Ultrasensitive immunodetection method based on adjacent nucleic acid amplification signal amplification

Abstract

The invention discloses an ultrasensitive immunodetection method based on adjacent nucleic acid amplification signal amplification in the technical field of medical detection. The ultrasensitive immunodetection method comprises the following steps: step 1, immune binding; step 2, enzyme digestion release: releasing a hairpin loop DNA single strand; step 3, complementation and enzyme ligation: adding T4 ligase to cyclize the circular DNA single chain; step 4, rolling circle amplification; and step 6, detecting, and quantifying and positioning by qPCR (quantitative polymerase chain reaction) and an in-situ oligonucleotide fluorescence labeling detection method. According to the present invention, the hairpin ring structure on the oligonucleotide DNA chain coupled to the antibody protects the complementary sequence so as to avoid the generation of the non-specific circular DNA single chain, and the rolling circle amplification technology is combined to amplify the detection signal, such that the detection background signal intensity is inhibited while the specificity of the detection signal is enhanced so as to further improve the detection sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical detection, and specifically relates to a ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification. Background Art

[0002] Immunoassay, that is, immunoassay (IA), is a method based on the specific binding of antigen and antibody, and converts the binding event into a detectable signal through a label (enzyme, fluorescence, radioactive isotope, etc.). Based on the specificity and sensitivity of the antigen-antibody reaction, its application scope covers all fields of medical testing. Any substance as long as it can obtain the corresponding specific antibody can be detected by immunoassay. However, with the development of medical detection technology, various problems of immunoassay technology have also emerged. The sensitivity of traditional immunoassay is limited by the antigen-antibody binding efficiency and signal amplification mechanism. Since each antibody only carries a limited number of label molecules, and the non-specific binding existing in the detection process will also mask the low-concentration signal, resulting in the problem of insufficient sensitivity of immunoassay. For example, the detection limit of ELISA is usually at the pg / mL level, and it is difficult to detect trace biomarkers (such as early tumor markers, low-abundance cytokines); in addition, due to the rapid depletion of the label caused by high-concentration targets, exceeding the linear detection range, the reading method also depends on visual judgment or simple optical equipment, with low resolution. Some immunoassay methods (such as colloidal gold test strips) can only provide qualitative or semi-quantitative results, and it is difficult to accurately cover targets in a wide concentration range, resulting in the current dilemma that immunoassay methods need to be improved.

[0003] Rolling circle amplification is an isothermal nucleic acid amplification technology that uses a circular DNA template and a DNA polymerase with strand displacement activity (such as Phi29) to continuously synthesize long single-stranded DNA (ssDNA) repeats. The mechanism of its signal amplification lies in that each circular template can generate ssDNA up to thousands of bases in length, serving as a signal carrier. Multiple probe binding sites can be designed on the signal carrier to achieve geometric signal amplification.

[0004] Existing proximity immunodetection methods using rolling circle amplification for signal amplification have become an option for highly sensitive immuno-medical detection. It is based on the proximity effect triggered by the sandwich immune complex formed by antibodies and antigens. By adding single-stranded DNA at both ends that is complementary to the oligonucleotide DNA probes conjugated to the antibodies and forming a circular structure, and then ligating by polymerase to form a circular DNA template, the binding site signals on the template are amplified using rolling circle amplification to achieve highly sensitive detection, with a detection limit that can be as low as the fM level, such as the PLA system described in the academic paper "Protein detection using proximity-dependent DNA ligation assays".

[0005] However, even by amplifying the immune binding signal through rolling circle amplification, the PLA system still has problems such as difficulty in reducing the detection background, which affects the detection limit, a relatively high possibility of non-specific binding, reducing the reliability of the detection signal, and even generating false positive signals. Therefore, it is necessary to propose a method for ultrasensitive immuno-detection based on proximity nucleic acid amplification signal amplification that can suppress the intensity of the detection background signal, protect the oligonucleotide DNA strand from non-specific binding, thereby improving the sensitivity of immuno-detection, broadening the detection limit range, and enabling automated detection. Summary of the Invention

[0006] To solve the above problems, the object of the present invention is to provide an ultrasensitive immuno-detection method based on proximity nucleic acid amplification signal amplification. The hairpin structure on the oligonucleotide DNA strand conjugated to the antibody protects the complementary sequence from generating non-specific circular single-stranded DNA, and combines with rolling circle amplification technology to amplify the detection signal, suppress the intensity of the detection background signal while enhancing the specificity of the detection signal, further improve the sensitivity of the detection, and contribute to the realization of automated detection.

[0007] To achieve the above object, the technical solution of the present invention is as follows: An ultrasensitive immuno-detection method based on proximity nucleic acid amplification signal amplification, comprising the following steps: Step 1, Immune binding: Add a first DNA antibody conjugate probe and a second DNA antibody conjugate probe to the sample to bind to the antigen to form an immune complex; Step 2, Enzymatic cleavage and release: Use a branched structure endonuclease to cut the 5' end branch of the DNA hairpin structure of the first DNA antibody conjugate probe and the second DNA antibody conjugate probe, and release the hairpin loop DNA single-stranded that is complementary to the root of the oligonucleotide DNA strand of the other conjugate probe; Step 3. Complementation and ligation. Under the influence of the proximity effect formed by antibody-immune binding, the oligonucleotide DNA strands cut from the first DNA-antibody conjugate probe and the second DNA-antibody conjugate probe are complementary to each other, forming a circular DNA single strand with two nicks that is complementary to the oligonucleotide DNA strands of both conjugate probes at the same time, and the single-strand ends at the nicks are adjacent. Add T4 ligase to circularize the circular DNA single strand; Step 4. Rolling circle amplification. Add phi29 polymerase for rolling circle amplification of the circular DNA single strand; Step 5. Detection. Analyze the antibody content by detecting the repeated specific sequences on the rolling circle amplification product through qPCR, and perform in-situ fluorescence imaging of the repeated specific sequences on the rolling circle amplification product in tissue cells by the in-situ oligonucleotide fluorescence labeling detection method.

[0008] The principle of the basic protocol is as follows: Hairpin structures are designed on the oligonucleotide strands of the first and second DNA-antibody conjugate probes, and the loop part contains complementary sequences. When the two antibody probes bind to the same antigen simultaneously (forming a "sandwich immune complex"), the spatial proximity causes the hairpin structure to be cleaved by the endonuclease, releasing the complementary strand. The released complementary strands form a circular DNA template through the proximity effect and are circularized by T4 ligase, ensuring that only specifically bound antigens can trigger the generation of circular templates and avoiding non-specific amplification.

[0009] The circularized DNA template undergoes RCA under the action of phi29 polymerase to generate a long ssDNA chain containing thousands of repeated units, achieving exponential signal amplification. qPCR realizes the precise detection of trace antigens through quantitative analysis of the repeated sequences in the amplification products. In-situ fluorescence imaging directly labels the amplification products in-situ in tissues or cells, retaining spatial information and being suitable for pathological analysis.

[0010] The beneficial effects of the basic protocol are as follows: 1. The linear amplification ability of RCA combined with the proximity-dependent template generation mechanism converts single antigen binding into a long-chain DNA signal carrier, with a detection limit reaching the fg / mL level, and the sensitivity is increased by more than 1000 times compared to traditional ELISA.

[0011] 2. The double antibody sandwich method ensures target selectivity and realizes antibody-antigen specific recognition. In addition, for the proximity-dependent template, the DNA cyclization reaction will only be initiated when the two antibody probes bind to the antigen simultaneously, double-verifying the presence of the target and achieving double guarantee of high specificity.

[0012] 3. Ultra-sensitive quantification is achieved through qPCR, which is suitable for the detection of trace markers in body fluids (serum, urine). The fluorescence-labeled RCA products of in-situ imaging can localize the distribution of antigens in cells or tissues and are used for cancer pathological staging or infection focus analysis, adapting to flexible use in a variety of scenarios.

[0013] 4. The present invention does not require expensive labeled antibodies (such as enzyme-labeled antibodies), reduces costs through a universal fluorescent probe. In addition, the RCA reaction is an isothermal amplification and does not require a thermal cycler, making it suitable for scenarios with limited resources and improving the cost-effectiveness of immunoassay methods.

[0014] Furthermore, the antibody proteins on the first DNA antibody conjugate probe and the second DNA antibody conjugate probe can bind to the antigen to form a sandwich immuno-complex.

[0015] The beneficial effects of the basic protocol are as follows: 1. Through the double antibody sandwich method (i.e., two antibody probes simultaneously bind to the antigen to form a sandwich structure), the high selectivity for the target antigen can be ensured, reducing the possibility of non-specific binding, thereby improving the accuracy of the detection results.

[0016] 2. The formation of the sandwich immuno-complex enables the oligonucleotide chains on the two antibody probes to approach each other, and then in the subsequent steps of enzymatic cleavage release, complementation, and ligation, it is easier to form a circular DNA template. This proximity effect promotes the occurrence of the rolling circle amplification (RCA) reaction, thereby achieving a significant signal amplification.

[0017] Furthermore, a single-stranded DNA is conjugated to the second DNA antibody conjugate probe. The single-stranded DNA has a hairpin loop structure and is a 3'-to-5' sequence.

[0018] The beneficial effects of the basic protocol are as follows: 1. The design of the hairpin loop structure enables the single-stranded DNA to self-close when not enzymatically cleaved, avoiding non-specific hybridization and amplification. When the antibody binds to the antigen, through specific enzymatic cleavage, the hairpin loop is opened and complementary DNA sequences are released, and these sequences can hybridize with each other to form a circular DNA template. Due to the precise design of the hairpin loop structure, the efficiency of the hybridization and cyclization processes is ensured, thereby improving the signal amplification efficiency of the rolling circle amplification (RCA).

[0019] 2. The design of the hairpin loop structure simplifies the operation steps. Before the rolling circle amplification, no additional DNA template preparation step is required because the hairpin loop structure can be directly used for hybridization and cyclization after being opened. This reduces the complexity of the operation and improves the detection efficiency.

[0020] Furthermore, a partial double-stranded DNA is conjugated to the first DNA antibody conjugate probe, and one of the single-stranded DNAs has a hairpin loop structure and is a 3'-to-5' sequence.

[0021] The beneficial effects of the basic scheme are as follows: The 3'-to-5' sequence design of the hairpin loop structure increases the specificity of hybridization. Only when the first DNA antibody-conjugated probe binds to the antigen and the hairpin loop structure is correctly opened can the sequence complementary to the other single-stranded DNA be released. This proximity dependence ensures that only specifically bound antigens can trigger subsequent rolling circle amplification reactions.

[0022] Furthermore, the hairpin loop structure sequence in the partially double-stranded DNA conjugated to the first DNA antibody-conjugated probe is reverse complementary to the root sequence of the single-stranded DNA conjugated to the second DNA antibody-conjugated probe, and the hairpin loop structure sequence of the single-stranded DNA conjugated to the second DNA antibody-conjugated probe is reverse complementary to the exposed single-stranded sequence in the partially double-stranded DNA conjugated to the first DNA antibody-conjugated probe.

[0023] The beneficial effects of the basic scheme are as follows: 1. Due to the reverse complementary design of the hairpin loop structure sequence, when the two antibody-conjugated probes bind to the antigen to form a sandwich structure, the DNA strands between them can undergo precise hybridization. This hybridization not only enhances the stability of the sandwich structure but also improves the specificity of detection. The reverse complementary sequence design enables only the probes that specifically bind to the antigen to undergo hybridization, thereby reducing the possibility of non-specific binding. This helps to reduce background signals and improve the accuracy of detection.

[0024] 2. Due to the design of the hairpin loop structure and the reverse complementary sequence, a circular DNA template can be directly generated after the formation of the sandwich structure without additional DNA template preparation steps. When the two antibody-conjugated probes bind to the antigen, the DNA strands between them form a circular DNA template through hybridization. Due to the presence of the hairpin loop structure and the design of the reverse complementary sequence, the formation of this circular DNA template is more efficient and stable.

[0025] Furthermore, the length of the hairpin loop structure single-stranded DNA in the partially double-stranded DNA conjugated to the first DNA antibody-conjugated probe and the single-stranded DNA conjugated to the second DNA antibody-conjugated probe is greater than or equal to 25 bp.

[0026] The beneficial effects of the basic scheme are as follows: 1. The length of the DNA probe is one of the key factors affecting hybridization efficiency. When the probe length is greater than or equal to 25 bp, they can hybridize more effectively with the target DNA sequence. Probes of this length can provide sufficient base pairs for stable complementary pairing, thereby improving the efficiency and accuracy of hybridization.

[0027] 2. A longer probe length helps reduce the possibility of non-specific hybridization. Shorter probes may be more likely to hybridize with incompletely matching sequences, resulting in an increase in background signal. Probes with a length greater than or equal to 25 bp can hybridize more strictly and selectively with the target sequence, thus reducing the interference of non-specific hybridization.

[0028] 3. In the hybridization reaction, if the probe structure is unstable or vulnerable to interference, it may lead to problems such as non-specific hybridization or probe degradation, resulting in false positive results. In particular, the endonuclease with a branched structure may cut off from the partial double-stranded DNA ends of the first DNA antibody-conjugated probe, while probes with a length greater than or equal to 25 bp can avoid these problems and improve the accuracy and reliability of detection.

[0029] Furthermore, the hairpin loop structure sequences in the partial double-stranded DNA conjugated by the first DNA antibody-conjugated probe and the hairpin loop structure sequences in the single-stranded DNA conjugated by the second DNA antibody-conjugated probe both have a length of 8 - 15 bp.

[0030] The beneficial effects of the basic protocol are: 1. When the length of the hairpin loop structure sequence is in the range of 8 - 15 bp, it can provide enough base pairs for stable complementary pairing and maintain the flexibility of the structure, which is beneficial to the hybridization reaction. The hairpin loop structure of this length can bind more effectively to the target DNA sequence and improve the hybridization efficiency.

[0031] 2. The moderate length of the hairpin loop structure sequence helps reduce the possibility of non-specific hybridization. Shorter hairpin loop structure sequences may be more likely to hybridize with incompletely matching sequences, resulting in an increase in background signal. While a length of 8 - 15 bp can hybridize more strictly and selectively with the target sequence, thus reducing the interference of non-specific hybridization.

[0032] Furthermore, the circular DNA template for rolling circle amplification consists of the hairpin loop structure sequences on the first DNA antibody-conjugated probe and the second DNA antibody-conjugated probe and their 5' end sequences.

[0033] The beneficial effects of the basic protocol are: 1. The circular DNA template composed of the hairpin loop structure sequence and its 5' end sequence has a stable structure and is easy to form. This circular template can bind more effectively to the primers and enzymes for rolling circle amplification, thus improving the efficiency and specificity of the amplification reaction. Due to the specific and complex structure of the circular DNA template, it can reduce non-specific binding and amplification with non-target sequences, thus reducing the possibility of background signal and false positive results.

[0034] 2. Traditional rolling circle amplification methods require the separate preparation of circular DNA templates, while the circular DNA template in this basic protocol is directly composed of the hairpin loop structure sequence on the antibody-coupled probe and its 5'-end sequence, eliminating the need for additional template preparation steps and thus simplifying the operation procedure. Due to the reduction of template preparation steps, the detection time of this method is also correspondingly shortened. This helps to improve work efficiency, especially in application scenarios where rapid detection is required.

[0035] Furthermore, the circular DNA template sequence contains primer binding sequences or fluorescent probe binding sequences for qPCR detection, and also contains fluorescent labeling sequences for in-situ fluorescence imaging detection.

[0036] The beneficial effects of the basic protocol are as follows: 1. Since the circular DNA template sequence contains sequences for both qPCR detection and in-situ fluorescence imaging detection, this protocol can achieve multiplex detection. This means that in a single experiment, two different detection methods can be used simultaneously to qualitatively and quantitatively analyze the target DNA, thus providing more comprehensive and accurate detection results.

[0037] 2. qPCR detection and in-situ fluorescence imaging detection each have their own unique advantages and application scenarios. The former occupies an important position in molecular biology research due to its high sensitivity and specificity, while the latter is favored in cell biology and histology research for its intuitive and visual characteristics. The design of this basic protocol enables these two detection methods to complement each other and adapt to different research needs and application scenarios.

[0038] 3. The primer binding sequences and fluorescent probe binding sequences in the circular DNA template sequence are carefully designed to enable highly specific binding to the target DNA sequence. This specific binding helps to reduce interference from non-specific amplification and background signals, thereby improving the accuracy and sensitivity of detection.

[0039] 4. In-situ fluorescence imaging detection directly observes the distribution and expression of the target DNA in cells or tissues through the fluorescent labeling sequence. This intuitiveness helps to more accurately judge the presence and quantity of the target DNA, while providing information about its spatial distribution and cellular localization.

[0040] Furthermore, the oligonucleotide DNA and antibody in the first DNA antibody-coupled probe and the second DNA antibody-coupled probe are both coupled by click chemistry.

[0041] The beneficial effects of the basic protocol are as follows: 1. Click chemistry is an efficient and rapid coupling method that can achieve efficient coupling between oligonucleotide DNA and antibody under mild conditions. This method not only has a fast reaction speed but also a high yield, helping to ensure the stability and reliability of the coupled probe.

[0042] 2. The chemical bonds formed by click chemistry usually have high stability and can maintain the structural integrity of the coupled probe in a complex biological environment. This helps to ensure the stability and persistence of the probe in the organism. The probe coupled by click chemistry has good biocompatibility and low toxicity to living cells. This helps to ensure the safety and effectiveness of the probe in the organism and avoid adverse effects on experimental or therapeutic results.

[0043] 3. Compared with traditional coupling methods, the coupling operation steps of click chemistry are simpler and faster. This helps to simplify the probe preparation process and improve work efficiency. Since the reaction conditions of click chemistry coupling are mild and the yield is high, the production cost of the probe can be reduced. This helps to promote the wide application and commercialization process of the probe. Description of the Drawings

[0044] Figure 1 It is a schematic structural diagram of the DNA antibody-coupled probe in the embodiment of the present invention.

[0045] Figure 2 It is a schematic diagram of the method for detecting traditional PLA probe and the ultrasensitive immunoassay of the present invention in the embodiment of the present invention.

[0046] The reference numerals in the accompanying drawings of the specification include: 1. The first DNA antibody-coupled probe; 2. The second DNA antibody-coupled probe; 3. Antibody; 4. Root; 5. Hairpin stem; 6. Hairpin loop. Detailed Embodiments

[0047] The following is further detailed through specific embodiments: Example 1

[0048] Basically as shown in the attached Figure 1 . Figure 2 An ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification includes the following steps: Step 1. Immune binding: Add the first DNA antibody-coupled probe 1 and the second DNA antibody-coupled probe 2 to the sample to bind to the antigen to form an immune complex; Step 2. Enzymatic cleavage and release: Use a branched structure endonuclease to cut the 5'-end branch of the DNA hairpin structure of the first DNA antibody-coupled probe 1 and the second DNA antibody-coupled probe 2, and release the hairpin loop 6 DNA single strand complementary to the root 4 of the oligonucleotide DNA strand of the other coupled probe; Step 3. Complementation and ligation. Under the influence of the proximity effect formed by the immune binding of Antibody 3, the oligonucleotide DNA strands cleaved from the first DNA antibody-conjugated probe 1 and the second DNA antibody-conjugated probe 2 are complementary to each other, forming a circular DNA single strand with two nicks that is complementary to the oligonucleotide DNA strands of both conjugated probes simultaneously, and the single-strand ends at the nicks are adjacent. Add T4 ligase to circularize the circular DNA single strand; Step 4. Rolling circle amplification. Add phi29 polymerase for rolling circle amplification of the circular DNA single strand; Step 5. Detection. Analyze the content of Antibody 3 by detecting the repeated specific sequences on the rolling circle amplification products through qPCR, and perform in-situ fluorescence imaging of the repeated specific sequences on the rolling circle amplification products in tissue cells by in-situ oligonucleotide fluorescence labeling detection method.

[0049] The specific implementation process is as follows: For qPCR quantitative detection, use human serum as the detection sample, and use rabbit or mouse monoclonal antibodies, as well as goat or rabbit polyclonal antibodies as the primary antibodies.

[0050] 1. Briefly centrifuge the human plasma sample at 10,000×g for 10 min or filter it through a 1.2 µm filter plate to remove cell debris. Add 10 - 20 μL of the treated sample to a 100 μL reaction system, which contains 10 nM of the monoclonal antibody DNA antibody-conjugated probe and 20 nM of the polyclonal antibody DNA antibody-conjugated probe. Incubate at room temperature for 1 hour with gentle oscillation at 300 rpm to form an immune complex. The buffer composition of the reaction system is 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween-20, 1% BSA.

[0051] 2. After the formation of the immune complex, add 90 µL of the digestion buffer and 5 U / µL of flap endonuclease 1 (FEN1) to a final concentration of 0.5 U / µL, and incubate at 37°C for 10 minutes to digest the 5'-end branch of the hairpin loop 6 and release the complementary sequence of the hairpin loop 6. The buffer composition of the digestion buffer is 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT.

[0052] 4. Add the termination buffer to terminate the digestion reaction, and remove the enzyme and the digestion buffer. Add 100 µL of the ligation buffer and 0.25 mg / mL BSA, and add T4 DNA ligase at a final concentration of 0.02 U / µL and incubate at 37°C for 30 minutes to ligate the circular DNA single strand.

[0053] 5. Add the stop buffer to terminate the ligation reaction. For the rolling circle amplification of the circular DNA template, use 0.1 U / µL phi29 DNA polymerase, 100 µL RCA buffer, and incubate at 30 °C for 60 minutes for isothermal amplification, followed by inactivation at 65 °C for 10 minutes to obtain the rolling circle amplification product. The RCA buffer consists of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, and 0.2 mM dNTPs.

[0054] 6. Collect the final eluate and perform qPCR quantitative detection. The primer and fluorescent probe complementary sequences for qPCR are designed for the repeat sequences of the RCA product. Establish the linear relationship between Ct value and concentration by using a known concentration antigen gradient dilution (0.1 fg / mL to 1 ng / mL).

[0055] Detection results: The limit of detection (LOD) of this method is 0.1 fg / mL (using PSA antigen as the model target), which is 10 5 times higher than that of the traditional ELISA (~10 pg / mL). Its linear range is from 0.1 fg / mL to 1 ng / mL (R² > 0.99), covering 6 orders of magnitude. In the cross-reaction test, there is no significant signal for 10 ng / mL of similar proteins (such as CEA, AFP, IgG). Through the double-antibody verification experiment, the RCA signal is generated only when the first DNA antibody conjugated with probe 1 and the second DNA antibody conjugated with probe 2 are both present, and there is no amplification in the single-probe control group.

[0056] In simulated samples containing 10% human serum, 5% hemoglobin, or 2% lipids, the recovery rate remains at 90 - 110%, as shown in Table 1 below.

[0057] Table 1. Recovery rate in complex samples

[0058] For in-situ fluorescence imaging detection, use human immortalized keratinocytes HaCat and tissue sections as detection samples.

[0059] 1. Seed HaCat cells at a density of approximately 120,000 cells / cm2 in 8-well Lab-Tek II chamber slides and culture for 1 - 2 days until 70 - 80% confluence. Seed BJ hTert cells at a density of approximately 47,000 cells / cm2 in the chamber slides and perform serum starvation overnight in DMEM. Subsequently, incubate BJ hTert cells with 50 ng / ml PDGF-BB in DMEM on ice for 1 hour, wash twice continuously with PBS, and then store at -20 °C until use.

[0060] HCT116 cells were seeded in 96-well plates at approximately 15,000 cells per well and cultured adherently for 48 hours. Subsequently, the cells were serum-starved in DMEM for 5 hours. The HCT116 cells were incubated with 100 ng / ml EGF at 37 °C for 3 min, washed twice consecutively with PBS, and used directly.

[0061] 2. Process tissue sections according to standard methods. The slides were dewaxed in 100% xylene (incubated continuously 3 times for 5 minutes, 5 minutes, and 1 minute respectively). Next, the tissue was rehydrated to reduce the ethanol concentration, starting with incubation in 99% ethanol twice for 3 minutes each, then in 95% ethanol twice for 5 minutes and 3 minutes respectively, and finally in 70% ethanol for 3 minutes. The slides were washed 3 times in water and antigen retrieval was performed. In a pressure cooker, the slides were boiled in 1× Dako Target Retrieval Solution at 2 atm pressure and 125 °C for 4 minutes, and then boiled at 90 °C for 3 minutes. Then the tissue sections were allowed to cool gradually to room temperature and washed with water, and the samples were stored in PBS until use.

[0062] The thawed cells were rehydrated in PBS at room temperature for 5 minutes, permeabilized with PBS solution containing 0.2% TritonX100 at room temperature for 5 minutes, and then washed in PBS. For EGFR and Shp2 assays, the cells were permeabilized with PBS solution containing 0.1% TritonX100 at room temperature for 5 minutes, then incubated with PBS solution containing 1% SDS for 5 minutes, and then washed thoroughly with PBS to obtain phosphorylation sites.

[0063] 50% Odyssey blocking buffer and 1× Tris-buffered saline (TBS) were added to the cells and tissue sections, and incubated at 37 °C for 30 minutes. The paired combinations of rabbit and mouse, and goat and rabbit antibodies were incubated with the samples overnight in a humidified chamber at 4 °C. After incubation, before adding the second conjugated probe, the slides were washed 3 times in TBS containing 0.05% Tween20 (TBS-T) for 3 minutes each time.

[0064] 3. Dilute the traditional in situ PLA probes and the first DNA antibody conjugated probe 1 and the second DNA antibody conjugated probe 2 of the present invention to 20, 66, 200, 600, and 1800 ng / ml, and add them to the sample solution buffered with TBS and incubate for 60 min. After incubation, wash 3 times with TBS-T for 3 minutes each time.

[0065] 4. Digest the two probes by adding restriction buffer (20 mM Tris-HCl (pH 7.6), 30 mM NaCl, 1 mM EDTA, 100 mM KCl and 1 nM dithiothreitol (DTT)) and adding 0.25 mg / ml BSA and 0.5 U / µL FEN1, and incubate at 37 °C for 45 minutes. Then wash the slide twice in TBS-T for 3 minutes each time. Subsequently, add 0.02 U / μl T4 DNA ligase in T4 DNA ligase buffer supplemented with 0.25 mg / ml BSA, and ligate the circular oligonucleotides designed on the two adjacent probes at 37 °C for 30 minutes, then wash the slide twice in TBS-T for 3 minutes each time.

[0066] 5. Add phi29 polymerase buffer and add 0.5 U / μl phi29 polymerase, supplement 0.25 mM dNTP and 0.25 mg / ml BSA, and perform isothermal amplification at 37 °C for 60 minutes. Then wash the slide twice in TBS-T for 3 minutes.

[0067] 6. Place the slide in PBS containing 0.025 μM fluorescently labeled detection oligonucleotide and 40 μg / ml Hoechst 33342, supplemented with 2.5 μg / ml salmon sperm DNA and 0.25 mg / ml BSA, and incubate at 37 °C for 30 minutes to observe the RCA products and cell nuclei. Before mounting with mounting medium, wash the slide twice in 1xTBS for 10 minutes each time and once in 0.2×TBS for 15 minutes.

[0068] 7. Observe the slide using a fluorescence microscope and image it. The filter is suitable for the fluorescence wavelengths of DAPI, FITC and Cy3. During image acquisition, the exposure time for DAPI is 40 ms, the exposure time for FITC is 510 ms, and the exposure time for Cy3 is 1.5 s.

[0069] Detection results: To achieve the best signal-to-noise ratio, a series of diluted DNA antibody-conjugated probes were used in the detection experiment. The DNA antibody-conjugated probes of traditional in situ PLA were tested at concentrations of 50, 100, 200, 500, 1200, and 1800 ng / ml. It was analyzed that the DNA antibody-conjugated probe of 500 ng / ml PLA was suitable for studying receptor signal transduction in cells, and 1800 ng / ml was suitable for fluorescence imaging of tissue sections. For the DNA antibody-conjugated probe of the present invention, the tested concentrations were 20, 50, 100, 200, 400, and 600 ng / ml, and the signal-to-noise ratio was the best at a concentration of 50 ng / ml. At higher concentrations of the DNA antibody-conjugated probe, the RCA products became very dense, so that the number of non-specific detection signals in the results increased significantly. This phenomenon is similar to that of the existing in situ PLA DNA antibody-conjugated probe. When one primary antibody was omitted, the amount of signal from cross-reactivity and non-specific binding was low. Omitting any enzyme would cause the disappearance of all signals in cell fluorescence imaging, indicating that the hairpin loop 6 structure could completely avoid the complementarity between non-specific circular DNA templates and probes.

[0070] Compare the efficiency of detecting phosphorylated proteins using traditional PLA probes and the probes of the present invention. Immortalized fibroblast BJ hTert cells were cultured by starvation or stimulated with platelet-derived growth factor-BB (PDGF-BB). Both DNA antibody-conjugated probes used anti-PDGFR and anti-pan-phosphorylation (pY100) as primary antibodies, and an increase in PDGFR-β phosphorylation was detected in the stimulated cells. At both probe concentrations (50 and 500 ng / ml), the probes of the present invention were able to show more phosphorylation processes than traditional in situ PLA. In addition, after stimulation with EGF in HCT116 colorectal cancer cells, the specific phosphorylation of tyrosine 1068 (pY1068) of epidermal growth factor receptor (EGFR) was quantified. Consistent with the results of previous experiments, the fluorescence signal intensity of the probes of the present invention was 5 times higher than that of the traditional PLA probe method under the condition of EGF-stimulated cells at a concentration of 50 ng / ml.

[0071] Example 2

[0072] The difference from the above example is that as shown in the appendix Figure 1 、 Figure 2As shown: The antibody 3 proteins on the first DNA-antibody conjugate probe 1 and the second DNA-antibody conjugate probe 2 can bind to the antigen to form a sandwich immune complex. The oligonucleotide DNA on the first DNA-antibody conjugate probe 1 and the second DNA-antibody conjugate probe 2 are both conjugated to the antibody 3 by click chemistry; a partial double-stranded DNA is conjugated to the first DNA-antibody conjugate probe 1, one of the DNA single strands is a hairpin loop 6 structure and has a 3'-end to 5'-end sequence; a DNA single strand is conjugated to the second DNA-antibody conjugate probe 2, the DNA single strand is a hairpin loop 6 structure and has a 3'-end to 5'-end sequence; the hairpin loop 6 structure sequence in the partial double-stranded DNA conjugated to the first DNA-antibody conjugate probe 1 is reverse complementary to the root 4 sequence of the DNA single strand conjugated to the second DNA-antibody conjugate probe 2, and the hairpin loop 6 structure sequence of the DNA single strand conjugated to the second DNA-antibody conjugate probe 2 is reverse complementary to the exposed single strand sequence in the partial double-stranded DNA conjugated to the first DNA-antibody conjugate probe 1; the hairpin loop 6 structure DNA single strand in the partial double-stranded DNA conjugated to the first DNA-antibody conjugate probe 1 and the DNA single strand conjugated to the second DNA-antibody conjugate probe 2 have a length greater than or equal to 25 bp; the hairpin loop 6 structure sequences in the partial double-stranded DNA conjugated to the first DNA-antibody conjugate probe 1 and the hairpin loop 6 structure sequences in the DNA single strand conjugated to the second DNA-antibody conjugate probe 2 both have a length of 8-15 bp; the circular DNA template for rolling circle amplification is composed of the hairpin loop 6 structure sequences on the first DNA-antibody conjugate probe 1 and the second DNA-antibody conjugate probe 2 and their 5'-end sequences, and the circular DNA template sequence contains a primer binding sequence or a fluorescent probe binding sequence for qPCR detection, and also contains a fluorescent labeling sequence for in-situ fluorescence imaging detection.

[0073] The specific implementation process is as follows: 1. Design two corresponding DNA sequences, including the root 4, hairpin stem 5, and hairpin loop 6 sequences. The hairpin loop 6 and root 4 sequences of the two DNA sequences are complementary to each other. Chemically synthesize and amplify using a 3'-end azide-modified primer, and purify and recover through gel electrophoresis to increase the concentration.

[0074] 2. In the modification method without copper-catalyzed cyclooctyne, use N-hydroxysuccinimide ester (NHS ester) to modify the amino group of antibody 3 to add a linking group to antibody 3, and add the recovered DNA strand for click chemical reaction. The buffer is PBS with a pH of 7.4. The molar ratio of antibody 3 to the DNA strand is 1:5. React overnight with light avoidance and oscillation at 4°C, and purify the DNA-antibody conjugate probe and freeze it at ultra-low temperature for standby.

[0075] Example 3

[0076] The difference from the above example is as shown in the attached Figure 1 、 Figure 2As shown: Traditional PLA probes have been proven to improve the sensitivity of protein detection in binding assays, where an antibody 3 is used to capture the target from the sample, followed by rolling circle amplification with two DNA antibody-conjugated probes. Proximity extension assay (PEA) relies on a smaller reaction distance and reduced background dilution, but cannot be washed before ligation or polymerization.

[0077] Compared with traditional in-situ PLA probes, the probes of the present invention showed the intensity of signal generation in cells and tissues, and the two methods were compared by detecting proteins in solution captured on solid supports. We immobilized the probes in microtiter wells to capture IL-6, and then performed a conventional ELISA assay with an enzyme-conjugated antibody reagent to detect the captured antigen using traditional in-situ PLA or the probes of the present invention. The results showed that compared with the traditional in-situ PLA design, the probe design of the present invention achieved a new limit of detection (LOD) and had better detection performance than ELISA. Diluting the antigen with 10% or 100% cell lysate also demonstrated that the probes of the present invention had better performance than traditional in-situ PLA probes and ELISA in binding assays.

[0078] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variation thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0079] The above are only embodiments of the present invention. Specific structures and common knowledge such as those well-known in the art are not described in detail herein. Those of ordinary skill in the art know all the common general technical knowledge in the technical field to which the invention belongs before the filing date or the priority date, can know all the prior art in this field, and have the ability to apply the conventional experimental means before this date. Those of ordinary skill in the art can, under the inspiration given in this application, complete and implement this solution in combination with their own abilities. Some typical well-known structures or well-known methods should not be an obstacle for those of ordinary skill in the art to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several modifications and improvements can be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be subject to the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. A super-sensitive immunoassay method based on proximity nucleic acid amplification signal amplification, characterized in that: Including the following steps: Step 1, Immune binding: Add the first DNA antibody-coupled probe (1) and the second DNA antibody-coupled probe (2) to the sample to bind to the antigen to form an immune complex; Step 2, Enzymatic cleavage and release: Use a branched structure endonuclease to cut the 5'-end branch of the DNA hairpin structure of the first DNA antibody-coupled probe (1) and the second DNA antibody-coupled probe (2), and release the hairpin loop (6) DNA single strand complementary to the root (4) of the oligonucleotide DNA strand of the other coupled probe; Step 3, Complementary pairing and ligation: Under the influence of the proximity effect formed by antibody immune binding, the oligonucleotide DNA strands cut from the first DNA antibody-coupled probe (1) and the second DNA antibody-coupled probe (2) are complementary to each other, forming a circular DNA single strand with two nicks that is complementary to the oligonucleotide DNA strands of both coupled probes at the same time, and the single-strand ends at the nicks are adjacent. Add T4 ligase to circularize the circular DNA single strand; Step 4, Rolling circle amplification: Add phi29 polymerase to perform rolling circle amplification of the circular DNA single strand; Step 5, Detection: Analyze the content of antibody (3) by detecting the repeated specific sequences on the rolling circle amplification product through qPCR, and perform in-situ fluorescence imaging of the repeated specific sequences on the rolling circle amplification product in tissue cells through the in-situ oligonucleotide fluorescence labeling detection method.

2. The ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification according to claim 1, wherein: The antibody (3) proteins on the first DNA antibody-coupled probe (1) and the second DNA antibody-coupled probe (2) can bind to the antigen to form a sandwich immune complex.

3. The ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification according to claim 2, wherein: The second DNA antibody-coupled probe (2) is coupled with a DNA single strand, and the DNA single strand is in the hairpin loop (6) structure and has a 3'-end to 5'-end sequence.

4. The ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification according to claim 2, wherein: The first DNA antibody-coupled probe (1) is coupled with a partial double-stranded DNA, and one of the DNA single strands is in the hairpin loop (6) structure and has a 3'-end to 5'-end sequence.

5. The ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification according to claim 2, characterized in that: The hairpin loop (6) structure sequence in the partial double-stranded DNA coupled to the first DNA antibody-coupled probe (1) is reverse complementary to the root (4) sequence of the DNA single strand coupled to the second DNA antibody-coupled probe (2), and the hairpin loop (6) structure sequence of the DNA single strand coupled to the second DNA antibody-coupled probe (2) is reverse complementary to the exposed single-strand sequence in the partial double-stranded DNA coupled to the first DNA antibody-coupled probe (1).

6. The ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification according to claim 5, characterized in that: The hairpin loop (6) structure DNA single strand in the partial double-stranded DNA coupled to the first DNA antibody-coupled probe (1) and the DNA single strand coupled to the second DNA antibody-coupled probe (2) are greater than or equal to 25 bp in length.

7. The ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification according to claim 6, wherein: The hairpin loop (6) structure sequences in the partial double-stranded DNA coupled to the first DNA antibody-coupled probe (1) and the DNA single strand coupled to the second DNA antibody-coupled probe (2) are both 8 - 15 bp in length.

8. The ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification according to claim 7, wherein: The circular DNA template for rolling circle amplification is composed of the hairpin loop (6) structure sequences on the first DNA antibody-coupled probe (1) and the second DNA antibody-coupled probe (2) and their 5'-end sequences.

9. The ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification according to claim 8, wherein: The circular DNA template sequence contains a primer binding sequence or a fluorescent probe binding sequence for qPCR detection, and also contains a fluorescent labeling sequence for in-situ fluorescence imaging detection.

10. The ultrasensitive immunoassay method based on proximity nucleic acid amplification signal amplification according to claim 3, characterized in that: Both the oligonucleotide DNA and the antibody (3) of the first DNA antibody conjugate probe (1) and the second DNA antibody conjugate probe (2) are conjugated by click chemistry.

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