CN114594270A - Label-free ratio fluorescence type antigen detection method - Google Patents

Abstract

The invention discloses a labeling-free ratio fluorescence type antigen detection method. The invention comprises the following steps: designing a single-stranded DNA comprising a CRISPR recognition DNA sequence and an aptamer of a target antigen, and constructing a component A by a plurality of continuous T base intervals between the single-stranded DNA and the aptamer of the target antigen; modifying an antibody of the target antigen on a 96-well plate by taking the target antigen as a target to construct a component B; connecting the surface-carboxylated silicon dioxide microspheres with a DNA chain with the tail end modified with amino, and complementarily pairing the auxiliary DNA with the amino to form a double chain to construct a component C; adding a component A, a component B, a component C, copper ions, ascorbic acid, a DAPI dye and exonuclease III into an object to be detected; detecting red fluorescence and blue fluorescence of the object to be detected, and detecting the target antigen by a ratio fluorescence method. The ratio fluorescence method compares the intensities of two kinds of fluorescence as signal parameters, and is more accurate, higher in sensitivity and better in selectivity; and the sensitivity is improved, the defect that the traditional method needs to label the antibody is overcome, and the detection cost is saved.

Description

Label-free ratio fluorescence type antigen detection method

Technical Field

The invention relates to the technical field of antigen detection, in particular to a labeling-free ratio fluorescence type antigen detection method.

Background

As a gold standard for immunoassay, enzyme-linked immunosorbent assay (ELISA) is an extremely powerful complex substance technology, which has been widely used in various fields, and a large number of commercial kits have been available for selection.

Traditional ELISA relies primarily on specific antigen-antibody immunoreactions, typically in combination with a native enzyme (horseradish peroxidase, HRP in most cases) catalytic substrate as a colorimetric signal. Indeed, ELISA is excellent in detecting protein targets in biomedical and other fields.

However, conventional ELISA also has some disadvantages, such as low sensitivity, high cost, time-consuming and cumbersome procedure. Typical ELISA assays are performed at concentrations above picomolar. However, in the early stages of the disease, the serum concentration of biomarkers ranges from sub-picomolar. The weak response may be attributed to the inherent characteristics of colorimetric methods and the relatively poor catalytic activity of HRP. Furthermore, the labeling of HRP on antibodies is not only a time consuming and cumbersome process, but may also hinder their function. Methods aimed at improving the performance of ELISA sensors have been to introduce novel signal-reading enzymes, such as alkaline phosphatase (ALP) and nanoenzymes, which have been applied to ELISA as alternatives to HRP. In particular, nanoenzymes have advantages over native enzymes (e.g., tunable catalytic activity, low cost, and better stability), greatly improving the analytical performance of ELISA. However, the nanoenzymes are still insufficient in selectivity and variety of catalytic reactions, mainly including oxidases, peroxidases and hydrolases, and limit their wide application.

The fluorescence method is higher than the spectrophotometry, so that fluorescent organic dye or nanometer material can be marked on the antibody instead of enzyme. In addition, other types of functional nanomaterials (such as photothermal nanomaterials) can be labeled on the antibody, so that the application range of ELISA is further widened.

Nevertheless, in order to meet the continuing need for clinical diagnostics, it is necessary to continue to develop some ELISA strategies that are highly sensitive, economical and completely label-free.

Disclosure of Invention

The invention aims to provide a label-free ratio fluorescence type antigen detection method, which is used for providing a label-free ratio method and a method with higher sensitivity for detecting antigens, and solves the technical problems of low sensitivity, high cost, time-consuming steps and complexity in the prior art.

The specific scheme is as follows:

a method for detecting antigen in a fluorescence type without labeling ratio comprises the following steps:

designing a single chain of a recognition DNA sequence containing CRISPR to enable the single chain to contain a section of aptamer of a target antigen, and constructing a component A through a plurality of continuous T basic groups and recognition DNA sequence intervals;

modifying an antibody of the target antigen on a 96-well plate by taking the target antigen as a target, and sealing a non-specific binding site by using bovine serum albumin to construct a component B;

connecting the surface-carboxylated silicon dioxide microspheres with the DNA chain modified with the amino at the tail end, and complementarily pairing the other auxiliary DNA with the DNA chain modified with the amino to form a double chain to construct a component C;

adding a component A, a component B, a component C, copper ions, ascorbic acid, a DAPI dye and exonuclease III into an object to be detected;

and detecting the red fluorescence and the blue fluorescence of the object to be detected, and detecting the target antigen.

Further, the detection of the red fluorescence and the blue fluorescence of the object to be detected for the detection of the target antigen mainly comprises:

if the target antigen is present in the test object:

the target antigen is linked to the aptamer of component a, while the target antigen is bound to the antibody of component B, and no single-chain DNA, i.e. no polyT and primer DNA, is present;

due to the absence of polyT, the copper ions and ascorbic acid cannot be combined to emit Cu nanoclusters, and red fluorescence is weak;

because primer DNA does not exist, the primer DNA does not have complementary pairing with the cohesive end of the component C, the component C cannot be cut by exonuclease III, the DAPI dye can be embedded into the double strand of the component C, and the blue fluorescence intensity is high;

then:

if the content of the target antigen in the object to be detected is lower, the stronger the red fluorescence is, and the weaker the blue fluorescence is;

then:

let X = blue fluorescence intensity: intensity of red fluorescence

The larger the X value is, the higher the content of the target antigen is; the smaller the value of X, the lower the content of the target antigen.

Compared with the prior art, the invention has the beneficial effects that:

1. according to the invention, the ratio fluorescence method is used for comparing the intensities of two kinds of fluorescence as signal parameters, so that the method is more accurate, higher in sensitivity, better in selectivity and wider in linear range.

2. The invention carries out antigen detection by constructing the component A, the component B, the component C, copper ions, ascorbic acid, DAPI dye and exonuclease III, improves the sensitivity, is a completely label-free method and simplifies the procedure;

3. the invention replaces the secondary antibody of the traditional method by the aptamer and only uses the primary antibody, so that the labeled secondary antibody is avoided, thereby saving the cost.

Drawings

FIG. 1 is a schematic view of the process of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Aptamers are synthetic oligonucleotide or peptide sequences that can fold into secondary and tertiary structures, binding with high affinity and specificity to the corresponding target. Due to their unique properties, aptamers are a more promising alternative than antibodies. First, aptamers from in vitro screening are easy to synthesize, overcoming the limitation of having to use cell lines or animals for antibody production. Therefore, the aptamer has low cost and good repeatability. Secondly, the aptamer can be simply modified according to different purposes due to simple chemical structure and less impurities, and can be conveniently combined with other analysis technologies. Finally, aptamers are more stable than antibodies, easier to store, and suitable for use at extreme pH or high temperatures.

As a popular gene editing tool, a Clustered regulated Short Palindromic Repeats (CRISPR) system consists of CRISPR-associated enzyme (Cas), single-stranded guide RNA (crRNA) and trigger DNA, and opens up a new way for developing a biosensing platform. For CRISPR/Cas 12a, the pre-assembled Cas 12a-crRNA complex recognizes its target double-stranded dna (dsDNA) to activate efficient behavior for cleavage of the target dsDNA and any single-stranded dna (ssdna).

The ratio of the fluorescence intensities of two different wavelengths is used as a signal parameter in the ratio fluorescence method, so that the ratio fluorescence method is more accurate, higher in sensitivity, better in selectivity and wider in linear range. Poly (thymine) (poly t) templated copper nanoclusters (Cu NCs) are widely used for biochemical sensing and imaging by virtue of their excellent properties, including large stokes shift, low toxicity, good biocompatibility, and low cost.

The three-dimensional DNA walker (3D DNA walker) fabricated on nanoparticles is a molecular machine that has found widespread applications in biosensing, biocomputing and delivery due to its exquisite programmability, significant mechanical motion and high directionality. For example, spherical nanoparticles (e.g., gold nanoparticles or silica microspheres) typically build such 3D DNA walkers by integrating three basic functions: a rail (a DNA probe loaded on a nano material substrate), walker DNA and a driving force. Its rapid kinetics and high continuity help to achieve high signal amplification over a given time period. Therefore, 3D DNA walker is considered an ideal tool for signal transduction and amplification.

We select Prostate Specific Antigen (PSA) as a target to construct a general immunosensor. As shown in fig. 1, antibodies to PSA were modified in 96-well plates, and then non-specific binding sites were blocked with Bovine Serum Albumin (BSA). The target will specifically bind to the antibody and then be introduced into the CRISPR/Cas 12a system that has been previously mixed. It is worth noting that the recognition DNA sequence of CRISPR was engineered to comprise an aptamer to PSA, separated from the recognition sequence by several consecutive T bases. After binding of the aptamer to PSA, CRISPR/Cas 12a is immobilized on the surface of the 96-well plate. And finally, adding polyT single-stranded DNA, which is cut into fragments of 2-4 nt by CRISPR/Cas 12a, so that after adding copper ions and ascorbic acid, red-fluorescence-emitting copper nanoclusters cannot be formed. The method can be applied to human serum samples to obtain satisfactory results.

The technical scheme adopted by the invention is as follows;

1. the poly (thymine) polyT is used as a template, and copper ions and ascorbic acid are added to prepare a Cu nano cluster which emits red fluorescence.

2.3 construction of the DNA walker: the surface carboxylated silicon dioxide microspheres are connected with a DNA chain (linker DNA) with the tail end modified with amino. The other helper DNA (H1) is complementary to the linker DNA and forms a double strand that has a protruding 3' end (also a sticky end) and is therefore not hydrolyzed by exonuclease III (Exo III). The DAPI dye will intercalate into the double strand and emit strong blue fluorescence. DAPI that is not incorporated into the duplex is poorly fluorescent. The primer (primer DNA) can be complementarily paired with the cohesive end of the above double strand, and at this time, a double strand whose 3' -end is blunt-ended and which can be hydrolyzed by Exo III to give a single strand is formed. primer DNA is released and continues to pair with the next duplex, cyclically amplifying the signal.

3. Immunosensing strategy: using Prostate Specific Antigen (PSA) as a target, a 96-well plate is first modified with PSA antibody, and Bovine Serum Albumin (BSA) is used to block the sites that do not adsorb antibody. The target PSA can be combined with an antibody, and the trigger DNA of the CRISPR is designed into a trigger sequence which is connected with an aptamer of the PSA through a continuous T base, so that the CRISPR is fixed on the surface of the PSA and can be activated, and all fragments with single chains of 2-4 bases can be cut after the activation. At this point, both polyT and primer DNA are added and sheared. When the target PSA exists in the system, the CRISPR is fixed, and no single strand exists, namely no polyT and primer DNA exists. If no polyT exists, the copper cluster cannot be synthesized, and the red fluorescence is weak; in the absence of primer DNA, ExoIII was unable to cleave the double strand on the silica microspheres, the fluorescence intensity of DAPI. Accordingly, a ratiometric fluorescence sensor was constructed.

Thus, it is clear;

if the content of the target antigen in the object to be detected is lower, the stronger the red fluorescence is, and the weaker the blue fluorescence is, namely: let X = blue fluorescence intensity: intensity of red fluorescence

The larger the X value is, the higher the content of the target antigen is; the smaller the value of X, the lower the content of the target antigen.

The method improves sensitivity. The method completely avoids marking simplifies the procedure and saves the cost.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (2)

1. A method for detecting an antigen in a fluorescence type with no labeling ratio, which is characterized by comprising the following steps:

designing a single chain of a recognition DNA sequence containing CRISPR to enable the single chain to contain a section of aptamer of a target antigen, and constructing a component A through a plurality of continuous T basic groups and recognition DNA sequence intervals;

modifying an antibody of the target antigen on a 96-well plate by taking the target antigen as a target, and sealing a non-specific binding site by using bovine serum albumin to construct a component B;

connecting the surface-carboxylated silicon dioxide microspheres with the DNA chain modified with the amino at the tail end, and complementarily pairing the other auxiliary DNA with the DNA chain modified with the amino to form a double chain to construct a component C;

adding a component A, a component B, a component C, copper ions, ascorbic acid, a DAPI dye and exonuclease III into an object to be detected;

detecting red fluorescence and blue fluorescence of the object to be detected, and detecting the target antigen by a ratio fluorescence method.

2. The method for detecting antigen of claim 1, wherein the detection of red fluorescence and blue fluorescence of the analyte for detection of the target antigen comprises:

if the target antigen is present in the test object:

the target antigen is linked to the aptamer of component A, and the target antigen is combined with the antibody of component B, so that no single-chain DNA exists, namely no polyT and primer DNA exists;

due to the absence of polyT, the polyT can not be combined with copper ions and ascorbic acid to emit Cu nanoclusters, and red fluorescence is weak;

because primer DNA does not exist, the primer DNA does not have complementary pairing with the cohesive end of the component C, the component C cannot be cut by exonuclease III, the DAPI dye can be embedded into the double strand of the component C, and the blue fluorescence intensity is high;

then:

if the content of the target antigen in the object to be detected is lower, the stronger the red fluorescence is, and the weaker the blue fluorescence is;

then:

let X = blue fluorescence intensity: intensity of red fluorescence

The larger the X value is, the higher the content of the target antigen is; the smaller the value of X, the lower the content of the target antigen.

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