A label-free ratiometric fluorescence method for detecting antigens

Abstract

The present invention discloses a label-free ratio fluorescence type antigen detection method. The present invention comprises the following steps: designing a single-stranded DNA, comprising a recognition DNA sequence for CRISPR, an aptamer for a target antigen, and the two are separated by several consecutive T bases to construct component A; using the target antigen as a target, modifying the antibody of the target antigen on a 96-well plate to construct component B; connecting the surface carboxylated silica microspheres to a DNA chain modified with an amino group at the end, and complementary pairing of the auxiliary DNA with the amino group DNA to form a double strand to construct component C; adding component A, component B, component C, copper ions and ascorbic acid, DAPI dye, and exonuclease III to the object to be detected; detecting the red fluorescence and blue fluorescence of the object to be detected, and detecting the target antigen by ratio fluorescence method. The ratio fluorescence method of the present invention compares the intensity of the two fluorescences as signal parameters, which is more accurate, more sensitive, and more selective; and improves the sensitivity, overcomes the disadvantages of the traditional method that requires labeled antibodies, and saves the detection cost.

Description

Technical Field

[0001] The invention relates to the technical field of antigen detection, in particular to a label-free ratio fluorescence type antigen detection method. Background Art

[0002] As the gold standard of immunoassay, enzyme-linked immunosorbent assay (ELISA) is an extremely powerful complex material technology that has been widely used in various fields, and there are also a large number of commercial kits to choose from.

[0003] Traditional ELISA mainly relies on specific antigen-antibody immune reaction, generally combined with natural enzyme (mostly horseradish peroxidase, HRP) catalyzed substrate as colorimetric signal. Undoubtedly, ELISA has excellent performance in detecting protein targets in biomedicine and other fields.

[0004] Nevertheless, conventional ELISA also has some disadvantages, such as low sensitivity, high cost, time-consuming and cumbersome steps. Typical ELISA detection concentrations are above the picomolar level. However, in the early stages of the disease, the serum concentration of biomarkers ranges from sub-picomolar. The weak response can be attributed to the inherent characteristics of the colorimetric method and the relatively poor catalytic activity of HRP. In addition, labeling HRP on antibodies is not only a time-consuming and cumbersome process, but may also hinder their functionality. Efforts to improve the performance of ELISA sensors include the introduction of novel signal readout enzymes, such as alkaline phosphatase (ALP) and nanozymes, which have been applied as alternatives to HRP in ELISA. In particular, nanozymes have advantages over natural enzymes (e.g., tunable catalytic activity, low cost, and better stability), which greatly improves the analytical performance of ELISA. However, nanozymes are still insufficient in the selectivity and variety of catalytic reactions, mainly including oxidases, peroxidases, and hydrolases, which limits their widespread application.

[0005] Since the fluorescence method is more sensitive than the spectrophotometric method, fluorescent organic dyes or nanomaterials can be used to label antibodies instead of enzymes. In addition, other types of functional nanomaterials (such as photothermal nanomaterials) can also be labeled on antibodies, further broadening the application scope of ELISA.

[0006] Nevertheless, to meet the ongoing needs of clinical diagnostics, highly sensitive, economical, and completely label-free ELISA strategies must continue to be developed. Summary of the invention

[0007] The purpose of the present invention is to provide a label-free ratiometric fluorescence method for detecting antigens, which is used to provide a label-free, ratiometric, and more sensitive method for detecting antigens to solve the technical problems of low sensitivity, high cost, time-consuming steps, and cumbersome procedures in the prior art.

[0008] The specific plan is as follows:

[0009] A label-free ratiometric fluorescence method for detecting antigens, comprising the following steps:

[0010] The single strand containing the CRISPR recognition DNA sequence is designed to contain an aptamer of the target antigen and to be separated from the recognition DNA sequence by several consecutive T bases to construct component A;

[0011] Using the target antigen as the target, modify the antibody of the target antigen on a 96-well plate, and use bovine serum albumin to block the nonspecific binding sites to construct component B;

[0012] The silica microspheres with carboxyl groups on the surface are connected to the DNA chains with amino groups modified at the ends, and another auxiliary DNA is complementary to the DNA chains with amino groups modified to form a double strand, thereby constructing component C;

[0013] Adding component A, component B, component C, copper ions and ascorbic acid, DAPI dye, and exonuclease III to the object to be detected;

[0014] Detect the red fluorescence and blue fluorescence of the object to be detected and perform target antigen detection.

[0015] Further, the red fluorescence and blue fluorescence of the object to be detected are detected to perform target antigen detection, which mainly includes:

[0016] If the target antigen is present in the sample to be detected:

[0017] Then the target antigen is connected to the aptamer of component A, and at the same time the target antigen is bound to the antibody of component B, and there is no single-stranded DNA, that is, there is no polyT and primer DNA;

[0018] Due to the absence of polyT, it cannot combine with copper ions and ascorbic acid to emit Cu nanoclusters, and the red fluorescence is weak;

[0019] Since there is no primer DNA, there is no complementary pairing between the primer DNA and the sticky end of component C, so component C cannot be cut by nuclease III, and the DAPI dye can be embedded in the double strand of component C, with strong blue fluorescence;

[0020] but:

[0021] If the target antigen content in the object to be detected is lower, the red fluorescence is stronger and the blue fluorescence is weaker;

[0022] but:

[0023] Let X = blue fluorescence intensity: red fluorescence intensity

[0024] The larger the X value, the higher the target antigen content; the smaller the X value, the lower the target antigen content.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention uses ratiometric fluorescence method to compare the intensity of two fluorescence as signal parameters, which is not only more accurate, but also has higher sensitivity, better selectivity and wider linear range.

[0027] 2. The present invention performs antigen detection by constructing component A, component B, component C, copper ions and ascorbic acid, DAPI dye, and exonuclease III, thereby improving sensitivity and being a completely label-free method, thereby simplifying the procedure;

[0028] 3. The present invention replaces the secondary antibody of the traditional method with a nucleic acid aptamer and only uses a primary antibody, thereby avoiding the labeling of the secondary antibody and saving costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the method of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Aptamers are synthetic oligonucleotides or peptide sequences that can be folded into secondary and tertiary structures and bind to the corresponding targets with high affinity and specificity. Due to their unique properties, aptamers have become a more promising alternative to antibodies. First, aptamers obtained by in vitro screening are easy to synthesize, overcoming the limitation that antibodies must be prepared using cell lines or animals. Therefore, aptamers are low-cost and have good reproducibility. Secondly, nucleic acid aptamers have simple chemical structures and few impurities, and can be easily modified according to different purposes, and can be easily combined with other analytical techniques. Finally, aptamers are more stable and easier to store than antibodies, and are suitable for applications at extreme pH values ​​or high temperatures.

[0032] As a popular gene editing tool, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, which consists of CRISPR-associated enzyme (Cas), single-stranded guide RNA (crRNA) and trigger DNA, has opened up new avenues for the development of biosensing platforms. For CRISPR / Cas 12a, the preassembled Cas12a-crRNA complex recognizes its target double-stranded DNA (dsDNA) to activate the efficient behavior of cutting the target dsDNA and any single-stranded DNA (ssDNA).

[0033] Ratiometric fluorescence uses the ratio of fluorescence intensities at two different wavelengths as the signal parameter, which is not only more accurate, but also has higher sensitivity, better selectivity, and a wider linear range. Poly(thymine) (polyT)-templated copper nanoclusters (CuNCs) are widely used in biochemical sensing and imaging due to their excellent properties, including large Stokes shift, low toxicity, good biocompatibility, and low cost.

[0034] A 3D DNA walker fabricated on nanoparticles is a molecular machine that has been widely used in biosensing, biocomputing, and delivery due to its exquisite programmability, remarkable mechanical motion, and high directionality. For example, spherical nanoparticles (e.g., gold nanoparticles or silica microspheres) are usually used to construct such 3D DNA walkers by integrating three basic functions: tracks (DNA probes loaded on the nanomaterial substrate), walker DNA, and driving force. Its fast kinetics and high continuity help to obtain high signal amplification times within a given period of time. Therefore, 3D DNA walkers are considered to be ideal tools for signal transduction and amplification.

[0035] We selected prostate-specific antigen (PSA) as the target and constructed a universal immunosensor. Figure 1As shown, the PSA antibody was modified on a 96-well plate, and then bovine serum albumin (BSA) was used to block nonspecific binding sites. The target will specifically bind to the antibody and then be introduced into the pre-mixed CRISPR / Cas 12a system. It is worth noting that we modified the recognition DNA sequence of CRISPR to include a PSA aptamer, which is separated from the recognition sequence by several consecutive T bases. After the aptamer binds to PSA, CRISPR / Cas 12a is fixed on the surface of the 96-well plate. Finally, polyT single-stranded DNA is added, which will be cut into 2-4 nt fragments by CRISPR / Cas 12a, so after adding copper ions and ascorbic acid, red fluorescent copper nanoclusters cannot be formed. This method can be applied to human serum samples to obtain satisfactory results.

[0036] The technical solution adopted by the present invention is as follows;

[0037] 1. Using poly(thymine) polyT as a template and adding copper ions and ascorbic acid, Cu nanoclusters can be prepared, emitting red fluorescence.

[0038] 2. Composition of 3D DNA walker: Silica microspheres with carboxyl groups on the surface are connected to a DNA chain (linker DNA) with amino groups at the end. Another auxiliary DNA (H1) can complementarily pair with the linker DNA to form a double-stranded chain. This double-stranded chain has a protruding 3' end (also a sticky end), so it will not be hydrolyzed by nuclease III (Exo III). DAPI dye will be embedded in the double-stranded chain, thereby emitting strong blue fluorescence. The fluorescence of DAPI that is not embedded in the double-stranded chain is very weak. Primer (primer DNA) can complementarily pair with the sticky end of the above double-stranded chain, at this time forming a double-stranded chain with a flat end at the 3' end, which can be hydrolyzed by Exo III, and the product is a single strand. The primer DNA is released and continues to pair with the next double-stranded chain, cyclically amplifying the signal.

[0039] 3. Immunosensing strategy: With prostate-specific antigen (PSA) as the target, the 96-well plate is first modified with PSA antibodies, and the sites without antibody adsorption are blocked with bovine serum albumin (BSA). The target PSA can bind to the antibody, and the trigger DNA of CRISPR is designed so that the trigger sequence is connected to the PSA aptamer through a continuous T base, so CRISPR is fixed on the PSA surface and can be activated. After activation, it can cut all single-stranded fragments of 2-4 bases. At this time, polyT and primerDNA are added, and both are cut. If the target PSA exists in the system, CRISPR will be fixed, and there will be no single strand, that is, there will be no polyT and primer DNA. If there is no polyT, copper clusters cannot be synthesized, and the red fluorescence is weak; if there is no primer DNA, ExoIII cannot cut the double strands on the silica microspheres, and the fluorescence of DAPI is strong. Based on this, a ratio fluorescence sensor is constructed.

[0040] From this, we can know that;

[0041] If the target antigen content in the object to be detected is lower, the red fluorescence is stronger and the blue fluorescence is weaker, that is: let X = blue fluorescence intensity: red fluorescence intensity

[0042] The larger the X value, the higher the target antigen content; the smaller the X value, the lower the target antigen content.

[0043] This method improves sensitivity. This completely label-free method simplifies the procedure and saves costs.

[0044] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0045] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A label-free ratiometric fluorescence method for detecting antigens, characterized in that: The following steps are involved: The single strand containing the CRISPR recognition DNA sequence is designed to contain an aptamer of the target antigen and to be separated from the recognition DNA sequence by several consecutive T bases to construct component A; Using the target antigen as the target, modify the antibody of the target antigen on a 96-well plate, and use bovine serum albumin to block the nonspecific binding sites to construct component B; After the aptamer binds to the target antigen, CRISPR / Cas12a is fixed on the surface of the target antigen and activated. After activation, it can cut all single-stranded fragments of 2-4 bases. At this time, adding polyT and primer DNA will be cut into fragments; The silica microspheres with carboxyl groups on the surface are connected to the DNA chains with amino groups modified at the ends, and another auxiliary DNA is complementary to the DNA chains with amino groups modified to form a double strand, thereby constructing component C; Another auxiliary DNA is complementary to the amino-modified DNA chain to form a double-stranded chain with sticky ends. The DAPI dye will be embedded in the double-stranded chain, thereby emitting strong blue fluorescence. The DAPI fluorescence that is not embedded in the double-stranded chain is very weak. Adding component A, component B, component C, copper ions, ascorbic acid, DAPI dye and exonuclease III to the object to be detected; Detect the red fluorescence and blue fluorescence of the object to be detected, and detect the target antigen by ratiometric fluorescence method; If the target antigen exists in the system, CRISPR / Cas12a will be fixed, that is, polyT and primer DNA will not exist. If polyT does not exist, Cu nanoclusters cannot be synthesized and the red fluorescence is weak. If primer DNA does not exist, nuclease III cannot cut the double-stranded chain on the silica microspheres, and the fluorescence of DAPI is strong.

2. A label-free ratio fluorescence antigen detection method according to claim 1, characterized in that: in, Detect the red fluorescence and blue fluorescence of the object to be detected and perform target antigen detection, mainly including: If the target antigen is present in the sample to be detected: Then the target antigen is connected to the aptamer of component A, and at the same time the target antigen is bound to the antibody of component B, and there is no single-stranded DNA, that is, there is no polyT and primer DNA; Due to the absence of polyT, it cannot combine with copper ions and ascorbic acid to emit Cu nanoclusters, and the red fluorescence is weak; Since there is no primer DNA, there is no complementary pairing between the primer DNA and the sticky end of component C, so component C cannot be cut by exonuclease III, and the DAPI dye can be embedded in the double strand of component C, with strong blue fluorescence; but: If the target antigen content in the object to be detected is lower, the red fluorescence is stronger and the blue fluorescence is weaker; but: Let X = blue fluorescence intensity: red fluorescence intensity The larger the X value, the higher the target antigen content; the smaller the X value, the lower the target antigen content.

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