CN107254550B

CN107254550B - SPR sensor for detecting HIV related gene and preparation and application thereof

Info

Publication number: CN107254550B
Application number: CN201710380620.6A
Authority: CN
Inventors: 颜玉蓉; 丁世家; 程伟; 杨建儒; 刁玮; 程文彬; 晏小玉; 马洪敏
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2017-05-25
Filing date: 2017-05-25
Publication date: 2020-09-04
Anticipated expiration: 2037-05-25
Also published as: CN107254550A

Abstract

The invention provides an SPR sensor for detecting HIV related genes and preparation and application thereof, wherein the preparation of the SPR sensor comprises the preparation of an entropy-driven strand displacement cyclic amplification system: (a) preparing a probe: preparing a triple-stranded composite product PRQ by adopting a DNA single strand P, R, Q; (b) taking the triple-stranded composite product PRQ obtained in the step (a), mixing the hybridization solution, the triple-stranded composite product PRQ, the single strand F and the target sequence T, and incubating to obtain a reaction solution; (c) fixing the capture probe on the surface of a working chip, placing the working chip in a surface plasma resonance instrument, and injecting the reaction liquid obtained in the step (b) to the surface of the working chip fixed with the capture probe to prepare the entropy-driven chain displacement cyclic amplification system. The invention successfully constructs the surface plasmon resonance sensor and the detection system which can be used for HIV related genes, and the sensor of the invention has high sensitivity, good stability and good reproducibility for the determination of HIV related genes.

Description

SPR sensor for detecting HIV related gene and preparation and application thereof

Technical Field

The invention relates to the field of nucleic acid detection and surface plasmon resonance sensing, in particular to an SPR (surface plasmon resonance) sensor for detecting HIV (human immunodeficiency virus) related genes and preparation and application thereof.

Background

Since the 1 st HIV-infected person was discovered in the continental countries in 1985, the global prevalence of Acquired Immune Deficiency Syndrome (AIDS) has become increasingly severe and severe. According to the data of the United nations AIDS planning administration, 110 million people die of AIDS in 2015 globally, and 210 million new AIDS virus infection cases are added in the 2015. The prevalence of AIDS presents a tremendous detriment to human health and socioeconomic performance. The main purposes of Human Immunodeficiency Virus (HIV) detection are to diagnose HIV pathogens, guide clinical medication and detect HIV carriers, and early detection and intervention helps control disease conditions and block transmission pathways in time. At present, the diagnosis of HIV infection in China mostly depends on serological detection, and HIV antibodies are mostly detected by adopting enzyme-linked immunosorbent assay (ELISA) combined with immunoblotting (WB). Although serological tests are specific and suitable for the detection of large sample quantities, such as clinical tests and blood screening tests, they require corresponding instrumentation and specially trained technicians. And the possibility of mutual interference cannot be completely excluded for technical reasons, and the sensitivity is poor. In addition, serological tests have drawbacks of their own: in the window period, virus invades but antibodies are not produced, the immunological method cannot detect the virus, and even if the fourth generation ELISA detection is carried out, the window period of 2-3 weeks still exists, and the requirement of early diagnosis cannot be met.

Thus, there is a need for earlier markers for the diagnosis of HIV infection. Viral nucleic acid is first detectable in infected individuals prior to antibody production, and is present at excessively high levels, and thereafter declines. Nucleic acid detection is independent of antibody production, and can significantly shorten the window period. The nucleic acid detection has the following characteristics: the method can be used for detecting the viruses (such as HBV, HCV, HIV and the like) which cannot be cultured and separated conventionally; the dosage of the sample is less, and the sample with low abundance content can be detected; thirdly, the antibody is not generated, and the detection can be carried out before the antibody in the acute phase of virus infection appears, so that the method is suitable for early diagnosis; fourthly, the virus gene can be genotyped, and the method is more valuable than serotyping; through the detection of virus drug resistance gene, the drug resistance of the virus can be predicted or found; sixthly, the method can be used for diagnosing congenital or perinatal acquired viral infection; high sensitivity (reaching ng and even fg level, theoretically detecting single virus gene), good specificity, quick and simple.

At present, nucleic acid detection is mainly performed by a fluorescent quantitative PCR method, which is to label and track PCR products through fluorescent dyes or fluorescent labeled specific probes, and monitor the reaction process on line, so as to realize real-time quantitative detection of nucleic acid to be detected. The nucleic acid detection kit can be applied to HIV virus nucleic acid detection, can well solve the problem of window-period missed diagnosis, can detect the virus only by virus invasion, and has strong specificity and high automation degree. However, the method still has the defects of complicated process, long detection time and the like.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide an SPR sensor for detecting HIV-associated genes and its preparation and application, which are used to solve the problems of the prior art, such as poor detection specificity, tedious process, and long detection time for HIV.

In order to achieve the above and other related objects, a first aspect of the present invention provides a method for preparing an SPR sensor for detecting HIV-associated genes, comprising preparing an entropy-driven strand displacement cyclic amplification system, specifically comprising the steps of:

(a) preparing a probe: dissolving, mixing and denaturing the DNA single strand P, R, Q to prepare a triple-stranded composite product PRQ for later use;

(b) taking the triple-stranded composite product PRQ obtained in the step (a), mixing the hybridization solution, the triple-stranded composite product PRQ, the single strand F and the target sequence T to obtain a mixed solution, incubating and reacting to obtain a reaction product;

(c) and (c) injecting the reaction product obtained in the step (b) to the surface of a working chip fixed with a capture probe to prepare the entropy-driven strand displacement cyclic amplification system.

Further, in step (a), the single strand P comprises a nucleotide sequence of:

5'-TTTTTTTTTTTTTTTTCCCTACTGCTAGAGATTTT-3'(SEQ ID NO.2)。

further, in step (a), the nucleotide sequence of the single-stranded R is:

5'-CCTACGTCTCCAACTAACTTACGG-3'(SEQ ID NO.3)。

further, in step (a), at least 3 bases of the single strand Q are complementarily paired with the capture probe.

Further, in step (a), at least 4 bases of the single strand Q are complementarily paired with the capture probe.

Further, in step (a), the single strand Q has 4-8 bases complementary to the capture probe.

Further, in step (a), the single strand Q has 6 bases complementary to the capture probe.

Further, in the step (a), when 6 bases on the single-stranded Q are complementarily matched with the capture probe, the nucleotide sequence of the single-stranded Q is Q6:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCC-3'(SEQ IDNO.4)。

further, in the step (a), when 4 bases on the single-stranded Q are complementarily matched with the capture probe, the nucleotide sequence of the single-stranded Q is Q4:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGAT-3'(SEQ IDNO.5)。

further, in the step (a), when 5 bases on the single-stranded Q are complementarily matched with the capture probe, the nucleotide sequence of the single-stranded Q is Q5:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATC-3'(SEQ IDNO.6)。

further, in the step (a), when 7 bases on the single-stranded Q are complementarily matched with the capture probe, the nucleotide sequence of the single-stranded Q is Q7:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCCT-3'(SEQID NO.7)。

further, in the step (a), when 8 bases on the single-stranded Q are complementarily matched with the capture probe, the nucleotide sequence of the single-stranded Q is Q8:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCCTA-3'(SEQID NO.8)。

further, in step (a), the molar ratio of the single strand P, R, Q in the mixture was 1.2:1.2: 1.

Further, in the step (a), the DNA single strands P, R, Q were dissolved in TE buffers, respectively, and then mixed.

Further, in step (a), the TE buffer comprises 10mM TrisHCl, 1mM EDTA, pH 8.0.

Further, in the step (b), the nucleotide sequence of the single strand F is:

5'-AACTGACTCCTACGTCTCCAACTAACTTACGGCCCTACTGCTAGAGATTTTATCAGAGTGGCTTC-3'(SEQ ID NO.9)。

further, in step (b), the target sequence T comprises the following nucleotide sequence:

5'-ACTGCTAGAGATTTTCCACAT-3'(SEQ ID NO.10)。

further, in step (b), the concentration of the target sequence T in the mixture is not less than 50 fM.

Further, in step (b), the concentration of the target sequence T in the mixture is more than or equal to 1.5 pM.

Further, in the step (b), the incubation time is more than or equal to 15 min;

further, in the step (b), the incubation time is 15-75 min;

further, in the step (b), the incubation temperature is more than or equal to 4 ℃;

further, in the step (b), the incubation temperature is 4-48 ℃;

further, in the step (b), the DNA single strand F and the target sequence T are respectively dissolved in TE buffer, and then the hybridization solution is mixed with the TE buffer respectively containing the triple-stranded composite product PRQ, the single strand F and the target sequence T to obtain the entropy-driven strand displacement cyclic amplification system.

Further, in step (b), the TE buffer comprises 10mM TrisHCl, 1mM EDTA, pH 8.0.

Further, in step (b), the hybridization solution comprises 10mM Tris, 480mM NaCl, 5mM MgCl₂The pH was 7.0.

Further, in step (b), the final concentration of the triple-stranded complex PRQ in the mixture was 10 nM.

Further, in step (b), the final concentration of single-stranded F in the mixture was 10 nM.

Further, in step (b), the target sequence T is added in an amount of 2.5% by volume based on the total volume of the mixture.

Further, in the step (c), the capture probe sequence immobilized on the working chip is shown as SEQ ID NO. 1.

The sequence of the capture probe is not limited to the sequence shown in SEQ ID NO.1, and may be adaptively designed according to different target sequences T.

Further, in the step (c), the capture probe is a dimercapto-modified capture probe, and the sequence thereof is as follows:

5'-HS-(CH₂)₆-GGCTGTTTTTTAGGATCCGAGTCAGTTTTTTTTCAGCC-(CH₂)₆-SH-3'。

the double sulfydryl modification can enable the chip to have better anti-interference performance, and compared with the traditional probe preparation method, the subsequent MCH sealing process is not needed.

Further, in the step (c), the capture probe is mixed with the fixing solution, the mixed solution is injected to the surface of the working chip, and the working chip fixed with the capture probe is prepared, wherein the molar concentration of the capture probe in the mixed solution is 500 nM-2000 nM.

Further, in the step (c), when the capture probe is immobilized, the immobilizing solution is selected from KH₂PO₄An aqueous solution.

Further, in the step (c), the injection volume is more than or equal to 10 mu L.

Further, in step (c), the injection volume is 10-50 μ L, border values included.

Further, in the step (c), the working chip was set in a surface plasmon resonance apparatus to contain 450mM NaCl, 30mM Na₃PO₄·12H₂O, 3mM EDTA-2Na, 0.25% Triton X-100 buffer solution as the running solution.

Further, in the step (c), the method of immobilizing the capture probe on the working chip comprises:

(1) surface treatment: carrying out surface treatment on the working chip to make the surface smooth;

(2) immobilizing a capture probe: and (3) placing the working chip in a surface plasma resonance instrument, dissolving the capture probe in the fixing solution, and injecting the capture probe to fix the capture probe on the surface of the working chip to obtain the working chip fixed with the capture probe.

Further, in the step (2), the molar concentration of the capture probe is 500nM to 2000 nM.

Further, in step (2), the capture probe was present at a molar concentration of 1000 nM.

The molar concentration of the capture probe is the final concentration of the capture probe in the mixture after the capture probe is dissolved in the immobilizing solution.

Further, in the step (2), the fixing liquid is selected from KH₂PO₄An aqueous solution.

Further, in the step (2), the fixing solution was KH with pH of 3.8 and a molar concentration of 1M₂PO₄An aqueous solution.

And further, the method also comprises a method for preparing the DNA tetrahedron nano molecules, which comprises the steps of preparing at least one layer of DNA tetrahedron, mixing the hybridization solution and the DNA tetrahedron for reaction, and injecting the obtained reaction solution to the surface of the working chip fixed with the capture probe to obtain the DNA tetrahedron nano molecule amplification detection system.

Further, the hybridization solution comprises 20mM Tris, 50mM MgCl₂。

Further, the preparation method of the first layer of DNA tetrahedral nano-molecules (T0) specifically comprises the following steps: mixing and denaturing the DNA single strands A0, B0, C0 and D0 to obtain a first layer of DNA tetrahedral nano molecules (T0).

Preferably, the nucleotide sequence of the single-stranded A0 is:

5'-GAAGCCACTCTGATACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTA-3'(SEQ ID NO.11)。

preferably, the nucleotide sequence of the single-stranded B0 is:

5'-CTGTCATCGGTCACTATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATAC-3'(SEQ ID NO.12)。

preferably, the nucleotide sequence of the single-stranded C0 is:

5'-CTGTCATCGGTCACTCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC-3'(SEQ ID NO.13)。

preferably, the nucleotide sequence of the single-stranded D0 is:

5'-CTGTCATCGGTCACTTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT-3'(SEQ ID NO.14)。

further, the single-stranded DNAs A0, B0, C0, and D0 were mixed in equimolar amounts.

Further, the DNA single strands A0, B0, C0 and D0 are respectively dissolved in TE buffer, and then the TEbuffer solutions of the four single strands are mixed for subsequent denaturation treatment.

Further, the preparation method also comprises a second layer of DNA tetrahedral nano-molecules (T1), and specifically comprises the following steps: mixing single-chain DNAA1, B1, C1 and D1 at equimolar concentration, denaturing, and rapidly cooling; and mixing the first layer of DNA tetrahedral nano molecules (T0) with the second layer of DNA tetrahedral nano molecules (T1), incubating to obtain a mixed solution containing the double-layer DNA tetrahedral nano molecules, and adding the mixed solution to the surface of the working chip to obtain the double-layer DNA tetrahedral nano molecule amplification detection system.

Further, the nucleotide sequence of the single-stranded A1 is as follows:

5'-GTGACCGATGACAGACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTA-3'(SEQ ID NO.15)。

further, the four single strands include single strand B1, the nucleotide sequence of the single strand B1 is:

5'-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATAC-3'(SEQ IDNO.16)。

further, the four single strands include single strand C1, the nucleotide sequence of the single strand C1 is:

5'-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC-3'(SEQ IDNO.17)。

further, the four single strands include single strand D1, the nucleotide sequence of the single strand D1 is:

5'-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT-3'(SEQ IDNO.18)。

further, the single-stranded DNAs A1, B1, C1, and D1 were mixed in equimolar amounts.

Further, the single-stranded DNAs A1, B1, C1 and D1 were dissolved in TE buffer, and then the four single-stranded TEbuffer solutions were mixed for subsequent denaturation.

Further, the first layer of DNA tetrahedral nano-molecules (T0) was mixed with the second layer of DNA tetrahedral nano-molecules (T1) in a molar ratio of 1: 3.

Further, the concentration of the double-layer DNA tetrahedral nano-molecules in the mixed solution is 1 mu M;

further, the sample injection volume of the mixed solution is more than or equal to 10 mu L;

further, the sample injection volume of the mixed solution is 10-50 μ L, including a boundary value.

Further, the sample injection flow rate was 2. mu.L.min^-1。

In a second aspect, the invention provides an SPR sensor made by the above method.

The third aspect of the present invention provides the use of the SPR sensor described above for detecting HIV-associated genes.

As described above, the SPR sensor for detecting HIV related genes and the preparation and application thereof have the following beneficial effects: the invention successfully constructs the surface plasmon resonance sensor and the detection system which can be used for HIV related genes, and the measurement of the HIV related genes by using the sensor of the invention shows the capabilities of high sensitivity, stability and good reproducibility. Compared with the prior art, the sensor of the invention has no enzyme, can monitor in real time, has low cost, simple and convenient operation, short detection period, high sensitivity and good specificity, and is expected to become a sensor with practical application value.

Drawings

FIG. 1 is a typical SPR process sensorgram of the present invention, which is a sample injection entropy driven strand displacement cyclic amplification system, a sample injection double-layer DNA tetrahedral nano-molecule and a regeneration liquid (NaOH), respectively.

FIG. 2 is a comparison graph of SPR sensing signals of blank, no PRQ compound added, no F chain added, no DNA tetrahedral nano-molecule added, only a single layer of DNA tetrahedral nano-molecule added, and double layer of DNA tetrahedral nano-molecule added.

FIG. 3 is a diagram of electrophoretic verification that a monolayer DNA tetrahedron T0 and T1 can form a double-layer DNA tetrahedron nano molecule.

FIG. 4 is a graph showing the result of the response signals of the surface plasmon resonance HIV-associated gene sensor constructed by Q chains of binding sites with different numbers of bases.

FIG. 5 is a graph showing the response signal results of a surface plasmon resonance HIV-associated gene sensor constructed by an entropy-driven strand displacement cyclic amplification system at different reaction times.

FIG. 6 is a graph showing the response signal results of a surface plasmon resonance HIV-associated gene sensor constructed by an entropy-driven strand displacement cyclic amplification system at different reaction temperatures.

FIG. 7 is a graph of the response signal results of a surface plasmon resonance HIV-associated gene sensor constructed with double-layered DNA tetrahedral nano-molecules of different sample sizes.

FIG. 8-a is an SPR response signal diagram obtained by detecting HIV-related gene standard solutions of 6 different concentrations (150nM, 15nM, 1.5nM, 150pM, 15pM, 1.5pM) in the present invention.

FIG. 8-b is a four parameter fit calibration S-plot for an embodiment of the present invention.

FIG. 9 is a diagram showing the result of the specificity analysis of the surface plasmon resonance HIV-associated gene detecting sensor prepared in the example of the present invention.

Fig. 10 is a schematic diagram of the present invention.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

It should be understood that the processing equipment or devices not specifically mentioned in the following examples are conventional in the art; all pressure values and ranges refer to absolute pressures.

Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

Example 1 preparation of surface plasmon resonance HIV-associated Gene detection sensor

1. Materials and methods

1.1 materials

The DNA sequence purified by HPLC was synthesized by Shanghai Bioengineering Co., Ltd. TE buffer (10mM Tris HCl, 1mM EDTA, pH 8.0) was purchased from Shanghai Bioengineering Co., Ltd, and DNA marker was purchased from Takara, Inc., Kh₂PO₄、NaCl、MgCl₂、Na₃PO₄·12H₂O, EDTA-2Na and other reagents were purchased from Chongqing Chemicals, Inc.

1.2 detection Instrument

The Biocore X type surface plasmon resonance apparatus is a product of Biocore AB, Sweden.

1.3 detection principle

As shown in FIG. 10, in the homogeneous reaction system, the target sequence T triggers the entropy-driven strand displacement cycle amplification reaction through the foothold located in the Q chain, so that the P single strand in the triple-stranded complex PRQ is released to form a TRQ triple-stranded complex, and a new foothold located in the middle of the Q chain is exposed. When the F chain exists, the F chain is combined with a new foothold to generate a double-chain product F-Q, and simultaneously, the R single chain and the target sequence T are released, and the target sequence T can enter the next round of circulating reaction for signal amplification. In the double-stranded product F-Q, a single-stranded DNA fragment is respectively present on the F chain and the Q chain, and can be combined with a stem-loop type capture probe (CaptureProbe) fixed on the surface of a chip through base complementary pairing. After the homogeneous reaction system is injected into a surface plasma resonance instrument, the F-Q is specifically combined with a Capture Probe (Capture Probe), and meanwhile, the combination site of the double-layer DNA tetrahedral nano-molecule on the F chain is exposed. After the pre-prepared double-layer DNA tetrahedral nano-molecules are injected into a surface plasma resonance instrument, the nano-molecules can be specifically combined with an F chain, so that an obviously amplified SPR signal response is generated. A calibration curve is drawn from SPR response signals generated by standard HIV-associated gene reactions and the level of HIV-associated gene in the sample is determined.

2. Preparation of working chip

(1) Surface treatment of bare gold chips: a bare gold chip with a gold film thickness of 50nm is used as a working chip, and piranha solution (95-98% concentrated sulfuric acid and 30% H) is applied to the surface of the bare gold chip₂O₂The volume ratio of the aqueous solution is 3:1), treating for 3 times, each time for 3min, washing with deionized water, and drying at room temperature.

(2) Immobilizing a capture probe: and (3) placing the working chip in a surface plasma resonance instrument, injecting a capture probe, enabling the capture probe to flow through the surface of the working chip through a channel, and fixing the capture probe on the surface of the chip through a gold-sulfur bond to obtain the working chip containing the capture probe. Before use, the working chip can be stored at low temperature of 4 ℃, and can be repeatedly used.

The molar concentration of the capture probe on the working chip can be 500nM to 2000nM, in this example 1000 nM. Mixing the capture probe with a fixing solution, and injecting the sample, wherein the fixing solution is KH with pH of 3.8 and molar concentration of 1M₂PO₄An aqueous solution.

The molar concentration of the capture probe is: the final concentration of capture probe in the mixture after mixing the capture probe with the fixative solution.

3. Use of surface plasmon resonance HIV-related gene detection sensor

(1) Placing a working chip on the surfaceIn a plasma resonance apparatus, the mixture is mixed with a solution containing 450mM NaCl and 30mM Na₃PO₄·12H₂O, 3mM EDTA-2Na, 0.25% Triton X-100, PH 7.4 buffer was used as the running solution, and the flow rate was set at 5 μ L/min until the baseline was stable.

(2) Dissolving the DNA single-chain P, R, Q in TE buffer respectively, mixing the DNA single-chain P, R, Q with the molar ratio of the single-chain P, R, Q of 1.2:1.2:1, denaturing, and returning to room temperature for later use; the DNA single strands F, T were dissolved in TE buffers respectively for use. The TEbuffer of this step contained 10mM Tris-HCl, 1mM EDTA, pH 8.0.

Containing 10mM Tris, 480mM NaCl, 5mM MgCl₂And (3) constructing an entropy-driven strand displacement cyclic amplification system by using a buffer solution with the pH value of 7.0 as a hybridization solution, wherein the composition of the mixed solution is as follows:

the entropy-driven chain displacement circulation system is reacted at 37 ℃ for 30min, and then is injected into a surface plasma resonance instrument (injected to the surface of a working chip through an injection channel), the injection amount is 30 mu L, and the flow rate is 2 mu L/min.

The target sequence T adopted in the experiment is artificially synthesized, and the specific sequence is 5'-ACTGCTAGAGATTTTCCACAT-3' (SEQ ID NO. 10).

(3) Respectively dissolving DNA single-chains A0, B0, C0 and D0 in TE buffer to obtain four solutions, wherein the molar concentrations of the single-chains in the solutions are equal, mixing the four single-chain solutions in equal volumes to enable the four single-chains to be mixed in equal molar volumes, after mixing, denaturing, and rapidly cooling (the solution is cooled from 95 ℃ to 4 ℃ within 1 min) to obtain a first layer of DNA tetrahedral nano-molecules (T0); respectively dissolving DNA single-chains A1, B1, C1 and D1 in TE buffer, mixing the four single-chains in an equimolar way, denaturing, and rapidly cooling (cooling the solution from 95 ℃ to 4 ℃ within 1 min) to obtain a second layer of DNA tetrahedral nano-molecules (T1); mixing the prepared first layer of DNA tetrahedral nano-molecules (T0) and the second layer of DNA tetrahedral nano-molecules (T1) according to the molar ratio of 1:3, adding hybridization solution, and reacting at 37 ℃ for 60min to prepare double-layer DNA tetrahedral nano-molecules for later use. The step ofThe hybridization solution contained 20mM Tris, 50mM MgCl₂pH 8.0. The sequence of the single-chain A0 is shown as SEQ ID NO.11, the sequence of the single-chain B0 is shown as SEQ ID NO.12, the sequence of the single-chain C0 is shown as SEQ ID NO.13, the sequence of the single-chain D0 is shown as SEQ ID NO.14, the sequence of the single-chain A1 is shown as SEQ ID NO.15, the sequence of the single-chain B1 is shown as SEQ ID NO.16, the sequence of the single-chain C1 is shown as SEQ ID NO.17, and the sequence of the single-chain D1 is shown as SEQ ID NO. 18.

(4) And (3) injecting the prepared double-layer DNA tetrahedron nano molecules into a surface plasma resonance instrument (injecting the sample to the surface of a working chip through a sample injection channel), wherein the sample injection amount is 30 mu L, and the flow rate is 2 mu L/min.

(5) 50mM NaOH aqueous solution was used as a regenerating solution, and 5. mu.L of the solution was injected at a flow rate of 2. mu.L/min.

(6) And taking the sum of the SPR response signals of two times of sample injection as the response signal of the corresponding sample.

(7) The working chip can be repeatedly used, so that the operations (1) to (5) can be repeated to carry out detection on a plurality of samples.

Example 2 verification of the feasibility of surface plasmon resonance HIV-associated Gene detection Sensors

1. And sequentially adding an entropy-driven strand displacement cyclic amplification system, double-layer DNA tetrahedral nano molecules and regenerated liquid NaOH.

The amount of capture probe was 100. mu.L, at a molar concentration of 1. mu.M.

The number of base-complementary pairs Q/C is 6, the sequence of the Q strand is Q6:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCC-3' (SEQ ID NO. 4). And mixing the hybridization solution, the triple-stranded compound PRQ, the single strand F and the target sequence T to obtain a mixed solution, wherein the concentrations of the triple-stranded compound PRQ and the single strand F in the mixed solution are both 250nM, the concentration of the target sequence T is 100nM, the entropy-driven strand displacement cyclic amplification reaction temperature is 37 ℃, the reaction time is 30min, and the sample injection amount is 30 muL. The concentration of the double-layer DNA tetrahedral nano-molecule is 1 mu M, and the sample size is 30 mu L. The concentration of the regeneration solution NaOH aqueous solution was 50mM, and the amount of the sample was 5. mu.L. The flow rates are all 2 mu L min^-1. The other operations were the same as in example 1.

FIG. 1 shows the SPR response signal of each step (including adding entropy-driven strand displacement cyclic amplification system, adding double-layer DNA tetrahedral nano-molecules, and adding regeneration liquid NaOH):

Δ RU1 is the SPR response signal level for the entropy-driven strand displacement cyclic amplification system.

Δ RU2 is the SPR response signal level of the double-layered DNA tetrahedral nano-molecule.

And the combined substances can be completely regenerated by the regeneration liquid NaOH.

2. And (3) carrying out feasibility analysis on a two-step amplification detection system.

The amount of capture probe was 100. mu.L at a concentration of 1. mu.M.

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCC-3' (SEQ ID NO. 4). The concentration of the complex PRQ and the single-stranded F in the mixture was 250nM, and the concentration of the target sequence T was 100 nM. The reaction temperature of entropy-driven strand displacement cyclic amplification is 37 ℃, the reaction time is 30min, and the sample injection amount is 30 mu L. The concentration of the monolayer DNA tetrahedral nano-molecule is 1 mu M, and the sample size is 30 mu L. The concentration of the double-layer DNA tetrahedral nano-molecule is 1 mu M, and the sample size is 30 mu L. The concentration of the regeneration solution NaOH aqueous solution was 50mM, and the amount of the sample was 5. mu.L. The flow rates are all 2 mu L min^-1. The other operations were the same as in example 1.

As shown in fig. 2, the curves are a-f curves from top to bottom:

a is an SPR response signal obtained when double-layer DNA tetrahedral nano molecules are added;

b is an SPR response signal obtained when only a single layer of DNA tetrahedral nano-molecule T0 is added;

c is an SPR response signal obtained when DNA tetrahedral nano-molecules are not added;

d is an SPR response signal obtained when F chains are not added;

e is the SPR response signal obtained without addition of PRQ complex;

f is the SPR response signal of the blank group (added with a two-step amplification system except the target sequence T);

experimental results show that a two-step amplification detection system is feasible.

3. Verification of double-layer DNA tetrahedral nano-molecules

The DNA tetrahedral nanomolecules obtained in example 1 were verified by PAGE electrophoresis.

As shown in fig. 3:

the band 1 is a Marker of 500 bp;

band 2 is 1. mu.M single stranded A0;

band 3 is 1. mu.M single-stranded A0+ B0;

band 4 is 1. mu.M single stranded C0+ D0;

band 5 is 1. mu.M single-stranded A0+ B0+ C0;

band 6 is 1. mu.M single-stranded B0+ C0+ D0;

lane 7 is 1 μ M T0;

lane 8 is 1 μ M T0+ T1;

the band 9 is a 1000bp Marker.

The electrophoresis result shows that the double-layer DNA tetrahedral nano-molecule is successfully assembled.

In FIG. 3, the samples in bands 2-7 were the respective sequences at the corresponding concentrations, mixed, denatured, and rapidly cooled (from 95 ℃ to 4 ℃ in 1 min), and the sample in band 8 was prepared by mixing T0 and T1 at a molar ratio of 1:3, and incubating at 37 ℃ for 1 h.

Example 3 surface plasmon resonance HIV-associated Gene detection sensor and Effect test of Using the same

We also conducted further experiments on the influence of several important conditions (i.e., four determination conditions, i.e., the number of base-complementary pairs of Q/C, the entropy-driven strand displacement cycle amplification reaction time, the reaction temperature, and the double-layer DNA tetrahedral nano-molecule sample injection amount) on the detection results. Five points were selected for each condition from low to high concentrations to perform a series of experiments.

1. In order to examine the influence of the number of the Q/C base complementary pairs on the surface plasmon resonance HIV related gene detection sensor, the surface plasmon resonance sensor is constructed by adopting different base pairing numbers in the experiment. As can be seen from FIG. 4, the signal-to-noise ratio varies depending on the number of base pairs, and when the number of Q/C base pairs is 6, the signal-to-noise ratio is at a maximum, indicating that this number of base pairs is optimum.

In FIG. 4, the amount of capture probe was 100. mu.L at a concentration of 1. mu.M. The concentrations of the complex PRQ and the single-chain F in the mixed solution were 250nM, and the concentration of the target sequence T was 100 nM. The reaction temperature of entropy-driven strand displacement cyclic amplification is 37 ℃, the reaction time is 60min, and the sample injection amount is 30 mu L. The concentration of the double-layer DNA tetrahedral nano-molecule is 1 mu M, and the sample size is 60 mu L. The concentration of the regeneration solution NaOH aqueous solution was 50mM, and the amount of the sample was 5. mu.L. The flow rates are all 2 mu L min^-1. The other operations were the same as in example 1.

When the number of Q/C base pairs is 6, the nucleotide sequence of the Q chain is as follows:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCC-3'(SEQ IDNO.4)。

when the number of Q/C base pairs is 4, the nucleotide sequence of the Q chain is as follows:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGAT-3'(SEQ IDNO.5)。

when the number of Q/C base pairs is 5, the nucleotide sequence of the Q chain is as follows:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATC-3'(SEQ IDNO.6)。

when the number of Q/C base pairs is 7, the nucleotide sequence of the Q chain is as follows:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCCT-3'(SEQID NO.7)。

when the number of Q/C base pairs is 8, the nucleotide sequence of the Q chain is as follows:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCCTA-3'(SEQID NO.8)。

the blank control group refers to the SPR response signal value added with the two-step amplification system except the target sequence T.

When the number of Q/C base pairs is 3, a better detection result can be obtained, and details are not repeated here.

2. In order to examine the influence of the entropy-driven strand displacement cyclic amplification reaction time on the surface plasmon resonance HIV-related gene detection sensor, a reaction system with different reaction times (15, 30, 45, 60 and 75min) is adopted in the experiment, and then surface plasmon resonance detection is carried out, as can be seen in FIG. 5, the optimal reaction time of the polymerization extension system is 30 min.

In FIG. 5, the amount of capture probe was 100. mu.L at a concentration of 1. mu.M.

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCC-3'(SEQ IDNO.4)。

the concentrations of the complex PRQ and the single-chain F in the mixed solution were 250nM, and the concentration of the target sequence T was 100 nM. The reaction temperature was 37 ℃ and the amount of sample was 30. mu.L for entropy-driven strand displacement cycle amplification. The concentration of the double-layer DNA tetrahedral nano-molecule is 1 mu M, and the sample size is 60 mu L. The regeneration solution was 50mM NaOH and 5. mu.L of NaOH was added. The flow rates are all 2 mu L min^-1. The other operations were the same as in example 1.

3. Similarly, in order to examine the influence of the entropy-driven strand displacement cyclic amplification reaction temperature on the surface plasmon resonance HIV-related gene detection sensor, different reaction temperatures (4 ℃, 25 ℃, 37 ℃, 42 ℃ and 48 ℃) are adopted in the experiment, and then surface plasmon resonance detection is carried out. As can be seen in FIG. 6, the hybridization temperature for the best signal-to-noise ratio was 37 ℃.

In FIG. 6, the amount of capture probe was 100. mu.L at a concentration of 1. mu.M. The number of base-complementary pairs Q/C is 6, the sequence of the Q strand is Q6:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCC-3' (SEQ ID NO. 4). The concentrations of the complex PRQ and the single-chain F in the mixed solution were 250nM, and the concentration of the target sequence T was 100 nM. The reaction time for the entropy-driven strand displacement cycle amplification was 30min, and the sample size was 30. mu.L. The concentration of the double-layer DNA tetrahedral nano-molecule is 1 mu M, and the sample size is 60 mu L. The regeneration solution was 50mM NaOH and 5. mu.L of NaOH was added. The flow rates are all 2 mu L min^-1. The other operations were the same as in example 1.

4. Similarly, in order to examine the influence of the double-layer DNA tetrahedral nano-molecule sample injection amount on the surface plasmon resonance HIV related gene detection sensor, different sample injection volumes (10, 20, 30, 40 and 50 mu L) are adopted in the experiment, and then the surface plasmon resonance detection is carried out. As can be seen in FIG. 7, the injection volume for the best signal-to-noise ratio was 30 μ L.

In FIG. 7, the amount of capture probe was 100. mu.L at a concentration of 1. mu.M. The number of base-complementary pairs Q/C is 6, the sequence of the Q strand is Q6:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCC-3' (SEQ ID NO. 4). The concentrations of the complex PRQ and the single-chain F in the mixed solution were 250nM, and the concentration of the target sequence T was 100 nM. The reaction temperature of entropy-driven strand displacement cyclic amplification is 37 ℃, the reaction time is 30min, and the sample injection amount is 30 mu L. The concentration of bilayer DNA tetrahedral nano-molecules was 1. mu.M. The regeneration solution was 50mM NaOH and 5. mu.L of NaOH was added. The flow rates are all 2 mu L min^-1. The other operations were the same as in example 1.

The blank control data refer to the values of SPR response signals to which the two-step amplification system other than the target sequence T was added.

In FIGS. 4-7, the signal-to-noise ratio refers to the ratio of the values of the SPR response signals of the experimental group and the control group under the same abscissa condition.

Performance analysis of surface plasmon resonance HIV-associated Gene detecting sensor prepared in example 4

In order to evaluate the performance of the surface plasmon resonance HIV-associated gene detection sensor, HIV-associated gene standards of various concentrations formulated in a hybridization solution (pH7.0) were analyzed under optimal experimental conditions.

The amount of capture probe was 100. mu.L at a concentration of 1. mu.M. The number of base-complementary pairs Q/C is 6, the sequence of the Q strand is Q6:

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCC-3' (SEQ ID NO. 4). The concentrations of the complex PRQ and the single-chain F in the mixed solution are both 250nM, and the concentrations a-g of the target sequence T are 150nM, 15nM, 1.5nM, 150pM, 15pM, 1.5pM and 0 in sequence. The reaction temperature of entropy-driven strand displacement cyclic amplification is 37 ℃, the reaction time is 30min, and the sample injection amount is 30 mu L. Double-layer DNA tetrahedral nano-moleculeThe concentration was 1. mu.M, and the amount of sample was 30. mu.L. The regeneration solution was 50mM NaOH and 5. mu.L of NaOH was added. The flow rates are all 2 mu L min^-1. The other operations were the same as in example 1.

Specifically, HIV-associated genes were diluted with hybridization solution (ph7.0) to 6 different concentrations (150nM, 15nM, 1.5nM, 150pM, 15pM, 1.5pM), 30 μ L of sample was injected, and a calibration sigmoid curve was drawn by fitting four parameters (four parameters include the number of Q/C base pairs, entropy-driven strand displacement cycle amplification reaction time, entropy-driven strand displacement cycle amplification reaction temperature, double-layer DNA tetrahedral nanomolecule injection amount), the detection range of the calibration curve was 1pM to 150nM, the regression equation was Y (Δ RU) 152.55 × lgc (nM) +863.36, and the correlation coefficient was 0.9906. And (3) taking the hybridization solution without the HIV related gene as a blank control, repeatedly detecting for 3 times, calculating the average value and the standard deviation, estimating the lowest detection limit according to the value corresponding to the blank signal average value plus 3 times of the standard deviation, calculating to obtain 50fM, wherein the detection result is shown in figure 8-a, and the standard curve is shown in figure 8-b.

Specific analysis of surface plasmon resonance HIV-associated Gene detection sensor prepared in example 5

The specificity of the surface plasmon resonance HIV-related gene detection sensor plays an important role in analyzing gene sequences in non-isolated biological samples, and mainly depends on the specificity of a recognition system. In order to evaluate the specificity of the present surface plasmon resonance HIV-associated gene detection sensor, single base mutation, double base mutation, and completely non-complementary sequence of HIV-associated gene were measured by using the surface plasmon resonance HIV-associated gene detection sensor constructed in example 4. The sequences are respectively as follows:

single base mismatches: 5'-ACTGCTAGAGATTTTCCACTT-3' (SEQ ID NO. 19).

Double base mismatches: 5'-ACTGCTAGAGATTTTACACTT-3' (SEQ ID NO. 20).

Are not complementary at all: 5'-TAGCTTATCAGACTGATGTTGA-3' (SEQ ID NO. 21).

5'-ATGTGGAAAATCTCTAGCAGTAGGGCCGTAAGTTAGTTGGAGACGTAGGCGGATCC-3' (SEQ ID NO. 4). The concentrations of the complex PRQ and the single-stranded F in the mixture were 250nM, and the concentration of each mismatch sequence was 100 nM. The reaction temperature of entropy-driven strand displacement cyclic amplification is 37 ℃, the reaction time is 30min, and the sample injection amount is 30 mu L. The concentration of the double-layer DNA tetrahedral nano-molecule is 1 mu M, and the sample size is 30 mu L. The regeneration solution was 50mM NaOH and 5. mu.L of NaOH was added. The flow rates are all 2 mu L min^-1。

As a result, as shown in FIG. 9, the SPR response signals of the single-base and double-base mutant sequences were significantly reduced compared to those of the HIV-related gene at the same concentration, and the completely non-complementary sequences were close to those corresponding to the blank. These results indicate that the prepared surface plasmon resonance HIV-related gene detection sensor has good specificity.

In FIG. 9, Target, SM, DM, NC and Blank sequentially show values of SPR response signals of the Target sequence T, the single-base mismatched sequence, the double-base mismatched sequence, the completely noncomplementary sequence and the Blank control group (the Blank control group is a two-step amplification system to which the Target sequence T is added) in example 1.

Stability and reproducibility analysis of surface plasmon resonance HIV-associated Gene detecting sensor prepared in example 6

The primary capture probe is fixed on the working chip, the surface plasmon resonance HIV related gene detection sensor constructed in the embodiment 4 is adopted for determination, a target sequence T of 100nM is taken as a sample, the sample is detected for 30 times, and the reduction of an SPR response signal is less than 10%, which indicates that the working chip can keep good performance after repeated use. The working chip is used for detecting the 1nM target sequence T under the optimal experimental condition (namely, the SPR sensor constructed in the embodiment 4 is adopted), and three parallel experiments are repeated, wherein the coefficient of variation is 6.5 percent, which shows that the surface plasmon resonance HIV related gene detection sensor prepared by the invention has good stability and reproducibility.

In conclusion, the invention has the following beneficial effects: (1) the invention develops a surface plasma resonance sensor based on entropy-driven strand displacement cyclic amplification system and double-layer DNA tetrahedral nano-molecule dual signal amplification, and the surface plasma resonance sensor can be used for sensitively, quickly and specifically detecting HIV related genes. Firstly, a double-sulfydryl-labeled stem-loop type capture probe is designed, and the probe can be combined with a double-stranded DNA product output by an entropy-driven strand displacement circulation amplification system. By design, the other end of the double stranded DNA product can be bound to a double-layered DNA tetrahedral nano-molecule. If HIV related genes do not exist in the sample, the entropy-driven strand displacement cyclic amplification reaction cannot be triggered, a double-stranded DNA product cannot be output, and then the double-stranded DNA product cannot be combined with a capture probe; when HIV related genes exist in a sample, an entropy-driven strand displacement cyclic amplification reaction is triggered, a double-stranded DNA product is output, and a target sequence T is liberated for first signal amplification. The double-stranded DNA product bound to the surface of the chip can be further bound with double-layer DNA tetrahedral nano-molecules to form secondary signal amplification. The surface plasma resonance sensor detects HIV related genes, a calibration curve is subjected to four-parameter fitting, the regression equation is Y (Delta RU) ═ 152.55 XlgC (nM) +863.36, the detection range is 1pM-150nM, the correlation coefficient is 0.9906, and the lowest detection limit is 50 fM. Certainly, according to the experimental result, the HIV related gene can be detected only based on the entropy-driven strand displacement cyclic amplification system, and on the basis, a single-layer or double-layer DNA tetrahedral nano-molecule amplification system is added, so that a better detection effect can be obtained.

(2) The specificity is good: the invention firstly prepares a double-sulfydryl-labeled stem-loop type capture probe, the probe is complementarily combined with a double-stranded DNA compound F-Q, the combining sites on the F-Q are respectively provided with a section on an F chain and a section on a Q chain, and the design greatly improves the specificity of recognition. The target sequence triggers the entropy-driven strand displacement reaction through the foothold, the whole process strictly follows the base complementary pairing principle, and the specificity is good. The double-stranded DNA compound F-Q is combined with DNA tetrahedral nano molecules through the tail end of single-stranded DNA, the whole process strictly follows the base complementary pairing principle, the influence of the adsorption effect of macromolecular particles on the result is reduced, and the specificity of target sequence detection is greatly improved.

(3) The detection speed is high: the invention utilizes entropy-driven strand displacement reaction and pre-prepared double-layer DNA tetrahedral nano molecules to carry out surface plasmon resonance detection on HIV related genes, the whole process only needs 1h, compared with other methods, the detection time is greatly shortened, and the detection speed is high.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

SEQUENCE LISTING

<110> Chongqing university of medical science

<120> SPR sensor for detecting HIV related gene and preparation and application thereof

<130>PCQYK172364

<160>21

<170>PatentIn version 3.5

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<223> Single-chain Q5

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<210>7

<211>57

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<213>Artificial

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<223> Single-chain Q7

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Claims

1. A preparation method of SPR sensor for detecting HIV related gene is characterized in that: the method comprises the following steps of preparing an entropy-driven strand displacement cyclic amplification system:

(a) preparing a probe: dissolving the DNA single strand P, the single strand R and the single strand Q, mixing and denaturing to prepare a triple-strand composite product PRQ for later use;

(b) taking the triple-stranded composite product PRQ obtained in the step (a), mixing the hybridization solution, the triple-stranded composite product PRQ, the single strand F and the target sequence T to obtain a mixed solution, and incubating to obtain a reaction solution;

(c) fixing a capture probe on the surface of a working chip, placing the working chip into a surface plasma resonance instrument, and injecting the reaction solution obtained in the step (b) onto the surface of the working chip fixed with the capture probe to prepare an entropy-driven strand displacement cyclic amplification system;

in the step (a), 4-8 bases on the single-chain Q are complementarily paired with a capture probe, the nucleotide sequence of the single-chain P is shown as SEQ ID NO.2, and the nucleotide sequence of the single-chain R is shown as SEQ ID NO. 3; in the step (b), the nucleotide sequence of the single-stranded F is shown as SEQ ID NO.9, and the nucleotide sequence of the target sequence T is shown as SEQ ID NO. 10; in the step (c), the capture probe sequence fixed on the working chip is shown in SEQ ID NO. 1;

when 4 bases on the single-stranded Q are complementarily matched with a capture probe, the nucleotide sequence of the single-stranded Q is shown as SEQ ID NO. 5;

when 5 bases on the single-stranded Q are complementarily matched with a capture probe, the nucleotide sequence of the single-stranded Q is shown as SEQ ID NO. 6;

when 6 bases on the single-stranded Q are complementarily matched with a capture probe, the nucleotide sequence of the single-stranded Q is shown as SEQ ID NO. 4;

when 7 bases on the single-stranded Q are complementarily matched with a capture probe, the nucleotide sequence of the single-stranded Q is shown as SEQ ID NO. 7;

when 8 bases on the single-stranded Q are complementarily matched with the capture probe, the nucleotide sequence of the single-stranded Q is shown as SEQ ID NO. 8.

2. The method of claim 1, wherein: in the step (a), the molar ratio of the single chain P, R, Q in the mixed solution is 1.2:1.2: 1;

and/or, in the step (a), the DNA single-strands P, R, Q are dissolved in the TE buffer respectively and then mixed;

and/or, in the step (b), the final concentration of the target sequence T in the mixed solution is more than or equal to 50 fM;

and/or, in the step (b), the incubation time is more than or equal to 15 min;

and/or, in the step (b), the incubation temperature is more than or equal to 4 ℃;

and/or in the step (b), respectively dissolving the DNA single chain F and the target sequence T in TE buffer, mixing the hybridization solution with the TE buffer respectively containing the triple-stranded composite product PRQ, the single chain F and the target sequence T to obtain a mixed solution, and incubating to obtain a reaction solution;

and/or, in step (b), the hybridization solution comprises 10mM Tris, 480mM NaCl, 5mM MgCl₂；

And/or in the step (b), the final concentration of the triple-stranded composite product PRQ in the mixed solution is 10 nM;

and/or in step (b), the final concentration of single-stranded F in the mixture is 10 nM;

and/or, in the step (b), the adding volume of the target sequence T is 2.5 percent of the total volume of the mixed solution;

and/or, in step (c), the capture probe is a dimercapto-modified capture probe;

and/or, in the step (c), mixing the capture probe with the fixing solution, injecting the mixed solution to the surface of the working chip to prepare the working chip fixed with the capture probe, wherein the molar concentration of the capture probe in the mixed solution is 500 nM-2000 nM;

and/or, in the step (c), the sample injection volume is more than or equal to 10 mu L;

and/or, in the step (c), the working chip is placed in a surface plasmon resonance apparatus to contain 450mM NaCl, 30mM Na₃PO₄.12H₂O, 3mM EDTA-2Na, 0.25% Triton X-100 buffer solution as the running solution.

3. The method of claim 2, wherein: in the step (b), the final concentration of the target sequence T in the mixed solution is more than or equal to 1.5 pM;

and/or, in the step (b), the incubation time is 15-75 min;

and/or, in step (b), the incubation temperature is 4-48 ℃;

and/or, in step (c), the capture probe is immobilized in a solution selected from KH₂PO₄An aqueous solution.

4. The preparation method according to claim 1, further comprising preparing at least one layer of DNA tetrahedron, mixing the hybridization solution and the DNA tetrahedron for reaction, and injecting the obtained reaction solution onto the surface of the working chip on which the capture probe is immobilized to obtain the DNA tetrahedron nano-molecule amplification detection system.

5. The method according to claim 4, wherein the hetero compound is used in preparation of a DNA tetrahedronThe cross-linking solution comprises 20mM Tris, 50mM MgCl₂。

6. The method according to claim 4, wherein the first layer of DNA tetrahedral nano-molecules comprises: mixing and denaturing the DNA single chain A0, the single chain B0, the single chain C0 and the single chain D0 to obtain the first layer of DNA tetrahedral nano molecules.

7. The preparation method according to claim 6, wherein the nucleotide sequence of the single-stranded A0 is shown in SEQ ID NO. 11;

the nucleotide sequence of the single-chain B0 is shown in SEQ ID NO. 12;

the nucleotide sequence of the single-stranded C0 is shown in SEQ ID NO. 13;

the nucleotide sequence of the single-stranded D0 is shown in SEQ ID NO. 14;

the single strand A0, the single strand B0, the single strand C0 and the single strand D0 are mixed in an equimolar way;

firstly, respectively dissolving the single chain A0, the single chain B0, the single chain C0 and the single chain D0 in TE buffer, then mixing TE buffer solutions respectively containing four single chains, and carrying out subsequent denaturation treatment.

8. The method according to claim 4, wherein the second layer of DNA tetrahedral nano-molecules comprises: mixing and denaturing the DNA single strand A1, the single strand B1, the single strand C1 and the single strand D1 to obtain a second layer of DNA tetrahedron; and mixing the hybridization solution, the first layer of DNA tetrahedral nano molecules and the second layer of DNA tetrahedral nano molecules, incubating to obtain a mixed solution of nucleotide sequence double-layer DNA tetrahedral nano molecules, and feeding the mixed solution to the surface of a working chip to obtain a double-layer DNA tetrahedral nano molecule amplification detection system.

9. The preparation method according to claim 8, wherein the nucleotide sequence of the single-stranded A1 is shown as SEQ ID No. 15;

the nucleotide sequence of the single-chain B1 is shown in SEQ ID NO. 16;

the nucleotide sequence of the single-stranded C1 is shown in SEQ ID NO. 17;

the nucleotide sequence of the single-stranded D1 is shown in SEQ ID NO. 18;

the DNA single strand A1, the single strand B1, the single strand C1 and the single strand D1 are mixed in an equimolar way;

dissolving DNA single-chain A1, single-chain B1, single-chain C1 and single-chain D1 in TE buffer respectively, mixing TE buffer solutions respectively containing four single-chains, and performing subsequent denaturation treatment;

and/or, the first layer of DNA tetrahedral nano-molecules is mixed with the second layer of DNA tetrahedral nano-molecules in a molar ratio of 1: 3;

and/or the concentration of the double-layer DNA tetrahedral nano-molecules in the mixed solution is 1 mu M;

and/or the sample injection volume of the mixed solution is more than or equal to 10 mu L.

10. The method according to claim 9, wherein the volume of the mixed solution is 10 to 50 μ L.

11. An SPR sensor manufactured by the manufacturing method according to any one of claims 1 to 10.

12. Use of the SPR sensor of claim 11 in the manufacture of a kit for detecting HIV-associated genes in a biological sample.