Single cell detection method

Abstract

The invention discloses a single cell detection method, which specifically recognizes a target cell through an antibody-DNA conjugate, and releases a nucleic acid marker by using a photolysis technology, thereby realizing accurate analysis of a single cell. The detection sensitivity is further improved through entropy-driven cyclic amplification and T7RNA polymerase transcription amplification, and high-sensitivity, low-cost and extensible single cell level detection is realized in combination with a specific cleavage amplification reaction of CRISPR / Cas12a. The electrochemical luminescence signal reading mode enables the detection result to be visual and suitable for high-throughput analysis, and can be widely applied to the fields of cancer research, pathogen screening, drug development, precision medical treatment and the like.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly to a single-cell detection method. Background Art

[0002] Single-cell analysis is a rapidly developing field in biomedical research. In cancer, immunology, and developmental biology research, revealing the behaviors and characteristics of individual cells is crucial for understanding complex biological systems. Single-cell detection techniques enable researchers to identify rare cell types, monitor dynamic cell processes, and explore disease molecular mechanisms with unprecedented precision.

[0003] Existing single-cell detection techniques have their respective drawbacks. For example, flow cytometry has limited detection sensitivity for rare cells or low-abundance molecules and requires a large sample size. Single-cell RNA sequencing (scRNA-seq) is costly, technically complex, and its destructive detection nature makes it impossible to perform repeated analysis or functional experiments on live cells. Microscopy-based techniques (such as fluorescence microscopy and immunohistochemistry) have low throughput and limited quantification capabilities. These bottlenecks in sensitivity, cost, complexity, and equipment dependence highlight the urgent need for new single-cell detection techniques.

[0004] As a molecular diagnostic tool, CRISPR-Cas12a exhibits unique advantages in biosensors due to its trans-cleavage activity. When the guide RNA (crRNA) binds to a specific DNA / RNA target and activates Cas12a, it can not only cleave the target sequence but also non-specifically cleave single-stranded DNA (ssDNA). This property has been ingeniously used for signal amplification to achieve highly sensitive detection of low-abundance targets. The split activation system represented by the SAHARA (Split Activator for Highly Accessible RNA Analysis) strategy triggers Cas12a activity through an RNA target and can achieve highly specific, multi-target detection (such as hepatitis C virus RNA and microRNA-155) at room temperature without traditional amplification steps such as PCR, reverse transcription, or strand displacement. This breakthrough provides a simple and efficient tool for molecular diagnosis in resource-limited settings. Summary of the Invention

[0005] In order to improve the sensitivity of single-cell detection and reduce the detection cost, the present invention proposes a single-cell detection method. The object of the present invention is achieved by the following technical solutions:

[0006] A single-cell detection method, comprising the following steps:

[0007] Step 1) Obtain an antibody-DNA1 conjugate according to the single cell to be detected; the antibody-DNA1 conjugate comprises an antibody for binding to the cell to be detected and a DNA1 nucleic acid binding to the antibody;

[0008] Step 2) Add the antibody-DNA1 conjugate to the sample containing the cell to be detected and incubate to allow the cell to be detected to bind to the antibody-DNA1 conjugate; then wash, and perform photolysis treatment on the washed cell to be detected to separate the DNA1 nucleic acid in the antibody-DNA1 conjugate bound to the cell to be detected from the antibody, obtaining a sample containing DNA1 nucleic acid;

[0009] Step 3) Add a blocker / T7 promoter / Scaffold triple-stranded complex and a fuel strand to the sample containing DNA1 nucleic acid, and perform an entropy-driven cyclic amplification reaction to obtain a product containing the T7 promoter;

[0010] Step 4) Add a transcription amplification reaction solution containing T7 RNA polymerase and a DNA template to the product containing the T7 promoter, and perform transcription amplification to obtain a transcription amplification reaction product;

[0011] Step 5) Add CRISPR / Cas12a, gRNA, a target strand / non-target strand duplex, and a DNA probe to the solution containing the RNA transcription product for reaction to obtain a reaction product; the DNA probe comprises a DNA probe nucleic acid and a luminescent group linked to the DNA probe nucleic acid;

[0012] Perform chemiluminescence detection on the reaction product, and calculate the concentration of the cell to be detected in the sample containing the cell to be detected according to the chemiluminescence detection result.

[0013] Optionally, in the antibody-DNA1 conjugate, the antibody is linked to the DNA1 nucleic acid through a photocleavable linker, and the chemical bond between the photocleavable linker and the DNA1 nucleic acid breaks at a specific wavelength to achieve controllable release. Preferably, the photocleavable linker adopts an o-nitrobenzyl ester compound (the cleavage wavelength is ultraviolet light 365 nm).

[0014] Optionally, in Step 5), the content of the single cell sample to be detected is obtained by the standard curve method, that is, a standard curve (the relationship curve between cell content and chemiluminescence signal) established by pre-detecting a series of cell standard samples with known concentrations by the same detection method as above is used. By comparing the chemiluminescence signal with the standard curve, the cell content can be quantitatively analyzed, and thus the concentration of this type of cell in the sample can be accurately obtained.

[0015] Optionally, the cell to be detected is a HEK293 cell, and the antibody is an antibody for binding to HEK293 cells, which is an antibody against the hERG potassium channel;

[0016] The nucleic acid sequence of DNA1 is shown in SEQ ID No.1.

[0017] Optionally, the sample containing the cell to be detected in step (ii) further contains fetal bovine serum, bovine serum albumin, and PBS buffer; the pH of the sample containing the cell to be detected is 7.2-7.6; the incubation time is 18-22 min;

[0018] The washing is to centrifuge the incubated sample to discard the supernatant, and then wash the cells successively with PBS containing fetal bovine serum and PBS containing serum albumin, and resuspend the cells with PBS with a pH of 7.2-7.6 containing bovine serum albumin after washing.

[0019] Optionally, the photocleavage is to expose the resuspended cells to light at 360-368 nm for 14-16 minutes.

[0020] Optionally, the entropy-driven cyclic amplification reaction process specifically includes:

[0021] Step S1: DNA1 nucleic acid binds to the blocker / T7 promoter / Scaffold triple-stranded complex, and the T7 promoter is released from the blocker / T7 promoter / Scaffold triple-stranded complex to obtain a T7 promoter and a blocker / DNA1 / Scaffold complex;

[0022] Step S2: The blocker / DNA1 / Scaffold complex binds to the fuel strand, and the blocker and DNA1 are released from the blocker / DNA1 / Scaffold complex to obtain a blocker, DNA1, and a fuel strand / Scaffold complex;

[0023] The obtained DNA1 returns to step S1 to bind to the blocker / T7 promoter / Scaffold triple-stranded complex again to obtain a T7 promoter;

[0024] Preferably, the temperature of the entropy-driven cyclic amplification reaction in step (iii) is 36-38 °C, and the reaction time is 20-30 minutes;

[0025] Preferably, the nucleic acid sequence of the blocker is shown in SEQ ID No.2, the nucleic acid sequence of the T7 promoter is shown in SEQ ID No.3, the nucleic acid sequence of the Scaffold is shown in SEQ ID No.4; the nucleic acid sequence of the fuel strand is shown in SEQ ID No.5.

[0026] Optionally, the blocker / T7 promoter / Scaffold triple-stranded complex in step (iii) is obtained by dissolving equal amounts of the blocker, T7 promoter and Scaffold in PBS, heating to 94-95 °C and incubating for 5-6 minutes, and then naturally cooling to room temperature.

[0027] Optionally, the transcription amplification reaction solution in step (iv) contains NTP, T7 RNA polymerase, DNA template, RNase and RNAPol reaction buffer;

[0028] The transcription amplification reaction is incubated at 36-38 °C for 40-42 minutes;

[0029] Preferably, the addition amount of T7 RNA polymerase is 0.2-0.33 U / μL;

[0030] Preferably, the RNA transcription product is as shown in SEQ ID No. 10.

[0031] Optionally, adding CRISPR / Cas12a, gRNA, target strand / non-target strand duplex and DNA probe to the solution containing the RNA transcription product for reaction, the method for obtaining the reaction product specifically includes:

[0032] The RNA transcription product in the solution containing the RNA transcription product binds to CRISPR / Cas12a, gRNA and the target strand / non-target strand duplex to form a CRISPR / Cas12a complex; the CRISPR / Cas12a complex cleaves the DNA probe to obtain the reaction product;

[0033] The nucleic acid sequence of the gRNA is as shown in SEQ ID No. 6; the nucleic acid sequence of the target strand in the target strand / non-target strand duplex is as shown in SEQ ID No. 7, and the nucleic acid sequence of the non-target strand is as shown in SEQ ID No. 8; the nucleic acid sequence of the DNA probe is as shown in SEQ ID No. 9.

[0034] Optionally, the reaction process in step (v) is to incubate at 20-22 °C for 20-21 minutes, and then heat at 36-38 °C for 20-30 minutes;

[0035] Preferably, the concentration of CRISPR / Cas12a is 10-20 nM.

[0036] Optionally, the luminescent group in the DNA probe is a ferrocene group;

[0037] Preferably, the ferrocene group is linked to the 5' end of the DNA probe nucleic acid.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] Through the synergistic effects of photocleavage, entropy-driven DNA strand displacement, CRISPR / Cas12a trans-cleavage and T7 RNA polymerase transcription amplification, the present invention achieves triple signal amplification. Photocleavage enables target-specific release, the entropy-driven reaction provides enzyme-free cyclic amplification, and the CRISPR / Cas12a and T7 systems achieve cascade amplification, with a total signal gain of 10-10 times. The limit of detection is as low as a single cell (LOD = 1 cell), and it can detect low-abundance targets on the surface of single cells (such as hERG potassium channels), solving the problem of missed detection caused by weak signals in traditional methods.

[0040] The detection steps of the present invention adopt a separation-free design, and all reaction steps are completed in a single liquid phase environment without the need for solid-phase carriers or complex washing steps (such as ELISA), reducing operation errors and sample loss. It has strong anti-interference ability, high binding efficiency of probes and targets in the homogeneous system, and significant background signal suppression (signal-to-noise ratio > 50), especially suitable for complex biological samples (such as cell lysates). The process from target recognition to ECL signal output can be completed within 2 hours, significantly shortening the detection time compared with traditional PCR or sequencing methods (> 6 hours).

[0041] 365 nm ultraviolet light can precisely trigger the cleavage of the antibody-DNA conjugate, avoiding interference from non-specific release. Entropy-driven selectively distinguishes single-base mismatches through thermodynamically controlled DNA strand displacement reactions, with a specificity > 99% (verified by irrelevant cell lines such as HeLa and A549). The present invention has a CRISPR / Cas12a double-lock verification: the guide RNA (gRNA) precisely recognizes the target sequence and activates the trans-cleavage activity of Cas12a, providing double-specificity guarantee (sequence matching + enzyme activity triggering). Therefore, the present invention has a multi-dimensional anti-missed detection mechanism and can achieve precise and specific control.

[0042] The amounts of CRISPR / Cas12a and T7 RNA polymerase in the present invention are optimized to the nanomolar level, with extremely low reagent consumption (a 60% cost reduction compared with commercial ELISA kits). The entropy-driven reaction and Cas12a activity are stable in the range of 25-37 °C without the need for precise temperature control equipment. The DNA probe and antibody conjugate can be prepared on a large scale by solid-phase synthesis, meeting the production specifications of in vitro diagnostic (IVD) reagents. Therefore, compared with the prior art, the present invention has the advantage of low detection cost.

[0043] The present invention adopts a modular design: the photolysis module, amplification module and detection module can be independently optimized and adapted to different targets (such as other ion channels, membrane proteins or nucleic acid markers). The applicable scenarios include basic research: single-cell heterogeneity analysis, dynamic monitoring of signal pathways. Clinical diagnosis: detection of circulating tumor cells (CTCs), screening of rare pathogens. Drug development: evaluation of the efficacy of hERG channel inhibitors at the single-cell level. ECL detection only requires a small electrochemical workstation (vs flow cytometer or sequencer), which is suitable for point-of-care testing (POCT) or areas with limited resources.

[0044] By integrating nanotechnology, synthetic biology technology and electrochemical technology, and through precise engineering regulation and innovation guided by clinical needs, the method of the present invention successfully solves the core pain points of insufficient sensitivity, complex operation and high cost in traditional single-cell analysis, and provides a transformative tool for precision medicine and basic research. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0046] Figure 1 It is a schematic diagram of the method flow of the present invention.

[0047] Figure 2 It is a graph of the experimental results of experimental parameter optimization.

[0048] Figure 3 It is a graph of the test results of detection performance.

[0049] Figure 4 It is a graph of the test results of specificity. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0050] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and do not limit the present invention.

[0051] Regarding the terms "comprising", "including", "having", "containing" and so on used herein, they are all open-ended terms, meaning including but not limited to.

[0052] The cells used in the present invention are obtained in the following manner:

[0053] A549, MCF-7 and CCRF-CEM cells were cultured in RPMI-1640 medium (GIBCO) in culture flasks; HeLa cells were cultured in DMEM medium (GIBCO) supplemented with 10% fetal calf serum (FCS, Sigma), penicillin (100 μg / mL) and streptomycin (100 μg / mL) in a humidified atmosphere of 5% CO₂ at 37°C. Cell counting was performed using a Petroff-Hausser cell counter (USA). HEK293 cells stably transfected with the hERG K⁺ channel were cultured in medium containing 0.4 mg / mL Zeocin. The cell number was again determined using a Petroff-Hausser counting chamber (USA), and images were taken using a TCS-SP5 laser scanning confocal microscope (Leica, Germany).

[0054] Example 1

[0055] This example provides a method for detecting HEK293 cells. The cells to be detected in this example are HEK293 cells, and the antibody is an antibody against the hERG potassium channel that binds to HEK293 cells. This antibody is a rabbit polyclonal antibody (Anti-hERG antibody, rabbit, polyclonal), provided by Sigma-Aldrich (Shanghai, China).

[0056] All nucleic acid probes, gRNAs, target strand / non-target strand duplexes, etc. used in the experiments of the present invention were synthesized by GenScript (China) to ensure the accuracy, stability, and repeatability of the experimental data. Specifically, DNA1 (SEQ ID No.1) and the antibody-DNA1 conjugate (the antibody is linked to the DNA1 nucleic acid through a photocleavable linker, and the chemical bond between the photocleavable linker and the DNA1 nucleic acid breaks at a specific wavelength to achieve controlled release. The photocleavable linker uses o-nitrobenzyl ester compounds, and the cleavage wavelength is ultraviolet light at 365 nm), Blocker (SEQ ID No.2), T7 promoter (SEQ ID No.3), Scaffold (SEQ ID No.4), fuel strand (SEQ ID No.5), gRNA (SEQ ID No.6), target strand / non-target strand duplex (SEQ ID No.7 & SEQ ID No.8), and DNA probe (the nucleic acid sequence is as shown in SEQ ID No.9, and the luminescent group in the DNA probe is a ferrocene group; the ferrocene group is linked to the 5'-end of the DNA probe nucleic acid through a -(CH2)6- group), DNA1a (SEQ ID No.11), DNA1b (SEQ ID No.12), and DNA1c (SEQ ID No.13) were all synthesized by GenScript, dissolved in PBS or TE buffer, and prepared at the concentration required for the experiment (20 - 100 μM). The CRISPR / Cas12a enzyme (from NEB, catalog number M0653T), T7 RNA polymerase (from Thermo Fisher, catalog number EP0111), RNase inhibitor (from NEB), RNAPol reaction buffer (from NEB), and NTP mixture (from Sigma-Aldrich, 10 mM each) required for the experiment were all purchased from the corresponding suppliers to ensure the efficiency of the enzymatic reaction. In addition, all experiments were operated in a ribonuclease-free experimental environment, nucleic acid probes were dissolved with DEPC-treated water, and Gibco brand PBS buffer (pH 7.4) was used to maintain the stability of the reaction system. The reagents described in the present invention have all passed quality control and are stored under appropriate low-temperature conditions (-80 °C or -20 °C) to ensure the repeatability of the experiment and the reliability of the detection results.

[0057] Table 1

[0058]

[0059] As Figure 1 shown, this example includes the following steps:

[0060] Step 1) Obtain an antibody-DNA1 conjugate according to the HEK293 cells to be detected; the antibody-DNA1 conjugate includes an antibody for binding to HEK293 cells (an antibody against the hERG potassium channel) and a DNA1 nucleic acid bound to the antibody; in the antibody-DNA1 conjugate, the antibody is linked to the DNA1 nucleic acid through a photocleavable linker, and the chemical bond between the photocleavable linker and the DNA1 nucleic acid breaks at a specific wavelength to achieve controlled release. The photocleavable linker uses an o-nitrobenzyl ester compound (the cleavage wavelength is ultraviolet light at 365 nm);

[0061] Step 2) Incubate 10 HEK293 cells with 10 μg / mL DNA-antibody in 200 μL of PBS buffer (pH 7.4; 136 mM NaCl, 2.7 mM KCl, 8.72 mM Na2HPO4, 1.41 mM KH2PO4) containing 2% fetal bovine serum (FBS) and 1% bovine serum albumin (BSA) for 20 minutes. After centrifuging at 300 g for 3 minutes to discard the supernatant, wash the cells successively with PBS containing 2% FBS and PBS containing 1% BSA, and then resuspend them in PBS (pH 7.4) containing 0.1% BSA. Resuspend the HEK293 cells modified with DNA1-antibody again in the same buffer and dilute them to different concentrations (100 μL system) with PBS buffer at different time gradients. The diluted cells are exposed to light at ~365 nm for 15 minutes, and after illumination, centrifuge at 300 g for 3 minutes to separate the DNA1 on the ion channels on the cell surface.

[0062] Step 3) Preparation of samples for entropy-driven cyclic amplification reaction: Dissolve an equal volume of blocker, T7 promoter, and Scaffold in PBS (0.1 M, pH = 7.4), heat to 95 °C and incubate for 5 minutes, and then cool naturally to room temperature to form a triple-stranded complex of blocker / T7 promoter / Scaffold. Add the fuel strand to the solution to initiate the entropy-driven cyclic amplification reaction, heat the reaction at 37 °C for 20 minutes to generate a large amount of T7 promoter. As Figure 1 shown, the entropy-driven cyclic amplification reaction specifically includes:

[0063] Step S1: The DNA1 nucleic acid binds to the triple-stranded complex of blocker / T7 promoter / Scaffold and releases the T7 promoter from the triple-stranded complex of blocker / T7 promoter / Scaffold to obtain a T7 promoter and a blocker / DNA1 / Scaffold complex.

[0064] Step S2: The blocker / DNA1 / Scaffold complex binds to the fuel strand, and the blocker and DNA1 are released from the blocker / DNA1 / Scaffold complex to obtain a blocker, DNA1, and a fuel strand / Scaffold complex.

[0065] The obtained DNA1 returns to Step S1 to bind to the blocker / T7 promoter / Scaffold triple complex again to obtain a T7 promoter.

[0066] Step (iv): Add 50 μL of the product containing the T7 promoter to a 100 μL amplification reaction system (containing 40 μM NTP, 30 U of T7 RNA polymerase, 1 μM DNA template, 20 U of RNase inhibitor, and 2 μL of 10×RNAPol reaction buffer), and incubate at 37°C for 40 minutes to complete transcription amplification to obtain a solution containing RNA transcription products.

[0067] Step (v): After transcription, add 10 nM CRISPR / Cas12a, 15 nM gRNA (dissolved in 1×NEBuffer, NEB), 15 nM target strand / non-target strand duplex (TS / NTS duplex), and 1 μL of 10 μM DNA probe to the system, incubate at 20°C for 20 minutes, and then heat at 37°C for 20 minutes. The RNA transcription products in the solution containing RNA transcription products bind to CRISPR / Cas12a, gRNA, and the target strand / non-target strand duplex to form a CRISPR / Cas12a complex; the CRISPR / Cas12a complex cleaves the DNA probe to obtain the reaction product; after the reaction, add 80 μL of deionized water to the reaction product, and measure the electrochemiluminescence (ECL) signal using an ITO electrode (Indium Tin Oxide electrode) for analysis. Calculate the concentration of the cells to be detected in the sample containing the cells to be detected according to the detection result of the electrochemiluminescence signal. Specifically, by comparing the ECL signal with a standard curve (the relationship curve between cell content and ECL signal) established in advance using the same detection method as above, quantitative analysis of the cells can be performed, and thus the concentration of the cells in the sample can be accurately obtained.

[0068] Among them, the ITO electrode, as a key electrochemiluminescence detection substrate, its role is mainly reflected in the signal transmission and amplification of the electrode-probe complex. Specifically, the DNA probe binds to the target product in the reaction system to form a stable signal amplification complex. Due to its high conductivity, optical transparency, and chemical stability, the ITO electrode can be used as a working electrode to excite the DNA probe to generate an electrochemiluminescence signal under the action of an external potential.

[0069] Example 2

[0070] In this example, the method in Example 1 was used to optimize the reaction time after incubation with CRISPR / Cas12a. The reaction time after incubation with CRISPR / Cas12a was modified to 0, 5, 10, 15, 20, 30, 50, 60 min respectively, and the other conditions were the same as those in Example 1. The results are as Figure 2 shown in A.

[0071] Figure 2 A shows the relationship between the ECL intensity and the cleavage time of CRISPR / Cas12a. The optimal incubation time of CRISPR / Cas12a is 20 minutes.

[0072] Example 3

[0073] In this example, the method in Example 1 was used to optimize the entropy-driven reaction time. The entropy-driven reaction time in step (iii) was modified to 0, 5, 10, 15, 20, 30, 40, 50, 60 min respectively, and the other conditions were the same as those in Example 1. The results are as Figure 2 shown in B.

[0074] Figure 2 B shows the relationship between the ECL intensity and the time of the entropy-driven process. The optimal time for the entropy-driven reaction is 20 minutes.

[0075] Example 4

[0076] In this example, the method in Example 1 was used to optimize the concentration of CRISPR / Cas12a. The concentration of CRISPR / Cas12a in the reaction was modified to 0, 1, 2.5, 5, 10, 20, 30 nM respectively, and the other conditions were the same as those in Example 1. The results are as Figure 2 shown in C.

[0077] Figure 2 C shows the influence of the CRISPR / Cas12a concentration on the ECL intensity. The optimal concentration of added CRISPR / Cas12a is 10 nM.

[0078] Example 5

[0079] In this example, the method in Example 1 was used to optimize the addition amount of T7 RNA polymerase. The addition amount of T7 RNA polymerase in the reaction was modified to 0, 5, 10, 20, 30, 50, 60 U respectively, and the other conditions were the same as those in Example 1. The results are as Figure 2 shown in C.

[0080] Figure 2D shows the effect of T7 RNA polymerase concentration on ECL intensity. The optimal concentration of added T7 RNA polymerase is 30 U.

[0081] Experimental Example

[0082] In this experimental example, the detection performance test of different concentrations of HEK293 cells (the samples contained 0, 1, 2, 5, 10, 100, 500, 1000, 10,000, 50,000, and 100,000 cells in sequence) was carried out by the method of Example 1, and the results are as Figure 3 shown:

[0083] Figure 3 A is the ECL intensity curve of detecting different concentrations of HEK293 cells by the method of Example 1 (a to k correspond to 0, 1, 2, 5, 10, 100, 500, 1000, 10,000, 50,000, and 100,000 cells respectively), showing that after three-stage amplification, the signal strongly increases from 1 to 10,000 cells. Figure 3 B is the linear relationship between the ECL intensity and the logarithm of the number of HEK293 cells, and the results show that the detection limit can reach a single cell.

[0084] In this experimental example, the DNA1 sequence used and the detected cells were replaced respectively, and other test methods were the same as those in Example 1 to specifically detect the detection method, including:

[0085] 1. Evaluation of the specificity of the biosensor by using mutant DNA constructs (DNA1a, DNA1b, and DNA1c) to replace the original DNA1 sequence.

[0086] 2. Evaluation of the selectivity of HEK293 cells by using different cell lines (HeLa, A549, MCF-7, CCRF-CEM) to replace HEK293 cells.

[0087] The specific characteristics of this biosensor in ion channel detection, based on the principles of photolysis and entropy-driven reactions, have been strictly evaluated, and two different control experiments have been adopted: (a) using a DNA sequence containing mutant bases (i.e., the mutant sequence in the DNA1 construct), (b) detecting four unrelated cell lines (HeLa, A549, MCF-7, CCRF-CEM) that lack the expression of hERG ion channels in their cell membranes. During this experimental process, three mutant DNA sequences (DNA1a, DNA1b, and DNA1c) were used as control sequences and incubated with the DNA1 / DNA2 double strand in 10,000 HEK293 cells. After two rounds of systematic amplification, the results showed that no significant electrochemiluminescence (ECL) signal was generated when using these control sequences, asFigure 4 As shown in A. The absence of this signal indicates that the ability of the biosensor to distinguish between paired and mismatched double-strands mainly depends on the hybridization event between DNA1 and DNA2, and this process is further enhanced by the selective enzymatic activity of CRISPR Cas12aI, which prefers to degrade fully paired double-strands rather than mismatched double-strands.

[0088] When studying ion channels in cell membranes, one of the main challenges researchers face is to accurately distinguish different cell lines, which is of great significance for improving our understanding of preclinical diagnosis and pathological mechanisms at the single-cell level. Therefore, the specificity of the biosensor was further evaluated by introducing other control cell lines that do not express the hERG ion channel. The experimental results showed that the ECL signals generated by these control cell lines were only slightly higher than the baseline control signal when no HEK293 cells were added, and were significantly lower than the signal of HEK293 cells, as Figure 4 shown in B. This highly selective signal can be attributed to the highly precise recognition ability of the antibody specifically targeting the hERG ion channel, highlighting the potential of the biosensor in targeted recognition.

[0089] These research results highlight the specificity of the biosensor in detecting the hERG ion channel in HEK293 cells. The sensor can effectively distinguish between target and non-target sequences, accurately identify cells expressing the hERG ion channel, and distinguish other cell lines, demonstrating its reliability in ion channel detection. This high degree of specificity is crucial for single-cell analysis applications because precise identification of ion channel expression can provide in-depth understanding of cell function and the mechanisms of various disease processes. Future research can explore the potential applications of this biosensor in clinical diagnosis, especially in the detection of rare cell populations or the field of personalized medicine, where its detailed understanding of ion channel expression at the single-cell level is expected to lay the foundation for more targeted and effective treatment strategies.

[0090] The present invention uses an antibody-DNA1 conjugate to specifically recognize specific molecules on the surface of target cells, and releases nucleic acid markers through photolysis technology to achieve precise detection at the cellular level. The entropy-driven amplification strategy of the present invention designs a Blocker / T7 promoter / Scaffold triple-stranded complex and a Fuel strand. Based on the entropy-driven cyclic amplification reaction, the released DNA1 is efficiently amplified to improve the detection sensitivity. And based on the amplification product containing the T7 promoter, T7 RNA polymerase is used for RNA transcription amplification to further amplify the detection signal. The present invention uses the CRISPR / Cas12a-gRNA system to specifically cleave the entropy-driven amplification product, and combines the activation mechanism of the target strand / non-target strand duplex to further improve the detection specificity and sensitivity. Finally, the signal is read out through the chemiluminescence reaction of the DNA probe, ensuring the high sensitivity and high signal-to-noise ratio of the detection method, and making it suitable for single-cell level analysis. Through multiple specific designs, including the targeting of the antibody-DNA1 conjugate, the sequence specificity of the entropy-driven amplification, the recognition of CRISPR / Cas12a-gRNA, and the highly sensitive detection of the electrochemiluminescence probe, the background interference is significantly reduced and the detection accuracy is improved.

[0091] As shown in Table 2, this solution of the present invention successfully solves the core pain points of insufficient sensitivity, complex operation, and high cost in traditional single-cell analysis through the integration of multidisciplinary technologies (nanotechnology, synthetic biology, electrochemistry), precise engineering regulation (reaction kinetics optimization), and innovation oriented by clinical needs (single-cell / low-abundance detection), providing a transformative tool for precision medicine and basic research. As shown in the following table, the present invention has obvious advantages compared with the prior art. This method can be applied to fields such as single-cell biomedical research, cancer detection, pathogen screening, drug development, etc., and has good generality and scalability, and can be used for the detection of other target molecules.

[0092] Table 2

[0093]

[0094] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A single-cell detection method, characterized in that, Comprising the following steps: Step 1) Obtain an antibody-DNA1 conjugate according to the single cell to be detected; the antibody-DNA1 conjugate comprises an antibody for binding to the cell to be detected and a DNA1 nucleic acid bound to the antibody; Step 2) Add the antibody-DNA1 conjugate to the sample containing the cell to be detected and incubate to allow the cell to be detected to bind to the antibody-DNA1 conjugate; then wash, and perform photolysis treatment on the washed cell to be detected to separate the DNA1 nucleic acid in the antibody-DNA1 conjugate bound to the cell to be detected from the antibody, obtaining a sample containing the DNA1 nucleic acid; Step 3) Add a blocker / T7 promoter / Scaffold triple-stranded complex and a fuel strand to the sample containing the DNA1 nucleic acid, and perform an entropy-driven cyclic amplification reaction to obtain a product containing the T7 promoter; Step 4) Add a transcription amplification reaction solution containing T7 RNA polymerase and a DNA template to the product containing the T7 promoter, and perform transcription amplification to obtain a solution containing RNA transcription products; Step 5) Add CRISPR / Cas12a, gRNA, a target strand / non-target strand duplex, and a DNA probe to the solution containing the RNA transcription products for reaction to obtain a reaction product; the DNA probe comprises a DNA probe nucleic acid and a luminescent group linked to the DNA probe nucleic acid; Perform chemiluminescence detection on the reaction product, and calculate the concentration of the cell to be detected in the sample containing the cell to be detected according to the chemiluminescence detection result.

2. The single-cell detection method according to claim 1, wherein The cell to be detected is a HEK293 cell, and the antibody is an antibody for binding to HEK293 cells, which is an antibody against the hERG potassium channel; The nucleic acid sequence of the DNA1 is shown as SEQ ID No.

1.

3. The single-cell detection method according to claim 1, characterized in that In the sample containing the cell to be detected in step 2), there are also fetal bovine serum, bovine serum albumin, and PBS buffer; the pH of the sample containing the cell to be detected is 7.2-7.6; the incubation time is 18-22 min; The washing is to centrifuge the incubated sample to discard the supernatant, and then wash the cells successively with PBS containing fetal bovine serum and PBS containing serum albumin, and resuspend with PBS with a pH of 7.2-7.6 containing bovine serum albumin after washing.

4. The single-cell detection method according to claim 1, characterized in that, The photolysis is to expose the resuspended cells to light at 360-368 nm for 14-16 minutes.

5. The single-cell detection method according to claim 1, characterized in that, The entropy-driven cyclic amplification reaction process specifically includes: Step S1: The DNA1 nucleic acid binds to the blocker / T7 promoter / Scaffold triple-stranded complex, and the T7 promoter is released from the blocker / T7 promoter / Scaffold triple-stranded complex, obtaining a T7 promoter and a blocker / DNA1 / Scaffold complex; Step S2: The blocker / DNA1 / Scaffold complex binds to the fuel strand, and the blocker and DNA1 are released from the blocker / DNA1 / Scaffold complex to obtain a blocker, DNA1, and a fuel strand / Scaffold complex; The obtained DNA1 returns to Step S1 to bind to the blocker / T7 promoter / Scaffold triple complex again to obtain a T7 promoter; Preferably, in step (iii), the temperature of the entropy-driven cyclic amplification reaction is 36-38 °C, and the reaction time is 20-30 minutes; Preferably, the nucleic acid sequence of the blocker is shown as SEQ ID No.2, the nucleic acid sequence of the T7 promoter is shown as SEQ ID No.3, the nucleic acid sequence of the Scaffold is shown as SEQ ID No.4; the nucleic acid sequence of the fuel strand is shown as SEQ ID No.

5.

6. The single-cell detection method according to claim 5, wherein The blocker / T7 promoter / Scaffold triple complex in step (iii) is obtained by dissolving equal amounts of the blocker, T7 promoter, and Scaffold in PBS, heating to 94-95 °C and incubating for 5-6 minutes, and then naturally cooling to room temperature.

7. The single-cell detection method according to claim 1, characterized in that, The transcription amplification reaction solution in step (iv) contains NTP, T7 RNA polymerase, DNA template, RNase, and RNAPol reaction buffer; The transcription amplification reaction is incubated at 36-38 °C for 40-42 minutes; Preferably, the addition amount of T7 RNA polymerase is 0.2-0.33 U / μL; Preferably, the RNA transcription product is shown as SEQ ID No.

10.

8. The single-cell detection method according to claim 1, wherein In step (v), the method of adding CRISPR / Cas12a, gRNA, target strand / non-target strand duplex, and DNA probe to the solution containing the RNA transcription product for reaction to obtain the reaction product specifically includes: The RNA transcription product in the solution containing the RNA transcription product binds to CRISPR / Cas12a, gRNA, and the target strand / non-target strand duplex to form a CRISPR / Cas12a complex; the CRISPR / Cas12a complex cleaves the DNA probe to obtain the reaction product; The nucleic acid sequence of the gRNA is shown as SEQ ID No.6; the nucleic acid sequence of the target strand in the target strand / non-target strand duplex is shown as SEQ ID No.7, and the nucleic acid sequence of the non-target strand is shown as SEQ ID No.8; the nucleic acid sequence of the DNA probe is shown as SEQ ID No.

9.

9. The single-cell detection method according to claim 1, wherein, The reaction process in step (v) is to incubate at 20-22 °C for 20-21 minutes, and then heat at 36-38 °C for 20-30 minutes; Preferably, the concentration of CRISPR / Cas12a is 10-20 nM.

10. The single-cell detection method according to claim 1, characterized in that, The luminescent group in the DNA probe is a ferrocene group; Preferably, the ferrocene group is linked to the 5' end of the DNA probe nucleic acid.

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