ECL sensor based on HCR nucleic acid amplification for MRSA detection

Abstract

The invention discloses an HCR nucleic acid amplification-based ECL sensor for MRSA detection. The HCR nucleic acid amplification-based ECL sensor can be used for sensitive detection of MRSA. Through the strong interaction force between the aptamer and the MRSA, the release of the trigger chain is completed and the sensor is constructed subsequently, so that the influence of the large-size MRSA on the electrode is eliminated. The structure generated in the HCR process is firmly fixed on the surface of the electrode through the tetrahedron frame, and the upper layer space of the tetrahedron is fully utilized. Meanwhile, the two DNA hairpins on the DNA tetrahedron framework simultaneously generate the HCR process, so that the loss of the ECL signal is effectively avoided, and a guarantee is provided for orderly enhancement of the ECL signal. The designed ECL sensor effectively realizes sensitive detection of MRSA, and a new thought can be provided for structural design of a nucleic acid framework.

Description

Technical Field

[0001] The present invention relates to the technical field of biological analysis and detection, and in particular to an electrochemiluminescence (ECL) biosensor based on HCR nucleic acid amplification and the quantitative detection of MRSA. Background Art

[0002] Morbidity and mortality caused by bacterial pathogens occur primarily in developing countries. Of the 57 million deaths annually, 15 million are caused by infectious diseases caused by bacterial pathogens. Escherichia coli, Pseudomonas aeruginosa, Bacillus, and Staphylococcus aureus cause urinary tract infections, gastroenteritis, skin diseases, and neonatal meningitis, respectively. The continued overuse of antibiotics in modern society has led to the development of multidrug resistance in many bacteria. These infections lead to increased mortality, severe illness, complications, and hospitalizations. In Europe, approximately 25,000 people die annually from drug-resistant bacterial infections. This resistance also leads to a dramatic increase in healthcare costs, with €9 billion spent annually on treating complications related to antibiotic resistance. Among various bacterial pathogens, Staphylococcus aureus can cause infections such as skin and soft tissue infections, keratitis, and pleural and pulmonary infections. Among these, methicillin-resistant Staphylococcus aureus (MRSA), a multidrug-resistant bacterium, has garnered significant attention, necessitating the development of sensors for MRSA detection.

[0003] Electrochemiluminescence (ECL) has the advantages of high sensitivity, low background signal and low cost. Due to its simple and versatile optical setting mode and controllability in time and space, ECL detection has attracted widespread attention. Among them, ECL biosensors have been widely used in DNA analysis, immunoassay and clinical diagnosis. 2+ Because of its advantages such as high stability, high luminescence signal, wide pH application range and electrochemical reversibility, it is widely used in the detection of ECL signals. Metal organic frameworks (MOFs) are a typical hybrid porous material composed of metal ions or clusters and organic ligands through coordination bonds. The material has the characteristics of adjustable pore size, high porosity, low density, easy functionalization, chemical stability and high thermal stability. In addition, MOFs have a wide range of applications and are a very promising material. Its applications include gas adsorption and separation, catalysis, energy storage, drug delivery and sensors. Among the many ECL signal material options, MOFs are often used as Ru(bpy)3 because of their adjustable pore size and high porosity. 2+ A carrier of signal molecules that generates an anodic signal by assembling it onto the electrode surface. Summary of the Invention

[0004] The present invention uses HCR nucleic acid amplification to enrich the Ru-MOF signaling material. The strong affinity between MRSA and the aptamer displaces the trigger strands T1 and T2. The trigger strands then open two hairpin structures on the tetrahedral framework, further completing the HCR process. The numerous sticky ends generated at both ends of the HCR are used to immobilize the signaling molecule, fully utilizing the upper space of the tetrahedral framework.

[0005] An ECL biosensor of the present invention is prepared by the following method: (1) pretreating the electrode; (2) plating a gold film on the surface of the GCE electrode; (3) modifying the synthesized DNA tetrahedral framework onto the surface of the gold electrode; (4) dripping the MRSA incubation solution onto the gold electrode modified with the tetrahedral framework, thereby opening the two hairpins H1 and H2 on the tetrahedral framework; (5) dripping a mixed solution of DNA hairpins H3, H4, H5 and H6 onto the electrode surface, and an HCR process occurs on the electrode; (6) dripping a DNA-modified Ru-MOF material onto the electrode surface, and the designed sticky ends on H3, H4, H5 and H6 will capture the DNA-modified Ru-MOF, thereby enriching a large amount of signal material on the electrode surface.

[0006] As a preferred embodiment, in the above-mentioned ECL biosensor, the pretreatment in step (1) includes polishing, cleaning and drying of the GCE electrode.

[0007] As a preferred embodiment, in the above-mentioned ECL biosensor, plating a gold film on the GCE electrode in step (2) refers to electrochemically depositing HAuCl4 on the electrode surface.

[0008] As a preferred embodiment, in the above-mentioned ECL biosensor, in step (3), the DNA chains TND-1, TND-2, TND-3, TND-4, S1, H1 and H2 form a tetrahedral framework after mixed annealing, and the mixed solution is dropped onto the electrode, and the tetrahedral framework is fixed to the gold electrode surface through Au-S bonds.

[0009] As a preferred embodiment, in the above-mentioned ECL biosensor, the MRSA incubation solution in step (4) is obtained by incubating the MRSA bacterial solution with the DNA double helix structure of the aptamer APT, wherein the DNA double helix structure of the aptamer APT is obtained by annealing the aptamer APT, the trigger chain T1 and the trigger chain T2 in the presence of magnesium ions; the concentration of the MRSA bacterial solution is 10 0 -10 7CFU / mL; MRSA binds to its aptamer chain APT (Anal. Chem. 2022, 94, 17205−17211), releasing target chains T1 and T2 that complement the aptamer base pair. The MRSA incubation solution is centrifuged, and the mixture containing T1 and T2 is dripped onto the electrode surface for incubation. T1 and T2 will respectively unwind the hairpins H1 and H2 on the tetrahedral framework. The supernatant is centrifuged to remove excess MRSA and ATP bound to MRSA, resulting in a MRSE incubation solution containing a large amount of T1 and T2. This incubation solution is dripped onto the electrode, where the T1 and T2 chains unwind the H1 and H2 hairpins. Annealing in this invention follows conventional annealing conditions.

[0010] As a preferred embodiment, in the above-mentioned ECL biosensor, in step (5), the sticky ends of the hairpins H1 and H2 opened in the previous step will open the hairpins H3 and H5 respectively, so that the sticky ends of H3 and H5 are exposed, and then the hairpins H4 and H6 are opened respectively, so that the sticky ends of H4 and H6 are exposed, and the sticky ends of H4 and H6 further open the hairpins H3 and H5 respectively; in this cycle, the HCR process is realized, so that the hairpins H3, H4, H5 and H6 are enriched on the electrode surface in large quantities.

[0011] As a preferred embodiment, in the above-mentioned ECL biosensor, the Ru-MOF material in step (6) is prepared by 2+ The DNA-modified Ru-MOF material is loaded onto UIO-66-NH2. MOF1 and MOF2 chains are fixed to the surface of UIO-66-NH2 via amide bonds. When the DNA-modified Ru-MOF solution is dripped onto the electrode surface, the sticky ends of H3, H4, H5, and H6 capture the Ru-MOF signal material. UIO-66-NH2 can be prepared in-house based on references or purchased from a professional manufacturer.

[0012] The TND-1, TND-2, TND-3, TND-4, S1, H1 and H2 sequences in step (3) are SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO.5 and SEQ NO.6, SEQ NO.7 in the sequence listing, respectively. Among them, TND-1, TND-2, TND-3, TND-4 and S1 are all single-stranded, TND-2, TND-3 and TND-4 are modified with thiol groups, and H1 and H2 are hairpin probes. The specific sequences are as follows: TND-1:TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCGGCT CTT CTT TTA GCT CGA TAA GCC TAG TND-2:5'SHC6-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TATTGG ACC CTC GCA T TND-3:5'SHC6-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGCGAG GGT CCA ATA C TND-4:5'SHC6-ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAGAGC CGC CAT AGT A S1:GCT CGT ATG ATG CGA GTA TCG AAC ATG CGT TCA TCT AGG CTT ATC GAGCTA H1:ATG AAC GCA TGT TCG ATT TTT GCG GTT GGG CAT GAT GTA CAG GTA AACATC ATG C H2:ACT CGC ATC ATA CGA GCA TTT CTG TGA TGC GGT TGA GTT CAT CGT CAACCG CA

[0013] The concentration of the MRSA incubation solution in step (4) is 10 0 -10 7 CFU / mL can be obtained by culturing MRSA on a plate; the APT, T1 and T2 sequences are SEQ NO. 8, SEQ NO. 9 and SEQ NO. 10 in the sequence listing, respectively, wherein APT, T1 and T2 anneal to form a DNA double helix structure as shown below: APT:ATG CGG TTG GTT GCG GTT GGG CAT GAT GTA TTT CTG TGA TGC GGT TGTTTT T T1:CAT CAT GCC CAA CCG CAA T2:CAA CCG CAT CAC AGA AAT

[0014] The sequences of H3, H4, H5 and H6 in step (5) are SEQ NO. 11, SEQ NO. 12, SEQ NO. 13 and SEQ NO. 14 in the sequence listing, respectively, as shown below: H3:ACA TCA TGC CGT AGG TTG CAT GAT GGT TAC CTG TGC ATA CAA GTG CATATC H4:GCA TAC AAG TGC ATA TCA ACC TAC GCC ATG ATG TAC AGG TAA ACA TCATGC H5:TCA ACC GCA TCT GTG AGT GCG GTT GAC GAT GAA CCT ATA CGT GAA CATACG H6:CTA TAC GTG AAC ATA CGC TCA CAG ATG CGG TTG AGT TCA TCG TCA ACCGCA

[0015] Step (5) Separate annealing of H3, H4, H5, and H6 can form stable structures, enhance molecular stability, and prevent degradation or nonspecific binding. After separate annealing, they are mixed to control the HCR reaction. If mixed annealing, HCR reaction will occur directly.

[0016] The sequences of MOF1 (M1) and MOF2 (M2) in step (6) are SEQ NO. 15 and SEQ NO. 16 in the sequence listing, respectively, as shown below: M1: 5'COOH -TTT TTT TTT TGG TTT AAA CCC AAC CCG M2: 5'COOH -TTT TTT TTT TCG TAT GTT CAC GTA TAG

[0017] As a preferred embodiment, the above-mentioned ECL biosensor can be prepared by the following method:

[0018] (1) Polish the electrode surface with Al2O3 powder, clean the electrode surface with ethanol and ultrapure water, and dry it at room temperature;

[0019] (2) Place the GCE electrode in a 25 mM HAuCl4 electrolyte and electrochemically deposit for 60 s;

[0020] (3) The thiol-modified TND-2, TND-3, and TND-4 were pretreated with TCEP, then mixed with TND-1, S1, hairpin H1, and hairpin H2 and annealed at 90°C to synthesize a DNA tetrahedral framework. The DNA tetrahedral framework was mixed with NaCl solution, and 10 μL of the mixed solution was added dropwise to the gold electrode surface and incubated at 4°C overnight (8-10 h). The concentration of the DNA tetrahedral framework in the mixed solution was 1 μM, and the MgCl solution was 0.1% (0.1%). 2+ The concentration of is 10 mM and the concentration of NaCl is 1 M;

[0021] (4) 10 0 -10 7 The double helix structure formed by MRSA bacterial solution with CFU / mL and APT, T1 and T2 was incubated at 37℃ for 1 h. 2+ The concentration of the solution was 10 mM, and then the supernatant was added dropwise to the electrode surface and incubated at 37 °C for 2 h.

[0022] (5) 10 μL of the mixed solution formed by annealing H3, H4, H5 and H6 was added to the electrode surface and incubated at 37 °C for 2 h. 2+ The concentration is 10 mM;

[0023] (6) 10 μL of DNA-encapsulated Ru-MOF solution was dropped onto the electrode surface and incubated at 37°C in the dark for 2 h. 2+ The concentration of 10 mM was used. After the incubation, the electrode system was tested for ECL signals with a photomultiplier tube voltage of 800 V, a scan rate of 0.1 V / s, and a scan range of 0 V-1.4 V.

[0024] The present invention relates to the field of biological analysis and detection, and in particular to an ECL biosensor and its preparation for realizing quantitative analysis of MRSA.

[0025] The principle of the present invention is analyzed as follows:

[0026] 1. Basis of tetrahedron frame design

[0027] In common nucleic acid sensing strategies, single-stranded nucleic acid structures are often used to modify the substrate material on the electrode. Compared to tetrahedral frameworks, single-stranded nucleic acid structures are far less stable in immobilizing signal molecules or materials. In this design, the TND-1 sequence within the tetrahedral framework consists of 75 bases, while the TND-2, TND-3, and TND-4 nucleic acid sequences each consist of 55 bases. During the formation of the DNA tetrahedral structure, the TND-1, TND-2, TND-3, and TND-4 sequences base-pair with each other. The 55 bases of TND-2, TND-3, and TND-4 are folded into three segments, each consisting of 17 bases. Two bases are designed between each segment to facilitate DNA bending—two unpaired bases are placed at the junction of the tetrahedron. The 5' end of the DNA is modified with a thiol group to form an Au-S bond with the gold electrode surface. Similarly, 55 bases in TND-1 participate in the construction of the bottom of the tetrahedral framework, and the remaining bases are used to capture the auxiliary chain S1, which is further complementary to the hairpin H1 and H2 bases. At this point, TND-1, TND-2, TND-3, TND-4, S1, H1 and H2 form a nucleic acid tetrahedral framework for modification on the surface of the gold electrode. This tetrahedral framework has three binding sites for forming Au-S bonds with the gold electrode, which can greatly increase the stability of the nucleic acid structure on the electrode and provide a reliable substrate for modification of signal materials.

[0028] 2. Basis for the design of the double helix incubation process between MRSA and aptamer (APT)

[0029] In the design of bacteria-related sensing strategies, there are many bacterial recognition substances, such as antibodies, bacteriophages, antibiotics, and some organic molecules. Aptamers, as one of the bacterial recognition substances, are widely used in some biosensing strategies. First, based on the nucleic acid aptamer sequence of MRSA, trigger chains T1 and T2 that are complementary to the aptamer bases are designed, and before incubation with MRSA, the DNA double helix structures of APT, T1, and T2 are synthesized by annealing. Secondly, MRSA is cultured on a plate, and the concentration of MRSA is confirmed by the dilution coating plate method, so as to further prepare 10 0 -10 7 Finally, during the incubation process with double-stranded DNA, MRSA competitively releases trigger chains T1 and T2 due to the stronger affinity between MRSA and aptamers.

[0030] 3. Basis for HCR nucleic acid amplification process design

[0031] Common nucleic acid amplification strategies include rolling circle amplification (RCA), hybrid chain amplification (HCR), and strand displacement amplification (SDA), etc., which aim to increase the intermediate products of the reaction and increase the binding sites of signal molecules. The trigger chains T1 and T2 displaced by MRSA open the target hairpins H1 and H2 on the tetrahedral framework (T1 first binds to the unpaired part of the H1 hairpin stem. Since the number of bases bound by T1 and H1 is greater than the number of bases complementary to the H1 hairpin itself, T1 and H1 bind to open H1). When hairpins H3, H4, H5 and H6 are added to the electrode surface, H1 with the exposed sticky end will bind to the unpaired part of the H3 hairpin stem and expose the sticky end of H3 (the sticky end of H1 is complementary to some bases of H3, and the number of pairings is greater than the number of bases complementary to the hairpin H3 itself, so H3 is opened). Then the sticky end of H3 will bind to H4 to expose the sticky end of H4, and then the sticky end of H4 will in turn bind to H3 to expose the sticky end of H3. In this cycle, hairpins H3 and H4 are continuously assembled on the electrode. Similarly, the sticky end of H2 will bind to H5 and expose the sticky end of H5, followed by H5 binding to H6, and then H6 binding to H5, and the assembly cycle continues. In the design process of the four hairpins, not only were sticky ends designed for hairpin self-assembly, but sticky ends for capturing signal materials were also added to each hairpin (after HCR of hairpins H3\H4\H5\H6, non-complementary paired bases will be exposed. These bases can perform base-pairing with the DNA chain connected to the signal material to capture the signal material). The HCR process allows the electrode surface to be assembled with a large number of four DNA hairpins with reserved sticky ends, which can enrich more signal materials to the electrode surface, thereby achieving signal amplification and realizing quantitative detection of MRSA through the intensity of ECL.

[0032] 4. Design Basis for DNA Functionalization on Ru-MOF

[0033] UIO-66-NH2 material was synthesized in a polytetrafluoroethylene reactor and loaded with Ru(bpy)3 2+ The molecules aggregate the signal molecules, and finally, the carboxyl-modified DNA is wrapped onto the Ru-MOF surface via amide bonds. The DNA sequence wrapped on the Ru-MOF surface complements the sticky terminal bases generated by HCR, enabling the aggregation of the signal material on the electrode surface. The degree of aggregation increases with the increase of HCR process, thus indirectly achieving a linear change in MRSA concentration and HCR process.

[0034] In the present invention, we designed an ECL sensor for quantitative detection of MRSA. We used the strong interaction between the aptamer and MRSA to release the trigger chain and participate in the subsequent construction of the sensor, thus getting rid of the influence of the larger volume of MRSA on the electrode. The structure generated by the HCR process is firmly fixed on the electrode surface through the tetrahedron framework, making full use of the upper space of the tetrahedron. At the same time, the simultaneous HCR process of two DNA hairpins on the DNA tetrahedron framework effectively avoids the loss of ECL signal and provides a guarantee for the orderly enhancement of ECL signal. The ECL sensor designed by us effectively realizes the sensitive detection of MRSA and can provide certain design ideas for the structural detection of nucleic acid framework. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the fabrication process of HCR strategy sensor for MRSA detection;

[0036] Figure 2 This is a gel electrophoresis diagram of DNA structure, which demonstrates the feasibility of the nucleic acid strategy through changes in DNA structure;

[0037] Figure 3 This is a transmission scan of MRSA and other bacteria;

[0038] Figure 4 Transmission and scanning electron microscopy imaging of UIO-66-NH2 and Ru-MOF;

[0039] Figure 5 shows the relationship between MRSA concentration and ECL intensity;

[0040] Figure 6 The selectivity of the biosensor for different bacteria. DETAILED DESCRIPTION

[0041] The following embodiments are further descriptions of the content of the present invention as an explanation of the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0042] Example 1

[0043] Preparation of tetrahedral framework nucleic acid:

[0044] Composition: 0.1 μM TND-1, 0.1 μM TND-2, 0.1 μM TND-3, 0.1 μM TND-4, 0.1 μM S1, 0.1 μM H1, 0.1 μM H2, 10 mM Mg 2+The solution was annealed at 90 °C for 10 min and then slowly cooled to allow DNA to self-assemble into a tetrahedral framework nucleic acid structure.

[0045] Synthesis and modification of Ru-MOF materials:

[0046] First, two different precursor solutions were prepared: Solution 1, in which 2-aminoterephthalic acid (43.4 mg, 0.24 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF); Solution 2, in which zirconium oxychloride octahydrate (21 mg, 0.066 mmol) was dissolved in 3 mL of DMF. These two solutions were then thoroughly mixed with 2.5 mL of acetic acid and transferred to a Teflon-sealed autoclave for reaction at 120°C for 24 hours. Finally, the resulting product was washed three times with DMF and three times with ethanol, then dried in a vacuum oven to obtain a solid powder, UIO-66-NH2.

[0047] Dissolve the UIO-66-NH2 solid powder in a mixture of 10 mL DMF and 10 mL ethanol, and add 2 mg of Ru(bpy)3Cl2 to the solution. Mix thoroughly and stir at 90°C overnight. The resulting solution is purified with DMF and ethanol, then placed in a vacuum drying oven at 80°C overnight. Finally, the Ru(bpy)3 loaded solution is obtained. 2+ UIO-66-NH2 is Ru-MOF.

[0048] DNA modification of Ru-MOF materials: First, prepare a 2 mg / mL Ru-MOF solution, then add 3 μM MOF1 chain (M1) and 3 μM MOF2 chain (M2) to the Ru-MOF solution containing 20 mM EDC and 10 mM NHS, and shake at room temperature and in the dark for 4 h. Finally, the reaction solution is centrifuged, washed, and redispersed in PBS solution.

[0049] Establishment of HCR nucleic acid amplification process on the electrode:

[0050] Polish the glassy carbon electrode (GCE) with 0.3 μm Al2O3 polishing powder, and rinse the electrode surface with ultrapure water for use. Turn on the electrochemical workstation, plate a layer of gold film on the surface of the GCE electrode at a voltage of -0.2 V, and rinse the surface of the gold electrode with ultrapure water to remove excess chloroauric acid solution. Add 100 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to the thiol-modified TND-2, TND-3, and TND-4 solutions for pretreatment, and then mix with TND-1, S1, hairpin H1, and hairpin H2 and anneal at 90 ° C to synthesize a DNA tetrahedral framework. The annealed tetrahedral framework solution was mixed with NaCl solution (maintaining the final concentrations of the two were 1 μM and 1 M, respectively, Mg 2+ 10 μL of the mixed solution was added dropwise to the gold electrode surface and incubated at 4°C overnight (8-10 h).

[0051] First, clean the electrode surface with PBS solution to remove excess tetrahedral framework. 0 -10 7 The double helix structure formed by MRSA bacterial solution with CFU / mL and APT, T1 and T2 was incubated at 37℃ for 1 h. 2+ The concentration of MgCl2 was 10 mM, and then the bacterial replacement solution was centrifuged and added to the electrode surface. The solution was incubated at 37 °C for 2 h. 2+ The concentration was 10 mM. Secondly, the excess bacterial replacement solution was removed with PBS solution, and 10 μL of the mixed solution of annealed H3, H4, H5 and H6 was added to the electrode and incubated at 37°C for 2 h. Finally, the DNA-modified Ru-MOF was added to the electrode and incubated in the dark at 37°C for 2 h. The experiment was analyzed by cyclic voltammetry (CV) and electrochemiluminescence (ECL), respectively. The relevant ECL performance was measured in 0.25 M tripropylamine (TPrA) in PBS solution (0.1 M, pH 7.4). Relevant parameters of the electrochemical workstation: photomultiplier tube voltage (800 V), scan voltage (0-1.4 V), scan rate (0.1 V / s).

[0052] like Figure 2 Shown is the analysis of DNA structure alone using polyacrylamide gel electrophoresis (PAGE).

[0053] Figure 2A: 10% PAGE imaging. Lane 1-19: TND1; TND2; TND3; TND4; S1; H1; H2; H3; H4; H5; H6; TND(1-2); TND(1-3); TND(1-4); TND(1-4)+S1 TND(1-4)+S1+H1; TND(1-4)+S1+H1+H2; TND(1-4)+S1+H1+H2+T1+T2; TND(1-4)+H1+H2+T1+T2+H3+H4+H5+H6;

[0054] Figure 2 Middle B: 10% PAGE image. Lanes 1-10: H1; H2; H3; H4; H5; H6; H1+T1; H1+T1+H3+H4; H2+T2; H2+T2+H5+H6.

[0055] We use gel electrophoresis to demonstrate the feasibility of this nucleic acid strategy. Figure 2 In A, Lane 1-11 represents each single nucleic acid strand involved in the construction of the nucleic acid sensor. Lane 12-17 is the process of building the tetrahedral framework. We can observe that as the nucleic acid framework is continuously assembled, its structure continues to grow, causing its migration speed in the gel to continue to slow down. The further upward shift of the Lane 18 band indicates that T1 and T2 successfully opened the hairpins H1 and H2 on the tetrahedral framework. In Lane 19, hairpins H3, H4, H5 and H6 successfully underwent the HCR process on the basis of the previous one, and the formed bands also shifted upward. However, because the nucleic acid structure is too large, it is difficult to observe the structure of the nucleic acid in the gel electrophoresis. Figure 2 The HCR process is further analyzed in Lane B. Lanes 1-6 depict the single-stranded DNA from H1 to H6. Lane 7 shows the double helix formed by T1 opening hairpin H1. Lane 8 depicts the HCR process between the sticky ends formed by T1 opening hairpin H1 and hairpins H3 and H4. Because hairpins H3 and H4 open mutually during HCR, the resulting DNA structure can include DNA of varying sizes, resulting in the "bamboo-joint" structure seen in Lane 8. Similarly, Lane 9 depicts the double helix formed by T2 and hairpin H2, while Lane 10 depicts the HCR process between the sticky ends formed by T2 opening hairpin H2 and hairpins H4 and H5. Overall, this demonstrates the successful construction of the tetrahedral framework and the successful HCR process, demonstrating the successful development of this bacterial sensor.

[0056] like Figure 3Figure A shows five different bacteria grown in liquid culture: methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli), Salmonella enteritidis (S. enteritidis), Bacillus subtilis (B. subtilis), and Salmonella typhimurium (S. typhimurium). Figure B shows the solid culture medium we prepared, in preparation for subsequent bacterial purification and plating. Figure C shows MRSA growth on solid culture medium, showing an overall golden yellow appearance with individual colonies of varying sizes. Figures (D-H) and (I-M) are transmission and scanning images of the five different bacteria. The "grape-like" morphology in Figures (D-I) demonstrates successful MRSA culture, laying the foundation for aptamer recognition. Similarly, the successful culture of other bacteria provides the basis for selective detection, demonstrating the successful design of our aptasensor.

[0057] like Figure 4 Transmission and scanning electron microscopy images of UIO-66-NH2 and Ru-MOF. Figure A is a transmission electron microscopy image of UIO-66-NH2 at different scales. UIO-66-NH2 is uniform in size, with an average size of about 200 µm. The overall shape is an octahedral morphology with sharp edges. Figures E and F are crystal structure images simulated by C4D software at different angles. It can be found that in space, the TEM image and the structure simulated by C4D are highly similar. In the scanning image, it can also be observed that the UIO-66-NH2 structure is relatively uniform in size. The EDS spectrum analysis of the material and its corresponding element mapping image clearly show the distribution of C, N, O, Zr, and Ru in the material. The above shows that UIO-66-NH2 is loaded with Ru(bpy)3 2+ The molecule does not change its shape and has a very stable structure, which also proves the feasibility of using this signal material in sensors.

[0058] like Figure 5A 、 5B As shown in Figure 5A, with the increase of the concentration of target bacteria (10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 CFU / mL), the corresponding ECL signal increases continuously, indicating that the ECL signal of the sensor is closely related to the concentration of bacteria. Figure 5B The bacterial concentration (100 -10 7 There is a good linear relationship between the concentration of 100 CFU / mL and the ECL signal intensity, with a correlation coefficient of 0.989, and a detection limit (LOD) of 1 CFU / mL. These indicate that the sensor has excellent detection performance in bacterial detection.

[0059] like Figure 6 As shown, the ECL signal of the MRSA-bacteria mixture is higher than that of the reference bacteria (Salmonella typhimurium, Bacillus subtilis, Escherichia coli, and Salmonella enteritidis), demonstrating the successful construction of a detection platform for MRSA. Staphylococcus aureus MRSA ATCC 43300, Salmonella typhimurium ATCC 14028, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 8739, and Salmonella enteritidis CMCC(B)50335 were purchased from Shanghai Luwei Technology.

[0060] It should be noted that the technical contents of the present invention described above are only for the purpose of explaining and illustrating the technical essence of the present invention to enable those skilled in the art to understand the technical essence of the present invention. Therefore, the technical contents described above are not intended to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be based on the claims. Those skilled in the art should be aware that any modifications, equivalent substitutions, and improvements based on the substantial spirit of the present invention shall fall within the substantial protection scope of the present invention.

Claims

1. An ECL sensor based on HCR nucleic acid amplification for MRSA detection was prepared by the following method: (1) Pretreatment of electrodes; (2) Plating a gold film on the surface of the GCE electrode; (3) Modifying the synthesized DNA tetrahedral framework onto the surface of the gold electrode; (4) The MRSA incubation solution is dropped onto the gold electrode modified with the tetrahedral framework, thereby opening the two hairpins H1 and H2 on the tetrahedral framework; (5) A mixed solution of DNA hairpins H3, H4, H5, and H6 is added dropwise to the electrode surface, and the HCR process occurs on the electrode; (6) The DNA-modified Ru-MOF material is added dropwise to the electrode surface. The sticky ends of H3, H4, H5, and H6 will capture the DNA-modified Ru-MOF material and enrich the signal material on the electrode surface.

2. The sensor according to claim 1, wherein: Step (1) pretreatment includes polishing, cleaning, and drying of the GCE electrode.

3. The sensor according to claim 1, wherein: In step (2), a gold film is plated on the GCE electrode by electrochemically depositing HAuCl4 on the electrode surface.

4. The sensor according to claim 1, wherein: The DNA tetrahedral framework in step (3) is formed by mixed annealing of DNA chains TND-1, TND-2, TND-3, TND-4, S1, hairpin H1 and hairpin H2, and the TND-2, TND-3 and TND-4 are modified with thiol groups; the modification to the gold electrode surface is to drop the DNA tetrahedral framework solution onto the electrode and fix the DNA tetrahedral framework to the gold electrode surface through Au-S bonds; the sequences of TND-1, TND-2, TND-3, TND-4, S1, H1 and H2 are SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7 in the sequence listing, respectively.

5. The sensor according to claim 1, wherein: Step (4) The MRSA incubation solution is obtained by incubating the MRSA bacterial solution with the DNA double helix structure of the aptamer APT, wherein the DNA double helix structure of the aptamer APT is obtained by annealing the aptamer APT, the trigger chain T1 and the trigger chain T2; the MRSA bacterial solution concentration is 10 0 -10 7 CFU / mL; through the binding of MRSA to the aptamer APT, target chains T1 and T2 that are complementary to the base pairing of the aptamer APT are released; the sequences of APT, T1 and T2 are SEQ ID No.8, SEQ ID No.9 and SEQ ID No.10 in the sequence listing, respectively.

6. The sensor according to claim 1, wherein: In step (5), H3, H4, H5 and H6 are annealed in advance and then mixed. When the mixed solution is dropped onto the electrode surface, the sticky ends of the hairpins H1 and H2 opened by the treatment in step (4) will open the hairpins H3 and H5 respectively, so that the sticky ends of H3 and H5 are exposed, and then the hairpins H4 and H6 are opened respectively, so that the sticky ends of H4 and H6 are exposed. The sticky ends of H4 and H6 further open the hairpins H3 and H5 respectively, and this cycle is repeated to realize the HCR process, thereby enriching the hairpins H3, H4, H5 and H6 in large quantities on the electrode surface; the sequences of H3, H4, H5 and H6 are SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 and SEQ ID No. 14 in the sequence listing, respectively.

7. The sensor according to claim 1, wherein: The Ru-MOF material in step (6) is prepared by 2+ The DNA-modified Ru-MOF material is obtained by fixing the MOF1 chain and the MOF2 chain to the surface of UIO-66-NH2 through amide bonds; the sequences of the MOF1 chain and the MOF2 chain are SEQ ID No.15 and SEQ ID No.16 in the sequence list, respectively.

8. The sensor according to any one of claims 1 to 7, characterized in that: Prepared by the following method: (1) Polishing, cleaning and drying of GCE electrodes; (2) The dried GCE electrode was placed in a 25 mM HAuCl4 electrolyte for electrochemical deposition; (3) The thiol-modified TND-2, TND-3, and TND-4 were pretreated with TCEP, and then mixed with TND-1, S1, hairpin H1, and hairpin H2 and annealed at 90°C to synthesize a DNA tetrahedral framework. The DNA tetrahedral framework was mixed with NaCl solution, and 10 μL of the mixed solution was added dropwise to the gold electrode surface and incubated at 4°C overnight. The concentration of the DNA tetrahedral framework in the mixed solution was 1 μM, and the MgCl content was 0.1%. 2+ The concentration of is 10 mM and the concentration of NaCl is 1 M; (4) 10 0 -10 7 The DNA double helix structure of the aptamer APT formed by APT, T1 and T2 was incubated at 37℃ for 1 h. 2+ The concentration was 10 mM; the solution was then centrifuged and added dropwise to the electrode surface and incubated at 37°C for 2 h; (5) 10 μL of the mixed solution of H3, H4, H5 and H6, which were annealed and then mixed, was added to the electrode surface and incubated at 37°C for 2 h. 2+ The concentration is 10 mM; (6) 10 μL of DNA-modified Ru-MOF solution was dropped onto the electrode surface and incubated at 37°C in the dark for 2 h. 2+ The concentration is 10 mM.

9. Use of the ECL sensor according to any one of claims 1 to 8 in MRSA detection.

10. The use according to claim 9, characterized in that The detection conditions of the ECL sensor were a photomultiplier tube voltage of 800 V, a scan rate of 0.1 V / s, and a scan range of 0 V–1.4 V.

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