CN113092754B

CN113092754B - Detection product for multi-mode analysis of HIV p24 antigen based on immunofluorescence and two-dimensional visualization and application thereof

Info

Publication number: CN113092754B
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: 2022-11-15
Anticipated expiration: 2041-04-09
Also published as: CN113092754A

Abstract

The invention provides an immunofluorescence and two-dimensional visual 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 the HIV p24 antigen and an antibody modified with biotin, 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 chain ²⁺ Forming a poly T template CuNPs, and selectively identifying Cu by combining with QDs ²⁺ And 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 POCT analysis of diseases in future and is convenient to popularize in primary hospitals.

Description

Detection product for multi-mode analysis of HIV p24 antigen based on immunofluorescence and two-dimensional visualization and application thereof

Technical Field

The invention relates to the technical field of biomedical diagnosis and analysis methods, in particular to a detection product for multi-mode analysis of HIV p24 antigen based on immunofluorescence and two-dimensional visualization 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 simultaneously identify 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 appears about 7 days earlier than the antibody and is one of the earliest protein biomarkers in the assay. Therefore, HIV-1 p24 antigen detection has important value in 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 p24 detection 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 this, an objective of the present invention is to provide a detection product for multi-modal analysis of HIV p24 antigen based on immunofluorescence and two-dimensional visualization, 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 chemiluminescence instruments are expensive, and are difficult to realize miniaturization and portability, so that the popularization of the electrochemical luminescence and chemiluminescence instruments in primary hospitals is limited.

The invention also aims to provide the application of the detection product for analyzing the HIV p24 antigen based on immunofluorescence and two-dimensional visualization multimode.

In order to achieve one of the above objects, the present invention provides a detection product for analyzing HIV p24 antigen based on immunofluorescence and two-dimensional visualization multimode, the method comprises performing antigen-antibody immune recognition reaction of HIV p24 antigen and 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 chain ²⁺ Forming poly T template Cu NPs, and selectively recognizing Cu by combining with QDs ²⁺ And poly-T template Cu NPs, and identifying a single target based on fluorescence signals of the poly-T template Cu NPs or the QDs, wherein the target is HIV p24 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 Cu ²⁺ 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 Cu NPs is 340nm.

According to a preferred embodiment, the immunofluorescence and two-dimensional visualization multimode analysis method is a two-dimensional HIV p24 antigen analysis method which utilizes the strong luminescence characteristics of the QDs and integrates the ink-jet printing technology to realize color and distance reading.

In order to achieve the second purpose, the invention provides an application of a detection product for analyzing HIV p24 antigen based on immunofluorescence and two-dimensional visualization multimode analysis, which comprises the step of applying any one of the immunofluorescence and two-dimensional visualization multimode analysis methods for ultra-sensitively recognizing HIV p24 antigen to p24 antigen analysis in serum of clinical HIV patients.

The invention provides a detection product for analyzing HIV p24 antigen based on immunofluorescence and two-dimensional visualization in a multi-mode manner and application thereof, and the detection product 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 (Cu NPs) which are used 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 discovered 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 template Cu NPs and QDs, and p24 analysis feasibility verification;

FIG. 3 is HIV p24 assay condition optimization with T40 template Cu NPs as the signaling molecule;

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

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

FIG. 6 is HIV p24 assay performance 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 should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. 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, a nucleic acid chain is connected to a biotin-modified second antibody on the premise of retaining antigen-antibody immunoassay, a luminous poly-T chain template Cu NPs and inorganic Quantum Dots (QDs) are introduced, and the Cu NPs and the inorganic Quantum Dots (QDs) can be selectively identified by combining the QDs ²⁺ And poly-T template Cu NPs, forming a Cu NPs and QDs multimode fluorescence analysis strategy of HIV p24, as shown in FIG. 1-Mode 1.

Subsequently, using the strong luminescence properties of QDs, and integrating inkjet printing technology, two-dimensional p24 analysis methods of color and distance reading were developed, which could offer more options for clinical POCT diagnosis and were 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 Cu NPs, with the goal of improving the sensitivity of p24 analysis, as shown in fig. 1-mode 2. TdT catalyzes the formation of poly-ssDNA from any single-stranded DNA (ssDNA) in the presence of a substrate (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 (10 mmol/L MOPS buffer, pH7.4, 100mmol/L NaNO) ₃ ，2.5mmol/L Mg(NO ₃ ) ₂ ) After mixing, incubation was carried out overnight at 4 ℃. The above 96-well plates were washed 3 times with 200. Mu.l of wash buffer (assay buffer containing 0.05% (v/v) Tween 20) for removal of unbound recognition antibody.

Subsequently, 100. Mu.l of blocking buffer (1% w/v, gmL) was added ^-1 BSA,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 clinical samples of HIV p24 protein or serum at various concentrations, 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 ) And 60 microliters of assay buffer were added to the 96-well plate and incubated at 37 ℃ for 1 hour for the formation of a primary anti-p 24-detection antibody-biotin sandwich complex, and washed three times.

2.2 When the T40 template Cu NPs are signal molecules

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 added ₄ And 20. Mu.L of 4mM Ascorbic Acid (AA), and 100. Mu.L MOPS assay buffer plusInto the above solution and incubated at room temperature for 10min for the generation of T40 template Cu NPs.

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

2.3 When QDs is a signal molecule

To the Cu NPs solution generated in the 2.2 reaction, 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 P24 analysis with TdT enzyme assistance

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 an 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 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 pipetted into two aliquots.

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 solution ₄ And 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 Cu NPs; the fluorescence intensity of the Cu NPs of the different solutions was monitored under excitation light at 340nm.

Synthesis of QDs

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

first, 0.5mmol of CdCl ₂ And 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 Na ₂ TeO ₃ And 50mg KBH ₄ Adding 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 was purified by precipitation (using n-propanol) and centrifugation (11000rpm, 30 min). The MPA-CdTe QDs synthesized above are stored at 4 deg.C before use.

4. Material characterization and multimode 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 Cu NPs are uniform sphere-like, have a particle size of about 4nm, and exhibit characteristic UV absorption peak at 340nm. The synthesized QDs have a particle size similar to Cu NPs, which have a lattice structure (fig. 2C and 2D). When Cu ²⁺ 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, cu NPs were successfully generated when T40 was the template, and the fluorescence signal for generating Cu NPs 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 using T40-Cu NPs 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 QDs ²⁺ And the phenomenon of DNA template Cu NPs were examined. As shown in FIG. 2G, cu ²⁺ The quenching effect on the fluorescence signal of QDs is obviously stronger than that of T40 template Cu NPs on the QDs (FIGS. 2G-a to c).

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

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

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 preliminary experiment result shows that the fg/mL level p24 analysis can be realized, and the DNA gel electrophoresis experiment also shows that the TdT enzyme assists in successfully generating poly T chains.

5.p24 assay Condition optimization

5.1 When the T40 template Cu NPs are signal molecules

Firstly, p24 analysis conditions are optimized when 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 M T40 concentration (fig. 3B and C); 3 μ M Cu ²⁺ When, the signal difference is maximum (fig. 3D and E); the highest fluorescence signal was generated for Cu NPs at 4mM ascorbic acid (FIG. 3F).

5.2 When QDs is a signal molecule

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 was completed within 7 minutes (fig. 4C).

5.3 TdT 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 30min ₁ -recognition reaction of biotin and SA (fig. 5C); recognition binding of P1-DNA-biotin to SA was also completed within 30min (fig. 5D); 0.25 μ MP1-DNA-biotin (FIGS. 5E and F), 5.6U TdT enzyme (FIGS. 5G and H); dTTP concentration of 15mM (FIGS. 5I and J); tdT enzyme extension reaction time of 20 minutes (fig. 5K); 100 μ M CuSO ₄ (FIGS. 5L and M); 4mM AA (FIG. 5N); and 10 minutes for Cu NPs generation, the maximum fluorescence signal or fluorescence signal difference, respectively, can be achieved.

6 p24 analytical Performance in different modes

6.1 When the T40 template Cu NPs are signal molecules

When Cu NPs are used as signal molecules, the quantitative capability of the system on p24 is examined. 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).

6.2 When QDs is a signal molecule

Subsequently QDs can be identified as Cu ²⁺ And T40-Cu NPs, as signal molecules, with QDs introduced into the p24 assay.

Considering the sensitivity of the assay when the fluorometer is used as a detector first, as shown in FIGS. 6C and D, similarly to the T40 template Cu NPs as a signal molecule, the fluorescence signal of QDs gradually decreases as the p24 concentration increases, and the logarithm of the concentration in the range of 1-1000pg/mL shows a good phenomenological relationship with the fluorescence signal with a detection limit of 0.25pg/mL (based on a 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 property 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 Cu NPs are generated, the lower the fluorescence signal of QDs, and the color visualization can achieve 100pg/mLp24 sensitivity.

Subsequently, the analytical ability of the test strip for ink-jet printing on 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.

6.3 TdT enzyme assisted

The analysis quantitative sensitivity of the 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 p24 concentration analysis of fg/mL scale was achieved with TdT assistance, and in the 0.1-10fg/mL concentration range, the logarithm of the concentration showed a good linear relationship with the fluorescence signal, with a detection limit as low as 0.04fg/mL, which was 4 orders of magnitude lower than when QDs were used as signal molecules (FIG. 6H).

Subsequently, the p24 assay strategy was examined for its ability to interfere with the protein as a potential interfering agent. As shown in FIGS. 6I and J, the decrease in the fluorescence signal of the system caused by various high concentrations (0.1 pg/mL) of interfering proteins was comparable to that of the blank solution and was almost negligible. Whereas low concentrations of p24 (10 and 100 fg/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 analysis 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 a clinical HIV patient, and the analysis result is compared with a 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, and as shown in FIGS. 7A and 7B, the p24 content was analyzed at pg/mL level, consistent well with the clinical electrochemiluminescence COI value results, and the linear correlation coefficient was 0.993 (FIG. 7C).

Subsequently, we monitored the serum p24 levels of the same patient at different times and found that the p24 levels in the patients decreased rapidly within 3-5 days as the time to 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 in normal and non-HIV patients were very low, 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 HIV-positive patients.

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. A detection product for analyzing HIV p24 antigen based on immunofluorescence and two-dimensional visualization multimode, which is characterized by comprising recognition antibodies, biotin-labeled detection antibodies, streptavidin, nucleic acid chains, polymerase and Cu ²⁺ And QDs; wherein HIV p24 antigen and biotin-modified detection antibody are subjected to antigen-antibody immune recognition reaction, and poly-T chain is introduced, or nucleic acid chain is introduced to carry out extension reaction with polymerase to generate poly-T chain, and the poly-T chain and Cu ²⁺ Forming poly T template Cu NPs, and selectively recognizing Cu by combining with QDs ²⁺ And poly-T template Cu NPs, identifying a single target based on fluorescence signals of the poly-T template Cu NPs or QDs, wherein the target is HIV p24 antigen;

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 fluorescence signal change analysis mode is constructed;

the immunofluorescence and two-dimensional visualization analysis is a two-dimensional HIV p24 antigen analysis method which utilizes the strong luminescence property of the QDs and integrates an ink-jet printing technology to realize color and distance reading.

2. The immunofluorescence and two-dimensional visualization based multi-modal assay detection product for HIV p24 antigen, according to claim 1, wherein the polymerase is TdT enzyme, phi29 polymerase, or elongase.

3. The immunofluorescence and two-dimensional visualization based multi-modal analysis detection product of HIV p24 antigen according to claim 2, wherein the nucleic acid strand is a DNA template.

4. The immunofluorescence and two-dimensional visualization based multi-modal assay HIV p24 antigen detection product according to claim 1, wherein the Cu ²⁺ Can be replaced by Ag ⁺ 。

5. The immunofluorescence and two-dimensional visualization based multimodal assay product for the detection of the p24 antigen of HIV according to claim 1, wherein, the QDs comprise CdTe QDs or CdSe QDs.