CN112630439A - Splitting aptamer sensor based on nanogold and preparation method and application thereof - Google Patents
Splitting aptamer sensor based on nanogold and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of sensors, and relates to a split aptamer sensor, in particular to a split aptamer sensor based on nanogold, and a preparation method and application thereof. The method is based on the energy transfer principle, and utilizes nanogold to quench FAM fluorescent groups; a high-sensitivity split aptamer sensor based on nanogold is constructed by combining a split aptamer recognition mechanism. Experimental optimization is carried out on the preparation conditions of the sensor, such as the amount of sodium citrate used in the combination process of AuNPs and P2, the ratio of AuNPs to P2, the stabilization time of a working solution and the like. The optimized working solution is used for detecting ATP, and the result shows that the detection range of the sensor is 30pmol/L to 15nmol/L, the high-sensitivity linear detection range is 30pmol/L to 3330pmol/L, the detection limit is 30pmol/L, and the sensor has good specificity and is a split aptamer sensor with great application potential.
Description
Technical Field
The invention belongs to the field of sensors, and relates to a split aptamer sensor, in particular to a split aptamer sensor based on nanogold, and a preparation method and application thereof.
Background
Adenosine Triphosphate (ATP) is a direct source of energy for various vital activities in organisms, and plays a key role in biochemical reactions and drug analysis. Common cardiovascular diseases, Parkinson's disease and Alzheimer's disease are closely related to ATP concentration, and the detection of ATP concentration has important significance in pathology and life science research. The traditional method for detecting ATP mainly depends on expensive instruments and equipment, such as high performance liquid chromatography, isotope tracer method and the like, although the sensitivity is higher, the popularization and the application of the ATP are limited by the defects of high sample preparation requirement, complex experimental operation, expensive instruments and the like.
The aptamer (aptamer) is an oligonucleotide fragment (RNA or DNA) which is screened by an in vitro screening technology SELEX (exponential enrichment ligand system evolution) and can be specifically bound with proteins or other small molecular substances, and has the advantages of easiness in synthesis, easiness in marking, stable property, no immunogenicity and toxicity and the like. After the aptamer is combined with a target, the conformation of the aptamer generally changes remarkably to form a hairpin, a stem-loop, a pseudoknot or a bulge structure, and a plurality of fluorescent aptamer sensors can be designed by utilizing the conformational change or structural modification after the aptamer is combined with the target and combining the high sensitivity of a fluorescence analysis technology. However, in complex matrices, structural changes are susceptible to interference factors that can produce false positive or non-specific signals that affect the specificity and sensitivity of the sensor. To this end, Stojanovic et al propose a method of fragmenting aptamers into two fragments P1 and P2 to form a P1-target-P2 sandwich in the presence of a target. Since these two independent oligonucleotide fragments lack secondary structure, no false positive or non-specific signal is generated. There are many methods for designing a fluorescent sensor by using a split aptamer, and young et al and Jiang et al quench a fluorescent signal modified on P1 by using graphene oxide or a carbon nanotube as a quencher; after the P2 and the target are added, a P1-target-P2 sandwich structure is formed, fluorescence is recovered, the concentration of the added target can be measured according to the quantity of recovered fluorescence signals, the detection limit of ATP is 20 mu mol/L, and the detection limit of theophylline is 155nmol/L and 4 nmol/L. The detection limit of the Wang M for detecting ATP by using nanogold quenching fluorescence recovery is 5 mu mol/L. The ATP is detected by combining a label-free mode with a split aptamer, and the detection limit is 2.67 nmol/L at least; DNA molecule design, exonuclease and the like are combined with a split aptamer to detect ATP, and the detection limit is 1-30 mu mol/L. The research shows that the detection limit of the splitting-type aptamer for detecting ATP is generally higher, and the splitting-type aptamer is not favorable for detecting low-concentration ATP, because the specificity of the aptamer is enhanced after splitting, but the affinity of the aptamer with a target molecule is relatively weakened, and when the concentration is too low, a trace amount of ATP molecules in a solution are difficult to capture due to steric hindrance. Researchers can effectively reduce the detection limit of the split aptamer sensor by enhancing the reaction processes such as various molecular modifications (the ATP detection limit is 2.4 nmol/L), chemical process changes (the ATP detection limit is 0.02 nmol/L) and the like, but the sensor is complex in preparation process, high in operation difficulty and relatively high in cost.
Disclosure of Invention
In order to solve the technical problems, the invention provides a split aptamer sensor based on nanogold and a preparation method and application thereof.
The technical scheme of the invention is realized as follows:
the preparation method of the split aptamer sensor based on the nanogold comprises the following steps:
(1) adding the nucleotide chain P2 into a Tris-HCl solution for reaction for 2h for activation to obtain a P2 solution;
(2) adding AuNPs into the P2 solution, reacting for 20min at normal temperature, adding sodium citrate solution, carrying out constant temperature shaking culture at 37 ℃ for 3 h, centrifuging, and taking a base solution to obtain an AuNPs @ P2 coupling compound;
(3) adding a nucleotide chain P1 into the AuNPs @ P2 coupler, and culturing at room temperature for 2h to obtain the splitting aptamer sensor working solution based on nanogold.
Preferably, in the step (1), the nucleotide chain P2 has a sequence of 5' -TGCGGAGGAAGGT- (CH)2) 6-SH-3'.
Further, in the step (1), the Tris-HCl solution has a pH =7 and a concentration of 10 mmol/L, and the P2 solution has a concentration of 5 μmol/L.
Preferably, in the step (2), the concentration of the AuNPs is 1nmol/L, and the concentration of the sodium citrate solution is 500 mmol/L, pH = 5.
Further, the volume ratio of the P2 solution to the AuNPs to the sodium citrate solution is 20:8: 1.
Further, in the step (2), the centrifugation condition is 14000 r/min for 15 min.
Preferably, in said step (3), the sequence of nucleotide chain P1 is shown as 5 '-FAM-ACCTGGGGGAGTAT-3', and the mass ratio of AuNPs @ P2 coupler to nucleotide chain P1 is 1: 1.
The split aptamer sensor prepared by the preparation method.
The split aptamer sensor is applied to detection of adenosine triphosphate in diagnosis and treatment of non-diseases.
The method comprises the following steps: adding 20 mu L of sample to be detected into the working solution, fixing the total volume of the solution to 600 mu L by using 500 mmol/LTris-HCl buffer solution, carrying out fluorescence detection on AuNPs @ P2-ATP-P1 compound containing ATP by using a fluorescence spectrophotometer, substituting the fluorescence intensity into a linear regression equation Y =66.54-7.31X, and obtaining the concentration of the sample to be detected, wherein the correlation coefficient R = 0.99.
The detection principle of the sensor is as follows: the ATP aptamer was split into two fragments P1 and P2. Modifying sulfydryl at the 3' end of P2, and modifying the surface of the nano gold by adopting a low pH method; the 5' end of P1 is modified with a fluorescent group FAM. When the detected substance ATP is not added, the P1 and the P2 are not combined due to the mutual exclusion of the basic groups, the fluorescent group FAM is far away from the nanogold, and the fluorescent signal is strong; after ATP is added, P1, P2 and ATP are specifically combined to form an AuNPs @ P2-ATP-P1 @ FAM sandwich structure, the distance between the FAM and the nanogold is shortened, due to the energy transfer phenomenon, a fluorescence signal is relatively weakened, the weakened light intensity is in direct proportion to the concentration of the added ATP, and the concentration of the added ATP can be calculated by measuring the reduction amount of the fluorescence signal.
The invention has the following beneficial effects:
1. the application utilizes nanogold as a quenching agent, combines a split type ATP aptamer recognition mechanism, designs a fluorescent aptamer sensor with low detection limit and simple operation, and realizes accurate detection of low-concentration ATP.
2. The method is based on the energy transfer principle, and utilizes nanogold to quench FAM fluorescent groups; a high-sensitivity split aptamer sensor based on nanogold is constructed by combining a split aptamer recognition mechanism. Experimental optimization is carried out on the preparation conditions of the sensor, such as the amount of sodium citrate used in the combination process of AuNPs and P2, the ratio of AuNPs to P2, the stabilization time of a working solution and the like. The optimized working solution is used for detecting ATP, and the result shows that the detection range of the sensor is 30pmol/L to 15nmol/L, the high-sensitivity linear detection range is 30pmol/L to 3330pmol/L, the detection limit is 30pmol/L, and the sensor has good specificity and is a split aptamer sensor with great application potential.
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 ATP detection by a split aptamer sensor based on nanogold.
FIG. 2 shows fluorescence spectra of the working solution before and after addition of ATP.
FIG. 3 is a fluorescence spectrum and a fluorescence intensity curve of a working solution when the amount of sodium citrate is different.
FIG. 4 is a graph showing the fluorescence spectrum and fluorescence intensity change curve of FAM when the pH value of the environmental solution is changed.
FIG. 5 is a fluorescence spectrum and a fluorescence intensity curve of a working solution when the ratio of AuNPs to P2 is different.
FIG. 6 is a fluorescence spectrum and a fluorescence intensity curve of a working solution when the stabilization time is different.
FIG. 7 shows fluorescence spectra and fluorescence intensity curves of working solutions when different concentrations of ATP (0.03-15nmol/L) were added.
FIG. 8 shows the result of specific detection of ATP.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Experimental Material
F-7000 spectrofluorometer (HITACHI, Japan); electronic balance and PE-20K pH meter (METTLER TOLEDO, Switzerland); HZQ-F200 constant temperature shaking incubator (Beijing Toyobo sea Instrument Co., Ltd.); 07HWS-2 digital display constant temperature magnetic stirrer (Hangzhou instrument motor Co., Ltd.); eppendorf centrifuge 5418 (Eppendorf AG, Germany).
Tris (2-carboxyethyl) phosphine (TCEP), disodium adenosine triphosphate, 20nm gold nanoparticles (AuNPs), trisodium citrate dihydrate, Tris (hydroxymethyl) aminomethane (Tris-HCL) were purchased from shanghai bioengineering gmbh. NaCl, MgCl2KCl, and glucose were all purchased from the exploration platform. All chemical reagents used in the experiment are Analytical Reagents (AR), and all solutions used are prepared by ultrapure water. Both oligonucleotide strands obtained in the laboratory were prepared by Shanghai Biotechnology engineering, Inc., and the sequences were as follows:
P1（ATP P1）:5′-FAM-ACCTGGGGGAGTAT-3′
P2（ATP P2）:5′-TGCGGAGGAAGGT-(CH2)6-SH-3′。
principle and feasibility of experiment
The principle of detecting ATP by using the split aptamer sensor based on nanogold is shown in figure 1. The ATP aptamer was split into two fragments P1 and P2. Modifying sulfydryl at the 3' end of P2, and modifying the surface of the nano gold by adopting a low pH method; the 5' end of P1 is modified with a fluorescent group FAM. When the detected substance ATP is not added, the P1 and the P2 are not combined due to the mutual exclusion of the basic groups, the fluorescent group FAM is far away from the nanogold, and the fluorescent signal is strong; after ATP is added, P1, P2 and ATP are specifically combined to form an AuNPs @ P2-ATP-P1 @ FAM sandwich structure, the distance between the FAM and the nanogold is shortened, due to the energy transfer phenomenon, a fluorescence signal is relatively weakened, the weakened light intensity is in direct proportion to the concentration of the added ATP, and the concentration of the added ATP can be calculated by measuring the reduction amount of the fluorescence signal.
Example 1
The preparation method of the split aptamer sensor based on the nanogold comprises the following steps:
mu. mol/L of thiol-modified P2 was activated in 10 mmol/L, pH =7 Tris-HCl for 2 h. And (3) taking 20 mu L of 5 mu mol/L P2 solution, adding 8 mu L of 1nmol/L AuNPs, reacting at normal temperature for 20min, adding 1 mu L of 500 mmol/L, pH =5 sodium citrate solution, carrying out shake culture at constant temperature of 37 ℃ for 3 h, and combining the aptamer P2 with the nanogold through an Au-S bond. And centrifuging at the rotating speed of 14000 r/min for 15min to remove redundant P2 chains, and taking the bottom liquid to obtain the AuNPs @ P2 coupling substance. 20 mu L and 5 mu mol/L of split aptamer P1 modified with fluorescent group FAM are added into the AuNPs @ P2 coupler, and the mixture is cultured at normal temperature for 2h to obtain a sensor working solution which can be stored at 4 ℃ for later use.
Detection of ATP: and taking 6 parts of the working solution, respectively adding 20 mu L of ATP with the concentrations of 1nmol/L, 15nmol/L, 65nmol/L, 100nmol/L, 200nmol/L and 450nmol/L, fixing the total volume of the solution to 600 mu L by using 500 mmol/LTris-HCl buffer solution, and performing fluorescence detection on AuNPs @ P2-ATP-P1 compounds containing the ATP with different concentrations by using a fluorescence spectrophotometer to obtain the detection range of the sensor.
The fluorescence signal intensities of the sensor working solution before and after ATP addition were measured separately, as shown in FIG. 2. Curve 1 is the fluorescence spectrum without ATP addition, and curves 2-3 are the fluorescence spectra after addition of 0.33nmol/L and 0.67nmol/L, respectively. As can be seen from the figure, after ATP is added, the fluorescence signal of the working solution is obviously quenched, and the quenching phenomenon is more obvious along with the increase of the concentration of the added ATP, so that the feasibility of the sensor is verified.
Example 2
The preparation method of the split aptamer sensor based on the nanogold comprises the following steps:
in the procedure of this example, as in example 1, only the addition volume of the sodium citrate solution was adjusted, and the sodium citrate solutions of 500 mmol/L, pH =5 were added in a volume of 0.5 μ L, 1 μ L, 1.5 μ L, 2 μ L, 2.5 μ L, and 3 μ L, respectively, to prepare working solutions having different initial fluorescence intensities, as shown in fig. 3. With increasing sodium citrate content, the initial fluorescence intensity of the working solution showed a tendency of increasing first and then decreasing, as shown in fig. 3 (b). This is because when the amount of sodium citrate is too small, a proper low pH solution environment cannot be formed, and the Au — S bond is less in a short time, resulting in a bare state of part of the AuNPs surface. The single-stranded base P1 is easily adsorbed on the surface of the nanogold, and draws the distance between the FAM and the AuNPs, so that energy transfer from the FAM to the surface of the AuNPs occurs, and fluorescence quenching is caused. With the increase of the amount of the sodium citrate, the fluorescence quenching phenomenon is gradually reduced, so that the fluorescence intensity of the working solution is in an enhancement trend. When the amount of the sodium citrate is too large, the pH of the solution is too low, and the self-fluorescence signal of the FAM is gradually reduced under the influence of the pH. As shown in fig. 4, the fluorescence intensity of FAM decreases significantly with the decrease of pH of the environmental solution when the concentration of FAM itself is constant. The optimal amount of sodium citrate is experimentally selected to be 1 muL by comprehensively considering the combination effect of AuNPs and P2 and the fluorescence characteristic of FAM.
Example 3
The preparation method of the split aptamer sensor based on the nanogold comprises the following steps:
the procedure of this example was the same as that of example 1, and working solutions with different ratios of AuNPs to P2 were prepared by adjusting the volume of AuNPs from 1. mu.L to 15. mu.L while keeping the concentration of P2 constant, and the initial fluorescence spectra and peak change are shown in FIG. 5 (a) and FIG. 5 (b). As can be seen from the graph, the light intensity of the working solution shows a trend of increasing and then decreasing with the increase of the AuNPs concentration. The working solution has the strongest fluorescence signal at 8. mu.L AuNPs volume. The reason for this is that, when the AuNPs concentration is less than 8 μ L, the fluorescence intensity of the working solution increases with the increase of the AuNPs concentration, because when the AuNPs concentration is too low, a large amount of P2 can be modified on a single AuNPs, and the surface of the AuNPs is almost completely surrounded by the P2 chain; at this time, the fluorescence signal in the working solution is derived from the FAM modified on free P1, AuNPs and P1 have little mutual influence, and the concentration of P1 is unchanged, so that the fluorescence intensity change of the working solution is small, as shown by 1. mu.L and 4. mu.L samples in FIG. 5 (b). When the AuNPs concentration continues to increase, part of the AuNPs surface is uncovered without being covered by P2, and the single-chain P1 is attracted to be close to the AuNPs surface. However, the P1 and P2 chains contain a large number of bases G, and the repulsion between the two chains keeps P1 away from AuNPs. When the attraction force and the repulsion force to the P1 reach a certain balance, the P1 may be kept a certain distance away from the AuNPs, so that part of the AuNPs generate a metal-enhanced fluorescence effect on the FAM, and the fluorescence signal intensity of the working solution is enhanced, as shown in 4 muL and 8 muL samples in FIG. 5 (b). When the concentration of AuNPs is increased more, the attraction force of the AuNPs to P1 is greater than the repulsion force of P2, most of P1 will approach the AuNPs, so that FAM and AuNPs are quenched by energy transfer, as shown in 8. mu.L, 12. mu.L and 15. mu.L samples in FIG. 5 (b). Therefore, when the concentration of P2 is kept constant in the preparation process of the sensor, the volume of AuNPs should be 8 muL.
Example 4
The preparation method of the split aptamer sensor based on the nanogold comprises the following steps:
in the procedure of this example, different stabilization times were measured in the ATP measurement, and the fluorescence spectrum of the working solution was measured every 10 minutes after the addition of P1, as shown in fig. 6, the fluorescence intensity of the working solution tended to increase first and then to stabilize with the increase of time, and the fluorescence was strong and remained substantially constant around 90 min. Therefore, in the preparation process of the sensor, the stable time of the working solution after the P1 is added is selected to be 90 min.
Application example
Sensitivity test:
using the working solution obtained by the method of preparing the working solution of example 1, 20. mu.L of ATP solutions with concentrations of 1nmol/L, 15nmol/L, 65nmol/L, 100nmol/L, 200nmol/L, and 450nmol/L were added, and the fluorescence spectrum was measured by fixing the volume to 600. mu.L with Tris-HCl buffer, as shown in FIG. 7 (a). The ATP concentration was plotted on the abscissa and the fluorescence peak at different concentrations on the ordinate, as shown in FIG. 7 (b). As the ATP concentration increases, the generation amount of the sandwich structure of AuNPs @ P2-ATP-P1 @ FAM increases, the fluorescence intensity of the reaction system continuously decreases, the measurement range is 30pmol/L to 15nmol/L, and the detection limit is 30 pmol/L. Wherein in the range of 0.03nmol/L to 3.33nmol/L, the linear regression equation of the fluorescence intensity and the ATP concentration is Y = -7.31X +66.54 (Y is the fluorescence intensity, and X is the ATP concentration), the correlation coefficient R =0.99, and the detection sensitivity is relatively high. The linear regression equation in the range of 3.33nmol/L to 15nmol/L is Y = -1.56X +47.58, the correlation coefficient R =0.99, and the detection sensitivity is relatively low.
Specific experiments:
to analyze the specificity of the split-type aptamer sensor, 20. mu.L of glucose, sodium ion, potassium ion, magnesium ion, UTP, GTP, CTP and ATP solutions were added to the working solution prepared in example 1, respectively, and the volume was adjusted to 600. mu.L with Tris-HCl buffer to measure the fluorescence spectrum. The change in the peak fluorescence value was measured before and after the addition of the test substance (F represents the fluorescence intensity before the addition of the test substance, and F0 represents the fluorescence intensity after the addition of the test substance). The experimental result is shown in fig. 8, compared with ATP, the amount of change of the fluorescence peak of the working solution is smaller after the test substances such as glucose, sodium ions, potassium ions, magnesium ions, UTP, GTP, and CTP are added, which indicates that the split-type aptamer sensor has good specificity to ATP.
Performing an effect analysis
There are two main methods for modifying DNA strands on AuNPs: salt aging and low pH methods. To reduce sensor preparation time, the low pH method was experimentally selected for DNA modification. AuNPs prepared by sodium citrate reduction method with negative surface charge[21]The DNA structure also has negative charge, and the low pH method is to realize protonation of base in DNA by using low pH and low salt[22]The repulsive force between AuNPs and DNA is reduced, and the rapid assembly of Au-S bonds is accelerated. The low pH and low salt environment are mainly provided by sodium citrate, so the content of the sodium citrate in the experiment influences the combination effect of the AuNPs and the P2. The combination of AuNPs and P2 and the fluorescence characteristic of FAM itself are considered, and the amount of sodium citrate is 1 muL.
When the concentration of AuNPs in the working solution is too high, the excessive AuNPs can adsorb single-chain P1 on the surface, so that energy transfer occurs when the distance between FAM and AuNPs is too close, fluorescence quenching occurs, and the initial fluorescence signal of the working solution can be greatly reduced at the moment, thereby influencing the measurement signal intensity and the detection sensitivity of the sensor. In order to optimize the ratio of AuNPs to P2 in the working solution, the volume of AuNPs should be 8 muL when the concentration of P2 is kept constant in the preparation process of the sensor.
The AuNPs @ P2 conjugate did not emit light, and the fluorescence signal in the working solution was derived from FAM modified at P1. The working solution can be obtained by adding P1 into the AuNPs @ P2 coupler, but the fluorescence signal of the coupler needs a certain time to be stable, and the stable time needs to be as short as possible in order to reduce the preparation time of the sensor. Experiments prove that in the preparation process of the sensor, the stable time of the working solution is selected to be 90min after the P1 is added.
Based on the energy transfer principle, nano gold is utilized to quench FAM fluorescent groups; a high-sensitivity split aptamer sensor based on nanogold is constructed by combining a split aptamer recognition mechanism. Experimental optimization is carried out on the preparation conditions of the sensor, such as the amount of sodium citrate used in the combination process of AuNPs and P2, the ratio of AuNPs to P2, the stabilization time of a working solution and the like. The optimized working solution is used for detecting ATP, and the result shows that the detection range of the sensor is 30pmol/L to 15nmol/L, the high-sensitivity linear detection range is 30pmol/L to 3330pmol/L, the detection limit is 30pmol/L, the sensor has good specificity, and the sensor is a split aptamer sensor with great application potential.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The preparation method of the split aptamer sensor based on the nanogold is characterized by comprising the following steps of:
(1) adding the nucleotide chain P2 into a Tris-HCl solution for reaction for 2h for activation to obtain a P2 solution;
(2) adding AuNPs into the P2 solution, reacting for 20min at normal temperature, adding sodium citrate solution, carrying out constant temperature shaking culture at 37 ℃ for 3 h, centrifuging, and taking a base solution to obtain an AuNPs @ P2 coupling compound;
(3) adding a nucleotide chain P1 into the AuNPs @ P2 coupler, and culturing at room temperature for 2h to obtain the splitting aptamer sensor working solution based on nanogold.
2. The method of claim 1, wherein the method comprises the steps of: in the step (1), the nucleotide chain P2 has a sequence of 5' -TGCGGAGGAAGGT- (CH)2) 6-SH-3'.
3. The method of claim 1, wherein the method comprises the steps of: in the step (1), the Tris-HCl solution has pH =7 and a concentration of 10 mmol/L, and the P2 solution has a concentration of 5 μmol/L.
4. The method of claim 1, wherein the method comprises the steps of: in the step (2), the concentration of AuNPs is 1nmol/L, and the concentration of the sodium citrate solution is 500 mmol/L, pH = 5.
5. The method of claim 4, wherein the method comprises the steps of: the volume ratio of the P2 solution to the AuNPs to the sodium citrate solution is 20:8: 1.
6. The method of claim 1, wherein the method comprises the steps of: in the step (2), the centrifugation condition is 14000 r/min for 15 min.
7. The method of claim 1, wherein the method comprises the steps of: in said step (3), the sequence of nucleotide chain P1 is represented by 5 '-FAM-ACCTGGGGGAGTAT-3', and the mass ratio of the AuNPs @ P2 conjugate to nucleotide chain P1 is 1: 1.
8. The split-type aptamer sensor prepared by the preparation method of any one of claims 1 to 7.
9. Use of the split-aptamer sensor of claim 8 for the detection of adenosine triphosphate in non-disease diagnostics and therapy.
10. Use according to claim 9, characterized in that the steps are as follows: adding 20 mu L of sample to be detected into the working solution, fixing the total volume of the solution to 600 mu L by using 500 mmol/LTris-HCl buffer solution, carrying out fluorescence detection on AuNPs @ P2-ATP-P1 compound containing ATP by using a fluorescence spectrophotometer, substituting the fluorescence intensity into a linear regression equation Y =66.54-7.31X, and obtaining the concentration of the sample to be detected, wherein the correlation coefficient R = 0.99.
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