A method for single cell detection

Abstract

The application discloses a single cell detection method, and the application realizes accurate analysis on single cells by specific recognition of target cells through antibody-DNA conjugates and release of nucleic acid markers by using photolysis technology. Through entropy-driven cyclic amplification and T7 RNA polymerase transcription amplification, the detection sensitivity is further improved, and the specific cutting amplification reaction of CRISPR / Cas12a is combined to realize high-sensitivity, low-cost and scalable single cell level detection. The electrochemiluminescence signal reading mode of the application makes the detection result intuitive and suitable for high-throughput analysis, and can be widely applied to the fields of cancer research, pathogen screening, drug development and precision medicine.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to a single-cell detection method. Background Technology

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

[0003] Existing single-cell detection technologies each have their limitations. For example, flow cytometry has limited sensitivity for detecting rare cells or low-abundance molecules and requires large sample volumes. Single-cell RNA sequencing (scRNA-seq) is costly, technically complex, and its destructive detection characteristics prevent it from being used for repeatable analysis or functional experiments on live cells. Microscopic imaging-based techniques (such as fluorescence microscopy and immunohistochemistry) have low throughput and limited quantitative capabilities. These bottlenecks in sensitivity, cost, complexity, and equipment dependence highlight the urgent need for novel single-cell detection technologies.

[0004] CRISPR-Cas12a, as a molecular diagnostic tool, exhibits unique advantages in biosensors due to its trans-cleavage activity. When guide RNA (crRNA) binds to specific DNA / RNA targets and activates Cas12a, it can not only cleave the target sequence but also non-specifically cleave single-stranded DNA (ssDNA). This characteristic has been cleverly used for signal amplification, enabling highly sensitive detection of low-abundance targets. Split activation systems, exemplified by the SAHARA (Split Activator for Highly Accessible RNA Analysis) strategy, trigger Cas12a activity through RNA targets, achieving 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 diagnostics in resource-constrained environments. Summary of the Invention

[0005] To improve the sensitivity and reduce the cost of single-cell detection, this invention proposes a single-cell detection method. The objective of this invention is achieved through the following technical solution:

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

[0007] Step 1) Obtain the antibody-DNA1 conjugate based on the single cell to be tested; the antibody-DNA1 conjugate contains an antibody for binding to the cell to be tested and DNA1 nucleic acid that binds to the antibody;

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

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

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

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

[0012] The reaction product is subjected to chemiluminescence detection, and the concentration of the target cells in the sample containing the target cells is calculated based on the chemiluminescence detection results.

[0013] Optionally, in the antibody-DNA1 conjugate, the antibody is linked to DNA1 nucleic acid via a photolytic linker. The chemical bond between the photolytic linker and the DNA1 nucleic acid is broken at a specific wavelength, achieving controlled release. Preferably, the photolytic linker is an o-nitrobenzyl ester compound (cleavage wavelength is 365 nm ultraviolet light).

[0014] Optionally, in step five, the content of the single-cell sample to be tested is obtained by the standard curve method. That is, the same detection method is used to pre-detect a series of cell standard samples with known concentrations to establish a standard curve (the relationship curve between cell content and chemiluminescence signal). By comparing the chemiluminescence signal with the standard curve, the cell content can be quantitatively analyzed, thereby accurately obtaining the concentration of the cell in the sample.

[0015] Optionally, the cell to be tested is an HEK293 cell, and the antibody is an antibody against the hERG potassium channel used to bind to HEK293 cells.

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

[0017] Optionally, the sample containing the cells to be tested in step two) further contains fetal bovine serum, bovine serum albumin, and PBS buffer; the pH of the sample containing the cells to be tested is 7.2–7.6; and the incubation time is 18–22 min.

[0018] The washing process involves centrifuging the incubated sample, discarding the supernatant, washing the cells sequentially with PBS containing fetal bovine serum and PBS containing serum albumin, and then resuspending them in PBS containing bovine serum albumin at a pH of 7.2–7.6.

[0019] Optionally, the photolysis involves exposing the resuspended cells to 360-368 nm light 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 scaffold triple-stranded complex, and the T7 promoter is released from the blocker / T7 promoter / Scaffold scaffold triple-stranded complex, to obtain the T7 promoter and the blocker / DNA1 / Scaffold scaffold complex.

[0022] Step S2: The blocker / DNA1 / Scaffold complex binds to the fuel chain, and the blocker and DNA1 are released from the blocker / DNA1 / Scaffold complex, yielding the blocker, DNA1, and fuel chain / Scaffold complex.

[0023] The obtained DNA1 is returned to step S1 and bound again to the blocker / T7 promoter / Scaffold triple-stranded complex to obtain the T7 promoter;

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

[0025] Preferably, the nucleic acid sequence of the blocking agent 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, and the nucleic acid sequence of the fuel chain is shown in SEQ ID No. 5.

[0026] Optionally, the three-stranded complex of the blocker / T7 promoter / Scaffold scaffold in step three) 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 four) includes NTP, T7 RNA polymerase, DNA template, RNase enzyme and RNAPol reaction buffer;

[0028] The transcription amplification reaction was incubated at 36–38°C for 40–42 minutes.

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

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

[0031] Optionally, methods for adding CRISPR / Cas12a, gRNA, target / non-target double strands, and DNA probes to a solution containing RNA transcription products to obtain the reaction products include:

[0032] The RNA transcript in the solution containing the RNA transcript binds to CRISPR / Cas12a, gRNA, and the target / 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 shown in SEQ ID No. 6; the nucleic acid sequence of the target strand in the target / non-target strand duplex is shown in SEQ ID No. 7 and the nucleic acid sequence of the non-target strand is shown in SEQ ID No. 8; the nucleic acid sequence of the DNA probe is shown in SEQ ID No. 9.

[0034] Optionally, in step five), the reaction process involves incubating at 20-22°C for 20-21 minutes, followed by heating at 36-38°C for 20-30 minutes.

[0035] Preferred choice: CRISPR / Cas12a concentration of 10–20 nM.

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

[0037] Preferably, the ferrocene group is attached 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] This invention achieves triple signal amplification through the synergistic effects of photocleavage, entropy-driven DNA strand displacement, CRISPR / Cas12a trans-cleavage, and T7 RNA polymerase transcriptional amplification. Photocleavage enables target-specific release, the entropy-driven reaction provides enzyme-free cyclic amplification, and the cascade amplification by the CRISPR / Cas12a and T7 systems results in a total signal gain of 10-10 times. With a detection limit as low as a single cell (LOD = 1 cell), it can detect low-abundance targets on the surface of single cells (such as hERG potassium ion channels), solving the problem of missed detection caused by weak signals in traditional methods.

[0040] The detection steps of this invention employ a separation-free design, with all reaction steps completed in a single liquid phase environment. This eliminates the need for solid-phase carriers or complex washing steps (such as in ELISA), reducing operational errors and sample loss. It exhibits strong anti-interference capabilities, with high probe-target binding efficiency in a homogeneous system and significant background signal suppression (signal-to-noise ratio >50), making it particularly 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 to traditional PCR or sequencing methods (>6 hours).

[0041] 365nm ultraviolet light can precisely trigger the breakage of antibody-DNA conjugates, avoiding interference from non-specific release. Entropy-driven, thermodynamically controlled DNA strand displacement reactions selectively distinguish single-base mismatches with a specificity >99% (verified by irrelevant cell lines such as HeLa and A549). This invention features CRISPR / Cas12a dual-lock verification: the guide RNA (gRNA) precisely recognizes the target sequence, activating the trans-cleavage activity of Cas12a, ensuring dual specificity (sequence matching + enzyme activity triggering). Therefore, this invention has a multi-dimensional anti-false positive mechanism, enabling precise and specific control.

[0042] The CRISPR / Cas12a and T7 RNA polymerase dosages of this invention are optimized to the nanomolar level, resulting in extremely low reagent consumption (60% cost reduction compared to commercial ELISA kits). The entropy-driven reaction and Cas12a activity are stable within the 25-37°C range, eliminating the need for precise temperature control equipment. DNA probes and antibody conjugates can be scalably prepared via solid-phase synthesis, meeting the standards for in vitro diagnostic (IVD) reagent production. Therefore, this invention offers the advantage of lower detection costs compared to existing technologies.

[0043] This invention employs a modular design: the photolysis module, amplification module, and detection module can be independently optimized to adapt to different targets (such as other ion channels, membrane proteins, or nucleic acid biomarkers). Application scenarios include basic research: single-cell heterogeneity analysis, dynamic monitoring of signaling pathways; clinical diagnosis: detection of circulating tumor cells (CTCs), screening for rare pathogens; and drug development: single-cell level efficacy evaluation of hERG channel inhibitors. ECL detection requires only a small electrochemical workstation (vs. flow cytometer or sequencer), making it suitable for point-of-care testing (POCT) or resource-limited areas.

[0044] The method of this invention, by integrating nanotechnology, synthetic biology, and electrochemical technology, and through precise engineering control and clinically demand-oriented innovation, successfully solves the core pain points of insufficient sensitivity, complex operation, and high cost in traditional single-cell analysis, providing a transformative tool for precision medicine and basic research. Attached Figure Description

[0045] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required for the specific embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0047] Figure 2 The experimental results were plotted to optimize the experimental parameters.

[0048] Figure 3 This is a graph showing the results of the performance test.

[0049] Figure 4 This is a graph showing the results of a specificity test. Detailed Implementation

[0050] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is only for describing particular embodiments and is not intended to limit the present invention.

[0051] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0052] The cells used in this invention were 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 bovine serum (FCS, Sigma), penicillin (100 μg / mL), and streptomycin (100 μg / mL) at 37°C in a humidified atmosphere of 5% CO2. Cell counts were performed using a Petroff-Hausser cell counter (USA). HEK293 cells stably transfected with hERG K+ channels were cultured in medium containing 0.4 mg / mL Zeocin. Cell numbers were again determined using a Petroff-Hausser counting chamber (USA), and images were captured using a TCS-SP5 laser scanning confocal microscope (Leica, Germany).

[0054] Example 1

[0055] This embodiment provides a method for detecting HEK293 cells. The cells to be detected in this embodiment are HEK293 cells, and the antibody used to bind to HEK293 cells is an anti-hERG potassium channel antibody. This antibody is a rabbit-derived polyclonal antibody (anti-hERG antibody, rabbit, polyclonal), provided by Sigma-Aldrich (Shanghai, China).

[0056] All nucleic acid probes, gRNAs, target / non-target double strands, etc. used in the experiments of this invention were synthesized by GenScript Biotech (GenScript, China) to ensure the accuracy, stability and reproducibility of the experimental data. Specifically, the following are included: DNA1 (SEQ ID No. 1) and antibody-DNA1 conjugate (the antibody is linked to DNA1 nucleic acid via a photolytic linker, and the chemical bond between the photolytic linker and DNA1 nucleic acid breaks at a specific wavelength, achieving controlled release. The photolytic linker is an o-nitrobenzyl ester compound, and the cleavage wavelength is 365 nm ultraviolet light), Blocker (SEQ ID No. 2), T7 promoter (SEQ ID No. 3), Scaffold (SEQ ID No. 4), fuel chain (SEQ ID No. 5), gRNA (SEQ ID No. 6), target / non-target double strand (SEQ ID No. 7 & SEQ ID No. 8), and DNA probe (the nucleic acid sequence is 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 via a -(CH2)6- group), DNA1a (SEQ ID No. 11), DNA1b (SEQ ID No. 12), and DNA1c (SEQ ID No. 13). All reagents (No. 13) were synthesized by GenScript Biotech, dissolved in PBS or TE buffer, and prepared at the required concentrations (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 experiments were purchased from their respective suppliers to ensure the high efficiency of the enzymatic reactions. Furthermore, all experiments were conducted in an RNase-free environment, using DEPC to treat water to dissolve the nucleic acid probes, and Gibco brand PBS buffer (pH 7.4) to maintain the stability of the reaction system. All reagents described in this invention have undergone quality control and are stored at suitable low temperatures (-80°C or -20°C) to ensure the reproducibility of the experiments and the reliability of the detection results.

[0057] Table 1

[0058]

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

[0060] Step 1) Obtain the antibody-DNA1 conjugate from the HEK293 cells to be tested. The antibody-DNA1 conjugate contains an antibody (anti-hERG potassium channel antibody) for binding to HEK293 cells and DNA1 nucleic acid bound to the antibody. In the antibody-DNA1 conjugate, the antibody is linked to the DNA1 nucleic acid via a photolytic linker. The chemical bond between the photolytic linker and the DNA1 nucleic acid is broken at a specific wavelength, achieving controlled release. The photolytic linker is an o-nitrobenzyl ester compound (lysis wavelength is 365 nm ultraviolet light).

[0061] Step 2) Ten HEK293 cells were incubated 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 NaHPO4, 1.41 mM KHPO4) containing 2% fetal bovine serum (FBS) and 1% bovine serum albumin (BSA) for 20 minutes. After centrifugation at 300g for 3 minutes and discarding the supernatant, the cells were washed sequentially with PBS containing 2% FBS and PBS containing 1% BSA, and then resuspended in PBS containing 0.1% BSA (pH 7.4). The HEK293 cells modified with DNA1-antibody were resuspended in the same buffer and diluted to different concentrations (100 μL system) with PBS buffer at different time gradients. The diluted cells were exposed to ~365 nm light for 15 minutes, followed by centrifugation at 300g for 3 minutes to separate DNA1 from the ion channels on the cell surface.

[0062] Step 3) Sample Preparation for Entropy-Driven Cyclic Amplification Reaction: An equal volume of the blocking agent, T7 promoter, and Scaffold scaffold were dissolved in PBS (0.1M, pH 7.4), heated to 95°C and incubated for 5 minutes. The mixture was then allowed to cool naturally to room temperature, forming a triple-stranded complex of the blocking agent / T7 promoter / Scaffold scaffold. A fuel chain was added to the solution to initiate the entropy-driven cyclic amplification reaction. The reaction was heated at 37°C for 20 minutes, generating a large number of T7 promoters. Figure 1 As shown, the entropy-driven cyclic amplification reaction specifically includes:

[0063] Step S1: DNA1 nucleic acid binds to the blocker / T7 promoter / Scaffold scaffold triple-stranded complex, and the T7 promoter is released from the blocker / T7 promoter / Scaffold scaffold triple-stranded complex, resulting in the T7 promoter and the blocker / DNA1 / Scaffold scaffold complex.

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

[0065] The obtained DNA1 is returned to step S1 and bound again to the blocker / T7 promoter / Scaffold triple-stranded complex to obtain the T7 promoter.

[0066] Step 4) Take 50 μL of the product containing the T7 promoter and add it to 100 μL of amplification reaction system (containing 40 μM NTP, 30 U T7 RNA polymerase, 1 μM DNA template, 20 U RNase inhibitor and 2 μL 10×RNAPol reaction buffer). Incubate at 37℃ for 40 minutes to complete transcription amplification and obtain a solution containing RNA transcript.

[0067] (Step 5) After transcription, add a final concentration of 10 nM CRISPR / Cas12a, 15 nM gRNA (dissolved in 1×NEBuffer, NEB), 15 nM target / non-target duplex (TS / NTS duplex), and 1 μL of 10 μM DNA probe to the system. Incubate at 20°C for 20 minutes, then heat at 37°C for 20 minutes. The RNA transcript in the solution containing the RNA transcript binds to CRISPR / Cas12a, gRNA, and target / non-target 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. Analyze the electrochemiluminescence (ECL) signal using an ITO electrode (Indium Tin Oxide). Calculate the concentration of the target cells in the sample containing the target cells based on the ECL signal detection results. Specifically, by comparing the ECL signal with a pre-established standard curve (the relationship between cell content and ECL signal) using the same detection method, the cells can be quantitatively analyzed, thereby accurately obtaining the concentration of the cells in the sample.

[0068] Among them, the ITO electrode serves as a key electrochemiluminescence detection substrate, primarily playing a role in signal transmission and amplification of the electrode-probe complex. Specifically, the DNA probe binds to the target product in the reaction system, forming a stable signal amplification complex. Due to its high conductivity, optical transparency, and chemical stability, the ITO electrode can be used as the working electrode, enabling the DNA probe to generate an electrochemiluminescence signal under an applied potential.

[0069] Example 2

[0070] This embodiment optimizes the reaction time after CRISPR / Cas12a incubation using the method described in Example 1. The reaction time after CRISPR / Cas12a incubation was modified to 0, 5, 10, 15, 20, 30, 50, and 60 min, respectively, while all other conditions remained the same as in Example 1. The results are as follows: Figure 2 As shown in Figure A.

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

[0072] Example 3

[0073] This embodiment optimizes the entropy-driven reaction time using the method described in Example 1. The entropy-driven reaction time in step three) is modified to 0, 5, 10, 15, 20, 30, 40, 50, and 60 min, respectively, while all other conditions remain the same as in Example 1. The results are as follows: Figure 2 As shown in B.

[0074] Figure 2 B represents the relationship between 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] This embodiment uses the method described in Example 1 to optimize the CRISPR / Cas12a concentration. The CRISPR / Cas12a concentration in the reaction was modified to 0, 1, 2.5, 5, 10, 20, and 30 nM, while all other conditions remained the same as in Example 1. The results are as follows: Figure 2 As shown in C.

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

[0078] Example 5

[0079] This embodiment optimizes the amount of T7 RNA polymerase added using the method described in Example 1. The amount of T7 RNA polymerase added in the reaction was modified to 0, 5, 10, 20, 30, 50, and 60 U, while all other conditions remained the same as in Example 1. The results are as follows: Figure 2 As shown in C.

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

[0081] Test case

[0082] In this experimental example, the detection performance of HEK293 cells at different concentrations (samples containing 0, 1, 2, 5, 10, 100, 500, 1000, 10,000, 50,000, and 100,000 cells sequentially) was tested using the method described in Example 1. The results are as follows: Figure 3 As shown:

[0083] Figure 3 A shows the ECL intensity curves for detecting different concentrations of HEK293 cells using 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), indicating that the signal increases strongly from 1 to 10,000 cells after the three-stage amplification. Figure 3 B represents the linear relationship between ECL intensity and the logarithm of HEK293 cell number, indicating that the detection limit can reach a single cell.

[0084] In this experimental example, the DNA1 sequence and the cells used for testing were replaced, while all other testing methods remained the same as in Example 1 to ensure the specificity of the detection method, specifically including:

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

[0086] 2. The selectivity of HEK293 cells was evaluated by replacing HEK293 cells with different cell lines (HeLa, A549, MCF-7, CCRF-CEM).

[0087] The biosensor's specificity in ion channel detection, based on the principles of photolysis and entropy-driven reactions, underwent rigorous evaluation using two different control experiments: (a) using DNA sequences containing mutated bases (i.e., the mutated sequence in the DNA1 construct), and (b) detecting four unrelated cell lines (HeLa, A549, MCF-7, and CCRF-CEM) lacking hERG ion channel expression in their cell membranes. In this experiment, three mutated DNA sequences (DNA1a, DNA1b, and DNA1c) were used as control sequences and co-incubated with the DNA1 / DNA2 double strand in 10,000 HEK293 cells. After two rounds of system amplification, the results showed that using these control sequences failed to produce significant electroluminescence (ECL) signals, such as... Figure 4 As shown in A. The absence of this signal indicates that the biosensor's ability to distinguish between paired and mismatched double strands depends primarily on hybridization events between DNA1 and DNA2, and this process is further enhanced by the selective enzymatic activity of CRISPR Cas12aI, which prefers to degrade perfectly paired double strands rather than mismatched ones.

[0088] One of the main challenges researchers face when studying ion channels in cell membranes is accurately distinguishing between different cell lines, which is crucial for improving our understanding of preclinical diagnosis and single-cell-level pathological mechanisms. Therefore, the specificity of this biosensor was further evaluated by introducing other control cell lines that do not express hERG ion channels. Experimental results showed that the ECL signal generated by these control cell lines was only slightly higher than the baseline control signal without HEK293 cells, and significantly lower than the signal generated by HEK293 cells. Figure 4 As shown in B. This highly selective signal can be attributed to the highly precise recognition capability of the antibody specifically targeting the hERG ion channel, highlighting the potential of this biosensor in targeted recognition.

[0089] These findings highlight the biosensor's specificity in detecting hERG ion channels in HEK293 cells. The sensor effectively distinguishes between target and non-target sequences, accurately identifies cells expressing hERG ion channels, and differentiates from other cell lines, demonstrating its reliability in ion channel detection. This high specificity is crucial for single-cell analysis applications, as precise identification of ion channel expression provides in-depth insights into cellular function and the mechanisms of various disease processes. Future research can explore the potential applications of this biosensor in clinical diagnostics, particularly in detecting rare cell populations or in personalized medicine. Its detailed understanding of ion channel expression at the single-cell level holds promise for laying the foundation for more targeted and effective treatment strategies.

[0090] This invention utilizes an antibody-DNA1 conjugate to specifically recognize specific molecules on the surface of target cells and releases nucleic acid markers via photolysis technology, achieving precise detection at the cellular level. The entropy-driven amplification strategy of this invention designs a Blocker / T7 promoter / Scaffold triple-stranded complex and a Fuel strand. Based on an entropy-driven cyclic amplification reaction, it efficiently amplifies the released DNA1, improving detection sensitivity. Furthermore, based on the amplification product containing the T7 promoter, RNA transcription amplification is performed using T7 RNA polymerase to further amplify the detection signal. This invention employs a CRISPR / Cas12a-gRNA system to specifically cleave the entropy-driven amplification product, combined with the activation mechanism of the target / non-target strand double strand, further improving detection specificity and sensitivity. Finally, signal readout is achieved through the chemiluminescence reaction of the DNA probe, ensuring high sensitivity and a high signal-to-noise ratio, making it suitable for single-cell level analysis. Through multiple specificity designs, including the targeting of the antibody-DNA1 conjugate, the specificity of the entropy-driven amplification sequence, the recognition of CRISPR / Cas12a-gRNA, and the high sensitivity of the electrochemiluminescence probe, background interference is significantly reduced, improving detection accuracy.

[0091] As shown in Table 2, this invention, through the integration of multidisciplinary technologies (nanotechnology, synthetic biology, electrochemistry), precise engineering control (reaction kinetics optimization), and clinically driven innovation (single-cell / low-abundance detection), successfully addresses the core pain points of traditional single-cell analysis, such as insufficient sensitivity, complex operation, and high cost, providing a transformative tool for precision medicine and basic research. As shown in the table below, this invention has significant advantages over existing technologies. This method can be applied to single-cell biomedical research, cancer detection, pathogen screening, drug development, and other fields, exhibiting good versatility and scalability, and can be used for the detection of other target molecules.

[0092] Table 2

[0093]

[0094] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting single cells, characterized in that, Includes the following steps: Step 1) Obtain the antibody-DNA1 conjugate based on the single cell to be tested; the antibody-DNA1 conjugate contains an antibody for binding to the cell to be tested and DNA1 nucleic acid that binds to the antibody; Step 2) Add the antibody-DNA1 conjugate to the sample containing the cells to be tested and incubate to allow the cells to be tested to bind to the antibody-DNA1 conjugate; then wash and perform photolysis on the washed cells to separate the DNA1 nucleic acid in the antibody-DNA1 conjugate bound to the cells from the antibody, to obtain a sample containing DNA1 nucleic acid. Step 3) Add the blocking agent / T7 promoter / Scaffold triple-stranded complex and fuel chain to the DNA1-containing nucleic acid sample, and perform an entropy-driven cyclic amplification reaction to obtain the product containing the T7 promoter; Step 4) Add a transcription amplification reaction solution containing T7 RNA polymerase and DNA template to the product containing the T7 promoter, and perform transcription amplification to obtain a solution containing RNA transcription product; Step 5) Add CRISPR / Cas12a, gRNA, target / non-target double strand and DNA probe to the solution containing RNA transcription product and react to obtain the reaction product; the DNA probe includes DNA probe nucleic acid and a luminescent group attached to the DNA probe nucleic acid; The reaction product is subjected to chemiluminescence detection, and the concentration of the target cells in the sample containing the target cells is calculated based on the chemiluminescence detection results. The cells to be tested are HEK293 cells, and the antibody used to bind to HEK293 cells is an antibody against hERG potassium channels. The nucleic acid sequence of DNA1 is shown in SEQ ID No. 1; In step five), the method of adding CRISPR / Cas12a, gRNA, target / non-target double strand, and DNA probe to a solution containing RNA transcription products to obtain the reaction product specifically includes: The RNA transcript in the solution containing the RNA transcript binds to CRISPR / Cas12a, gRNA, and the target / 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 in SEQ ID No. 6; the nucleic acid sequence of the target strand in the target / non-target strand duplex is shown in SEQ ID No. 7 and the nucleic acid sequence of the non-target strand is shown in SEQ ID No. 8; the nucleic acid sequence of the DNA probe is shown in SEQ ID No.

9.

2. The single-cell detection method according to claim 1, characterized in that, The sample containing the cells to be tested in step two) also contains fetal bovine serum, bovine serum albumin, and PBS buffer; the pH of the sample containing the cells to be tested is 7.2-7.6; the incubation time is 18-22 min; The washing process involves centrifuging the incubated sample, discarding the supernatant, washing the cells sequentially with PBS containing fetal bovine serum and PBS containing serum albumin, and then resuspending them in PBS containing bovine serum albumin at a pH of 7.2-7.

6.

3. The single-cell detection method according to claim 1, characterized in that, The photolysis involves exposing resuspended cells to 360-368 nm light for 14-16 minutes.

4. The single-cell detection method according to claim 1, characterized in that, The entropy-driven cyclic amplification reaction process specifically includes: Step S1: DNA1 nucleic acid binds to the blocker / T7 promoter / Scaffold scaffold triple-stranded complex, and the T7 promoter is released from the blocker / T7 promoter / Scaffold scaffold triple-stranded complex, to obtain the T7 promoter and the blocker / DNA1 / Scaffold scaffold complex. Step S2: The blocker / DNA1 / Scaffold complex binds to the fuel chain, and the blocker and DNA1 are released from the blocker / DNA1 / Scaffold complex, yielding the blocker, DNA1, and fuel chain / Scaffold complex. The obtained DNA1 is returned to step S1 and bound again to the blocker / T7 promoter / Scaffold triple-stranded complex to obtain the T7 promoter; The entropy-driven cyclic amplification reaction in step three) is carried out at a temperature of 36-38°C for 20-30 minutes. The nucleic acid sequence of the blocking agent 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 scaffold is shown in SEQ ID No. 4, and the nucleic acid sequence of the fuel chain is shown in SEQ ID No.

5.

5. The single-cell detection method according to claim 4, characterized in that, The triple-stranded complex of the blocker / T7 promoter / Scaffold scaffold in step three) 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.

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

10.

7. The single-cell detection method according to claim 1, characterized in that, The reaction process in step five is to incubate at 20-22°C for 20-21 minutes, and then heat at 36-38°C for 20-30 minutes; The concentration of CRISPR / Cas12a is 10~20 nM.

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

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