CN113092754A

CN113092754A - Immunofluorescence and two-dimensional visualization multi-mode analysis method for ultra-sensitively identifying HIV p24 antigen and application thereof

Info

Publication number: CN113092754A
Application number: CN202110383687.1A
Authority: CN
Inventors: 陈飘飘; 唐卓芸; 应斌武; 陶传敏
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2021-04-09
Filing date: 2021-04-09
Publication date: 2021-07-09
Anticipated expiration: 2041-04-09
Also published as: CN113092754B

Abstract

The invention provides an immunofluorescence and two-dimensional visualization multi-mode analysis method for ultra-sensitively recognizing HIV p24 antigen and application thereof, relating to the technical field of biomedical diagnosis and analysis methods, wherein the analysis method comprises the steps of carrying out antigen-antibody immune recognition reaction on HIV p24 antigen and biotin-modified antibody, introducing a poly-T chain, or introducing a nucleic acid chain to carry out auxiliary extension reaction with polymerase to generate the poly-T chain, and carrying out the poly-T chain and Cu chain²⁺Forming poly T template CuNPs, and combining with QDs to selectively recognize Cu²⁺And a phenomenon of poly-T template CuNPs, and identifying a single target object based on fluorescence signals of the poly-T template Cu NPs or QDs, wherein the target object is a HIVp24 antigen. The invention improves the detection limit, constructs a two-dimensional visualization strategy, lays a foundation for the POCT analysis of diseases in the future and is convenient to popularize in primary hospitals.

Description

Immunofluorescence and two-dimensional visualization multi-mode analysis method for ultra-sensitively identifying HIV p24 antigen and application thereof

Technical Field

The invention relates to the technical field of biomedical diagnosis and analysis methods, in particular to an immunofluorescence and two-dimensional visualization multi-mode analysis method for ultra-sensitively identifying HIV p24 antigen and application thereof.

Background

Acquired Immune Deficiency Syndrome (AIDS) is a serious infectious disease caused by HIV that threatens human health seriously, and the death number and death rate of the AIDS are the first of the legal infectious diseases in our country. As reported by the world health organization, aids virus carriers (PLWH) have exceeded 3790 thousands by 2018, with only 75% of people knowing their own infection status. HIV infection can be divided into three stages: acute HIV Infection (AHI), asymptomatic infection and AIDS. Among them, AHI is the most dangerous stage of infection, with a risk of infection 26 times higher than during asymptomatic infection. The 90-90-90 targets proposed by the United nations AIDS planning agency/world health organization and the 'Know your status' theme of the world AIDS day emphasize that screening for HIV infection has profound significance for the prevention and control of AIDS, especially for screening for AHI. Early screening allows for early diagnosis and effective treatment, which can improve overall survival and reduce transmission.

Fourth generation antigen antibody reagents are now commercially available kits and are widely used in clinical assays to identify both HIV-1 p24 antigen and anti-HIV IgM and IgG antibodies, thereby reducing the "window period" to about 2 weeks. However, this agent cannot distinguish between antigen and antibody, resulting in unclear disease stage. The HIV-1 p24 antigen appeared about 7 days earlier than the antibody and was one of the earliest protein biomarkers in the assay. Therefore, the detection of HIV-1 p24 antigen has important value in the early detection of diseases.

The existing HIV p24 detection method mainly utilizes an immunoassay principle, and an electrochemical active substance ruthenium tripyridine or horse radish peroxidase (HRP, catalytic substrate TMB) is marked on a detection antibody for color development, so that the detection of p24 is realized by monitoring an electrochemical luminescence signal and an ultraviolet absorption signal respectively. However, the above electrochemical luminescence method has high instrument cost, low analysis sensitivity of the ultraviolet absorption spectrometer, and is easily interfered by the detection medium; most importantly, the method depends on the existing detection instrument, and portable visual rapid analysis is difficult to realize.

Even though some HIV p24 analysis methods have been reported to visualize color readings, they are only identification in this dimension of color. For some color blindness people, even color weakness people, the accuracy and effectiveness of the method will be greatly influenced. Therefore, the development of a multidimensional visualization analysis method is significant.

Disclosure of Invention

In view of the above, an objective of the present invention is to provide an immunofluorescence and two-dimensional visualization multimodal analysis method for ultra-sensitive recognition of HIV p24 antigen, so as to solve the following disadvantages of the existing clinical HIV p24 analysis method: 1. the immune recognition-electrochemiluminescence/chemiluminescence/ultraviolet absorption spectrum has low detection sensitivity, and can only realize the detection limit of pg/mL level; 2. the dependence on instruments is large, and POCT analysis is difficult to realize; 3. the electrochemical luminescence and the chemiluminescence are expensive and difficult to miniaturize and carry, and the popularization of the electrochemical luminescence and the chemiluminescence in primary hospitals is limited.

The invention also aims to provide application of the immunofluorescence and two-dimensional visualization multimode analysis method for ultra-sensitively recognizing the HIV p24 antigen.

In order to achieve one of the above objects, the present invention provides an immunofluorescence and two-dimensional visualization multimodal assay method for ultra-sensitive recognition of HIV p24 antigen, the method comprising an antigen-antibody immunological recognition reaction of HIV p24 antigen with biotin-modified antibody, introducing poly-T chain, or introducing nucleic acid chain to react with polymerase-assisted extension to form poly-T chain, which reacts with Cu to form poly-T chain²⁺Forming poly T template Cu NPs, and selectively recognizing Cu by combining with QDs²⁺And poly-T template Cu NPs, and identifying a single target based on fluorescence signals of the poly-T template Cu NPs or QDs, wherein the target is the HIVp24 antigen.

According to a preferred embodiment, when QDs are used as signal molecules, a two-dimensional change analysis mode based on color and test strip distance is constructed, and when poly-T template Cu NPs are used as signal molecules, a change analysis mode based on fluorescence signals is constructed.

According to a preferred embodiment, the hydrolase is a TdT enzyme, phi29 polymerase or an elongase.

According to a preferred embodiment, the nucleic acid strands are DNA templates, ssDNA of different sequences and double stranded DNA.

According to a preferred embodiment, the Cu²⁺Can be replaced by Ag⁺。

According to a preferred embodiment, said QDs comprise CdTe QDs or CdSe QDs.

According to a preferred embodiment, the excitation wavelength of the poly-T template CuNPs is 340 nm.

According to a preferred embodiment, the immunofluorescence and two-dimensional visualization multimodal assay method is a two-dimensional HIV p24 antigen assay method using the strong luminescence properties of the QDs, integrated with inkjet printing technology, to achieve color and distance readings.

In order to achieve the second purpose, the invention provides application of an immunofluorescence and two-dimensional visualization multimode analysis method for ultra-sensitively recognizing HIV p24 antigen, wherein the application comprises applying any one of the immunofluorescence and two-dimensional visualization multimode analysis method for ultra-sensitively recognizing HIV p24 antigen to p24 antigen analysis in serum of clinical HIV patients.

The invention provides an immunofluorescence and two-dimensional visualization multi-mode analysis method for ultra-sensitively identifying HIV p24 antigen and application thereof, and the method has the following technical effects:

(1) on the premise of retaining antigen-antibody immune recognition reaction, the invention introduces nucleic acid poly-T chain template copper nanoparticles (CuNPs) as signal molecules and recognition media to construct a multi-mode two-dimensional visual fluorescence analysis method, and when a nucleic acid-free signal amplification technology is introduced, the sensitivity similar to that of the existing clinical method can be realized.

(2) Meanwhile, selective cation exchange reaction based on QDs found in the early stage is integrated, and POCT analysis of color and distance reading is realized.

(3) In addition, the method uses a fluorescence analysis technology, and various portable miniaturized fluorescence instruments are used, so that the invention not only provides a new method for clinical immunoassay, but also lays a foundation for POCT analysis of diseases in the future.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram of immunological two-dimensional visualization and fluorescence analysis of HIV p24 based on QDs selective recognition reaction and TdT enzyme assistance;

FIG. 2 is a representation of poly T templates CuNPs and QDs, and validation of feasibility of p24 analysis;

FIG. 3 shows the optimization of HIV p24 analysis conditions when T40 template CuNPs are used as signal molecules;

FIG. 4 is a selective cation exchange assisted HIV p24 assay condition optimization;

FIG. 5 is TdT enzyme-assisted optimization of HIV p24 assay conditions;

FIG. 6 is the performance of the HIV p24 assay in different assay formats;

FIG. 7 is the results of serum sample analysis of clinical HIV patients.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

1. Principle of the invention

Based on the current situation of diagnosis and research of clinical HIV, on the premise of retaining antigen-antibody immunoassay, a nucleic acid chain is connected to a biotin (biotin) -modified second antibody, luminescent poly-T chain templates CuNPs and inorganic Quantum Dots (QDs) are introduced, and the Cu can be selectively identified by combining with the QDs²⁺And poly-T template CuNPs, the CuNPs and QDs multimode fluorescence analysis strategy of HIV p24 was developed, as shown in FIG. 1-Mode 1.

Subsequently, using the strong luminescence properties of QDs, and integrating inkjet printing technology, a two-dimensional p24 analysis method of color and distance reading was developed, which could offer more options for clinical POCT diagnosis and could be easily generalized in primary hospitals and remote villages, etc.

Furthermore, in view of the importance of early diagnosis of HIV, a terminal deoxyribonuclease (TdT enzyme) -assisted nucleic acid amplification technique was introduced, which aids in the generation of poly-T chains as a template for the generation of CuNPs, with the goal of improving the sensitivity of the p24 assay, as shown in fig. 1-mode 2. TdT catalyzes the formation of poly-ssDNA from any single stranded DNA (ssDNA) in the presence of substrates (dNTPs). The TdT enzyme has no special requirements on the sequence of a substrate ssDNA, so the TdT enzyme is easy to be used in various bioanalytical methods, and has low cost and high enzyme activity.

P24 analytical procedure

2.1 immunoassay step:

first, 10 microliters of 20. mu.g/mL recognition antibody (Ab)₁) And 30. mu.L of assay buffer (10mmol/LMOPS buffer, pH7.4, 100mmol/L NaNO)₃，2.5mmol/L Mg(NO₃)₂) After mixing, incubation was carried out overnight at 4 ℃. The above 96-well plate was washed 3 times with 200. mu.l of washing buffer (assay buffer containing 0.05% (v/v) Tween 20) for removing unbound recognition antibody.

Subsequently, 100. mu.l of blocking buffer (1% w/v, g mL) was added^-1BSA, 10mmol/L pH7.4, MOPS buffer) was incubated at 37 ℃ for 1 hour for blocking the remaining empty space in the 96-well plate and washed 3 times. Subsequently, 40 microliters of different concentrations of HIV p24 protein or serum clinical samples, as well as 60 microliters of assay buffer were added to 96-well plates, incubated for 1 hour at 37 ℃, and washed three times.

Finally, 40 microliters of 10. mu.g/mL biotin-labeled detection antibody (biotin-Ab)₂) Add to 96-well plate with 60. mu.l assay buffer and incubate at 37 ℃ for 1 hour for the formation of a primary anti-p 24-detection antibody-biotin sandwich complex and wash three times.

2.2T40 template CuNPs as signal molecule

To the mixture described in 2.1, 40. mu.L of 1. mu.g/mL Streptavidin (SA) and 60. mu.L MOPS assay buffer were added and incubated for 1h at room temperature; followed by 3 washes with washing buffer to form an anti-p 24-detection antibody-biotin-SA sandwich complex.

Then, 25. mu.L of 0.5. mu. M T40-DNA and 21. mu.L of LMOPS assay buffer were added to the above solution, and reacted at room temperature for 30min, and 45. mu.L of the supernatant solution was aspirated.

Subsequently, 40. mu.L of 3. mu.M CuSO was added₄And 20 μ L of 4mM Ascorbic Acid (AA), and 100 μ L LMOPS assay buffer were added to the above solution and incubated at room temperature for 10min for generation of T40 template CuNPs.

Finally, the intensity of the CuNPs fluorescence was monitored under 340nm light excitation.

2.3QDs as Signal molecules

To the CuNPs solution generated by the reaction described in 2.2, 2.75. mu.L of QDs (stock solution diluted 10-fold) was added and incubated at room temperature for 7min to complete the cation exchange reaction.

Finally, changes in the fluorescence intensity of QDs were monitored under 365nm light excitation.

2.4 analysis of p24 with the aid of TdT enzyme

To the mixture described in 2.1, 40. mu.L of 1. mu.g/mL SA and 60. mu.L MOPS assay buffer were added and incubated for 1h at room temperature.

Subsequently, 3 washes with washing buffer formed a single anti-p 24-detection antibody-biotin-SA sandwich complex.

Subsequently, 40. mu.L of 0.25. mu. M P1-DNA-biotin and 21. mu.L of MOPS assay buffer were added and incubated at room temperature for 30min to form an anti-P24-detection antibody-biotin-SA-biotin-P1-DNA sandwich complex, and 30. mu.L of the supernatant solution was aspirated and divided into two portions.

Then, 5. mu.L of 30mM dTTP, 5. mu.L of 10 XTdT enzyme buffer, and 0.4. mu.L of TdT enzyme were added to the above 15. mu.L solution, and incubated at 37 ℃ for 20 minutes to form poly T chains, after which the above solution was incubated at 75 ℃ for 10 minutes to inactivate the enzyme.

Finally, 40. mu.L of 100. mu.M CuSO was added to the above solution₄And 20 μ L of 4mM AA, and 100 μ L of MOPS assay buffer, and incubated at room temperature for 10min to generate poly-T template CuNPs; the CuNPs fluorescence intensity of the different solutions was monitored under excitation light at 340 nm.

Synthesis of QDs

CdTe QDs are synthesized according to a one-pot method:

first, 0.5mmol of CdCl₂And 0.20g of trisodium citrate dissolved in 50 ml of water, 52. mu.L of mercaptopropionic acid (MPA) added to the above solution. The above mixture solution was adjusted to pH 10.5 using NaOH solution.

Then, 0.1mmol of Na₂TeO₃And 50mg KBH₄Adding into the above solution, refluxing for 1 hr until the solution is red, and under ultraviolet lamp, it shows strong red fluorescence.

Finally, the CdTe QDs solution is purified by precipitation (using n-propanol) and centrifugation (11000rpm,30 minutes). The MPA-CdTe QDs synthesized above are stored at 4 deg.C before use.

4. Material characterization and multimodal p24 analytical feasibility

Before verifying the analytical feasibility of the different analytical patterns p24, poly-T chain templates Cu NPs and QDs involved in the experiment were first synthesized and characterized. As shown in FIGS. 2A and 2B, the synthesized CuNPs are uniform sphere-like, have a particle size of about 4nm, and exhibit a characteristic UV absorption peak at 340 nm. The synthesized QDs have a particle size similar to CuNPs, which have a lattice structure (fig. 2C and 2D). When Cu²⁺When mixed with QDs, the QDs undergoes significant agglomeration, i.e., a cation exchange reaction occurs, yielding CuTe.

Subsequently, the feasibility of p24 analysis was examined for T40-Cu NPs as signal molecules. As shown in FIG. 2F, CuNPs were successfully generated when T40 was the template, and the fluorescence signal for CuNPs was changed when the amount of T40 template was changed (compare FIGS. 2F-a and b). When the p24 content was varied, the fluorescence signal of the solution gradually decreased with increasing p24 content (FIGS. 2F-c to F). Namely, the above experimental results show that the p24 analysis strategy of T40-CuNPs as signal molecules is feasible.

Before verifying the feasibility of p24 analysis when QDs are used as signal molecules, the method firstly identifies Cu selectively for QDs²⁺And the phenomenon of DNA template Cu NPs were examined. As shown in FIG. 2G, Cu²⁺The quenching effect on the fluorescence signal of the QDs is obviously stronger than that of T40 template Cu NPs on the QDs (FIG. 2G)-a to c).

In addition, the above selective recognition phenomenon can be recognized by naked eyes under an ultraviolet lamp, such as the inset in fig. 2G, which can read the color of the solution in the test tube, and the distance change of the designed ink-jet printing test strip.

Subsequently, varying the p24 concentration while monitoring the change in fluorescence signal of QDs in solution, it was found that the fluorescence signal gradually decreased as the p24 concentration increased. That is, the strategy was applicable to p24 analysis, and p24 analysis was available at the pg/mL concentration level.

The feasibility of the TdT enzyme-assisted p24 assay strategy was also verified, as shown in fig. 2H and 2I. The analysis sensitivity of p24 under the assistance of TdT enzyme is greatly improved, the result of a preliminary experiment shows that the analysis of fg/mL grade p24 can be realized, and the DNA gel electrophoresis experiment also shows that the TdT enzyme assists the successful generation of poly T chain.

Optimization of p24 analysis conditions

5.1T40 template CuNPs as signal molecule

Firstly, the p24 analysis condition is optimized when the T40 template Cu NPs are signal molecules. As shown in fig. 3, among poly-T chains with different lengths, T40 chain shows better signal-to-noise ratio (fig. 3A); maximum fluorescence signal difference was obtained at 0.5 μ MT40 concentration (fig. 3B and C); 3 μ M Cu²⁺When, the signal difference is maximum (fig. 3D and E); the highest fluorescence signal was generated for CuNPs at 4mM ascorbic acid (FIG. 3F).

5.2QDs as Signal molecules

When QDs are signal molecules, the conditions for immunoassay and the like are the same as those described above, and the conditions for selective cation exchange reaction are shown in FIG. 4. The maximum fluorescence signal difference was obtained when 2.75. mu.L was diluted 10-fold of QDs (FIGS. 4A and B); the cation exchange reaction can be completed within 7 minutes (fig. 4C).

5.3TdT enzyme-assisted

As shown in FIG. 5, the maximum signal difference was obtained at a Streptavidin (SA) concentration of 1. mu.g/mL (FIGS. 5A and B); ab can be completed in 30 minutes₁-recognition reaction of biotin and SA (fig. 5C); recognition binding of P1-DNA-biotin to SA was also completed within 30 minutes (fig. 5D); 0.25 μ MP1-DNA-biotin (FIGS. 5E and F), 5.6U TdT enzyme (FIGS. 5G and H); d of 15mMTTP concentration (fig. 5I and J); TdT enzyme extension reaction time of 20 minutes (fig. 5K); 100 μ M CuSO₄(FIGS. 5L and M); 4mM AA (FIG. 5N); and 10 minutes for Cu NPs generation, the maximum fluorescence signal or difference in fluorescence signal, respectively, can be achieved.

6 p24 analytical Performance in different modes

6.1T40 template CuNPs as signal molecule

The quantitative capability of this system to p24 was examined when Cu NPs were used as signal molecules. As shown in FIG. 6A, the fluorescence signal of the system gradually decreases as the concentration of p24 increases. As shown in FIG. 6B, after comparing and fitting the data, it can be found that the log of the concentration shows a good linear relationship with the fluorescence signal in the concentration range of 10pg/mL to 10ng/mL, and the detection limit is 3pg/mL (based on triple signal-to-noise ratio).

6.2QDs as Signal molecules

Subsequently, QDs can be identified as Cu²⁺And T40-CuNPs, as signal molecules, with QDs introduced into the p24 assay.

Considering the sensitivity of the assay when the fluorometer is used as the detector first, as shown in FIGS. 6C and D, similarly to when the T40 template CuNPs is used as the signal molecule, the fluorescence signal of QDs is gradually reduced when the concentration of p24 is increased, and the logarithm of the concentration in the range of 1-1000pg/mL shows a good phenomenological relationship with the fluorescence signal, with the detection limit of 0.25pg/mL (based on the triple signal ratio). That is, the QDs-based cation exchange reaction improves the detection sensitivity by 10 times.

Furthermore, in view of the high luminescence properties of QDs in combination with inkjet printing technology, a two-dimensional visualization p24 analysis strategy was constructed, as shown in fig. 6E, the solution color becomes lighter as the p24 concentration increases, i.e., the less CuNPs are generated, the lower the fluorescence signal of QDs, and the color visualization can achieve 100pg/mL p24 sensitivity.

Subsequently, the analytical ability of the inkjet-printed test strip for p24 was examined, and as shown in fig. 6F, the quenching reaction of QDs on the test strip became weaker and closer as the concentration of p24 increased. Comparison shows that the blank solution and the 10pg/mL p24 concentration can be easily distinguished at distance, i.e., the distance analysis sensitivity is comparable to when the fluorometer is used as a detector.

6.3TdT enzyme-assisted

The analysis quantitative sensitivity of the above strategy is similar to that of the existing clinical electrochemical luminescence method, and in order to further improve the analysis sensitivity and provide more choices for clinical ultra-early HIV screening, TdT enzyme extension reaction is introduced, and poly T chain is generated by TdT enzyme catalysis, so that the ultra-sensitive analysis of p24 is realized.

As shown in FIG. 6G, a TdT-assisted assay of p24 concentration at fg/mL level was achieved, and in the 0.1-10fg/mL concentration range, the log of the concentration showed a good linear relationship with the fluorescence signal, with a detection limit as low as 0.04fg/mL, a 4-order reduction over QDs as signal molecules (FIG. 6H).

Subsequently, the p24 assay strategy was examined for its ability to interfere with the enzyme, using co-existing proteins in serum as potential interferents. As shown in FIGS. 6I and J, the decrease in the fluorescence signal of the system caused by various high concentrations (0.1pg/mL) of interfering proteins was comparable to that of the blank solution and was almost negligible. Whereas low concentrations of p24(10 and 100fg/mL) caused a significant signal reduction. That is, this strategy has high p24 selectivity, which is mainly due to the high specific recognition ability between antigen and antibody.

7 applicability of this p24 analytical strategy in clinical HIV patient samples

Under the excitation of high sensitivity and selectivity of the TdT enzyme-assisted p24 analysis strategy, the system is used for p24 analysis in serum of clinical HIV patients, and the analysis result is compared with the clinical electrochemiluminescence strategy to verify the accuracy and the applicability of the method.

First, 20 HIV early stage patients were selected and tested for p24 content in their sera, as shown in FIGS. 7A and 7B, the p24 content was analyzed at pg/mL level and was consistent well with the clinical electrochemiluminescence COI value results with a linear correlation coefficient of 0.993 (FIG. 7C).

Subsequently, we monitored the serum p24 levels in the same patient at different times and found that the p24 levels in patients decreased rapidly within 3-5 days as the time of diagnosis was extended, which is consistent with the results of the current clinical study (FIG. 7D).

Finally, to ensure the accuracy of the method and its utility in clinical samples, 10 normal human sera, 16 non-HIV patient sera, and 3 HIV-positive patient sera were selected and analyzed for p24 content. As shown in FIG. 7E, the serum levels of p24 were very low in normal and non-HIV patients, and the effect on the fluorescence signal in the system was similar to that of the blank solution, and was significantly lower than the decrease in fluorescence signal caused by the serum of the HIV-positive patient.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. An immunofluorescence and two-dimensional visualization multimode analysis method for ultra-sensitively recognizing HIV p24 antigen, which is characterized in that the method comprises the steps of carrying out antigen-antibody immunological recognition reaction on HIV p24 antigen and biotin-modified antibody, introducing a poly T chain, or introducing a nucleic acid chain to react with polymerase-assisted extension to generate a poly T chain, wherein the poly T chain and Cu are subjected to polymerase-assisted extension reaction²⁺Forming poly T template Cu NPs, and selectively recognizing Cu by combining with QDs²⁺And poly-T-template Cu NPs, and recognizing a single target based on fluorescence signals of the poly-T-template Cu NPs or QDs, wherein the target is HIV p24 antigen.

2. The immunofluorescence and two-dimensional visualization multimode analysis method for ultrasensitive recognition of HIV p24 antigen according to claim 1, wherein a two-dimensional change analysis mode based on color and test strip distance is constructed with QDs as signal molecules, and an analysis mode based on fluorescence signal change is constructed with poly-T template Cu NPs as signal molecules.

3. The immunofluorescence and two-dimensional visualization multimodal assay for the ultrasensitive recognition of HIV p24 antigen according to claim 2, wherein the polymerase is TdT enzyme, phi29 polymerase or elongase.

4. The immunofluorescence and two-dimensional visualization multimode assay for the ultra-sensitive recognition of HIV p24 antigen according to any one of claims 1 to 3, wherein the nucleic acid strand is DNA template, ssDNA of different sequence and double-stranded DNA.

5. The immunofluorescence and two-dimensional visualization multimodal assay for the ultrasensitive recognition of the HIV p24 antigen according to claim 1, wherein the Cu²⁺Can be replaced by Ag⁺。

6. The immunofluorescence and two-dimensional visualization multimodal assay for the ultrasensitive recognition of HIV p24 antigen according to claim 1, wherein the QDs comprise CdTeQDs or CdSeQDs.

7. The immunofluorescence and two-dimensional visualization multimodal assay for the ultrasensitive recognition of the HIV p24 antigen according to claim 1, wherein the excitation wavelength of the poly-T template Cu NPs is 340 nm.

8. The immunofluorescence and two-dimensional visualization multimodal assay for ultrasensitive recognition of HIV p24 antigen according to claim 1, wherein the immunofluorescence two-dimensional visualization assay is a two-dimensional HIV p24 antigen assay that utilizes the strong luminescence property of QDs, integrates inkjet printing technology, and realizes color and distance reading.

9. Use of an immunofluorescence and two-dimensional visualization multimodal assay for the ultrasensitive recognition of the HIV p24 antigen, comprising applying the immunofluorescence two-dimensional visualization assay for the ultrasensitive recognition of the HIV p24 antigen according to any one of claims 1 to 8 to the analysis of the p24 antigen in the serum of clinical HIV-infected patients.