A fluorescence assay for detecting ape1 enzyme using a three-dimensional dna walker biosensor

Abstract

The application discloses a method for fluorescent analysis of APE1 enzyme based on a three-dimensional multi-legged DNA walker constructed by double gold balls. AuNPs as a basis to construct two systems, one of which is to modify the walker chain to AuNPs according to 500:1 to serve as a walker, named AuNP-W; the other is to modify the Sub chain to AuNPs according to 1000:1 to serve as a track, named AuNP-T. In addition, endogenous ATP serves as the driving force for the continuous walking of the DNA walker. 2+ buffer to anneal AuNP-W and Block chain to form double-stranded AuNP-W / B; use TAE / Mg 2+ buffer to anneal AuNP-T and ATP aptamer chain Apt (containing FAM fluorophore) to form double-stranded AuNP-S / A; mix AuNP-W / B, AuNP-S / A and ATP according to a certain proportion to obtain a biosensor for fluorescent analysis and detection of APE1 enzyme, add APE1 enzyme with different concentrations, and perform real-time fluorescent quantitative analysis under the condition of 37 DEG C to measure the fluorescence curves corresponding to APE1 enzyme with different concentrations; add the sample to be measured into the biosensor for fluorescent analysis and detection of APE1, set the same detection conditions, measure the fluorescence curves of different samples to be measured, and obtain the specific detection results of APE1 enzyme.

Description

Technical Field

[0001] This invention pertains to biochemical analysis methods, specifically relating to a three-dimensional multi-legged DNA walker biosensor for the fluorescence analysis and detection of APE1 enzyme. Background Technology

[0002] Depurine / depyrimidine endonucleases, also known as redox effector 1 (APE1), are important DNA repair proteins that are overexpressed in most tumor cells and are considered biomarkers for cancer risk assessment, diagnosis, prognosis, and treatment efficacy. Despite APE1's significant role in cancer diagnosis, sensing changes in APE1 abundance and monitoring enzyme activity in living cells remains a challenge. The realization of DNA walkers stems from the excellent programmability of DNA. The progressive movement of DNA walkers offers the possibility of performing multiple tasks and signal amplification, providing a valuable platform for molecular transport, biosensors, and biosynthesis. In the developed DNA walker systems, different driving forces propel the progressive movement of DNA walkers, such as nucleases, DNAzymes, strand substitution, or a few special functions, but all require the addition of some exogenous cofactors. ATP is the primary energy source for most cellular processes and is ubiquitous in the cytoplasm and nucleoplasm of every cell. As an endogenous driving force, it can effectively amplify the DNA walker signal without any auxiliary action, thus providing an advantage for imaging APE1 within cells. Combined with sensitive fluorescence analysis methods, this offers a unique approach for the detection and imaging of the APE1 enzyme. Summary of the Invention

[0003] In view of this, the present invention provides a three-dimensional multi-legged DNA walker biosensor for the fluorescence analysis and detection of APE1 enzyme. Specifically, the following technical solution is provided:

[0004] 1. A three-dimensional multi-legged DNA walker biosensor for detecting APE1 enzyme by fluorescence analysis, comprising the following steps:

[0005] 1) AuNPs were modified with n-butanol at a molar ratio of 500:1 to thiol DNA-walker, centrifuged and washed three times, and then dissolved in ultrapure water to obtain AuNP-W.

[0006] 2) Using the n-butanol method, AuNPs were modified with a molar ratio of thiol DNA-Sub to AuNPs of 1000:1. After centrifugation and washing three times, AuNP-S was obtained by dissolving in pure water.

[0007] 3) Using TAE / Mg 2+The buffer solution was used to anneal the AuNP-W obtained in step 1) with the blocking chain Block to form a double-stranded AuNP-W / B;

[0008] 4) Using TAE / Mg 2+ The buffer solution is used to anneal the AuNP-S obtained in step 2) with the ATP aptamer chain Apt (containing the FAM fluorophore) to form a double-stranded AuNP-S / A;

[0009] 5) Mix AuNP-W / B and AuNP-S / A obtained in steps 3) and 4) with ATP to obtain a biosensor for detecting APE1 enzyme by fluorescence analysis;

[0010] 6) Add different concentrations of APE1 enzyme to a biosensor for fluorescence analysis to detect APE1 enzyme, place it in an RT-qPCR reactor at 37°C, and measure the fluorescence curves of different concentrations of APE1 enzyme.

[0011] 7) Add the sample to be tested into the biosensor for detecting APE1 enzyme obtained in step 5), incubate at 37°C, set the same detection conditions as in step 6), measure the fluorescence curves of different samples to obtain specific detection results of APE1 enzyme.

[0012] Furthermore, in step 6), the reaction time of the double gold spheres AuNP-W / B, AuNP-S / A, and APE1 enzyme with ATP at 37°C is 1h–3h.

[0013] Furthermore, in step 6), the reaction time of the double gold spheres AuNP-W / B, AuNP-S / A, and APE1 enzyme with ATP at 37°C is 3 hours.

[0014] Furthermore, the thiol-modified DNA walker sequence is SEQ ID NO.1; the closed strand Block sequence is SEQ ID NO.2; the ATP aptamer chain Apt sequence is SEQ ID NO.4; and the DNASub sequence is SEQ ID NO.6.

[0015] Furthermore, the TAE / Mg described in step 1) 2+ The buffer solution consisted of 40 mM Tris, 20 mM acetate, 1 mM EDTA, and 10 mM Mg. 2+ pH=8.

[0016] Further, in step 1), the AuNPs were modified by the n-butanol method. In a 200 μL system, 1.8 mL of n-butanol was added, and the mixture was stirred rapidly by vortexing until homogeneous. Then, 0.4 mL of 0.5×TBE was added, and the mixture was stirred rapidly by vortexing until homogeneous. The mixture was then centrifuged at 2000 g, washed three times, and the product was dissolved in ultrapure water.

[0017] Furthermore, in step 3), AuNP-W is hybridized with the closed-chain Block, with Block in excess by 1.2 times; and AuNP-T and Apt are in equimolar amounts.

[0018] Furthermore, the amount of ATP used in step 5) is 1-10 mM.

[0019] Furthermore, the amount of ATP used in step 5) is 1 mM.

[0020] Furthermore, the ratio of AuNP-W / B and AuNP-S / A in step 5) is 1:4.

[0021] Furthermore, the APE1 enzyme in step 6) has a range of 0.1-1000 U / ml.

[0022] Furthermore, the parameters for RT-qPCR in step 6) are set as follows: Temperature: 37℃, Acquisition: Green.

[0023] The beneficial effects of this invention are as follows: Based on the good biocompatibility and fluorescence quenching effect of AuNPs, combined with the high efficiency and stability of three-dimensional DNA walkers, a biosensor for the fluorescence detection of APE1 enzyme was constructed using ATP-driven DNA walker signal amplification. Taking advantage of the rapid fluorescence response, a DNA walker system composed of two gold spheres was designed. AuNP-W acts as a multi-legged DNA walker carrier, while AuNP-T acts as the walker's walking track, enabling AuNP-W to move around AuNP-T. Before target recognition, a block is integrated into AuNP-W to seal all walker chains. Additionally, the ATP aptamer Apt (containing a FAM fluorophore) is hybridized into AuNP-S to obtain AuNP-S / A (also simply AuNP-T). The FAM fluorophore is close to the AuNPs, resulting in a FRET effect that quenches the FAM fluorescence. When the target APE1 is recognized, the AP site in the Block / walker double strand is cleaved, unlocking the multi-legged walker (AuNP-W), which reacts with the Apt on the AuNP-T. Subsequently, with the assistance of ATP, the walker is released again to proceed to the next walking reaction. In this process, the binding of ATP to the aptamer acts as the driving force for the continuous movement of the DNA walker. Due to strand displacement, the FAM group on the Apt has moved away from the AuNPs, the FRET effect disappears, and fluorescence is restored. In the entire experimental protocol, the strategy of using ATP to drive the DNA walker and combining it with the FRET effect to achieve fluorescence quenching and recovery can be used for the detection of APE1 enzyme. When the target APE1 enzyme is absent, the AP site in the Block / walker double strand is not cleaved, and the subsequent reaction cannot be triggered, so no fluorescence recovery signal is generated. This invention combines DNA walker signal amplification with fluorescence analysis, incorporates endogenous ATP as the driving force for continuous walking, and uses APE1 enzyme as the trigger switch for walker activation. The process is simple, easy to implement, and has a rapid response. It has been successfully applied to the detection of APE1 enzyme and is expected to monitor APE1 enzyme activity in cells, laying the foundation for in vitro detection in intracellular imaging and has potential application value. Attached Figure Description

[0024] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:

[0025] Figure 1 This is a schematic diagram of the three-dimensional DNA walker constructed based on AuNPs for fluorescence analysis of the APE1 enzyme according to the present invention;

[0026] Figure 2Polyacrylamide gel electrophoresis image to verify the feasibility of the experimental principle;

[0027] Figure 3 Polyacrylamide gel electrophoresis image of adjusting Sub / Apt background signal to optimize ATP concentration;

[0028] Figure 4 Polyacrylamide gel electrophoresis image optimized for Apt sequence;

[0029] Figure 5 Real-time fluorescence quantitative curves to verify the feasibility of in vitro ATP-driven 3D DNA walker experiments;

[0030] Figure 6 To optimize the real-time fluorescence quantitative curve of the Sub sequence;

[0031] Figure 7 Real-time fluorescence quantitative PCR curves for different APE1 enzyme concentrations;

[0032] Figure 8 A fitting plot showing the relationship between fluorescence values ​​and different APE1 enzyme concentrations;

[0033] Figure 9 Selectivity analysis for an ATP-driven 3D DNA walker fluorescent biosensor;

[0034] Figure 10 Actual sample analysis for the ATP-driven 3D DNA walker fluorescent biosensor. Detailed Implementation

[0035] A three-dimensional multi-legged DNA walker biosensor for detecting APE1 enzyme using fluorescence analysis, such as Figure 1As shown, all AuNP-W were initially blocked with an excessive amount of Block, which could not trigger the Block chain breakage and subsequent reactions in the absence of the target APE1 enzyme. All AuNP-S hybridized to the Apt chain, and the Apt end had a modified FAM fluorophore. After hybridization with Sub, AuNPs quenched the FAM fluorescence and did not produce a fluorescence signal. In the presence of the target enzyme APE1, APE1 recognizes the AP site, the Block chain is cleaved, and the base complementarity is insufficient to continue blocking the walker chain, thus releasing AuNP-W. The walker chain can then begin hybridization through the hybridization domain at the end of the Apt. Chain displacement completely displaces the Apt. At this point, the FAM groups on the Apt have moved away from the AuNPs, and fluorescence gradually recovers. Next, under the action of ATP, the displaced Apts in the walker / Apt are carried away, causing the walker to be released again. This cycle repeats. APE1 plays the role of initiating the DNA walker, while ATP drives the walker to continue walking. A large number of FAM fluorescent groups detach from the AuNPs, causing them to change from a quenched state to a fluorescent state, thus restoring fluorescence and changing the signal from off to on.

[0036] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0037] Example 1: Pretreatment of probe DNA

[0038] First, the powdered primer DNA ordered from Shanghai Sangon Biotech was centrifuged at 4000 rpm for 1 min before use, and then dissolved in ultrapure water as required to form a 100 μM DNA solution. When using, it was then mixed with TAE / Mg... 2+ Buffer preparation: The buffer composition is 40 mM Tris, 20 mM acetic acid, 1 mM EDTA, and 10 mM Mg. 2+ pH=8.

[0039] The walker sequence is 5'-CCC AGG TTA GCT GAT GTT TTT TTT TTT TTT TTT TTT TTTTTT TTT TTT TTT TTT TTT T-SH-3' (SEQ ID NO. 1);

[0040] The block sequence is 5'-CAT CAG CXAACC TGG G-3' (SEQ ID NO.2);

[0041] The Apt-0 sequence is 5'-AGC TAACCT GGG GGA GTATTG CGG AGG AAG GTT T-FAM-3' (SEQ ID NO.3);

[0042] The sequence of Apt-1 (Apt) is 5'-CAG CTAACC TGG GGG AGT ATT GCG GAG GAA GGT TT-FAM-3' (SEQ ID NO. 4);

[0043] The Apt-2 sequence is 5'-TCA GCT AAC CTG GGG GAG TAT TGC GGA GGA AGG TTT-FAM-3' (SEQ ID NO.5);

[0044] The Sub-0 (Sub) sequence is 5'-SH-TTT TTT TCT TCC TCC GCT TTT TTC CCC CAG GT-3' (SEQ ID NO.6);

[0045] The Sub-1 sequence is 5'-SH-TTT TTT TTT TCC TCC GCT TTT TTC CCC CAG GT-3' (SEQ ID NO. 7);

[0046] The Sub-2 sequence is 5'-TTT TTT TTA ACC TCC GCT TTT TTC CCC CAG GT-3' (SEQ ID NO. 8);

[0047] Example 2: Feasibility verification of the polyacrylamide gel electrophoresis protocol.

[0048] First, the Block and Walker strands were annealed to form a stable double-stranded structure. Then, the double strands were digested with enzymes. The results of 10% PAGE are as follows. Figure 2 As shown. Figure 2Bands 1 and 2 represent Block single strand and walker single strand, respectively; band 3 represents the Block / walker hybrid double-stranded structure, located at approximately 50 bp; band 4 verifies the background signal value of walker, that is, when Block / walker double strand and Apt coexist, after reacting at 37℃ for 1 h, the bands still show obvious Block / walker double strand and Apt single strand bands, indicating that walker can hybridize well with Block and will not produce false positive signals due to the presence of Apt. Subsequently, in band 5, APE1 enzyme was added to the Block / walker double strand. After incubation at 37°C for 1 hour, we observed that the Block / walker double strand completely disappeared, replaced entirely by walker strands at a position below 50 bp. This demonstrated that the APE1 enzyme successfully cleaved the AP site in the Block / walker double strand, causing the Block to detach from the walker and become two short strands, releasing all the walker strands for subsequent experiments. Finally, in band 6, we confirmed that the walker's ability to bind to Apt is a result of the presence of the APE1 enzyme. When the Block / walker double strand, APE1 enzyme, and Apt single strand were mixed and reacted, both the Block / walker double strand and the Apt single strand disappeared, and a walker / Apt double strand structure appeared at a position close to 60 bp. This indicates that after APE1 cleavage, the Block could no longer bind to the walker, and the released walker bound to Apt. In summary, the PAGE results show that our target APE1 can recognize the AP site in the double strand, and the released walker can proceed to the next reaction. The results of polyacrylamide gel electrophoresis validated the feasibility of our protocol.

[0049] Example 3 ATP Concentration Optimization

[0050] Experiments revealed that the binding of Sub to Apt is unstable at excessively high ATP concentrations. This is because five non-complementary bases remain between Sub and Apt, which facilitate the walker's replacement of Apt. To reduce the background signal between Sub and Apt, the ATP concentration was optimized to decrease the likelihood of Apt being directly carried away by ATP. Assuming Sub and Apt first hybridize to form a double strand, different concentrations of ATP were added, and the 10% PAGE results showed... Figure 3At a high concentration of 10 mM ATP, the Sub / Apt double helix is ​​extremely unstable; most of the Sub / Apt double helix has unraveled, with only a small portion remaining, and a large number of Apt chains are generated at the 20 bp mark. This indicates that the double helix cannot exist completely stably under 10 mM ATP conditions. Therefore, by progressively decreasing the ATP concentration, starting from 7.5 mM ATP, the situation shifts to one where more Sub / Apt double helixes exist and fewer Apt single chains. Figure 3 It can be observed that as the ATP concentration decreases, the number of Sub / Apt double strands gradually increases, while the strength of single-stranded Apt gradually decreases. After reaching 2.5 mM ATP, the Apt strands bound by ATP can be ignored. Further decreasing the ATP concentration has a smaller impact on the system.

[0051] Example 4: Apt Sequence Optimization

[0052] After adjusting the background signal for ATP binding of the Sub / Apt double strand, it is necessary to balance the ability of Walker to replace Apt. Therefore, we optimized the sequence structure of Apt. Apt-0 (SEQ ID NO.3) is a hybridization domain with 5 exposed bases at the end, which can initiate binding with Walker. Apt-1 (SEQ ID NO.4) adds one hybridization base at the 5' end of Apt-0, resulting in 6 bases that can bind with Walker. Apt-2 (SEQ ID NO.5) adds two hybridization bases at the 5' end of Apt-0, resulting in 7 bases that can bind with Walker.

[0053] The optimization results of the Apt sequence are as follows: Figure 4As shown in the PAGE, bands 1 to 3 represent the efficiency of ATP displacement after walker / Apt binding. In our system, ATP acts as the driving force for walker movement, and we want as much Apt as possible to bind to ATP in the walker / Apt double helix, so that walker can be repeatedly released for cyclical movement. With changes in the Apt sequence, the binding ability of Apt to walker gradually increases. When Apt changes from Apt-0 to Apt-2, the number of Apt strands that can be bound by ATP decreases, and a large amount of walker / Apt double helix remains, which is an undesirable result. Therefore, we choose Apt-0 or Apt-1 to participate in subsequent reactions. Bands 4, 5, and 6 verify whether walker can displace Apt from the Sub / Apt double helix in the presence of the same concentration of walker. The results in the figure show that as the number of bases bound to walker increases, Apt is more easily displaced by walker. Bands 7 to 9 represent the background signal magnitudes of Sub / Apt-0, Sub / Apt-1, and Sub / Apt-2 at the same ATP concentration, respectively. We found that the background signals of Sub / Apt-0, Sub / Apt-1, and Sub / Apt-2 were not significantly different, because this part mainly depends on the fact that the bases bound to Sub and Apt are the same. Finally, bands 10-11 verified that when walker, Sub / Apt, and ATP were mixed and incubated at 37°C, the final results showed that Apt-1 had a low background signal, could drive walker / Apt-1 with ATP, and could also allow walker to replace Apt-1 in Sub / Apt-1. Ultimately, we selected the sequence Apt-1 (denoted as Apt) for subsequent fluorescence analysis experiments.

[0054] Example 5 Subsequence Optimization

[0055] Previously, the ATP concentration was optimized to reduce background signal. However, due to the sensitivity of fluorescence measurements, a certain background fluorescence value was still observed in RT-qPCR. We need to further optimize the Sub sequence to enhance the binding affinity between Sub and Apt, thereby reducing the background fluorescence value. Furthermore, we want the optimized Sub sequence to not affect Walker's ability to replace Apt. Therefore, we measured the fluorescence curves of the experimental group (complete AuNP-W and AuNP-T) under the simultaneous action of APE1 and ATP, and the control group (AuNP-W and AuNP-T) under the action of ATP alone, under different Sub sequences. We compared three Sub sequences: Sub-2 (7nt, sequence SEQ ID NO. 8), Sub-1 (9nt, sequence SEQ ID NO. 7), and Sub-0 (10nt, sequence SEQ ID NO. 6), which refer to the number of bases capable of hybridizing with Apt on the 5' side.

[0056] The optimization results of the subsequence are as follows: Figure 5 As shown, the real-time fluorescence quantitative PCR curves of the experimental and control groups for the three Sub sequences are displayed. The results show that the fluorescence of the three Sub sequences in the experimental groups reached a uniform level at 180 min, but the fluorescence of the control group in the presence of ATP showed a difference. We can observe that as the number of hybridizations between Sub and Apt increases, the hybridization binding ability becomes stronger, and the corresponding background fluorescence decreases, with Sub-0 (10nt) showing the lowest background fluorescence. Therefore, in the following experiments, we used Sub-0 (10nt) for all subsequent reactions, denoted as the Sub chain.

[0057] Example 6: RT-qPCR real-time monitoring fluorescence analysis to verify experimental feasibility

[0058] To verify the feasibility of the ATP-driven 3D DNA walker experiment, we monitored the fluorescence changes of the entire system in real time using RT-qPCR. The experimental results are shown in... Figure 6 In this study, we used an AuNP-W to AuNP-T ratio of 1:4 and 1 mM ATP to verify the results in the presence of both APE1 and ATP, the presence of only APE1, the presence of only ATP, and a blank control without any added substances. The real-time fluorescence quantification curves showed that the blank control containing only AuNP-W and AuNP-T exhibited almost no fluorescence. Figure 6 -d), this is because FAM is completely quenched by AuNPs; when APE1 or ATP is added, fluorescence recovers slightly ( Figure 6-b, c) This is because after APE1 cleaves the AP site of the block, the walker replaces a portion of the Apt-FAM chain. After moving away from the gold nanoparticles, fluorescence is partially restored. In the system with added ATP, a small portion of the Sub / Apt chain unwinds due to the action of ATP, and a small portion of the Apt-FAM is carried into the solution phase by ATP, resulting in fluorescence recovery. However, when APE1 and ATP are added simultaneously ( Figure 6 -a) Fluorescence was fully recovered, and the recovery rate was fast at first, followed by a gradual slowdown in the curve. This indicates that our target APE1 recognized the AP site. After the block was broken, walker chains were released. During the process of walker replacing Apt-FAM, under the driving force of ATP, multi-legged walker walking was realized, and a large number of Apt-FAM chains were released into the solution, proving the feasibility of our scheme. The fluorescence amount was positively correlated with the target amount.

[0059] Example 7 Performance Analysis of the DNA Walker Biosensor

[0060] like Figure 7 As shown, APE1 at concentrations of 0 U / ml, 0.1 U / ml, 1 U / ml, 10 U / ml, 100 U / ml, 500 U / ml, and 1000 U / ml were analyzed under the same conditions. Within the target concentration range of 0-1000 U / ml, we observed that AuNP-W continuously moved over time, and the fluorescence gradually increased. Furthermore, the fluorescence value gradually increased with increasing APE1 enzyme concentration. The experimental results visually demonstrate the real-time quantitative fluorescence curves corresponding to different concentrations of APE1. Figure 8 The fluorescence values ​​corresponding to different target concentrations at 210 min were analyzed, among which... Figure 8 The inset is a linear graph showing the relationship between fluorescence signal and target concentration in the range of 0-1000 U / ml. Following the standard calculation method (LOD = 3σ / S, where σ is the standard deviation and S is the slope), the limit of detection is found to be 0.033 U / ml. Figure 8 illustration)

[0061] Example 8 Experimental Selectivity Analysis

[0062] During the experimental reaction, the APE1 enzyme was replaced with 100 U / ml UDG enzyme, T5 Exo enzyme, Lambda Exo, and ExoⅠ. After 3 hours of reaction, the fluorescence was measured using a fluorescence spectrophotometer, and the results are as follows. Figure 9 As shown, the 3D DNA walker still has a high response to the APE1 enzyme, while the other different types of enzymes have little effect on the whole system, which confirms that the present invention has good specificity.

[0063] Example 9: Analysis of Actual Samples

[0064] Two cell lysis buffers were used for the experiment: one was a 5% lysis buffer for HeLa cells (cancer cells), and the other was a 5% lysis buffer for L929 cells (normal cells). During the reaction, the APE1 enzyme was replaced with 5% cell lysis buffer, and the reaction was carried out under the same conditions. Fluorescence was measured at 210 min. Figure 10 The experimental results showed that the fluorescence value measured in HeLa cell lysate was much higher than that in normal L929 cell lysate. This is because APE1 is overexpressed in HeLa cells. The results indicate that the ATP-driven 3D DNA walker fluorescent biosensor can distinguish lysates of different cells in vitro, confirming that our designed fluorescence scheme can effectively distinguish cancer cells and play an important role in subsequent intracellular imaging experiments.

[0065] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for non-disease diagnostic purposes using a three-dimensional multi-legged DNA walker biosensor for fluorescence analysis of APE1 enzyme, characterized in that, The steps are as follows: 1) AuNPs were modified with n-butanol at a molar ratio of 500:1 to thiol DNA-walker, centrifuged and washed three times, and then dissolved in ultrapure water to obtain AuNP-W; 2) AuNPs were modified using the n-butanol method with a molar ratio of thiol DNA-Sub to AuNPs of 1000:

1. After centrifugation and washing three times, AuNP-S was obtained by dissolving in ultrapure water. 3) Using TAE / Mg 2+ The buffer solution was used to anneal the AuNP-W obtained in step 1) with the blocking chain Block to form a double-stranded AuNP-W / B; 4) Using TAE / Mg 2+ The buffer solution anneals the AuNP-S obtained in step 2) with the ATP aptamer chain Apt to form a double-stranded AuNP-S / A; 5) Mix AuNP-W / B and AuNP-S / A obtained in steps 3) and 4) with ATP to obtain a biosensor for detecting APE1 enzyme by fluorescence analysis; 6) Add different concentrations of APE1 enzyme to the biosensor for detecting APE1 by fluorescence analysis, and react in RT-qPCR at 37℃ to obtain the fluorescence curves for detecting different concentrations of APE1 enzyme. 7) Add the sample to be tested into the biosensor for detecting APE1 enzyme obtained in step 5), incubate at 37°C, set the same detection conditions as in step 6), measure the fluorescence curves of different samples to obtain the specific detection results of APE1 enzyme. The thiol-modified DNA walker sequence is SEQ ID NO.1; the blocking strand Block sequence is SEQ ID NO.2; the ATP aptamer chain Apt sequence is SEQ ID NO.4; and the DNA Sub sequence is SEQ ID NO.

6.

2. The method for non-disease diagnostic purposes using a three-dimensional multi-legged DNA walker biosensor for fluorescence analysis of APE1 enzyme, as described in claim 1, is characterized in that... The reaction time of the double gold spheres AuNP-W / B, AuNP-S / A, APE1 enzyme and ATP at 37°C in step 6) is 1 h – 3 h.

3. The method for non-disease diagnostic purposes of APE1 using a three-dimensional multi-legged DNA walker biosensor for fluorescence analysis according to claim 2, characterized in that, In step 6), the reaction time of the double gold spheres AuNP-W / B, AuNP-S / A, APE1 enzyme, and ATP at 37°C is 3 h.

4. The method for non-disease diagnostic purposes using a three-dimensional multi-legged DNA walker biosensor for fluorescence analysis of APE1 enzyme, as described in claim 1, is characterized in that... The TAE / Mg described in steps 3) and 4) 2+ The buffer solution consisted of 40 mM Tris, 20 mM acetate, 1 mM EDTA, and 10 mM Mg. 2+ pH=8.

5. The method for non-disease diagnostic purposes of APE1 detection using a three-dimensional multi-legged DNA walker biosensor with fluorescence analysis according to claim 1, characterized in that, For the n-butanol modification of AuNPs described in steps 1) and 2), DNA-walker and AuNPs or DNA-Sub and AuNPs were added in a 200 μL system according to the ratio, followed by 1.8 mL of n-butanol. The mixture was then rapidly vortexed and stirred until homogeneous. 0.4 mL of 0.5 × TBE was added and rapidly vortexed and stirred until homogeneous. The mixture was then centrifuged at 2000 g and washed three times. The product was dissolved in ultrapure water.

6. The method for non-disease diagnostic purposes of APE1 using a three-dimensional multi-legged DNA walker biosensor for fluorescence analysis according to claim 1, characterized in that, Step 3) describes hybridization of AuNP-W with the closed-chain Block, with the Block chain being 1.2 times overloaded.

7. The method for non-disease diagnostic purposes of APE1 using a three-dimensional multi-legged DNA walker biosensor for fluorescence analysis according to claim 1, characterized in that, The equimolar amounts of AuNP-S and Apt mentioned in step 4).

8. The method for non-disease diagnostic purposes of APE1 using a three-dimensional multi-legged DNA walker biosensor for fluorescence analysis according to claim 1, characterized in that, The amount of ATP used in step 5) is 1-10 mM.

9. The method for non-disease diagnostic purposes of APE1 using a three-dimensional multi-legged DNA walker biosensor for fluorescence analysis according to claim 1, characterized in that, The amount of ATP used in step 5) is 1 mM.

10. The method for non-disease diagnostic purposes of a three-dimensional multilegged DNA walker biosensor for fluorescence analysis of APE1 according to claim 1, characterized in that, The ratio of AuNP-W / B and AuNP-S / A in step 5) is 1:

4.

11. The method for non-disease diagnostic purposes of APE1 using a three-dimensional multi-legged DNA walker biosensor for fluorescence analysis according to claim 1, characterized in that, The APE1 enzyme in step 6) has a range of 0.1-1000 U / ml.

12. The method for non-disease diagnostic purposes of APE1 using a three-dimensional multi-legged DNA walker biosensor for fluorescence analysis according to claim 1, characterized in that, Step 6) The RT-qPCR parameters are set as follows: Temperature: 37℃, Acquisition: Green.

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