Pyridinedicarboxylic acid electrochemical sensor based on intramolecular DNA nano-machine

Abstract

The invention discloses a construction method, a construction structure and application of a picolinic acid electrochemical sensor based on an intramolecular DNA nano machine. The construction method comprises the following steps: preparing an X-type DNA nano structure, constructing a sandwich composite probe SSC, constructing an electrochemical sensing interface for detecting picolinic acid, and quantitatively detecting picolinic acid in a bacterial sample. According to the electrochemical sensor for detecting the dipicolinic acid, the reaction probe is immobilized by using an X-type nano bracket XSP, an intramolecular DNA nano machine is constructed, efficient signal output is performed through catalytic oxidation reduction cycle amplification, sensitive detection of the dipicolinic acid released by bacterial spores is realized, and the detection limit is as low as 7.4 pM. The research has important application value in the aspects of prevention, diagnosis and treatment of diseases such as anthrax.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biosensors, specifically to the construction method, structure, and application of a pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines. Background Technology

[0002] Pyridine dicarboxylic acid (DPA) is a unique component of *Bacillus anthracis* spores, comprising 5%–15% of their dry weight, and is exclusive to this bacterium. Therefore, DPA is a key biomarker for identifying *Bacillus anthracis*. *Bacillus anthracis* is an aerobic facultative anaerobic bacterium with Gram-positive, rod-shaped structures, capable of forming spores and causing anthrax. The spores of this bacterium can survive in harsh environments, remaining dormant until suitable germination conditions are encountered. Inhalation of more than 10... 4 Without timely medical intervention, an infection by a single Bacillus anthracis spore can lead to death within 24–48 hours. Therefore, B. anthracis spores are considered one of the most dangerous biological warfare agents due to their potential for large-scale destruction. Thus, achieving efficient detection of DPA is crucial for detecting B. anthracis spores and preventing anthrax epidemics.

[0003] Currently, various detection techniques are available for DPA detection, including mass spectrometry, high-performance liquid chromatography, and surface-enhanced Raman spectroscopy. However, these techniques not only require expensive equipment and skilled operators, but also suffer from complex and time-consuming processes. Furthermore, some studies have reported the use of lanthanides (Tb)... 3+ Eu 3+ 、Sm 3 + ), heavy metals (Cd 2+ While luminescent systems for DPA detection can be constructed using toxic organic dyes (BODIPY derivatives), these substances are all harmful to the environment. To address these issues, this invention assembles and positions reaction probes using DNA nanostructures, constructing intramolecular DNA nanomachines for highly efficient amplified detection of DPA. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method, structure and application of an electrochemical sensor for pyridine dicarboxylic acid based on intramolecular DNA nanomachines. The invention utilizes an X-type nanoscaffold (XSP) to immobilize a reaction probe, constructs an intramolecular DNA nanomachine, and achieves efficient signal output through catalytic redox cycle amplification, thereby realizing sensitive detection of DPA released from bacterial spores.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is: a pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines, the innovation of which is that the pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines includes the following steps:

[0006] Step S1: First, the gold electrode is polished and electrochemically treated. The capture probe CP is modified onto the surface of the gold electrode AuE and blocked with mercaptohexanol MCH to obtain MCH / CP / AuE.

[0007] The sequence of the CP is 5'-TCTACACATAATTCAGCTAGGATTATG TTT TTT-SH-(CH2)6-3';

[0008] Step S2: PAA / CeO2 material is prepared by precipitation method, and hairpin probe HP2-2 is modified on the surface of PAA / CeO2 to obtain HP2-2 / PAA / CeO2;

[0009] The HP2-2 sequence is 5'-GAC CATACG TACAGCACC GCTATT CAT CGG TAA GCTTCT ACA CAT AAT CAA CAT CAG CTT ACA GTA GAT GTT GATTAT GTG TAGA-(CH2)7-NH2-3';

[0010] Step S3: Anneal probes HP1-1, HP1-2, and HP2-1 separately, then mix HP1-1, HP1-2, HP2-1, HP2-2 / PAA / CeO2 and self-assemble to obtain XSP;

[0011] The sequence of HP1-1 is 5'-CCGATGAATAGC GGT CAGATC CGTACC TAC TCA ACATCG TCT GTA AGC TGA TGT TGA TTA TGT GTA GAA GCT TACAGAC-3'; the sequence of HP1-2 is 5'-GTC GTT CGCAATACG GCT GTACGT ATG GTC TCA ACA TCG TCT GTA AGC TGA TGT TGATTA TGT GTA GAA GCT TACAGAC-3'; and the sequence of HP2-1 is 5'-GTAGGTACG GAT CTG CGTATTGCG AAC GAC TAA GCT TCT ACA CAT AAT CAA CAT CAG CTT ACA GTA GAT GTT GATTATGTG TAGA-3'.

[0012] Step S4: Add AP, TP, and Zr 4+ The sandwich complex SSC was formed by mixing and incubating. Then, XSP, SSC and different concentrations of pyridine dicarboxylic acid were mixed and transferred to the MCH / CP / AuE surface for reaction.

[0013] The sequence of the AP is 5'-CTTAAC ATC CATAGG-PO3. 2- -3'; the sequence of the TP is 5'-PO3. 2- -AGC TTACAGACGATG TT-3';

[0014] Step S5: Add p-aminophenyl phosphate (p-APP) and nicotinamide adenine dinucleotide (NADH) to perform a redox cycle and detect the differential pulse voltammetry (DPV) signal.

[0015] Preferably, the operation of preparing the sensing interface in step S1 is as follows: first, polish the gold electrode with aluminum oxide powder, place the electrode in a dilute sulfuric acid solution for cyclic voltammetry scanning, dry it with nitrogen gas, react the capture probe CP with tris(2-carboxyethyl)phosphine for 1 hour, drop it onto the electrode surface and react in the dark for 12 hours, rinse it, and then drop mercaptohexanol MCH onto the electrode and seal it for 30 minutes to obtain MCH / CP / AuE.

[0016] Preferably, the experimental procedure in step S2 is as follows: A trivalent cerium inorganic salt (e.g., cerium nitrate) is dissolved and mixed with polyacrylic acid (PAA) in ultrapure water. Ammonia is then added and the mixture is stirred for 24 hours to obtain a light yellow precipitate. This precipitate is centrifuged and washed with water until the pH of the supernatant reaches 7. Simultaneously, HP2-2 is heated at 95°C for 10 minutes, followed by cooling to room temperature at a rate of 1°C / min. The resulting PAA / CeO2 is then mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and reacted for half an hour to activate the carboxylic acid groups. HP2-2 and N-hydroxysuccinimide (NHS) are then added and reacted for 6 hours to obtain HP2-2 / PAA / CeO2.

[0017] Preferably, the experimental operation of step S3 is as follows: HP1-1, HP1-2, and HP2-1 are heated at 95°C for 10 minutes, then cooled to room temperature at a rate of 1°C / minute, and then HP2-2 / PAA / CeO2 are added in equal proportion and reacted at 37°C for 2 hours to obtain XSP through self-assembly.

[0018] Preferably, the experimental operation in step S4 is to mix AP, TP, and Zr. 4+ Equimolar mixtures were placed at 4°C and reacted for 2 hours to form a sandwich complex (SSC). Subsequently, equimolar amounts of XSP and SSC were mixed with different concentrations of pyridine dicarboxylic acid and transferred to an MCH / CP / AuE surface and reacted at room temperature for 40 minutes.

[0019] Preferably, the experimental operation in step S5 is as follows: the sensing interface after the reaction in S4 is placed in a mixture of p-APP and NADH (molar ratio of 1:5), reacted at room temperature for 10 minutes, and the DPV response signal from -0.1V to 0.3V is recorded.

[0020] The construction structure of the pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines is described, and the construction method of the pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines is used. The construction structure of the pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines is an electrochemical sensor obtained by using the construction method of the pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines.

[0021] The application of the pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines is described in the context of detecting DPA content in bacterial spore samples.

[0022] The advantages of this invention are as follows: Compared with the prior art, this invention utilizes X-type nanoscaffolds (XSP) to immobilize reactive probes, constructs intramolecular DNA nanomachines, and achieves efficient signal output through catalytic redox cycle amplification, realizing sensitive detection of DPA in bacterial spore samples. It provides a method, structure, and application for constructing a highly efficient electrochemical sensor for detecting biomarkers, which has important application value in the prevention, diagnosis, and treatment of diseases such as anthrax. Attached Figure Description

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0024] Figure 1 This is a schematic diagram illustrating the principle of the pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines of this invention.

[0025] Figure 2 The following are CeO2 characterization diagrams of the pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines of this invention: A, Transmission electron microscopy image of PAA / CeO2; B, X-ray photoelectron spectroscopy of PAA / CeO2; C, X-ray diffraction patterns of CeO2 and PAA / CeO2; D, UV absorption spectra of CeO2, PAA / CeO2, and DNA / CeO2.

[0026] Figure 3 The gel electrophoretic characterization of the pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines of this invention is as follows: A. XSP self-assembly characterization: lane 1, HP1-1; lane 2, HP1-1 / HP2-1; lane 3, HP1-1 / HP2-1 / HP1-2; lane 4, HP1-1 / HP2-1 / HP1-2 / HP2-2; B. Intramolecular DNA nanomachine characterization: lane 1, AP; lane 2, TP; lane 3, AP+TP; lane 4, AP+TP+Zr. 4+ Lane 5, SSC+DPA; Lane 6, SSC+XSP; Lane 7, SSC+XSP+DPA.

[0027] Figure 4 The detection performance characterization of the pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines of this invention includes: A) Current response induced by different concentrations of DPA in the intramolecular DNA nanomachines; B) Linear relationship between current and the logarithm of DPA concentration in the intramolecular DNA nanomachines; C) Quantitative detection response of intermolecular DNA nanomachines; D) Selectivity of intramolecular DNA nanomachines.

[0028] Figure 5This invention relates to the actual sample detection of the pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines: A. DPV response caused by different samples: a. LB culture medium; b. B. subtilis spore suspension after incubation at 70°C; c. B. subtilis spore suspension without incubation at 70°C; B. DPA concentration released by B. subtilis spores without germination time. Detailed Implementation

[0029] The pyridine dicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachines of the present invention includes the following steps:

[0030] Step S1: First, the gold electrode is polished and electrochemically treated. The capture probe CP is modified onto the surface of the gold electrode AuE and blocked with mercaptohexanol MCH to obtain MCH / CP / AuE; wherein, the sequence of the CP is 5'-TCT ACA CATAAT TCA GCT AGG ATT ATG TTT TTT-SH-(CH2)6-3'.

[0031] Step S2: PAA / CeO2 material is prepared by precipitation method, and hairpin probe HP2-2 is modified on the surface of PAA / CeO2 to obtain HP2-2 / PAA / CeO2; wherein, the sequence of HP2-2 is 5'-GAC CATACG TACAGC ACC GCTATT CATCGG TAAGCT TCTACACATAAT CAACAT CAG CTTACAGTAGAT GTT GATTAT GTG TAGA-(CH2)7-NH2-3';

[0032] Step S3: Anneal probes HP1-1, HP1-2, and HP2-1 separately, then mix HP1-1, HP1-2, HP2-1, HP2-2 / PAA / CeO2 and self-assemble to obtain XSP; wherein, the sequence of HP1-1 is 5'-CCGATGAATAGC GGTCAGATC CGTACC TAC TCAACA TCG TCT GTA AGC TGATGT TGATTATGT GTAGAAGCT TACAGA C-3'; the sequence of HP1-2 is 5'-GTC GTT CGC AATACG GCT GTACGTATG GTC TCAACATCG TCTGTAAGC TGA TGT TGATTATGT GTA GAA GCT TAC AGA C-3'; the sequence of HP2-1 is 5'-GTAGGTACG GAT CTG CGTATT GCGAAC GAC TAA GCT TCT ACA CAT AAT CAA CAT CAG CTT ACAGTA GAT GTT GAT TAT GTG TAG A-3';

[0033] Step S4: Add AP, TP, and Zr 4+ A sandwich complex (SSC) was formed by mixing and incubation. Subsequently, XSP, SSC, and different concentrations of pyridine dicarboxylic acid were mixed and transferred to the MCH / CP / AuE surface for reaction. The sequence of the AP is 5'-CTT AAC ATCCAT AGG-PO3. 2- -3'; the sequence of the TP is 5'-PO3. 2- -AGC TTACAGACGATG TT-3';

[0034] Step S5: Add p-aminophenyl phosphate (p-APP) and nicotinamide adenine dinucleotide (NADH) to perform a redox cycle and detect the differential pulse voltammetry (DPV) signal.

[0035] This invention utilizes X-shaped nanoscaffolds (XSP) to immobilize reactive probes, constructing intramolecular DNA nanomachines. Through catalytic redox cycle amplification, efficient signal output is achieved, enabling sensitive detection of DPA in bacterial spore samples. This invention provides a method, structure, and application for constructing a highly efficient electrochemical sensor for detecting biomarkers, which has significant application value in the prevention, diagnosis, and treatment of diseases such as anthrax.

[0036] The operation of preparing the sensing interface in step S1 above is as follows: First, polish the gold electrode with alumina powder, place the electrode in a dilute sulfuric acid solution for cyclic voltammetry scanning, dry it with nitrogen gas, react the capture probe CP with tris(2-carboxyethyl)phosphine for 1 hour, drop it onto the electrode surface and react in the dark for 12 hours, rinse it, and then drop mercaptohexanol MCH onto the electrode and seal it for 30 minutes to obtain MCH / CP / AuE.

[0037] The experimental procedure in step S2 above is as follows: A trivalent cerium inorganic salt (e.g., cerium nitrate) is dissolved and mixed with polyacrylic acid (PAA) in ultrapure water. Ammonia is then added and the mixture is stirred for 24 hours to obtain a light yellow precipitate. This precipitate is centrifuged and washed with water until the pH of the supernatant reaches 7. Simultaneously, HP2-2 is heated at 95°C for 10 minutes, followed by cooling to room temperature at a rate of 1°C / min. The resulting PAA / CeO2 is then mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and reacted for half an hour to activate the carboxylic acid groups. HP2-2 and N-hydroxysuccinimide (NHS) are then added and reacted for 6 hours to obtain HP2-2 / PAA / CeO2.

[0038] The experimental procedure in step S3 above is as follows: HP1-1, HP1-2, and HP2-1 are heated at 95°C for 10 minutes, then cooled to room temperature at a rate of 1°C / minute, and then HP2-2 / PAA / CeO2 are added in equal proportion and reacted at 37°C for 2 hours to obtain XSP through self-assembly.

[0039] The experimental operation in step S4 above is to mix AP, TP, and Zr. 4+ Equimolar mixtures were placed at 4°C and reacted for 2 hours to form a sandwich complex (SSC). Subsequently, equimolar amounts of XSP and SSC were mixed with different concentrations of pyridine dicarboxylic acid and transferred to an MCH / CP / AuE surface and reacted at room temperature for 40 minutes.

[0040] The experimental procedure in step S5 above is as follows: the sensing interface after the reaction in S4 is placed in a mixture of p-APP and NADH (molar ratio of 1:5), reacted at room temperature for 10 minutes, and the DPV response signal from -0.1V to 0.3V is recorded.

[0041] The electrochemical sensor for DPA detection described above was constructed using a method based on intramolecular DNA nanomachines to create a pyridine dicarboxylic acid electrochemical sensor. The application of this intramolecular DNA nanomachine-based pyridine dicarboxylic acid electrochemical sensor is in the detection of DPA content in bacterial spore samples.

[0042] The specific principle is as follows:

[0043] The method for constructing the sensor in this invention is as follows: Figure 1 As shown, polyacrylic acid-coated cerium dioxide nanoparticles (PAA / CeO2) were synthesized via a precipitation method using ammonia as a precipitant. These nanoparticles possess carboxyl functional groups on their surface, enabling them to couple with DNA probes. Hairpin probe pairs (HP1, HP2) assembled on an X-shaped nanostructure (XSP) were used to initiate intramolecular reactions. It should be noted that HP1-1 and HP1-2 have identical hairpin region sequences and participate in DNA nanomachine reactions through the same mechanism; therefore, they are collectively referred to as HP1 in this paper. Similarly, HP2-1 and HP2-2 are collectively referred to as HP2 in this discussion. Based on two phosphorylated oligonucleotide probes (AP, TP) and Zr... 4+ PO3 between 2 --Zr 4+ -PO3 2 - Coordination covalent interactions were used to prepare a sandwich-structured complex (SSC). Upon addition of the target compound DPA, DPA reacted with Zr... 4+ Forming a stronger chelating effect will destroy PO3. 2 --Zr 4+ -PO3 2 The --bond releases TP from the SSC structure. The released TP hybridizes with HP1 via a toehold-mediated chain substitution reaction, exposing its complementary sequence to HP2. The exposed HP1 sequence then binds to adjacent HP2 molecules via intramolecular hybridization, prompting TP chain release. Furthermore, the released TP can trigger a conformational rearrangement of another HP1, which subsequently binds to HP2 via intramolecular hybridization, ultimately generating numerous HP1 / HP2 double strands within the XSP structure. After the XSP conformational rearrangement, the single-stranded regions of the HP1 / HP2 double strands are exposed, which further hybridize with the capture probe (CP) on the sensor surface, bringing CeO2 nanoparticles closer to the electrode surface. Upon addition of the enzyme substrate p-aminophenyl phosphate (p-APP) and the co-reactant reduced nicotinamide adenine dinucleotide (NADH), CeO2 catalyzes the conversion of p-APP to p-aminophenol (p-AP); p-AP then undergoes electrochemical oxidation at the sensing interface to generate p-quinone imine (p-QI). With the synergistic effect of NADH, the p-QI generated by electrochemical oxidation is reduced back to p-AP, thus forming a redox cycle between p-QI and p-AP. Based on the dual effect of the intramolecular DNA nanomachines and the redox cycle, a significantly amplified current signal can be obtained, enabling highly sensitive detection of DPA.

[0044] Transmission electron microscopy (TEM) characterization results Figure 2A) shows that the polyacrylic acid / cerium dioxide (PAA / CeO2) nanoparticles exhibit good dispersibility, with a particle size range of 2-5 nm and an average diameter of 3 nm. The elemental composition of PAA / CeO2 was further investigated using X-ray photoelectron spectroscopy (XPS). Figure 2 As shown in Figure B, the characteristic peaks of C 1s, O 1s and Ce 3d appear at 285 eV, 529 eV and 916 eV, respectively. Figure 2 C is a typical X-ray diffraction (XRD) pattern of the prepared CeO2 nanoparticles, showing four distinct characteristic peaks. These peaks correspond to the diffraction of the (111), (200), (220), and (311) crystal planes of cerium dioxide at 2θ angles of 28.5°, 33.1°, 47.5°, and 56.3°, respectively, and are in perfect agreement with the characteristic peaks of the cerium dioxide standard card (PDF No. 00-004-0539). After being coated with PAA, the XRD characteristic peaks of the CeO2 nanoparticles showed no significant change. Figure 2 D represents the UV-Vis absorption spectra of different CeO2 nanoparticles. Samples CeO2, PAA / CeO2, and DNA / PAA / CeO2 all exhibit absorption peaks around 300 nm, and PAA modification did not significantly affect their UV-Vis absorption spectra. Furthermore, the absorption peak at 260 nm for DNA / PAA / CeO2 indicates that the DNA probe has successfully bound to the surface of the CeO2 nanoparticles.

[0045] The assembly process of XSP and its relationship to DNA nanomachines were investigated using polyacrylamide gel electrophoresis (PAGE). Figure 3 As shown in Figure A, HP1-1 exhibits a clear band (lane 1). Hybridization between HP1-1 and HP2-1 reduces electrophoretic mobility (lane 2). Further addition of HP1-2 and continuous incubation further reduces the product's mobility (lane 3), which is attributed to the increase in product molecular weight. After the addition of HP2-2, lane 4 shows a single band with the lowest mobility, indicating the formation of XSP nanostructures during annealing. Furthermore, Figure 3 B demonstrates the response of DPA-activated intramolecular DNA nanomachines. Lanes 1 and 2 show single bands of AP and TP, respectively. Mixing AP and TP showed no significant change in their bands (lane 3). When Zr is present... 4+When AP and TP were mixed and incubated, a new band with reduced migration appeared (lane 4), indicating that the SSC nanostructure had been formed. When DPA was mixed with SSC, the AP and TP bands reappeared (lane 5). When DPA was absent, the DNA bands of SSC and XSP showed no significant change after mixing (lane 6), indicating that the intramolecular DNA nanomachines could not be activated. Upon further incubation with DPA, the SSC band disappeared, while AP and TP bands appeared (lane 7), and the XSP band migration decreased slightly, indicating that the XSP structure underwent conformational rearrangement through toehold chain substitution.

[0046] To examine the quantitative detection capability of this intramolecular DNA nanomachine, we recorded and analyzed the current response induced by different concentrations of DPA. Figure 4 As can be seen from Figure A, the differential pulse voltammetry (DPV) current gradually increases with the increase of the target analyte DPA concentration. Within the concentration range of 20 pM to 100 μM, the current exhibits an excellent linear relationship with the logarithm of the DPA concentration. Figure 4 B), whose linear equation is i = 2.088 + 0.1763lgc(R). 2 =0.9978), the calculated limit of detection (LOD) of the sensor is 7.4 pM. Furthermore, intermolecular DNA nanomachines were constructed using free hairpin probes HP1 and HP2 in solution, and compared with intramolecular DNA machines. For example... Figure 4 The results show that the sensing response value of the intermolecular DNA machine is lower than that of the intramolecular sensor, and its linear range is three orders of magnitude narrower. This confirms that the intramolecular DNA nanomachines have an exponential signal amplification effect and ultra-high sensitivity for DPA detection. Meanwhile, nicotinamide adenine dinucleotide (NA), pyrophosphate (PA), dihydroxytriazine carboxylic acid (DHTA), L-arginine (L-Arg), L-carnitine (L-Car), and glutamate (Glu) were selected as interfering agents to investigate the selectivity of the sensor. Figure 4 The results from D show that, compared to the current response of the target analyte DPA (1 μM), the high concentration of interfering substances (both at 50 μM) produced a very small current response. Furthermore, the current signal generated by the mixed solution of DPA and the aforementioned interfering substances (Mix) is similar to that of a single DPA solution, indicating that the sensor has good specificity.

[0047] This study explores the application value of this sensor in practical biological sample detection by detecting the DPA content released by Bacillus subtilis spores. Figure 5As shown in Figure A, the DPV current increased significantly after the addition of heat-treated spore suspension (curve b vs. curve a). In contrast, if the spore suspension was not heat-treated, only a negligible current response was observed (curve c), indicating that spores only generate DPA after heat treatment. Furthermore, as the germination time increased from 0 to 2.5 hours, the amount of DPA secreted by the spores showed a gradual increasing trend. Figure 5 (B) These results further indicate that the bacterial germination process can be roughly divided into three stages based on the DPA release pattern, providing a reference for the study of microbial maturation processes. In the first 10 minutes, the bacteria enter a rapid germination stage, at which point the DPA concentration reaches 23.7 μM, accounting for approximately one-third of the total release. During this stage, optimal growth conditions, including sufficient nutrients, the germination agent (L-alanine), and suitable reaction temperature, facilitate the rapid reproduction of *B. subtilis*, thus the large amount of DPA released by this bacterium further promotes spore germination. In the following 10 to 60 minutes, the germination process gradually slows down, and the DPA concentration increases to 53.1 μM. In the final stage (1.5 to 2.5 hours), due to resource scarcity, bacterial growth is limited, the DPA release rate decreases significantly, and the process eventually enters a stationary phase. Real-time monitoring of this process holds promise for controlling bacterial spore germination, demonstrating the method's good applicability in practical sample detection.

[0048] It should be noted that in the description of this specification, the terms are used for descriptive purposes and to distinguish similar objects only, and there is no order between them, nor should they be construed as indicating or implying relative importance. Furthermore, in the description of this specification, unless otherwise stated, "a plurality of" means two or more.

[0049] Any numerical values ​​cited in this document include all lower and upper values ​​that increase by one unit from the lower limit to the upper limit, with at least a two-unit interval between any lower value and any higher value. These are merely examples intended to be explicit, and it can be assumed that all possible combinations of the numerical values ​​listed between the minimum and maximum values ​​are explicitly described in the specification in a similar manner.

[0050] It should be understood that the above description is for illustrative purposes and not for limitation. Many embodiments and applications beyond the provided examples will be apparent to those skilled in the art upon reading the above description. Therefore, the scope of this teaching should not be determined by reference to the above description, but rather by reference to the appended claims and the full scope of their equivalents. For purposes of completeness, all articles and references, including patent applications and publications, are incorporated herein by reference. The omission of any aspect of the subject matter disclosed herein in the preceding claims is not intended as a waiver of that subject matter, nor should it be construed as an indication that the inventors have not considered that subject matter as part of the disclosed inventive subject matter.

Claims

1. An intramolecular DNA nanomachine-based pyridinedicarboxylic acid electrochemical sensor, characterized in that: The pyridinedicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachine comprises the following steps: Step S1: first polishing and electrochemical processing of the gold electrode, modifying the capture probe CP to the surface of the gold electrode AuE, and blocking with mercaptohexanol MCH to obtain MCH / CP / AuE; The sequence of the CP is 5'-TCTACACATAATTCAGCTAGGATTATG TTT TTT-SH-(CH2)6-3'; Step S2: preparing PAA / CeO2 material by precipitation method, and modifying the hairpin probe HP2-2 on the surface of PAA / CeO2 to obtain HP2-2 / PAA / CeO2; The sequence of the HP2-2 is 5'-GAC CATACG TACAGCACC GCTATT CAT CGG TAA GCT TCTACA CAT AAT CAA CAT CAG CTT ACA GTA GAT GTT GATTAT GTG TAGA-(CH2)7-NH2-3'; Step S3: annealing the probes HP1-1, HP1-2 and HP2-1 separately, then mixing HP1-1, HP1-2, HP2-1 and HP2-2 / PAA / CeO2 to obtain XSP by self-assembly; The sequence of the HP1-1 is 5'-CCGATGAATAGC GGT CAGATC CGTACC TAC TCA ACA TCGTCT GTA AGC TGA TGT TGA TTA TGT GTA GAA GCT TACAGAC-3'; the sequence of the HP1-2 is 5'-GTC GTT CGCAATACG GCT GTACGT ATG GTC TCA ACA TCG TCT GTA AGC TGA TGT TGA TTATGT GTA GAA GCT TACAGAC-3'; and the sequence of the HP2-1 is 5'-GTAGGTACG GAT CTG CGTATT GCGAAC GAC TAA GCT TCT ACA CAT AAT CAA CAT CAG CTT ACA GTA GAT GTT GATTAT GTGTAGA-3'; Step S4: The AP, TP, Zr 4+ The mixed incubation forms a sandwich complex SSC, followed by mixing XSP, SSC with different concentrations of pyridinedicarboxylic acid, and transferring to the MCH / CP / AuE surface for reaction; wherein the sequence of the AP is 5'-CTTAAC ATC CATAGG-P03 2- -3'; and the sequence of the TP is 5'-P03 2- -AGCTTACAGACGATG TT-3' Step S5: adding p-APP and NADH to carry out redox cycle, and detecting the differential pulse voltammetry (DPV) signal.

2. The intramolecular DNA nanomachine-based pyridinedicarboxylic acid electrochemical sensor of claim 1, wherein: The operation of preparing the sensing interface in step S1 is as follows: first, polishing the gold electrode with aluminum oxide powder, then placing the electrode in a dilute sulfuric acid solution for cyclic voltammetry scanning, blowing dry with nitrogen, reacting the capture probe CP with tris(2-carboxyethyl)phosphine for 1 hour, adding dropwise to the electrode surface and reacting in the dark for 12 hours, then washing and adding dropwise the mercaptohexanol MCH to the electrode for 30 minutes to obtain MCH / CP / AuE.

3. The intramolecular DNA nanomachine-based pyridinedicarboxylic acid electrochemical sensor of claim 1, wherein: The experimental operation of step S2 is as follows: dissolving and mixing the trivalent cerium inorganic salt (such as cerium nitrate) and polyacrylic acid PAA in ultrapure water, then adding ammonia water and stirring for 24 hours to obtain a light yellow precipitate, which is centrifuged and washed with water until the supernatant pH is 7. At the same time, HP2-2 is heated at 95℃ for 10 minutes, and then cooled to room temperature at a rate of 1℃ / min. Then, the obtained PAA / CeO2 is mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) for half an hour to activate the carboxyl group, and then HP2-2 and N-hydroxysuccinimide (NHS) are added and mixed for 6 hours to obtain HP2-2 / PAA / CeO2.

4. The intramolecular DNA nanomachine-based pyridinedicarboxylic acid electrochemical sensor of claim 1, wherein: The experimental operation of step S3 is as follows: heating HP1-1, HP1-2 and HP2-1 at 95℃ for 10 minutes, then cooling to room temperature at a rate of 1℃ / min, then adding HP2-2 / PAA / CeO2 in equal proportions and mixing at 37℃ for 2 hours to obtain XSP by self-assembly.

5. The intramolecular DNA nanomachine-based pyridinedicarboxylic acid electrochemical sensor of claim 1, wherein: The experimental operation of the step S4 is to mix AP, TP, Zr 4+ The sandwich complex SSC was formed by mixing equimolar at 4℃ for 2 hours. Then, equimolar of XSP, SSC and different concentrations of picolinic acid were mixed and transferred to the MCH / CP / AuE surface for reaction at room temperature for 40 minutes.

6. The intramolecular DNA nanomachine-based pyridinedicarboxylic acid electrochemical sensor of claim 1, wherein: The experimental operation of step S5 is as follows: placing the sensing interface after S4 reaction in a mixture of p-APP and NADH (molar ratio 1:5), and reacting at room temperature for 10 minutes to record the DPV response signal at -0.1V to 0.3V.

7. A construction structure of an intramolecular DNA nanomachine-based pyridinedicarboxylic acid electrochemical sensor, using the intramolecular DNA nanomachine-based pyridinedicarboxylic acid electrochemical sensor according to any one of claims 1 to 6, characterized by: The construction structure of the pyridinedicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachine is an electrochemical sensor obtained by the construction method of the pyridinedicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachine.

8. Use of the intramolecular DNA nanomachine-based pyridinedicarboxylic acid electrochemical sensor, according to the construction of the intramolecular DNA nanomachine-based pyridinedicarboxylic acid electrochemical sensor according to claim 7, characterized in that: The application of the pyridinedicarboxylic acid electrochemical sensor based on intramolecular DNA nanomachine is the application of the electrochemical sensor in detecting the content of pyridinedicarboxylic acid in a bacterial sample.

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