CN114621958B - Single-stranded DNA aptamer sequence for specifically recognizing ATP and application thereof - Google Patents

Abstract

The invention discloses a group of single-stranded DNA (ssDNA) aptamers capable of specifically recognizing ATP and application thereof, belonging to the fields of biochemistry and molecular biology. According to the invention, a double-stranded DNA library is fixed on magnetic beads through a magnetic bead-SELEX technology, ATP is added to compete for replacement of sequences with affinity, five sequences with higher affinities of ap1, ap3, ap4, ap7 and ap9 are finally obtained through several rounds of forward screening, several rounds of reverse screening, high-throughput sequencing and later selection, and an aptamer ap1-1 with the length of 32 nucleotides is obtained after truncated optimization of ap1, and the affinity of the aptamer to ATP is improved compared with that of the aptamer before truncated, and ATP and other structural analogues such as ADP and AMP can be effectively distinguished, so that the aptamer has high specificity to ATP and is selected as an optimal ATP aptamer. The invention provides a recognition element with excellent performance and a detection method for ATP detection.

Description

Single-stranded DNA aptamer sequence that specifically recognizes ATP and its application

Technical field

The invention belongs to the field of biochemistry and molecular biology, and in particular refers to a single-stranded DNA aptamer sequence that specifically recognizes ATP and its application.

Background technique

Adenosine triphosphate (ATP), composed of adenine, ribose and three phosphate groups, is a high-energy compound ubiquitous in all living cells. ATP, as the starting material for nucleic acid synthesis, is formed during the oxidation reaction of carbohydrates and glycolysis. It participates in the regulation of many biochemical processes and is the mediator of synapses. At the same time, ATP is also involved in the metabolic process. By interacting with actomyosin, it is broken down into adenosine diphosphate (ADP) and phosphate groups. In this case, energy is released, and most of the energy is used by the muscles to execute machinery. Works to synthesize proteins, urea and metabolic intermediates. Therefore, ATP is the provider of energy required for various life activities of organisms, and also plays a vital role in the regulation and integration of cell metabolism. Dynamic changes in ATP levels in the body reflect the development of disease or cancer, such as PA Kinson's disease, cardiovascular disease, etc. Therefore, ATP is an important detection indicator in biological response detection.

At present, the ATP detection methods reported at home and abroad mainly include bioluminescence method, high performance liquid chromatography, fluorescence method, biosensing method, spectrophotometry, etc. The advantages of bioluminescence are high sensitivity, strong selectivity, and relative ease of use, but it requires expensive and unstable bioluminescent agents. High-performance liquid chromatography can quickly separate and measure ATP and its analogues at the same time, but the equipment is expensive and difficult to popularize, it requires high experimental personnel, the pretreatment of samples is complex, and the analysis is time-consuming, making it difficult to meet the needs of lightweight and efficient on-site detection. Aptamers are widely used in the field of ATP detection due to their low cost and high stability. ATP aptamers are one of the model aptamers widely used in biosensing. Szostak et al. first reported the RNA aptamer of ATP in 1993, and screened the first DNA aptamer in 1995. However, the currently commonly used classic ATP DNA aptamers have poor specificity and cannot distinguish the phosphate substituent at the 5' position of adenosine, that is, they cannot distinguish ATP from ADP, AMP, and cAMP, which poses a problem for the accurate measurement of ATP and the energy charge of organisms. Status assessment has had a dramatic impact.

Systematic evolution of ligands by exponential enrichment (SELEX) is an in vitro screening of oligonucleotide fragments that can specifically bind to various target substances from random oligonucleotide libraries. molecular biology techniques. The target substance can be a protein, cell, small molecule, metal ion, nucleic acid or drug. The oligonucleotide fragment obtained by screening is called an aptamer. The basic principle of SELEX technology is to construct a synthetic random oligonucleotide library, incubate it with the target substance, and retain the sequences that interact with the target substance. After multiple rounds of amplification and screening, high affinity and specificity can be obtained. Nucleic acid aptamers. Nucleic acid aptamers have the advantages of high affinity, strong specificity and stability, low cost, and easy modification.

Contents of the invention

In order to solve the above technical problems, the present invention provides an ssDNA aptamer that specifically recognizes ATP. Through magnetic bead SELEX technology, a high-affinity oligonucleotide sequence that specifically binds to the target molecule is obtained through magnetic separation. After 10 rounds of screening and corresponding After reverse screening, high-throughput sequencing was performed to obtain the aptamer sequence, and the optimal aptamer sequence was obtained through sequence truncation optimization. These aptamers are new recognition elements for ATP. They have the advantages of good stability, high sensitivity, low cost, easy preparation, modification and labeling, and high specificity, and can be used in the construction of a variety of detection methods.

The first object of the present invention is to provide a single-stranded DNA aptamer sequence that specifically recognizes ATP. The ssDNA aptamer is ap1, ap3, ap4, ap7, ap9 or ap1-1. The ap1, ap3 , ap4, ap7, ap9 or apl-1 sequences are shown by SEQ ID NO.1-SEQ ID NO.6.

The specific sequence is: ap1 (SEQ ID NO. 1): 5′-TAGGGAATTCGTCGACGGATCCCGTGGCGTCTGCAACGGAAAAGAATTTATCTTGTCCTGCAGGTCGACGCATGCGCCG-3′

ap3(SEQ ID NO.2):5′-TAGGGAATTCGTCGACGGATCCCGAAGGACAGAAAGATACATCTGATGACGATTACACTGCAGGTCGACGCATGCGCCG-3′

ap4 (SEQ ID NO.3):5′-TAGGGAATTCGTCGACGGATCCTACCCGTTGCTGCAGGATCCTGAGATCGCCTCTGTCTGCAGGTCGACGCATGCGCCG-3′

ap7 (SEQ ID NO.4):5′-TAGGGAATTCGTCGACGGATCCATCCCCACGACGTCAAGGCCGCGTGCCGGTAGGGCTGCAGGTCGACGCATGCGCCG-3′

ap9 (SEQ ID NO.5):5′-TAGGGAATTCGTCGACGGATCCAAAAGCGTCTGCTGTGACGGGACAAAACCGGTGCTCTGCAGGTCGACGCATGCGCCG-3′

ap1-1 (SEQ ID NO. 6): 5′-TGGCGTCTGCATGCAGGTCGACGCATGCGCCG-3′.

In one embodiment of the present invention, the ssDNA aptamer can be modified with a group that improves stability, and/or a fluorescent group that provides a detection signal, an isotope, an electrochemical label, an enzyme label, and/or with a Modified by the affinity ligand and sulfhydryl group forming the composition.

The second object of the present invention is to provide the application of the ssDNA aptamer in specifically recognizing ATP.

The third object of the present invention is to provide a composition for specifically recognizing ATP, including one or more of the ssDNA aptamers.

The fourth object of the present invention is to provide a kit for specifically recognizing ATP, including one or more of the ssDNA aptamers.

The fifth object of the present invention is to provide a test strip for specifically recognizing ATP, including one or more of the ssDNA aptamers.

The sixth object of the present invention is to provide a chip for specifically recognizing ATP, including one or more of the ssDNA aptamers.

The seventh object of the present invention is to provide a fluorescent biosensor for specifically recognizing ATP, which includes one or more of the ssDNA aptamers; and also includes a fluorescent group and a fluorescence quencher.

In one embodiment of the invention, the fluorescence quenching agent is graphene oxide, gold nanoparticles, manganese dioxide nanosheets, 4-(4'-dimethylaminoazophenyl) benzoic acid or dimethyl Aminoazobenzoyl.

In one embodiment of the invention, the fluorescent group is selected from 6-carboxyfluorescein, CY3, hexachloro-6-methylfluorescein, 6-carboxytetramethylrhodamine, ROX, CY5, isothiocyanine Acid fluorescein or 5-carboxyfluorescein.

The eighth object of the present invention is to provide a screening method for single-stranded DNA aptamers that specifically recognize ATP, specifically including the following steps:

(a) ssDNA library: 5′-TAGGGAATTCGTCGACGGATCC-N35-CTGCAGGTCGACGCATGCGCCG-3′, where N represents any one of the bases A, T, C, and G;

Forward primer I-1: 5′-FAM-TAGGGAATTCGTCGACGGAT-3′;

Forward primer I-2: 5′-TAGGGAATTCGTCGACGGAT-3′;

Reverse primer II-1: 5′-biotin-CGGCGCATGCGTCGACCTG-3′;

Reverse primer II-2: 5′-CGGCGCATGCGTCGACCTG-3′;

(b) PCR amplify the ssDNA library using primers I-2 and II-1 in step (a) according to the following conditions: 1 pmol of ssDNA, 10 pmol of forward primer, 10 pmol of reverse primer, 12.5 μL Max DNA polymerase, 9.5 μL of deionized water; the working temperature cycle is 95°C for 300s, 95°C for 30s, 55°C for 30s, and 72°C for 15s, and the number of amplification cycles is 15;

(c) Streptavidin magnetic beads: particle size 1-2 μm, concentration 5 mg·mL ^-1 , washed 3-7 times with PBS buffer;

(d) Combine the streptavidin magnetic beads in step (c) with the DNA amplified in step (b) under appropriate conditions. The appropriate conditions include room temperature 20-30°C and a binding time of 1-3 hours;

(e) After the mixture in step (d) is separated using a magnetic stand, remove the supernatant, wash it 3-7 times with binding Tris buffer, then mix it with ATP solution, and incubate it at room temperature 20-30°C for 1-3 hours. ;

(f) After the mixture in step (e) is separated using a magnetic stand, take the supernatant and collect the ssDNA sequence bound to ATP in the supernatant;

(g) Repeat steps (b) to (f) 4-8 times;

(h) Introduce counter-screening, separate the mixture in step (d) using a magnetic stand, remove the supernatant, and wash 3-7 times with binding Tris buffer. Then mix it with ADP and AMP solutions, incubate at room temperature 20-30°C for 1-3 hours, and then magnetically separate to remove the supernatant. Then add ATP solution and incubate at room temperature 20-30°C for 1-3 hours. After magnetic separation, collect the ssDNA sequence bound to ATP in the supernatant. Repeat reverse screening 2-6 times;

(i) Repeat steps (b) to (f) 1-5 times;

(j) Collect the mixture of ATP and ssDNA obtained through the above steps, use forward primer I-2 and reverse primer II-2 to perform PCR amplification, and then perform high-throughput sequencing.

The present invention further constructs an aptamer-based fluorescent biosensor to detect ATP. This detection method combines graphene oxide with strand displacement amplification technology. When ATP is present, the template DNA containing the aptamer sequence can be competitively bound from the graphene oxide to induce strand displacement amplification technology. occurs, thereby turning on the molecular beacon, and the fluorescence intensity increases. In the absence of ATP, the aptamer will be adsorbed by graphene oxide and cannot proceed to subsequent steps. The sensor converts ATP concentration detection into fluorescence intensity measurement.

The present invention uses magnetic bead-SELEX technology to modify biotin at the 5' end of the antisense strand in the double-stranded DNA molecule, and utilizes the interaction between streptavidin and biotin to modify the double-stranded DNA molecule on the magnetic beads. The surface is then incubated with the target molecule. The ssDNA sequence with high affinity to the target molecule is competitively bound and freed in the system. Through magnetic separation, the ssDNA is isolated and used as a secondary library for the next round of screening. After multiple After rounds of screening, the ssDNA sequence finally retained has a higher affinity for ATP. At the same time, counter-screening methods were introduced in the last few rounds of screening, using ADP, AMP and other interference substances as counter-screening targets, and removing sequences that can bind both ADP and AMP from the enriched sequences with affinity for ATP, which greatly improved the efficiency. The specificity of the aptamer was finally obtained with high affinity and high specificity ATP aptamer. The aptamer was further truncated and optimized based on the secondary structure and molecular docking simulation results, and finally the optimal aptamer ap1-1 was obtained. The present invention provides a highly specific aptamer sequence with good stability, high affinity, easy preparation, modification and labeling for ATP detection.

The above technical solution of the present invention has the following advantages compared with the existing technology:

The present invention uses magnetic bead-SELEX technology to fix the oligonucleotide library to magnetic beads, and through competitive displacement and amplification enrichment, combined with repeated and strict reverse screening, ssDNA aptamers that bind highly specifically to ATP are obtained. The detection provides highly specific detection recognition elements and possible detection methods with good stability, high sensitivity, low cost, easy preparation, modification and labeling.

Description of the drawings

In order to make the content of the present invention easier to understand clearly, the present invention will be further described in detail below based on specific embodiments of the present invention and in conjunction with the accompanying drawings, wherein

Figure 1: Schematic diagram of magnetic bead-SELEX screening of ATP-specific aptamers.

Figure 2: Fitting curve graph for determining Kd values of aptamer sequences ap1, ap3, ap4, ap7, ap9 and ap1-1.

Figure 3: Fluorescence method to characterize the specificity of ap1-1 and ATP aptamers reported in the literature.

Figure 4: Standard curve for detecting ATP using fluorescent aptamer biosensor method.

Figure 5: Specificity verification of fluorescent aptamer biosensors.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples are not intended to limit the present invention.

Buffer material information used in the present invention:

PBS buffer: 137mmol·L ^-1 NaCl, 2.7mmol·L ^-1 KCl, pH 7.4;

Tris buffer: 50mmol·L ^-1 Tris-HCl, 5mmol·L ^-1 KCl, 100mmol·L ^-1 NaCl, 1mmol·L ^{- 1} MgCl ₂ , pH 7.4.

Example 1: Construction of random ssDNA library and its primers

(a) Construct a random ssDNA library of 79 bases in length

5′-TAGGGAATTCGTCGACGGATCC-N35-CTGCAGGTCGACGCATGCGC CG-3′, where N represents any one of the bases A, T, C, and G.

(b) Synthesize forward primer:

Forward primer 1: 5′-TAGGGAATTC GTCGACGGAT-3′;

Forward primer 2: 5′-FAM-TAGGGAATTC GTCGACGGAT-3′;

(c) Synthesize reverse primer:

Reverse primer 1: 5′-CGGCGCATGC GTCGACCTG-3′;

Reverse primer 2: 5′-biotin-CGGCGCATGC GTCGACCTG-3′.

Example 2: In vitro screening of nucleic acid aptamers

In order to screen out ssDNA aptamers with high affinity and specificity for ATP, a total of 10 rounds of nucleic acid aptamer screening were conducted, with rounds 1-6 being forward screening, rounds 7-9 introducing reverse screening, and round 10 again. Positive screening stabilizes the screening process.

(a) The 25 μL PCR amplification system is shown in Table 1.

Table 1 Forward primer 1 10μmol·L ^-1 1μL reverse primer 2 10μmol·L ^-1 1μL Template DNA 1μmol·L ^-1 1μL 2×PrimeSTARMax Premix 12.5μL ddH ₂ O Add to 25μL system

Amplification conditions: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; extension at 72°C for 15 seconds; extension at 72°C for 5 minutes; 15 cycles.

(b) Main steps of in vitro screening: After washing the streptavidin-coupled magnetic beads with PBS buffer 5 times, dissolve 100 μL of the initial library PCR product in 100 μL of binding buffer, and then add 50 μL of streptavidin. After the coupled magnetic beads were gently shaken at room temperature for 2 hours, place them on a magnetic separator, remove the supernatant, wash the magnetic beads 5 times with binding buffer, and then add 3 μL of 100 mmol·L ^-1 ATP. Solution, continue to shake gently for 2 hours at room temperature, and place it on a magnetic separator for 3 minutes. After that, collect the supernatant and put it into the next round of screening. As the number of screening rounds increases, in order to obtain high-affinity aptamers , gradually reducing target concentration to increase screening pressure. At the same time, in order to improve the specificity of aptamers, reverse screening was introduced starting from the seventh round.

(c) Counter screening: Perform PCR amplification of the secondary library prepared in the previous round, mix 100 μL of PCR product with 100 μL of binding buffer, then add 50 μL of streptavidin-coupled magnetic beads, and incubate at room temperature. After gently shaking for 2 hours, place it on a magnetic separator, remove the supernatant, wash the magnetic beads 5 times with binding buffer, then add 3 μL of 100 mmol·L ^-1 ADP and AMP solution, and continue gently at room temperature. Shake gently for 2h, discard the supernatant after magnetic separation, wash 3 times with binding buffer, then add 3μL of 100mmol·L ^-1 ATP solution, continue shaking at room temperature for 2h, and place it on the magnetic separator for 3min. , the supernatant was collected as a secondary library for the next round of screening.

(d) Determination of the number of screenings: PCR amplify the ssDNA secondary library obtained in each round of screening using forward primer 2 and reverse primer 2. Take 40 μL of streptavidin-modified magnetic beads, wash them three times with binding buffer, add 20 μL of PCR product and 80 μL of binding buffer, mix evenly, and incubate on a shaker at room temperature for 1 hour. Subsequently, place it on a magnetic separation rack, discard the supernatant, and wash three times with binding buffer to remove dsDNA that is not bound to the magnetic beads. Subsequently, 30 μL of 0.1 mol·L ^-1 NaOH was added, and incubated in a 37°C water bath for 1 hour. The dsDNA was denatured and melted, and could be repeatedly eluted twice. After magnetic separation, the supernatant was FAM-modified ssDNA. Take 4 pmol of FAM-modified ssDNA, add 100 μL of binding buffer, mix evenly, denature at 95°C for 10 minutes, quickly ice bath for 5 minutes, and then leave at room temperature for 10 minutes. In the experimental group, 2 μL of 1 mmol·L ^-1 ATP solution was added, and in the control group, an equal volume of binding buffer was added, and the system was supplemented with binding buffer to 250 μL, and the system was shaken and incubated at 25°C for 1 h. Subsequently, 5 μL of graphene oxide solution was added to the experimental group and the control group respectively, mixed evenly, and placed on a shaker for shaking and incubation for 50 min. Centrifuge the mixture at 12,000 rpm for 12 min, take 200 μL of the supernatant, place it in a 96-well plate and use a multifunctional microplate reader to measure the fluorescence value (excitation wavelength 488 nm, emission wavelength 525 nm). The difference in fluorescence value between the experimental group and the control group is the fluorescence intensity of the ssDNA sequence combined with ATP. Screening can be stopped until the fluorescence value reaches a stable level.

(e) The next round of screening was repeated according to the above screening method. A total of 10 rounds of nucleic acid aptamer screening were conducted. Rounds 1-6 were forward screening, rounds 7-9 introduced reverse screening, and round 10 was another positive screening for fluorescence. Strength tends to stabilize. Therefore, screening can be stopped after the 10th round of screening.

Example 3: Screening and sequencing of ssDNA clones

ssDNA cloning and sequencing: The ssDNA obtained after the final round of screening is amplified by PCR using forward primer 1 and reverse primer 1, and the amplified product is sent to Shanghai Sangon for high-throughput sequencing.

Example 4: Determination of the dissociation constant K _d value of the aptamer sequence using fluorescence method

The high-throughput sequencing results in Example 3 were analyzed and processed, and ap1, ap3, ap4, ap7, ap9, and the truncated sequence ap1-1 were selected for measurement of dissociation constant (K _d ). Add aptamer sequences of different concentrations modified with the fluorescent group 6-carboxyfluorescein (FAM) into the binding buffer, replenish the volume with binding buffer to 200 μL, denature at 95°C for 10 min, and quickly ice bath for 10 min. Then add 1 μL of 100 mmol·L ^-1 ATP solution into the solution, shake slightly, and react at room temperature for 1 h and 40 min. Then add graphene oxide and incubate at room temperature for 40 min. Centrifuge the mixture at 12,000 rpm for 12 min and take the supernatant. Finally, all the supernatant was added to a 96-well plate and its fluorescence intensity was measured with a microplate reader. The content of the aptamer sequence is directly proportional to the fluorescence intensity.

According to the equation: y=Bmax×free ssDNA/(K _d +free ssDNA), the K _d value of each aptamer sequence can be analyzed. In the equation, y represents the proportion of ATP bound by the nucleic acid aptamer to the total ATP, that is, the degree of saturation; Bmax represents the number of maximum binding sites, and free ssDNA represents the concentration of free ssDNA that is not bound to ATP. The fitting curve is shown in Figure 2. The measured K _d values of ap1, ap3, ap4, ap7, ap9 and ap1-1 are 81.66±10.52nmol·L ^-1 , 238.07±91.52nmol·L ^-1 and 101.71± respectively. 11.89nmol·L ^-1 , 187.44±62.36nmol·L ^-1 , 167.99±16.38nmol·L ^-1 and 70.61±20.91nmol·L ^-1 , all have high affinity, among which the truncated ap1-1 has high affinity with ATP. Has the highest affinity.

The sequences of ap1, ap3, ap4, ap7, ap9 or ap1-1 are respectively:

ap1:

5′-TAGGGAATTCGTCGACGGATCCCGTGGCGTCTGCAACGGAAAAGAATTTATCTTGTCCTGCAGGTCGACGCATGCGCCG-3′

ap3:

5′-TAGGGAATTCGTCGACGGATCCCGAAGGACAGAAAGATACATCTGATGACGATTACACTGCAGGTCGACGCATGCGCCG-3′

ap4:

5′-TAGGGAATTCGTCGACGGATCCTACCCGTTGCTGCAGGATCCTGAGATCGCCTCTGTCTGCAGGTCGACGCATGCGCCG-3′

ap7:

5′-TAGGGAATTCGTCGACGGATCCATCCCCACGACGTCAAGGCCGCGTGCCGTAGGGCTGCAGGTCGACGCATGCGCCG-3′

ap9:

5′-TAGGGAATTCGTCGACGGATCCAAAAGCGTCTGCTGTGACGGGACAAAACCGGTGCTCTGCAGGTCGACGCATGCGCCG-3′

ap1-1:5′-TGGCGTCTGCATGCAGGTCGACGCATGCGCCG-3′

Example 5: Fluorescence method to verify the specificity of apl-1

Add 200 μL of binding buffer to the aptamer modified with the fluorescent group 6-carboxyfluorescein (FAM) at the same concentration, denature at 95°C for 10 min, and quickly incubate on ice for 10 min. Then add 1 μL of 100 mmol·L ^-1 ATP, ADP and AMP solution, shake slightly, react at room temperature for 1 h and 40 min, then add graphene oxide, incubate at room temperature for 40 min, centrifuge the mixture at 12000 rpm for 12 min, and take the supernatant. Finally, all the supernatant was added to a 96-well plate and its fluorescence intensity was measured with a microplate reader. Compared with the 27nt classic ATP aptamer reported in the literature, ap1-1 can clearly distinguish ATP, ADP and AMP (Figure 3); among them, the literature refers to Huizenga, DE; Szostak, JWA DNA aptamer that binds adenosineandATP. Biochemistry 1995 ,34,656-665,10.1021/bi00002a033.

Example 6: Construction of fluorescent aptamer biosensor to detect ATP standard

The sensor introduces strand displacement amplification technology to achieve effective signal amplification. When ATP is present, ATP can competitively bind the template DNA from graphene oxide. After the template DNA hybridizes with the primer, strand displacement amplification is induced. The reaction occurs, which in turn turns on the molecular beacon, and the fluorescence intensity increases. In the absence of ATP, the aptamer will be adsorbed by graphene oxide and cannot proceed to subsequent steps. Mix 2 μL of 100 μmol·L ^-1 template DNA with the binding buffer, heat at 95°C for 10 min, and then quickly ice bath for 10 min. Add 5 μL of GO solution, mix well, and incubate with shaking at 25°C for 40 min. Subsequently, ATP solutions of different concentrations were added, and the incubation was continued with shaking at 25°C for 1 h and 40 min. Centrifuge the incubated mixed solution at 12,000 rpm for 15 min, and recover the supernatant. Take 5 μL of the supernatant and mix it with 3 μmol·L ^-1 primer, 5U Bsm DNA polymerase, 8UNb.bpu10I endonuclease, 1×Buffer R and 250 μmol·L ^-1 dNTP. The total system is 30 μL. Mix the system, incubate at 37°C for 90 minutes, and then incubate at 80°C for 20 minutes to inactivate the enzyme. Finally, add 1 μL of 100 μmol·L ^-1 molecular beacon to 25 μL of the above strand displacement amplification product, and add buffer to make up the system to 100 μL. Place the mixed solution in a metal bath for denaturation at 95°C for 5 minutes, and then incubate at 25°C for 1 hour and 30 minutes. use The multifunctional microplate reader measures the fluorescence intensity at an excitation wavelength of 488nm and an emission wavelength of 525nm. When the ATP concentration is between 0.1 μmol·L ^-1 and 25 μmol·L ^-1 , there is a good linear relationship between fluorescence intensity and ATP concentration. The linear regression equation is y=102.34x+3188.89 (R ² =0.9958), where x represents the concentration of ATP (μmol·L ^-1 ) and y represents the fluorescence intensity. The detection limit of this sensor is 33.85 nmol·L ^-1 (Figure 4).

Example 7: Specificity verification of fluorescent aptamer biosensor

In order to verify the specificity of the fluorescent aptamer biosensor, ATP analogues including ADP, AMP, GTP, UTP, and CTP were used as control groups. The experimental method was as described in Example 6. As shown in Figure 5, under optimal detection conditions, the relative fluorescence intensity in the presence of ATP was significantly higher than that of other control groups. Therefore, the constructed fluorescent aptamer sensor has high specificity for ATP.

Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other changes or modifications may be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

SEQUENCE LISTING

<110> Jiangnan University

<120> Single-stranded DNA aptamer sequence that specifically recognizes ATP and its application

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Claims (10)

1. An ssDNA aptamer that specifically recognizes ATP, characterized in that: the ssDNA aptamer is selected from apl-1, and the sequence of apl-1 is shown in SEQ ID NO. 6. 2. The ssDNA aptamer specifically recognizing ATP according to claim 1, characterized in that: the ssDNA aptamer can be provided with a group that improves stability, and/or a fluorescent group or isotope that provides a detection signal. , electrochemical labels, enzyme labels, and/or modified by affinity ligands and sulfhydryl groups used to form the composition. 3. Application of the ssDNA aptamer described in claim 1 or 2 in specifically recognizing ATP. 4. A composition for specifically recognizing ATP, characterized by comprising the ssDNA aptamer according to claim 1 or 2. 5. A kit for specifically recognizing ATP, characterized by comprising the ssDNA aptamer according to claim 1 or 2. 6. A test strip for specifically identifying ATP, characterized by comprising the ssDNA aptamer according to claim 1 or 2. 7. A chip for specifically recognizing ATP, characterized by comprising the ssDNA aptamer according to claim 1 or 2. 8. A fluorescent biosensor for specifically recognizing ATP, characterized by: comprising the ssDNA aptamer according to claim 1 or 2; and further comprising a fluorescent group and a fluorescence quencher. 9. The fluorescent biosensor according to claim 8, wherein the fluorescence quenching agent is graphene oxide, gold nanoparticles, manganese dioxide nanosheets, 4-(4'-dimethylaminoazo phenyl)benzoic acid or dimethylaminoazobenzoyl. 10. The fluorescent biosensor according to claim 8, characterized in that the fluorescent group is selected from the group consisting of 6-carboxyfluorescein, CY3, hexachloro-6-methylfluorescein, and 6-carboxytetramethylrhodamine. , ROX, CY5, fluorescein isothiocyanate or 5-carboxyfluorescein.