Homogeneous chemiluminescence imaging detection method based on nucleic acid nanometer machine signal amplification
By combining immunoaffinity probes, DNA nanomachines, and a homogeneous chemiluminescence method regulated by hemin dimer/monomer nucleic acid switches, the complexity and weak signal of existing heterogeneous chemiluminescence analysis methods are solved, enabling rapid and sensitive detection of protein targets.
Patent Information
- Application Number
- CN202511261161.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-04
- Publication Date
- 2025-12-09
AI Technical Summary
Existing HRP-based heterogeneous chemiluminescence analysis methods require complex interface modifications, cumbersome separation and washing steps, and lengthy detection times, making it difficult to meet the growing clinical testing needs. At the same time, the chemiluminescence signal catalyzed by hemin-DNA is weak and not conducive to the accuracy of detection.
By employing an immunoaffinity probe combined with the ortho-position effect, DNA nanomachine signal amplification, and hemin dimer/monomer nucleic acid switching regulation strategy, and utilizing imidazole-enhanced L012-hydrogen peroxide chemiluminescence system, homogeneous chemiluminescence imaging detection is achieved.
It achieves rapid, accurate, and sensitive detection of protein targets, is simple to operate, has a short reaction time, requires no washing or separation steps, has a significant signal amplification effect, and has high controllability and versatility.
Smart Images
Figure CN121090841A_ABST
Abstract
Description
I. Technical Field
[0001] This invention introduces a homogeneous chemiluminescence imaging detection method based on nucleic acid nanomachine signal amplification. II. Background Technology
[0002] Chemiluminescence analysis methods offer advantages such as low cost and high sensitivity because they do not require an external light source, avoid interference from autofluorescence in biological samples, and do not require large instruments. Currently, almost all clinically used chemiluminescence detection techniques for protein targets are based on horseradish peroxidase (HRP) heterogeneous immunoassays. These heterogeneous immunoassays first involve modifying a solid interface, such as a 96-well plate or magnetic beads, with a primary antibody capable of immunorecognizing the target protein to capture it. Then, a secondary antibody labeled with HRP is used to immunorecognize and label the captured target protein. Finally, a chemiluminescent signal is generated through a chemical reaction of luminol and hydrogen peroxide catalyzed by HRP, allowing for the quantitative analysis of the target protein. While heterogeneous immunoassays offer high specificity and sensitivity, the complex interface modification, cumbersome separation and washing steps, and lengthy detection time make them unsuitable for the growing clinical testing needs.
[0003] To overcome the shortcomings of HRP-based heterogeneous chemiluminescence analysis methods, scientists have developed a series of peroxidase mimics, such as G-quadruplex / heme mimics. However, G-quadruplex / heme mimics require specific DNA sequences, and heme itself has poor water solubility and is prone to aggregation, which inhibits its activity and is not conducive to practical applications. Modifying heme with DNA (hemin-DNA) can greatly increase the water solubility of heme, and its activity can be controlled and regulated through DNA sequence hybridization / unwinding. However, the chemiluminescence signal catalyzed by hemin-DNA is weak and flash-like, which is detrimental to detection accuracy. Therefore, enhancing the signal of hemin-DNA-catalyzed chemiluminescence systems and developing long-term chemiluminescence signal output are key issues for improving the practical application of hemin-DNA-catalyzed chemiluminescence systems.
[0004] This invention, based on the imidazole-enhanced chemiluminescence mechanism and combined with the ortho-induction effect, DNA nanomachine signal amplification strategy, and hemin dimer / monomer nucleic acid switching regulation strategy, develops a rapid, accurate, and sensitive homogeneous chemiluminescence imaging detection method for the analysis and detection of protein targets. This method can complete the detection of target proteins within 30 minutes and has significant clinical application potential. III. Summary of the Invention
[0005] The present invention utilizes a pair of immunoaffinity probes to perform immune recognition of target proteins, and combines the adjacent effect, DNA nanomachine signal amplification, hemin dimer / monomer nucleic acid switching regulation strategy, and imidazole-enhanced L012-hydrogen peroxide chemiluminescence system to develop a homogeneous chemiluminescence imaging detection method, which realizes rapid and sensitive detection of protein biomarkers.
[0006] This invention is achieved through the following technical solution:
[0007] The test solution contains the following components:
[0008] Immunoaffinity probes: mainly contain a pair of antibody 1-DNA1 conjugates and antibody 2-DNA2 conjugates that can simultaneously recognize the target.
[0009] Double-stranded DNA probes: hybridization products of the blocking strand and the promoter strand ( Figure 1 A).
[0010] DNA nanomachines: DNA nanostructures formed by the alternating assembly of complexes C1 and C2, which contain closed primer chains, onto the DNA backbone. Figure 1 B).
[0011] Hemin dimer nucleic acid probe: A double-stranded nucleic acid product formed by hybridization of a short-chain hemin monomer nucleic acid (hemin-DNA3) and a long-chain hemin monomer nucleic acid (hemin-DNA4), with complete hybridization at the hemin-modified ends of DNA3 and DNA4, resulting in a dimerized state of hemin and exhibiting low peroxidase activity. Figure 1 C).
[0012] The chemiluminescent substrate was a freshly prepared mixed solution of L012, imidazole, and hydrogen peroxide.
[0013] like Figure 2 As shown, after mixing the sample with the detection solution, the target protein can be immunorecognized by the affinity probe, converting the protein signal into a nucleic acid signal of the promoter chain. The promoter chain can trigger a cascade hybridization chain reaction on the DNA nanomachine, releasing a large number of primer chains to amplify the nucleic acid signal. The released primer chains can open the hemin dimer nucleic acid probe, converting the inhibited hemin dimer nucleic acid into hemin monomer nucleic acid with high peroxidase catalytic activity, which in turn catalyzes the luminescent substrate to produce a strong and continuous chemiluminescent signal. By constructing a linear relationship between the chemiluminescent signal intensity and the target concentration, the standard curve method is used to achieve quantitative detection of the target.
[0014] The above detection method includes the following detection steps:
[0015] (1) Add the sample to be tested to the test solution, mix well, and incubate at 37°C for 30 minutes;
[0016] (2) Add the freshly prepared chemiluminescent substrate and immediately measure the chemiluminescence intensity using a chemiluminescence imager.
[0017] The homogeneous chemiluminescence imaging detection method based on nucleic acid nanomachine signal amplification designed in this invention has the following target protein detection principle:
[0018] After mixing the sample with the detection solution, the target protein in the sample can be simultaneously recognized by a pair of immunoaffinity probes, forming an antibody 1-DNA1 / target protein / antibody 2-DNA2 complex. Through the ortho-position effect, DNA1 and DNA2 hybridize and self-assemble to form a new nucleic acid complex. This complex can undergo a chain substitution reaction with the double-stranded DNA probe, releasing the promoter chain and further initiating a cascade hybridization chain reaction on the nucleic acid backbone of the DNA nanomachine, releasing a large number of primer chains. Figure 3 The primer chain can undergo a hybridization chain substitution reaction with the hemin dimer nucleic acid probe to generate hemin monomer nucleic acids (hemin-DNA3 and hemin-DNA4 / primer chain). The hemin monomer nucleic acid exhibits good peroxidase activity, catalyzing the reaction between L012 and hydrogen peroxide in the chemiluminescent substrate to generate a chemiluminescent signal. Imidazole, as a chemiluminescence enhancer, can enhance the catalytic activity of the hemin group and reduce the free energy of the L012 oxidation reaction, thereby enhancing the signal of this chemiluminescent system while maintaining a longer luminescence time. Figure 4 Therefore, based on this enhanced continuous chemiluminescence system, combined with the ortho-induction effect, DNA nanomachines, and hemin dimer / monomer nuclease activity switch, accurate and sensitive quantitative detection of targets can be achieved.
[0019] Compared with the prior art, the present invention has the following characteristics:
[0020] This invention proposes a homogeneous chemiluminescence imaging detection method based on nucleic acid nanomachine signal amplification. Compared with commonly used heterogeneous immunoassay methods in clinical practice, it has the following characteristics:
[0021] (1) The method of the present invention is a homogeneous immunoassay method, which does not require washing, separation and other steps, is simple to operate, has a short reaction time, and can achieve rapid detection;
[0022] (2) Utilize the cascade hybridization reaction on DNA nanomachines to amplify signals and achieve ultrasensitive analysis and detection;
[0023] (3) The chemiluminescence switching strategy based on hemin dimer / monomer nucleic acid has high controllability and the switch does not require a specific nucleic acid sequence, thus having strong versatility. IV. Description of the attached drawings
[0024] Figure 1 Schematic diagram of double-stranded DNA probe, DNA nanomachine, and hemin dimer nucleic acid probe.
[0025] Figure 2 Schematic diagram of the reaction process for chemiluminescence imaging analysis and detection of target proteins.
[0026] Figure 3 Schematic diagram of the local cascade hybridization chain reaction of DNA nanomachines.
[0027] Figure 4 Imidazole-enhanced hemin monomer nucleic acid catalysis of L012-hydrogen peroxide chemiluminescence kinetics.
[0028] Figure 5 Optimization diagram of the detection method of the present invention.
[0029] Figure 6 The standard curve of the detection method of this invention for PCSK9-antibody.
[0030] Figure 7 The specificity test diagram of the detection method of the present invention for PCSK9-antibody. V. Detailed Implementation Methods
[0031] Example 1: Combined with Appendix Figure 1 This demonstrates the effectiveness of the invented homogeneous chemiluminescence imaging detection method for detecting the specific antibody PCSK9-antibody.
[0032] First, the conditions of the hemin monomer nucleic acid-catalyzed imidazole-L012-hydrogen peroxide system were optimized, including pH, imidazole concentration, hydrogen peroxide concentration, and L012 concentration. The results are as follows: Figure 5 As shown.
[0033] Mix 1 μL of sample solution containing PCSK9 antibody with 9 μL of detection solution (containing a certain concentration of immunoaffinity probe, double-stranded DNA probe, DNA nanomachines, and hemin dimer nucleic acid probe). Incubate at 37°C for 30 minutes. Then add 40 μL of luminescent substrate (containing 0.125 mM L012, 5 mM imidazole, and 1.25 mM hydrogen peroxide) to the mixture. Detect the sample using a cold light source camera (exposure time 30 seconds). Plot a standard curve between chemiluminescence intensity and PCSK9 antibody concentration. Figure 6 It was used to detect the concentration of PCSK9 antibody in unknown samples.
[0034] The homogeneous chemiluminescence imaging detection method of the present invention has good selectivity for the detection of PCSK9-antibody. Figure 7 When different proteins were analyzed and detected, only the target PCSK9-antibody sample produced a strong chemiluminescent signal, which proved the specificity of the method of the present invention.
Claims
1. A homogeneous chemiluminescence immunoassay method based on nucleic acid nanomachine signal amplification, characterized in that... The detection method uses a solution containing a pair of immunoaffinity probes, a double-stranded DNA probe, a DNA nanomachine, and a hemin dimer nucleic acid probe as the detection solution. The pair of immunoaffinity probes are antibody 1-DNA1 and antibody 2-DNA2 conjugates, and the double-stranded DNA probe is a double-stranded nucleic acid with a blocking strand / promoter strand.
2. The detection method according to claim 1, characterized in that, After the detection solution and sample solution are mixed, the target protein in the sample can be recognized by the immunoaffinity probe sandwich, forming an antibody 1-DNA1 / target protein / antibody 2-DNA2 complex. Based on the ortho-position effect, DNA1 hybridizes with DNA2 and undergoes a chain substitution reaction with the double-stranded DNA probe, releasing the promoter chain and triggering a cascade hybridization reaction on the DNA nanomachine, which in turn releases a large number of primer chains. The primer chains undergo a chain substitution reaction with the hemin dimer nucleic acid probe to produce hemin monomer nucleic acid with good peroxidase activity. After adding the chemiluminescent substrate, a luminescent signal is generated, thereby realizing the quantitative analysis of the target protein.
3. The detection method according to claim 1, characterized in that, The hemin dimer nucleic acid probe is a double-stranded nucleic acid hemin-DNA3 / hemin-DNA4 formed by hybridization of hemin-DNA3 and hemin-DNA4. The hemin-modified ends of DNA3 and DNA4 are completely hybridized, and the hemin is in a dimerized state, exhibiting low peroxidase activity.
4. The detection method according to claim 2, characterized in that, The hemin monomer nucleic acid is a hemin-DNA3 single strand, a hemin-DNA4 single strand, and a hemin-DNA3 / primer chain hybridization product.
5. The detection method according to claim 2, characterized in that, The chemiluminescent substrates include imidazole, L012, and hydrogen peroxide.
6. The detection method according to claims 1 to 5, characterized in that... The following testing steps are required: Step 1: Add the sample to be tested to the detection solution, mix well, and incubate at 37°C for 30 minutes; Step 2: Add the chemiluminescent substrate and immediately perform imaging detection using a chemiluminescence imager; Step 3: Use the standard curve method to determine the concentration of the target protein in the sample.