated water, 2 μL palindromic allosteric hairpin probe HP (1 μM), 1 μL different concentrations of miRNA-21, 2 μL 10× Bsm DNA polymerase buffer, 2 μL dNTPs (25 mM) and 2 μL Bsm DNA polymerase (2.5 U) per 20 μL.

[0102] Step three, dsDNA activates the CRISPR / Cas12a system.

[0103] The method for activating the CRISPR / Cas12a system by dsDNA comprises the following steps: mixing reaction liquid 1 and Cas 12a, crRNA (the sequence is shown in SEQ.ID NO.7), NE buffer TMr2.1, DEPC treated water and ssDNA gene reporter probe (ssDNA Reporter, the sequence is shown in SEQ.ID NO.4) to obtain a reaction system, and incubating at 37℃ for 1h. The fluorescence spectrum is obtained by using F97Pro fluorescence spectrophotometer (Lengguang, Shanghai). The excitation wavelength is set to 490nm, the emission spectrum is recorded in the range of 500-650nm, and the peak fluorescence intensity at 520nm is used to evaluate the performance.

[0104] The concentration of Cas 12a is 1 μM; the concentration of crRNA is 1 μM; and the concentration of ssDNA-Reporter is 5 μM.

[0105] In the reaction system, 40 μL contains: 20 μL of reaction solution 1, 8 μL of NE buffer r2.1, 7 μL of DEPC treated water, 1 μL of Cas 12a (1 μM), 1 μL of crRNA (1 μM) and 2 μL of ssDNA-Reporter (5 μM).

[0106] Step four, fixing thiolated reporter DNA (SH-PHO) on the gold electrode

[0107] The thiolated reporter probe SH-PHO (the sequence is shown in SEQ. ID NO. 3) diluted with TCEP buffer was added dropwise on the gold electrode to assemble for 30 min in the dark, and after washing and air-drying, 6-mercapto-1-hexanol MCH was continuously added dropwise, and the non-specific adsorption sites were blocked in the dark for 1 h. Then the electrode surface was washed with ddH2O and air-dried.

[0108] The TCEP buffer includes: 0.1M NaCl, 1mM EDTA, 1M Tris-HCl (pH=7.4); the concentration of TCEP is 9.8mM; the concentration of SH-PHO is 5μM; and the concentration of MCH is 0.04%.

[0109] Step five, cutting the thiolated reporter DNA (SH-PHO) fixed on the gold electrode

[0110] The method for cutting the thiolated reporter DNA fixed on the gold electrode includes: mixing Cas 12a, crRNA, NE buffer TM r2.1, DEPC treated water and reaction solution 1 to obtain reaction solution 2, adding the reaction solution 2 dropwise on the assembled gold electrode, and incubating in a 37℃ constant temperature incubator for 50 min, and then washing the electrode surface with ddH2O and air-drying.

[0111] The concentration of Cas 12a is 1 μM; the concentration of crRNA is 1 μM; and 38 μL of the reaction system includes: 20 μL of reaction solution 1, 8 μL of NE buffer r2.1, 7 μL of DEPC treated water, 1 μL of Cas 12a (1 μM) and 1 μL of crRNA (1 μM).

[0112] Step six, TdT mediated poly-guanine (poly-G) extension in the presence of dGTP

[0113] The exposed 3'-hydroxyl end initiates a terminal deoxynucleotidyl transferase (TdT)-mediated poly-G extension method, which comprises: incubating 5 μL of the reaction solution 2 on the gold electrode in step five, and the specific method comprises: adding the TdT extension solution on the gold electrode obtained by the reaction, and placing the gold electrode in a 37℃ constant temperature oven for incubation for 30 min to perform an extension reaction, poly-G is extended on the surface of the gold electrode, and then the electrode surface is washed with ddH2O and dried.

[0114] The TdT extension solution comprises: TdT buffer, CoCl2, dGTP and TdT, wherein the TdT buffer is 1×TdT buffer; the concentration of CoCl2 is 25 mM; the concentration of dGTP is 10 mM; and the concentration of TdT is 1 U / μL.

[0115] In the TdT extension solution, 5 μL comprises: 1.5 μL of 1×TdT buffer, 1.5 μL of CoCl2 (25 mM), 1 μL of dGTP (10 mM) and 1 μL of TdT (1 U / μL).

[0116] Step seven, poly-G is combined with methylene blue MB.

[0117] The embodiment of the application also provides that the methylene blue selectively binds to the poly-G of the G-rich sequence, and the method comprises:

[0118] 5 μL of a 50 μM MB diluent is added to the surface of the gold electrode obtained by the extension reaction in step six, the methylene blue selectively binds to the poly-G of the G-rich sequence, and the three-electrode system is assembled at room temperature for 10 min in the dark, and then the electrode surface is washed with 1×PBS (pH=7.4) and dried.

[0119] Further, the gold electrode obtained by the reaction is placed in an electrolyte, the gold electrode is used as a working electrode, a platinum wire electrode is used as a counter electrode, and an Ag / AgCl2 electrode is used as a reference electrode, the three-electrode system is immersed in a corresponding detection solution at room temperature for electrochemical detection, and the reaction product can generate a strong voltammetric signal proportional to the original miRNA-21 concentration.

[0120] The electrochemical detection includes differential pulse voltammetry curves (potential is 0.1 to-0.5, potential increment is 0.004 V, and amplitude is 50 mV), cyclic voltammetry curves (scanning parameters are-0.2 to 0.6, and the scanning speed is 0.1 V / s), and EIS curves.

[0121] Feasibility demonstration of a programmable biphasic electrochemical biosensor for detecting miRNA-21

[0122] Firstly, the trans-cleavage activity of CRISPR / Cas12a initiated by homogeneous dsDNA amplification product was detected, aiming to verify whether the dsDNA amplicon activated the CRISPR / Cas12a complex (Cas12a), which specifically included: using a fluorophore-quencher (FQ) labeled ssDNA reporter probe (ssDNA-Reporter, sequence as shown in SEQ. ID NO. 4) to carry out fluorescence-based assay.

[0123] The results are shown in Figure 8A The comparison results of the fluorescence response of the ssDNA reporter containing miRNA-21 and not containing miRNA-21 in the homogeneous polymerization reaction showed that a strong signal was only observed in the presence of the target, indicating that the solution-generated dsDNA successfully activated Cas12, that is, the presence of miRNA-21 resulted in a significant increase in fluorescence intensity, indicating that the solution-generated dsDNA amplicon successfully activated Cas12a; in contrast, in the absence of the target miRNA-21, almost no fluorescence was detected, indicating that the ssDNA reporter remained intact and Cas12a was not active.

[0124] Figure 8B The results of the reaction without adding Bsm DNA polymerase under the same other conditions are shown as a negative control, and the results show that no fluorescence signal was observed even in the presence of miRNA-21. This result indicates that the formation of dsDNA amplicon by hairpin polymerization is an important prerequisite for Casl2a activation.

[0125] The above fluorescence results verify that the homogeneous amplification reaction of the activation interface cleavage mechanism in this embodiment is successfully performed.

[0126] Secondly, the feasibility of the biphasic electrochemical sensing system was also verified, which specifically included verification experiments integrating DPV and gel electrophoresis PAGE.

[0127] As Figure 2AAs shown, only in the presence of miRNA-21, a significant current response was observed, indicating that the uniform amplification process was successfully initiated to generate dsDNA amplicon, followed by the activation of CRISPR / Cas12a. The signal increase was due to the Cas12a-mediated cleavage of surface-immobilized reporter gene, followed by TdT-mediated poly-G synthesis and MB intercalation, resulting in a strong DPV signal (curve b). In contrast, the absence of miRNA-21 failed to generate dsDNA, rendering Cas12a inactive, and the reporter probe intact, thus only producing minimal background signal (curve a). Further, this example also verified the important role of Cas12a in signal generation, under the above experimental conditions, a control experiment was performed without the addition of Cas12a-crRNA complex, and the results showed that no significant current response was detected, regardless of the presence of target miRNA-induced dsDNA amplicon (curve c and curve d). This confirmed that Casl2a is essential to initiate the downstream signal cascade, and there is no observable background leakage in the absence of Casl2a.

[0128] Further confirmation of the generation of dsDNA amplicon in solution by the results of gel electrophoresis PAGE experiment, Figure 2B The PAGE plot showing HP and miRNA-21 forming duplexes by hybridization and polymerization reaction is shown in FIG. 6, investigating the allosteric polymerization and amplification cascade being specifically activated by miRNA-21, and highlighting the important role of target recognition and enzymatic extension in driving the formation of dsDNA amplicon, as shown, in lane a, only HP was present, generating a single different band corresponding to its compact stem-loop structure; lane b contained only miRNA-21, showing a single band at different positions, representing free target strands; lane c was a mixture of HP and miRNA-21, and the results showed multiple bands of higher molecular weight, indicating that target binding induced the hairpin to unfold and expose palindromic ends, which mediated the spontaneous intermolecular assembly into long concatenated DNA scaffolds; in contrast, lane d was the result of HP co-incubated with Bsm polymerase and dNTPs but without containing target miRNA-21, and the electrophoretic pattern was the same as lane a, indicating that there was no non-specific or background polymerization during the reaction process; lane e was in a complete reaction mixture containing HP, miRNA-21, Bsm polymerase and dNTPs, and the observation of different high molecular weight bands corresponding to the 100 bp marker of dsDNA amplicon confirmed the generation of dsDNA amplicon, indicating the successful bidirectional strand extension and linker amplification, compared with lane c, the preservation of miRNA-21 band indicated the effective target release and recycling during the amplification cycle, and other slowly migrating bands might represent unresolved multimer assemblies or intermediates.

[0129] The above integrated DPV and gel electrophoresis results collectively demonstrate that the allosteric polymerization and amplification cascade is specifically activated by miRNA-21, indicating the important role of target recognition and enzymatic extension in driving the formation of dsDNA amplicons.

[0130] Thirdly, this embodiment investigates the extension function of terminal deoxynucleotidyl transferase (TdT) on short-chain oligonucleotides with 3'-hydroxyl terminal. Since the G-rich sequence generated by extension is a single-stranded structure and is difficult to be stained by 4S GelRed, this embodiment introduces a short-chain complementary sequence composed of 20 cytosine bases (C20, sequence as shown in SEQ. ID NO. 6), which realizes the visual detection of poly-G chain by forming a double-stranded structure; at the same time, this embodiment also designs a short-chain S3 containing only three nucleotides (sequence as shown in SEQ. ID NO. 5) for simulating the trans-cleavage product of Cas12a-mediated SH-PHO probe. Referring to Figure 2B , the band diagram of PAGE of TdT-mediated ploy-G extension is shown in the figure, and the experimental results show that: in lane a, no extension product is observed for the SH-PHO probe terminated by 3'-phosphate group, confirming that the polymerization of TdT must rely on the free 3'-hydroxyl terminal; lane b shows that even the S3 short chain containing only three nucleotides (with 3'-hydroxyl terminal) can still be an effective substrate for TdT-mediated poly-G extension, and the clear poly-G / C20 double-stranded signal indicates efficient poly-G synthesis. This result verifies the basic activity of TdT under the given conditions, and proves that it is suitable for surface-limited template-free polymerization reaction system.

[0131] Fourthly, this embodiment also evaluates the influence of terminal nucleotide composition on signal amplification efficiency, which specifically includes: using four types of deoxynucleotides, including dATP, dTTP, dCTP and dGTP, as the substrate for TdT-mediated template-free polymerization.

[0132] The electrochemical signal is recorded by DPV after the biphasic reaction cascade. In all cases, the homogeneous reaction phase produces dsDNA amplicons, which can activate Cas12a to cleave the SH-PHO anchored on the gold electrode, thereby exposing the 3'-hydroxyl terminal, and the exposed terminal uses a single type of dNTP as the starting site for TdT-mediated poly-N extension; then MB is added to enable DPV signal generation.

[0133] The results are shown in Figures 4A-4C , using dATP( Figure 4A ), dTTP( Figure 4B ) and dCTP( Figure 4C) respectively, not only failed to enhance the electrochemical signal, but even led to lower current in the presence of the target than in the absence of the target. This signal suppression can be caused by the elongated poly-A, poly-T and poly-C strands hindering electron transfer at the electrode interface; the corresponding current enhancement ratios (calculated as I miRNA-21 / I0) were 0.66, 0.73 and 0.51, respectively, where I miRNA-21 and I0represent the currents measured in the presence and absence of the target, respectively.

[0134] Figure 4D It is shown that the use of dGTP to synthesize poly-G results in a sharp amplification of the current, producing a signal enhancement ratio of 7.71, more than ten times higher than in other nucleotide conditions.

[0135] Fifth, the performance of the electrochemical biosensor was characterized using CV and EIS systems

[0136] The performance of the electrochemical biosensor was also characterized using CV and EIS systems in this example, and the results showed that all CV measurements were performed in 2.5 mM K3[Fe(CN)6], 2.5 mM K4[Fe(CN)6]·3H2O and 0.1 M KCl (pH 7.0, 25°C), while EIS data were recorded under the same conditions of 1.0 mM K3[Fe(CN)6], 1.0 mM K4[Fe(CN)6]·3H2O and 0.1 M KCl.

[0137] As Figure 3C is a flow chart of the gradual assembly process in the surface modification of the gold electrode in the electrochemical biosensor, the six modification stages a-f in the figure are as follows: bare AuE (a) is first surface-modified with thiolated reporter SH-PHO (curve b), followed by the addition of MCH to block non-specific adsorption sites (c), then the homogeneous reaction product produced in the presence of target miRNA-21 containing dsDNA amplicon and Cas12a-crRNA complex is co-incubated to cleave SH-PHO fixed on the gold electrode, the trans-cleavage of SH-PHO and the exposure of free 3'-hydroxyl end on the electrode (curve d), the template-independent extension of the cleaved DNA end in the presence of TdT and dGTP to produce a poly-G tail (curve e), and finally the addition of methylene blue which binds to the G structure on the poly-G tail (curve f).

[0138] In turn, based on the six stages a-f in Figure 3C CV and EIS characterization was performed, respectively, to obtain six curves a-f.

[0139] Figure 3BEIS measurements shown in FIG. 6 are the interfacial resistance changes during the fabrication process of the sensor. The Nyquist plot of bare AuE (curve a) shows a small semicircle, consistent with fast electron transfer; the charge transfer resistance Retincreases sharply after SH-PHO immobilization (curve b) and further increases after MCH passivation (curve c), after exposure to activated Cas12a and target-induced dsDNA (curve d), Retdecreases due to reporter cleavage, after poly-G elongation (curve e), the interfacial resistance continues to increase, and a further sharp decrease in Retis observed after MB binding (curve f), consistent with enhanced interfacial electron transfer via MB-mediated redox cycling.

[0140] Figure 3A The CV curves shown in FIG. 7 demonstrate that bare AuE (curve a) exhibits a well-defined pair of redox peaks with low charge transfer resistance, indicating fast electron transfer kinetics at a clean gold surface. After modification with thiolated SH-PHO, the redox peak current is significantly reduced (curve b), reflecting the formation of a negatively charged non-conductive DNA layer that introduces electrostatic repulsion and steric hindrance; subsequent blocking of the AuE surface with MCH (curve c) leads to a further decrease in current, indicating a more compact packing of the surface monolayer with reduced surface defects, which further hinders the access of redox probes to the electrode. After co-incubation with the homogeneous reaction product produced in the presence of dsDNA amplicon and Cas12a-crRNA complex of target miRNA-21, curve d, a significant increase in current is observed, and this recovery of signal is attributed to the PAM-containing dsDNA product activating Cas12a, leading to the trans-cleavage of SH-PHO and the exposure of free 3'-hydroxyl termini at the electrode; the addition of TdT and dGTP (curve e) allows the template-independent extension of the cleaved DNA termini, resulting in a poly-G tail; however, the poly-G tether further increases the interfacial resistance, resulting in a significant decrease in the CV current response due to the hindered electron transfer at the electrode surface; finally, the introduction of MB (curve f) leads to a significant increase in peak current, as MB selectively binds to the poly-G structure via π-π stacking and electrostatic interactions, and the accumulation of electroactive MB at the electrode surface significantly enhances the redox signal, thus completing the signal amplification cascade and enabling the sensitive detection of target miRNA-21 concentration.

[0141] Clearly, the CV redox peak currents and the corresponding Nyquist plots obtained from EIS provide evidence for the successful stepwise assembly of the biosensor and its robust, target-dependent modulation of electrochemical signal via the synergistic combination of uniform amplification and interfacial signal generation.

[0142] Storage stability of the programmable biphasic electrochemical biosensor for detecting miRNA-21 under test conditions for 7 consecutive days

[0143] To evaluate the storage stability of the electrochemical biosensor, a batch of modified electrodes was stored at 4°C and tested once a day for 7 days. Three representative concentrations of the target miRNA-21 (2.5 nM, 2.5 pM, 2.5 fM) were evaluated.

[0144] The results, as shown in Figure 10 , recorded DPV signals (I / I0) remained highly stable during storage, with the modified electrodes retaining more than 89.36% of the initial response after 7 days of storage at 4°C, and the relative standard deviation (RSD) was always less than 5%. These results demonstrate that the biosensor has stable performance in storage for at least a week, supporting its acceptable stability in practical applications.

[0145] Optimization of conditions

[0146] Further, in order to achieve the best analysis performance, the I miRNA-21 / I0 value was used as an evaluation index to evaluate the influence of the Bsm DNA polymerase dosage, homogeneous hairpin polymerization reaction time, and Cas12a-crRNA incubation time on the electrode surface on the electrochemical signal output.

[0147] Firstly, this embodiment provides the influence of Bsm DNA polymerase concentration on the homogeneous hairpin polymerization reaction process.

[0148] In the reaction system, the dosage of Bsm DNA polymerase was 0.5 U, 1 U, 1.5 U, 2.0 U, 2.5 U, and 3.0 U, respectively; and the results, other conditions being the same as the above experimental conditions.

[0149] The results, as shown in Figure 9A , the response value of the current signal I miRNA-21 first increased with the increase of enzyme concentration, reached the highest value when the enzyme concentration reached 2.5 U, and then the signal change was not obvious with the increase of concentration. Therefore, 2.5 U was selected as the optimal enzyme dosage for subsequent experiments.

[0150] Then, this embodiment also studied the influence of the reaction time of the homogeneous hairpin polymerization on the reaction process, and monitored the signal intensity of the current signal I miRNA-21 in turn for reaction times of 20 min, 30 min, 40 min, 50 min, 60 min, and 70 min.

[0151] The results, as shown in Figure 9B , the response intensity of the current signal I miRNA-21 tended to be stable after 60 minutes, indicating that the polymerization and chain extension were completed. Therefore, 60 minutes was selected as the optimal reaction duration for this stage.

[0152] Finally, the incubation time of the Cas12a-crRNA complex on the electrode surface was investigated, and the current signal I was monitored sequentially at incubation times of 20 min, 30 min, 40 min, 50 min, 60 min, and 70 min. miRNA-21 signal strength, Figure 9C The results showed that 50 minutes of incubation produced the highest signal, while longer incubation times did not produce further enhancement, likely due to saturation of the cleavage reaction. Therefore, 50 minutes was chosen as the optimal Cas12a activation period for the electrode interface reaction. These optimized conditions were used in all subsequent experiments to ensure high sensitivity, reproducibility, and robust signal output of the biphasic biosensor.

[0153] Sensitivity analysis

[0154] To further verify the analytical performance of the proposed biphasic electrochemical biosensor, this embodiment also evaluated its sensitivity for miRNA-21 detection. Specifically, a series of miRNA-21 samples with final concentrations increasing from 25 aM to 2.5 nM were prepared, covering more than eight orders of magnitude. (See [reference needed]). Figure 5A Electrochemical energy was measured using differential pulse voltammetry in the concentration range of miRNA-21 from 25 aM to 0.5 nM. As shown in the series of DPV curves in the figure, the current intensity increases with increasing concentration.

[0155] See Figure 5B I at different miRNA-21 concentrations miRNA-21 / I0 illustrates the intuitive visualization of the dynamic response of the sensing platform, specifically the thermal image of the current signal intensity at different concentration levels, using the current ratio I... miRNA-21 The electrochemical signal represented by / I0 gradually increases with increasing miRNA-21 concentration, indicating effective signal amplification driven by homogeneous hairpin polymerization and interfacial multi-G transduction mechanisms.

[0156] Figure 5C Statistical analysis of the sensor response to NTC for all miRNA-21 test concentrations showed that the statistical significance between each test concentration and the no-target control (NTC) was not assessed using the Student t-test. Notably, even when the miRNA-21 concentration was as low as 25 aM, the signal produced was higher than that of NTC (P < 0.0001), thus demonstrating the ultrasensitivity of the biphasic electrochemical biosensor.

[0157] Figure 5D This demonstrates the relative current signal I miRNA-21 / I0versus the logarithm of miRNA-21 concentration to construct a calibration curve, error bars represent standard deviation (n = 3), linear regression model was followed in the range of 25 aM to 2.5 nM, resulting in the equation: I miRNA-21 / I0= 0.721gC miRNA-21 + 4.41, correlation coefficient R 2 = 0.9900, proving that the biphasic electrochemical biosensor still has high sensitivity for quantitative detection and analysis at ultra-low target concentration.

[0158] The limit of detection (LOD) was calculated according to the IUPAC definition, i.e. the signal corresponding to the blank average (NTC) plus three times the standard deviation (average + 3σ). Thus, the LOD was determined to be 25 aM, demonstrating the remarkable sensitivity achieved by the synergistic cascade of uniform amplification and interfacial electrochemical signal readout. Comparative analysis showed that the sensitivity of this platform exceeds previously reported electrochemical and fluorescent miRNA biosensors, while requiring minimal reaction time

[0159] In summary, the programmable allosteric hairpin polymerization, CRISPR / Cas12a activation and TdT-mediated poly-G amplification integration provided in this embodiment constitutes an efficient cascade signal amplification strategy for ultra-trace molecular diagnosis.

[0160] Specificity analysis

[0161] To further confirm the analysis performance of the biphasic electrochemical biosensor proposed in the above scheme, this embodiment also evaluates the specificity of the miRNA-21 detection. Specifically, it includes: using non-targeted miRNAs and miRNA-21 analogs containing single or multiple base mutations to systematically evaluate the sequence specificity of the proposed biosensor.

[0162] Non-targeted miRNA evaluation includes: using structurally similar non-targeted miRNAs for interference testing.

[0163] Electrochemical reactions between miRNA-21 (2.5 pM, sequence as shown in SEQ. ID NO. 2) and five non-targeted miRNAs (each 25 pM), including miRNA-16 (sequence as shown in SEQ. ID NO. 16), miRNA-141 (sequence as shown in SEQ. ID NO. 15), miRNA-155 (sequence as shown in SEQ. ID NO. 14), miRNA-1290 (sequence as shown in SEQ. ID NO. 13) and miRNA-19a (sequence as shown in SEQ. ID NO. 14).

[0164] Reference Figure 6AThe DPV responses obtained for miRNA-21 (2.5 pM) and 5 non-target miRNAs (25 pM each), including miRNA-16, miRNA-141, miRNA-155, miRNA-1290 and miRNA-19a, showed only a clear signal for the target; Figure 6B The signal intensity ratios (I target / I0) for all 6 miRNAs tested are shown, and the results demonstrate the high selectivity of the sensor system for miRNA-21; Figure 6C The heatmap visualization of the relative signal output for all 6 miRNAs tested shows only miRNA-21 produced a significant signal increase (p<0.0001), while all non-targets produced reactions close to baseline, confirming the high sequence specificity of the recognition and amplification cascade.

[0165] Systematic evaluation of miRNA-21 analogs containing single or multiple base mutations included: a mismatch discrimination test using engineered point mutant variants of miRNA-21, including variants of miRNA-21 carrying 1-4 base mismatches, respectively, denoted as MT1 (sequence as shown in SEQ. ID NO. 8), MT2 (sequence as shown in SEQ. ID NO. 9), MT3 (sequence as shown in SEQ. ID NO. 10) and MT4 (sequence as shown in SEQ. ID NO. 11).

[0166] The 4 miRNA-21 mutants MT1-MT4 carrying 1 to 4 base mismatches were each configured at a concentration of 2.5 nM, and the test results of the mutants were compared with the test results of miRNA-21, and the results are shown in Figures 6D-6F The single base mismatch variant MT1 also produced a signal with an I mut / I0value close to 2, while MT2, MT3 and MT4, which have mutant sequences with 2, 3 and 4 mismatched bases, respectively, produced I mut / I0values all below 1.5, hardly distinguishable from the negative control.

[0167] The results of this example show that the stringent hybridization and structural specificity provides a very high fidelity of base mismatch discrimination and target recognition, supporting the potential of the platform for accurate diagnosis in complex matrices.

[0168] Practicality (sample analysis)

[0169] To evaluate the applicability of the electrochemical biosensing platform in real-world settings, this example evaluated the performance of the programmable biphasic electrochemical biosensor in human blood samples from breast cancer patients and healthy volunteers.

[0170] The whole blood miRNA extraction kit was purchased from HaiGene Biotech Co., Ltd. (product number: B1803), and the miRNA in the human blood sample in the actual sample was extracted and purified using the kit.

[0171] The RT-qPCR kit was PrimerScript™ RT reagent Kit with gDNA Eraser (Perferct Real Time), including: - RNase-free water, 5x PrimeScript buffer and PrimeScript RT Enzyme Mix I.

[0172] The RT-qPCR procedure includes: adding 4 μΐ of RNase-free water, 2 μΐ of 5x PrimeScript buffer, 0.5 μΐ of PrimeScript RT Enzyme Mix I, 1 μΐ of purified whole blood miRNA, 0.5 μΐ of miRNA-21-RT primer (sequence as shown in SEQ. ID. NO. 19) and 2 μΐ of U6-RT primer (sequence as shown in SEQ. ID. NO. 22) in a PCR tube, mixing, 37 °C for 15 min, heating to 80 °C for 5 s, and then 4 °C for infinity; adding 21 μΐ of diluted 2x Taq Pro Universal SYBR qPCR Master mixture dye (dilution ratio of DEPC-treated water: 2x Taq Pro Universal SYBR qPCR Master mixture = 5:2) in an eight-tube tube; continuing to add 2 μΐ of 10 μΜ U6 primer mix (forward primer U6-Forward prime + reverse primer U6-Reverse prime, volume ratio of 1:1, wherein the sequence of the forward primer U6-Forward prime is shown in SEQ. ID. NO. 17, and the sequence of the reverse primer U6-Reverse prime is shown in SEQ. ID. NO. 18) and 2 μΐ of 10 μΜ miRNA-21 primer mix (reverse primer miRNA-21-Reverse primer + forward primer miRNA-21-Forward primer, volume ratio of 1:1, wherein the sequence of the forward primer miRNA-21-Forward prime is shown in SEQ. ID. NO. 20, and the sequence of the reverse primer miRNA-21-Reverse prime is shown in SEQ. ID. NO. 21) in the above solution; finally adding 1 μΐ of the above reverse transcription product in the above solution, mixing and centrifuging, and placing in a qPCR instrument: pre-denaturation at 95 °C for 30 s for one cycle; then 95 °C for 5 s + 60 °C for 30 s for 40 cycles; and finally 95 °C for 15 s + 60 °C for 60 s + 95 °C for 15 s for one cycle of melting curve.

[0173] As shown in Figure 7A Total miRNA was extracted from whole blood samples and miRNA-21 levels were analyzed in parallel by the proposed electrochemical method and reverse transcription quantitative PCR (RT-qPCR) for comparison.

[0174] Figures 7B-7CThe DPV results are shown, in which a significantly increased signal intensity was detected in samples from breast cancer patients compared to healthy controls. Electrochemical signals reveal a clear distinction between disease states, with miRNA-21 being upregulated in breast cancer patients compared to healthy individuals.

[0175] Figure 7D The study showed RT-qPCR analysis, which confirmed the presence of miRNA-21(2-) in patient samples. ΔΔCt The elevated expression levels of miRNA and the high consistency between electrochemical and qPCR results validated the robustness and diagnostic reliability of the biphasic electrochemical biosensor in targeting circulating miRNAs in real biological samples.

[0176] Figure 7D The figure shows the ROC curves of the biphasic electrochemical biosensor and RT-qPCR for detecting miRNA-21 in actual samples. As can be seen from the figure, the biphasic electrochemical biosensor provided by this invention (This work in the figure) exhibits superior results compared to the existing RT-qPCR technology. The ROC area value (AUC) of this invention is 0.9956, while the ROC area value (AUC) of RT-qPCR technology is 0.9822. This result demonstrates the excellent diagnostic performance of the biphasic electrochemical biosensor provided by this invention in distinguishing between breast cancer patients and healthy individuals, making it of significant potential and application prospects in the development and application of diagnostic reagents or drugs for miRNA-21 molecule-related diseases.

[0177] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0178] The above are merely preferred embodiments of the present invention and do not limit the scope of protection of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any changes, modifications, substitutions, integrations, and parameter alterations to these embodiments within the spirit and principles of the present invention, achieved through conventional substitutions or by achieving the same function without departing from the principles and spirit of the present invention, fall within the scope of protection of the present invention.

Claims

1. A programmable biphasic electrochemical biosensor, characterized in that, At least comprising: a double-stranded DNA amplicon; including a hairpin polymerization reaction triggered by a target through a palindromic allosteric hairpin probe HP, generating the double-stranded DNA amplicon; a CRISPR / Cas12a system; a gold electrode; the gold electrode has a thiolated reporter gene fixed on the surface thereof; Under homogeneous conditions, the CRISPR / Cas12a system activated by the double-stranded DNA amplicon transcleaves the thiolated reporter gene, and then a poly-G sequence rich in G is extended on the interface of the gold electrode, the sequence rich in G binds with methylene blue to generate a redox active signal that is proportional to the concentration of miRNA molecules, thereby realizing detection of miRNA molecules.

2. The programmable bipolar electrochemical biosensor of claim 1, wherein, The stem of the palindromic allosteric hairpin probe HP comprises 16 bp of palindromic base pairs, and when a rigid structure is formed in the presence of miRNA-21, the two ends of the palindromic allosteric hairpin probe HP are paired with miRNA-21 through base complementation to form an HP-miRNA-21 complex.

3. The programmable bipolar electrochemical biosensor of claim 1, wherein, The preparation method of the palindromic allosteric hairpin probe HP comprises the following steps: dissolving DNA single strands of the palindromic allosteric hairpin probe HP into TAE / Na + buffer solution to obtain a mixed solution; then the mixed solution is heated at 95 DEG C for 5 min, and then cooled to room temperature to form a hairpin structure, thereby obtaining the palindromic allosteric hairpin probe HP. And / or, the sequence of the DNA single strand is shown in SEQ. ID NO.

1.

4. The programmable bipolar electrochemical biosensor of claim 1, wherein, The homogeneous interface comprises a thiolated reporter gene fixed on the gold electrode; The fixing method comprises: dropping the thiolated reporter gene probe diluted with a TCEP buffer solution on the surface of the gold electrode, assembling in the dark, and then continuing to drop MCH to block non-specific adsorption sites in the dark, washing and air-drying to obtain the gold electrode with the thiolated reporter gene fixed thereon. Preferably, the TCEP buffer solution comprises: 0.1M NaCl, 1mM EDTA, 1M Tris-HCl with pH=7.

4.

5. The programmable bipolar electrochemical biosensor of claim 1, wherein, The hairpin polymerization reaction comprises: mixing the palindromic allosteric hairpin probe HP, 1×TE buffer solution, DEPC-treated water, Bsm DNA enzyme, Bsm DNA enzyme buffer solution, dNTPs and miRNA-21, and incubating at 37°C for 1h, and then heating to 80°C for 10min to terminate the polymerization reaction, to obtain reaction solution 1. And / or, the transcleavage comprises: adding the reaction solution 1 and the CRISPR / Cas12a system, NE buffer TM r2.1, DEPC water is mixed uniformly, incubated at 37°C for 1h to obtain reaction solution 2; The CRISPR / Cas12a system is composed of Cas12a, crRNA and ssDNA reporter probe; Reaction solution 2 is dropped on the gold electrode with the thiolated reporter gene fixed thereon, and cutting is performed at 30°C for 50min.

6. The programmable bipolar electrochemical biosensor of claim 5, wherein, In the presence of dGTP, TdT extension solution is dropped on the gold electrode, incubation is performed, and under the action of terminal deoxynucleotidyl transferase TdT initiated at the 3'-hydroxyl end, poly-G is extended on the surface of the gold electrode to obtain a gold electrode with a sequence rich in G; The TdT extension solution comprises TdT buffer solution, CoCl2, dGTP and TdT. Preferably, 5μL of the TdT extension solution comprises: 1.5μL of 1×TdT buffer solution, 1.5μL of 25mM CoCl2, 1μL of 10mMdGTP, and 1μL of 1U / μL TdT.

7. The programmable bipolar electrochemical biosensor of claim 6, wherein, Methylene blue MB diluent is dropped on the gold electrode with the sequence rich in G, and assembly is performed in the dark at room temperature for 10min, and methylene blue is selectively combined with the sequence rich in G to generate a redox active signal that is proportional to the concentration of miRNA molecules.

8. Use of the programmable biphasic electrochemical biosensor according to any one of claims 1 to 7 for the detection of miRNA molecules.

9. A method for detecting a miRNA molecule based on a programmable biphasic electrochemical biosensor, characterized by, The programmable biphasic electrochemical biosensor according to any one of claims 1 to 8 is co-incubated with a target miRNA molecule, i.e. the miRNA molecule is quantitatively detected; preferably, the miRNA molecule is miRNA-21.

10. A diagnostic kit comprising at least the programmable biphasic electrochemical biosensor according to any one of claims 1 to 8.

Images