CN103439296B - Based on the aptamer sensor construction method of the two amplifying technique of Au NPs and DNA circulation and the application in adenosine detects thereof - Google Patents

Abstract

The invention discloses the construction of an aptamer sensor based on Au NPs and DNA circulation double amplification technology and its application in adenosine detection, belonging to the technical field of aptamer sensing. The detection system consists of adenosine aptamer, probe DNA partially hybridized with the aptamer, Au NPs-labeled hairpin DNA and hairpin DNA assembled on the surface of the gold sheet. When there is no adenosine, the Au NPs-labeled DNA maintains the hairpin structure and cannot hybridize with the hairpin DNA on the surface of the gold sheet, and cannot capture the Au NPs on the surface of the gold sheet. When adenosine is present, adenosine binds to its aptamer, and the probe DNA is released from the aptamer/probe DNA duplex and hybridizes with the Au NPs-labeled hairpin DNA to open the loop. The Au NPs-labeled DNA hybridizes with the DNA on the surface of the gold sheet to capture the Au NPs on the gold sheet, and the replaced probe DNA triggers the next round of DNA strand replacement reaction. In this cycle, a single adenosine molecule can trigger a large number of Au NPs to assemble on the gold sheet , to obtain enhanced SPR signal for highly sensitive detection of adenosine.

Description

Construction method of aptamer sensor based on double amplification technology of Au NPs and DNA cycle and its application in adenosine detection

technical field

The invention relates to the construction of an aptamer sensor based on Au NPs and DNA circulation double amplification technology and its application in adenosine detection, belonging to the technical field of aptamer sensing.

Background technique

Aptamer refers to a small single-stranded oligonucleotide sequence synthesized by in vitro screening and synthesis by exponential enrichment ligand system evolution technology, which has high affinity and specificity recognition ability for target molecules. Compared with antibodies as recognition elements, aptamers have the advantages of wide range of ligands, easy synthesis, easy labeling, and good chemical stability, and are widely used in the recognition and detection of target molecules. So far, various colorimetric sensors, fluorescent sensors, electrochemical sensors, luminescent sensors, quartz crystal microbalance sensors, and surface plasmon resonance (SPR) sensors have been reported using nucleic acid aptamers as recognition elements. Among them, the aptasensor based on SPR detection method has good application prospects due to its advantages of no need for labeling, high sensitivity and real-time monitoring. Most SPR aptasensors use the principles of sandwich assay, target-triggered conformational change, or target-triggered strand replacement and competition to detect biomolecules. Although these sensors have high selectivity and sensitivity for the detection of target molecules, most of them output signals according to a 1:1 signal:target ratio, which cannot achieve high-sensitivity detection of low-concentration substances, especially small molecules and nucleic acids. Therefore, it is of great significance to develop highly sensitive SPR sensing technology for the detection of low concentration biomolecules.

In recent years, nucleic acid molecular amplification technology has been widely used to improve the detection sensitivity of aptasensors, such as target-induced replacement polymerization, rolling circle amplification, polymerase chain reaction, endonuclease-based signal amplification reaction, etc. Although these methods improve the detection sensitivity, they often require the use of protease, which not only increases the analysis cost, but also requires precise control of the reaction conditions of the protease, which greatly limits the application. It is particularly important to develop enzyme-free amplification methods for highly sensitive detection of biomolecules. Enzyme-free amplification technologies based on hybridization and strand replacement (such as hybridization-strand amplification reactions, entropy-driven catalysis, and target-catalyzed hairpin assembly) have broad application prospects due to the inherent characteristics of the model, easy expansion, and no need for proteases. However, there have been no reports of SPR aptasensors based on enzyme-free amplification technology for high-sensitivity detection of small molecules.

Contents of the invention

The purpose of the present invention is to provide an aptamer sensor construction based on Au NPs and DNA cycle double amplification technology and its application in adenosine detection, which has the advantages of sensitive detection and good selectivity.

The present invention is realized in this way, in the adenosine aptamer, the probe DNA (c-DNA1) hybridized with the aptamer part, the hairpin structure DNA (Au-H-DNA1) labeled with Au NPs, pre-assembled In the experimental system of sulfhydrylated hairpin structure DNA (H-DNA2) on the SPR gold chip, when there is no target molecule adenosine, the H-DNA1 on Au-H-DNA1 maintains the hairpin structure and cannot be combined with the pre-assembled The hairpin structure H-DNA2 hybridization on the surface of gold flakes cannot capture Au NPs on the surface of gold flakes. However, when the target molecule adenosine exists, the specific binding of adenosine to the aptamer promotes the folding of the aptamer and changes the conformation, so that c-DNA1 is released from the aptamer/c-DNA1 double strand and binds with Au-H -DNA1 is hybridized to open the ring, and the Au-H-DNA1 after the ring is hybridized with the H-DNA2 pre-assembled on the surface of the gold sheet to capture Au NPs on the gold sheet, and at the same time replace c-DNA1, and the replaced c-DNA1 Then trigger the next round of DNA strand replacement cycle. Through such a DNA strand replacement cycle, a single adenosine molecule can trigger the assembly of a large number of Au-H-DNA1 on the SPR gold sheet. The amplification effect of Au NPs on the SPR signal greatly enhanced the SPR measurement signal, and the detection limit of adenosine was as low as pM level. At the same time, the high specificity of the aptamer makes the sensor have good anti-interference ability. The SPR aptamer sensor constructed based on the double amplification strategy of Au NPs on the SPR signal coupling amplification effect and target-induced DNA strand replacement cycle provides a universal platform for highly sensitive and selective detection of low-concentration biological small molecules. It has a good application prospect.

In order to successfully construct the aptasensor, the present invention adopts the following technical solutions:

The construction of aptasensor based on Au NPs and DNA loop double amplification technology includes the following steps:

(1) Preparation of Au NPs: Add 50 mL of HAuCl ₄ solution with a mass percentage of 0.01% into a 100 mL round-bottomed flask, heat to boiling, then quickly add 1 mL of HAuCl 4 solution with a mass percentage of 5 % trisodium citrate solution, continue to stir and keep boiling, stop heating when the color of the solution changes from yellow to deep wine red, and naturally cool to room temperature under stirring, and obtain stable and monodisperse Au NPs with an average particle size of 13 nm, Store at 4 °C for later use;

(2) Preparation of Au NPs-H-DNA1: thiolated H-DNA1 was activated on the column with 100 μL of freshly prepared 1 mM dithiothreitol solution, and mixed with 1 mL of Au NPs solution prepared in step (1), Shake and incubate on a shaker for 12 h, disperse the product in phosphate (PBS) buffer solution, and place at 25 °C for 40 h; centrifuge at 14,000 rpm for 10 min, discard the supernatant, and wash the red oily precipitate with PBS after centrifugation The buffer solution was washed and centrifuged 3 times to remove the H-DNA1 that did not react with Au NPs; the obtained Au NPs-H-DNA1 was resuspended in PBS buffer solution and stored at 4 °C for later use;

(3) Construction of the SPR aptamer sensing interface: Soak the gold sheet in a mixed solution of H ₂ SO ₄ :H ₂ O ₂ with a volume ratio of 7:3 for 2 min, rinse with secondary water, and immerse in 10 mM After 2 h in mercaptohexanol solution, rinse with secondary water and blow dry with nitrogen, put it into the SPR detection cell; inject 50 μL, 1.2 μM solution of thiolated hairpin structure H-DNA2 to react for 2 h, and prepare the preassembled The sensing interface of H-DNA2;

In the above steps, in step (2), the dithiothreitol solution is prepared with a phosphate buffer solution with a concentration of 170 mM and a pH of 8.0. The concentration of the phosphate buffer solution is 10 mM, pH is 7.4, and contains 0.1 M NaCl. In step (3), the mercaptohexanol solution is prepared with absolute ethanol. The thiolated hairpin structure DNA solution is prepared with a phosphate buffer solution with a concentration of 10 mM, a pH of 7.4, and 0.1 M NaCl.

The application of the aptamer sensor based on Au NPs and DNA loop double amplification technology refers to its application in the detection of adenosine: 20 μL, 1.0 μM of adenosine aptamer and the same ratio of probe DNA at 37 °C Incubate for 2 h to form aptamer/c-DNA1 double strands; add 20 μL of adenosine solution of different concentrations and incubate at 37 °C for 2 h, adenosine specifically binds to its aptamer to separate the probe DNA from the aptamer 25 μL of the above solution was added to 25 μL of Au NPs-H-DNA1 solution, and the mixed solution was injected into the SPR detection cell to combine with the H pre-assembled on the sensing interface. - DNA2 was reacted for 1 h; the reaction solution was discharged with a peristaltic pump and injected into 50 μL of secondary water for washing; with the increase of adenosine concentration, the Au NPs captured on the sensing interface gradually increased, and the SPR response signal increased rapidly, and the SPR The angle change has a good linear relationship with the concentration of adenosine in the range of 0.5-50 pM, and the detection limit is 0.21 pM, which can be used for ultra-sensitive and ultra-low concentration detection of adenosine.

The technical effect of the present invention is: the present invention combines the coupling amplification effect of Au NPs on the SPR signal and the target-induced DNA strand replacement amplification technology, and develops an enzyme-free SPR aptamer sensor for high sensitivity and selection of adenosine Sex detection. The advantages are as follows: (1) Using target-induced DNA strand replacement cyclic amplification technology, a single adenosine molecule can trigger a large number of Au NPs to assemble on the SPR sensing interface through multiple cyclic amplifications; (2) Au NPs have high molecular weight and high Moreover, the SPR gold film and Au NPs also have an electric field coupling resonance effect, which enables Au NPs to greatly improve the SPR response signal, thereby improving the detection sensitivity; (3) using the double amplification method of the present invention, it can be realized The detection of adenosine at the pM level is 3 orders of magnitude lower than the detection limit of adenosine based on single-fold amplification such as Au NPs amplification or DNA strand replacement cycle amplification; (4) High specificity of the aptamer to its target properties, so that the sensor constructed in the present invention has good selectivity to adenosine.

Description of drawings

Figure 1 is a schematic diagram of the construction of the SPR aptamer sensor and its detection of adenosine.

Figure 2 is the UV-Vis absorption spectra of (a) Au NPs and (b) Au-H-DNA1.

Figure 3 is the (A) cyclic voltammetry curve and (B) AC impedance curve of (a) bare electrode, (b) MCH, (c) H-DNA2 and (d) Au-H-DNA1/H-DNA2 modified electrode . The inset is the equivalent circuit diagram; R _s , Z _w , _Ret and C _dl are solution resistance, Warburg resistance, electron transfer resistance and double layer capacitance, respectively.

Figure 4 is the effect of (A) strand replacement cycle time, (B) H-DNA2 concentration, (C) H-DNA1 concentration on the performance of the SPR sensor.

Figure 5 is the SPR response curves of detecting 50 pM adenosine in different ways: (a) strand replacement cycle amplification, (b) Au NPs amplification, (c) Au NPs and strand replacement cycle dual amplification.

Figure 6 is the SEM images of (A) bare gold flakes, (B) gold flakes after Au NPs and strand replacement cycle dual amplification detection of 50 pM adenosine and (C) Au NPs amplification detection of 50 pM adenosine .

Figure 7 is (A) SPR curves of Au NPs and strand displacement cycle double amplification for detection of adenosine: a–i are 0, 0.5, 1, 5, 10, 30, 40, 50 and 100 pM adenosine, respectively. (B) Calibration curves for adenosine detection: (a) dual amplification of Au NPs with strand replacement cycle, (b) amplification of Au NPs and (c) amplification of strand replacement cycle.

Figure 8 is the SPR response curves of the sensor to adenosine, uridine, guanosine and cytidine constructed based on Au NPs and strand replacement cycle dual amplification detection.

Detailed ways

The present invention will be further elaborated below in conjunction with accompanying drawing and specific embodiment, and the present invention is not limited thereto;

Example 1

(1) Preparation of Au NPs: Add 50 mL of HAuCl ₄ solution with a mass percentage of 0.01% into a 100 mL round-bottomed flask, heat to boiling, then quickly add 1 mL of HAuCl 4 solution with a mass percentage of 5 % trisodium citrate solution, continue to stir and keep boiling, stop heating when the color of the solution changes from yellow to deep wine red, and naturally cool to room temperature under stirring, and obtain stable and monodisperse Au NPs with an average particle size of 13 nm, Store at 4 °C for later use;

(2) Preparation of Au NPs-H-DNA1: thiolated H-DNA1 was activated on the column with 100 μL of freshly prepared 1 mM dithiothreitol solution, and mixed with 1 mL of Au NPs solution prepared in step (1), Shake and incubate on a shaker for 12 h, disperse the product in PBS buffer solution, and place at 25 °C for 40 h; centrifuge at 14,000 rpm for 10 min, discard the supernatant, and wash the red oily precipitate with PBS buffer solution after centrifugation. Centrifuge 3 times to remove H-DNA1 that has not reacted with Au NPs; resuspend the obtained Au NPs-H-DNA1 in PBS buffer solution, and store at 4 °C for later use;

The preparation of Au NPs-labeled H-DNA1 probe was characterized by UV-vis spectroscopy, and the results are shown in Figure 2. The absorption peak at 520 nm in curve a indicates that the synthesized Au NPs have a particle size of about 13 nm. When thiolated H-DNA1 was modified on the surface of Au NPs, the absorption peak at 520 nm shifted red to 526 nm, indicating that the thiolated H-DNA1 interacted with Au NPs to form the Au-H-DNA1 probe , and the formed Au-H-DNA1 was very stable in 0.1 M NaCl solution.

Example 2

Preparation process of SPR aptasensor

(1) Soak the gold sheet in the mixed solution of H ₂ SO ₄ :H ₂ O ₂ with a volume ratio of 7:3 for 2 min, rinse it with secondary water, immerse it in 10 mM mercaptohexanol solution for 2 h, and wash it with distilled water After rinsed with water and dried with nitrogen, it was loaded into the SPR detection cell. Inject 50 μL, 1.2 μM H-DNA2 solution and react for 2 h to prepare the sensing interface pre-assembled with H-DNA2;

(2) Incubate 20 μL, 1.0 μM adenosine aptamer and the same ratio of c-DNA1 at 37 °C for 2 h, add 20 μL of adenosine solution of different concentrations, and incubate at 37 °C for 2 h; take 25 Add μL of the above solution to 25 μL Au NPs-H-DNA1 solution, inject the mixed solution into the SPR detection cell to react with the H-DNA2 pre-assembled on the sensing interface for 1 h; use a peristaltic pump to discharge the reaction solution And inject 50 μL of secondary water for cleaning;

The preparation process of the SPR sensor was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. It can be seen from Figure 3A that a pair of reversible [Fe(CN) ₆ ] ^3-/4- redox peaks (curve a) appears on the bare gold electrode; when a layer of MCH is assembled on the electrode surface, [Fe(CN) ₆ ] The redox peak current of ^3-/4- decreases (curve b); after modifying H-DNA2 on the surface of gold electrode through gold-sulfhydryl bond, the peak current further decreases (curve c); when 50 pM adenosine After reacting with the adenosine aptamer/c-DNA duplex for 2 h, Au NPs-H-DNA1 was added and injected into the SPR detection cell for incubation for 1 h, the oxidation of [Fe(CN) ₆ ] ^3-/4- on the electrode surface The reduction current further decreases (curve d), because a large amount of Au-H-DNA1 is captured by H-DNA2 immobilized on the surface of the SPR gold sheet, which increases the negative charge density on the electrode surface and hinders the [Fe(CN) ₆ ] ^3−/4− electron transfer to the electrode surface. Figure 3B is the EIS characterization of different modified electrodes. The electron transfer resistance ( R _et ) of the bare gold electrode is only 0.2 KΩ (curve a); when MCH is assembled on the electrode surface, R _et increases to 12 KΩ (curve b) ; and after H-DNA2 was assembled through gold-sulfhydryl bonds, R _et increased to 16.8 KΩ (curve c); when 50 pM adenosine was present, the DNA strand replacement cycle was initiated, making a large number of Au NPs-H-DNA1 open ring and was trapped on the surface of the _SPR gold flake, Ret increased significantly to 24.4 KΩ (curve d). The EIS is consistent with the CV results, indicating that the sensing interface has been successfully constructed according to the predetermined scheme.

Example 3

Detection of Adenosine by SPR Aptamer Sensor

(1) Optimization of strand replacement cycle time, H-DNA2 concentration, and H-DNA1 concentration

Figure 4A shows the SPR response of the sensor to 50 pM and 0 pM adenosine at different strand displacement cycle reaction times. It can be seen from the figure that for 50 pM adenosine, the intensity of the SPR response increases with the prolongation of the strand replacement cycle reaction time, and tends to be stable after 1 h; when there is no adenosine, the SPR response intensity increases with the prolongation of the reaction time The intensity increases slightly accordingly. Therefore, the selection strand replacement cycle reaction was 1 h. Figure 4B shows the effect of H-DNA2 concentration on the SPR sensing response. When the concentration of H-DNA2 was 1.2 μM, the signal-to-noise ratio of the sensor was optimal, further increasing the concentration of H-DNA2, the SPR response decreased slightly, which was due to the fact that although the density of capture probes assembled on the gold chip increased, the high-density The hairpin structure probe increases the steric hindrance and is not conducive to the hybridization of H-DNA2 and Au-H-DNA1. Therefore, 1.2 μM was chosen as the optimal concentration of H-DNA2 in the experiment. Figure 4C shows the effect of H-DNA1 concentration on the SPR sensing response. As the concentration of H-DNA1 increased (0.5–0.9 μM), both the background signal of the sensor and its SPR signal to 50 pM adenosine increased, further increasing the concentration of H-DNA1, the SPR signal decreased, which was due to the modification in Au NPs The H-DNA1 on the surface is too dense, and its steric hindrance effect inhibits the hybridization efficiency. Therefore, the optimal concentration of H-DNA1 was selected as 0.9 μM.

(2) In order to verify the amplification effect of the double amplification effect of Au NPs and strand replacement cycle on the detection of adenosine, the SPR response of the sensor constructed only based on Au NPs or strand replacement cycle amplification technology to 50 pM adenosine was investigated. The results are as follows: Figure 5 shows. It can be seen from the figure that the SPR response of strand replacement cycle amplification to 50 pM adenosine is only 14.1 m ^o (curve a), while the SPR response of Au NPs amplification to 50 pM adenosine is 80.3 m ^o (curve b), which is based on The amplification response of strand replacement cycle is 5.7 times; the SPR response of Au NPs and strand replacement cycle dual amplification method to 50 pM adenosine is 996 m (curve c), which is 12.5 times of the amplification response based on Au NPs, indicating that Au NPs and strand replacement The cyclic double amplification method has obvious amplification and enhancement effect on the detection of adenosine. This is due to the fact that a large amount of Au-H-DNA1 is assembled on the surface of the SPR gold sheet due to the strand replacement cycle mode, and the electronic coupling between the Au NPs and the gold film makes the SPR signal be amplified efficiently. In the amplification method of Au NPs, a short strand of DNA (c-DNA2) replaced the hairpin H-DNA2 and was immobilized on the surface of the SPR gold sheet. c-DNA2 contains 12 bases and can hybridize with the 12 bases near the 3' end of Au-H-DNA1. However, the hybridization process cannot separate the c-DNA1 strand from the Au-H-DNA1/c-DNA1 double strand Therefore, Au-H-DNA1 and c-DNA2 are hybridized and immobilized on the SPR gold sheet at a stoichiometric ratio of 1:1, which cannot further amplify the SPR signal;

The surface morphology of the gold flakes after detecting 50 pM adenosine in three different magnification methods was characterized by scanning electron microscopy, and the results are shown in Figure 6. The surface of SPR gold flakes is relatively smooth (Fig. 6A). When 50 pM adenosine was detected by double amplification method of Au NPs and strand replacement cycle, a large number of Au NPs were evenly loaded on the surface of gold flakes (Fig. 6B), indicating that through the chain replacement cycle mode A large amount of Au-H-DNA1 in the solution is hybridized with H-DNA2 on the surface of the gold sheet and captured on the SPR gold sheet. When Au NPs amplification method was used to detect 50 pM adenosine, there were only a small amount of Au NPs on the surface of the gold flakes (Figure 6C), and the coverage rate was much lower than that in Figure 6B, which was due to the interaction between Au-H-DNA1 and the adenosine immobilized on the gold flake surface. The short-strand c-DNA2 can only hybridize in a 1:1 ratio, which limits the amount of Au-H-DNA1 immobilized on the gold chip.

(3) Under optimized conditions, adenosine was detected as shown in Figure 1, and the SPR curve is shown in Figure 7A. The SPR angle change (ΔAngle) increases with the concentration of adenosine, and ΔAngle has a good linear relationship with the concentration of adenosine (C) in the range of 0.5-50 pM, and the linear regression equation is Angle (m ^o ) = 164.28 + 16.31 C (R=0.9949), with a detection limit of 0.21 pM. Under the same experimental conditions, the response to adenosine based only on Au NPs or strand replacement cyclic amplification was investigated, and the results are shown in Figure 7B. It can be seen from the figure that the SPR response of adenosine detection based on Au NPs or strand replacement cycle amplification method is significantly lower than that of Au NPs and strand replacement cycle dual amplification method, and the detection limits are 0.52 nM and 1.13 nM, respectively. It can be seen that the dual amplification method of Au NPs and strand replacement cycle can be used for highly sensitive detection of adenosine.

(4) Using the other three nucleosides in the nucleoside family (uridine, guanosine, and cytidine) as interfering substances, the selectivity of the sensor for adenosine detection was investigated, and the results are shown in Figure 8. It can be seen from the figure that under the same experimental conditions, the sensor has the largest SPR response to 50 pM adenosine, but almost no response to 50 pM uridine, guanosine and cytidine, indicating that the sensor constructed by the present invention has good detection performance on adenosine. selectivity.

Claims (6)

1. A method for constructing an aptamer sensor based on Au NPs and DNA circulation double amplification technology, characterized in that the construction comprises the following steps: (1) Preparation of Au NPs: Add 50 mL of HAuCl ₄ solution with a mass percentage of 0.01% into a 100 mL round-bottomed flask, heat to boiling, then quickly add 1 mL of HAuCl 4 solution with a mass percentage of 5 % trisodium citrate solution, continue to stir and keep boiling, stop heating when the color of the solution changes from yellow to deep wine red, and naturally cool to room temperature under stirring, and obtain stable and monodisperse Au NPs with an average particle size of 13 nm, Store at 4 °C for later use; (2) Preparation of Au NPs-labeled hairpin DNA: thiolated DNA was activated on the column with 100 μL of freshly prepared 1 mM dithiothreitol solution, mixed with 1 mL of Au NPs solution prepared in step (1), and placed in Shake and incubate for 12 h on a shaker, disperse the product in phosphate buffer solution, and place at 25 °C for 40 h; centrifuge at 14,000 rpm for 10 min, discard the supernatant, and wash the red oily precipitate with phosphate buffer solution after centrifugation. Centrifuge 3 times to remove DNA that has not reacted with Au NPs; resuspend the obtained Au NPs-labeled hairpin DNA in phosphate buffer solution, and store at 4 °C for later use; (3) Construction of SPR aptasensor: Soak the gold sheet in a mixed solution of H ₂ SO ₄ :H ₂ O ₂ with a volume ratio of 7:3 for 2 min, rinse it with secondary water, and immerse it in 10 mM mercaptohexyl After 2 h in alcohol solution, wash with secondary water and blow dry with nitrogen, put it into the SPR detection cell; inject 50 μL, 1.2 μM thiolated hairpin structure DNA solution and react for 2 h to prepare pre-assembled thiolated hairpins Structural DNA sensing interface; incubate the adenosine aptamer with the same ratio of probe DNA at 37 °C for 2 h to form aptamer/probe DNA double strands; add adenosine solution and incubate at 37 °C for 2 h, Adenosine specifically binds to its aptamer to release the probe DNA from the aptamer/probe DNA double strand; add the Au NPs labeled hairpin DNA solution prepared in step (2), and inject the mixed solution into the SPR The detection cell was allowed to react with the sensing interface pre-assembled with thiolated hairpin structure DNA for 1 h, and the reaction solution was discharged with a peristaltic pump and injected with secondary water for cleaning. 2. The method for constructing an aptamer sensor based on Au NPs and DNA circulation double amplification technology according to claim 1, characterized in that in step (2), the concentration of the dithiothreitol solution is 170 mM, Phosphate buffered saline solution with pH 8.0. 3. The aptasensor construction method based on Au NPs and DNA cycle double amplification technology according to claim 1, characterized in that in step (2), the phosphate buffer solution has a concentration of 10 mM and a pH of 7.4 , containing 0.1 M NaCl. 4. The method for constructing an aptamer sensor based on Au NPs and DNA cycle double amplification technology according to claim 1, characterized in that in step (3), the mercaptohexanol solution is prepared with absolute ethanol. 5. The aptasensor construction method based on Au NPs and DNA cycle double amplification technology according to claim 1, characterized in that in step (3), the concentration of the thiolated hairpin structure DNA solution is 10 mM , pH 7.4 and phosphate buffer solution containing 0.1 M NaCl. 6. Application of aptamer sensor based on Au NPs and DNA cycle double amplification technology in adenosine detection: characterized in that 20 μL, 1.0 μM adenosine aptamer and the same ratio of probe DNA were incubated at 37 °C After 2 h, aptamer/probe DNA double strands were formed; 20 μL of adenosine solution of different concentrations was added and incubated at 37 °C for 2 h, adenosine specifically bound to its aptamer and the probe DNA was separated from the aptamer/probe. The probe DNA double-strand is released; take 25 μL of adenosine and aptamer/probe DNA double-strand reaction solution and add it to 25 μL Au NPs-labeled hairpin DNA solution, and inject the mixed solution into the SPR detection cell react with the hairpin DNA pre-assembled on the sensing interface for 1 h; discharge the reaction solution with a peristaltic pump and inject 50 μL of secondary water for cleaning; as the concentration of adenosine increases, the SPR sensor The Au NPs at the interface gradually increased, the SPR response signal increased rapidly, the SPR angle change and the adenosine concentration showed a good linear relationship in the range of 0.5-50 pM, and the detection limit was 0.21 pM, which can be used for ultra-sensitive and ultra-low detection of adenosine Concentration detection.