ECL sensor based on RCA nucleic acid amplification for MRSA detection

Abstract

The invention discloses an ECL sensor based on RCA nucleic acid amplification for MRSA detection. The ECL sensor can be used for sensitive detection of MRSA. According to the invention, RCA nucleic acid amplification is combined with a DNA tetrahedral framework, and an ECL sensor for MRSA detection is designed. Firstly, a tetrahedral frame is fixed on the surface of a gold electrode, so that a stable basic condition is provided for the generation of RCA; secondly, two DNA hairpins on a DNA four-side frame are opened through combination and replacement of an MRSA aptamer, the upper layer space of the tetrahedral frame is fully utilized in the step, and the AuNCs capturing capacity of the sensor is improved. In the detection of the MRSA, the lowest detection concentration of the sensor is 1 CFU / mL, different bacteria can be specifically distinguished, and the excellent data shows that the sensor has great potential in the detection of the bacteria.

Description

Technical Field

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

[0002] Among Gram-positive bacteria, multidrug-resistant Staphylococcus aureus (MRSA) poses a significant threat to human health. Methicillin-resistant Staphylococcus aureus (MRSA) commonly causes soft tissue infections, septic arthritis, pneumonia, endocarditis, and bacteremia. Although the incidence of MRSA-induced bacteremia has decreased in recent years, the clinical treatment outcomes for MRSA remain relatively poor compared to methicillin-susceptible S. aureus (MSSA). The mortality rate of bacteremia caused by S. aureus is 20%-30%, with 10%-30% of patients with bacteremia also developing infective endocarditis. Furthermore, spontaneous meningitis caused by MRSA has an extremely high mortality rate.

[0003] The polymerase chain reaction (PCR) amplifies bacterial RNA and DNA, enabling trace detection. It is the most widely used technique for RNA and DNA amplification. Multiplex PCR testing for MRSA is now possible, but due to significant disparities in healthcare access, clinical testing is difficult in less developed countries. Furthermore, despite its large amplification capacity and high sensitivity, PCR is susceptible to contamination, resulting in false-positive signals. These factors limit its widespread application in detection. Electrochemiluminescence (ECL) offers advantages such as low background, high sensitivity, and simple equipment, making it a powerful technique widely used for analytical detection. Aptamers, which are simple, rapid, and easy to use in the design of ECL-related sensors, have become a common technique for pathogen detection. Rolling circle amplification (RCA) is an important research tool in medicine, materials science, and molecular biology. Researchers can rationally design the RCA process to produce a desired, customized, long chain of repetitive DNA sequences. This RCA product can then combine with complementary oligonucleotides already attached to a material to generate multifunctional materials with diverse properties. These materials include biotin, enzymes, antibodies, nanoparticles, fluorescent dyes and electrochemical labels. The composite structures formed are widely used in immunosensing, biorecognition, bioimaging and sensitive detection. In addition to rationally optimizing the ECL sensing process through changes in nucleic acid structure, the performance of ECL signal materials plays a vital role in the entire sensor system. In previous studies, commonly used ECL signal materials include luminol, Ru(bpy)3 2+While many ECL materials have been investigated, including quantum dots, perovskite nanocrystals, and carbon nitride nanosheets, most still suffer from low stability, low sensitivity, and biotoxicity. Therefore, it is necessary to explore stable, efficient, and biocompatible ECL signaling materials. Gold nanoclusters (AuNCs) have excellent stability, good biocompatibility, and favorable optical and electrochemical properties. These features have made AuNCs popular among ECL researchers, and a growing number of ECL sensors based on AuNCs have been reported. Among these, the ECL system of AuNCs and triethylamine (TEA) has been extensively studied. Summary of the Invention

[0004] The present invention uses RCA nucleic acid amplification to enrich AuNCs signal material. The strong affinity between MRSA and the aptamer displaces trigger strands T1 and T2. The trigger strands open two hairpins H1 and H2 on the tetrahedral framework. Under the action of phi29 polymerase, the RCA process is further completed, generating two long chains with a large number of repetitive DNA sequences. The repetitive DNA sequences on these long chains complement each other with the DNA sequences coated on the material surface, thereby enriching the signal material on the electrode surface and enhancing the ECL signal.

[0005] An ECL biosensor of the present invention is prepared by the following method: (1) pretreating a GCE 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) dropping a replacement solution of the aptamer APT and MRSA onto the surface of the gold electrode, and the trigger chains T1 and T2 in the solution open the two hairpins H1 and H2 on the tetrahedral framework; (5) dropping a mixed solution of two DNA rings, phi29 polymerase and dNTPs onto the electrode surface, and an RCA process occurs on the electrode surface; (6) dropping a solution of DNA-wrapped AuNCs, i.e., DNA-modified AuNCs, onto the electrode surface, and the generated long repetitive DNA chains will capture the DNA-wrapped AuNCs, thereby enriching the 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, step (2) deposits gold on the electrode surface by electrochemically reducing HAuCl4 solution.

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

[0009] As a preferred embodiment, in the above-mentioned ECL biosensor, the replacement solution of the aptamer APT and MRSA 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. The purpose of annealing is to convert the DNA into a single-stranded state, thereby allowing the three chains to bind. The concentration of the MRSA bacterial solution is 10 0 -10 7 CFU / mL can be obtained by culturing MRSA on plates; MRSA binds to the aptamer APT (Anal. Chem. 2022, 94, 49, 17205–17211), releasing trigger chains T1 and T2 that are complementary to the bases of the aptamer APT. Centrifugation separates the upper mixed solution containing T1 and T2, which is then added dropwise to the electrode surface. The trigger chains T1 and T2 will respectively open the hairpins H1 and H2 on the tetrahedral framework. T1 first binds to the unpaired portion 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; the same is true for T2 opening H2. 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.

[0010] As a preferred solution, in the above-mentioned ECL biosensor, in step (5), the sticky ends of H1 and H2 on the tetrahedral framework will capture DNA ring 1 and DNA ring 2 respectively. In the above step (4), after T1 opens H1, the newly exposed single-stranded portion of H1 (sticky end) will complement and bind with the partial sequence on ring 1, thereby achieving H1 capturing ring 1; H2 capturing ring 2 in the same way. Under the action of phi29 polymerase, the DNA hairpin will be removed from the 3 ’ The RCA process is carried out at the end, generating a large number of long chains with repeated DNA sequences, which provide binding sites for the enrichment of signal materials. The polymerized rings repeatedly roll on the polymerized new chains, resulting in long chains with repeated ring sequences.

[0011] As a preferred embodiment, in the aforementioned ECL biosensor, step (6) first synthesizes amino-functionalized AuNCs as the signal material, and then coats two DNA gold cluster chains onto the surface of the AuNCs, i.e., coating the AuNCs with DNA chains Au-DNA1 and Au-DNA2, respectively, to obtain DNA-modified AuNCs. The DNA-modified AuNCs solution is then dripped onto the electrode surface, where the long chains with repetitive DNA sequences on the electrode capture the DNA-coated AuNCs.

[0012] The sequences of TND-1, TND-2, TND-3, TND-4, S1, H1 and H2 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, as shown below: 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 TTG CGG TTG GGC ATG ATG TAA ACC AAG GTAAAC ATC ATG C H2: ACT CGC ATC ATA CGA GCA TTT CTG TGA TGC GGT TGA TCG TTC ATC GTCAAC CGC ATC The APT, T1 and T2 sequences in step (4) are SEQ NO. 8, SEQ NO. 9 and SEQ NO. 10 in the sequence listing, respectively, 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 The primer 1, primer 2, padlock 1 and padlock 2 sequences in step (5) are SEQ NO. 11, SEQ NO. 12, SEQ NO. 13 and SEQ NO. 14 in the sequence table, respectively, as shown below: Primer1:TAA ACC AAG GTA AAC ATC ATG C Primer2: ATC GTT CAT CGT CAA CCG CAT C Padlock1: 5'P-ACC TTG GTT TAA ACC CAA CCC GCC CTA CCC AAG CAT GAT GTTT Padlock2: 5'P-CAT TGA ACG ATA ACG TAT GTT CAC GTA TAG AAG ATG CGG TTGA The Au-DNA1 and Au-DNA2 sequences in step (6) are SEQ NO. 15 and SEQ NO. 16 in the sequence listing, respectively, as shown below: Au-DNA1: 5'COOH-TTT TTT TTT TGG TTT AAA CCC AAC CCG Au-DNA2: 5'COOH-TTT TTT TTT TGA TAT GCA CTT GTA TGC As a preferred embodiment, the above-mentioned ECL biosensor can be prepared by the following method: (1) Polish the electrode surface with Al2O3 powder and clean the electrode surface by ultrasonic treatment in ultrapure water and ethanol; (2) The GCE electrode was placed in a 25 mM HAuCl4 solution and electrochemically deposited for 60 s.

[0013] (3) The thiol-modified TND-2, TND-3, and TND-4 were pretreated with 10 mM TCEP for 1 h, and then TND-1, TND-2, TND-3, TND-4, S1, H1, and H2 were mixed and annealed at 90°C to form a tetrahedral framework mixture. The mixed solution was thoroughly mixed with 1 M NaCl solution, and 10 μL of the solution was pipetted onto the surface of the gold electrode and incubated at 4°C overnight. The final concentration of DNA was 1 μM, and Mg was 20 μg / mL. 2+ The final concentration was 10 mM; (4) Use the dilution plate method to obtain the concentration of MRSA in the original culture medium, and set the final concentration at 10 0 -10 7 The double helix structures formed by MRSA within the CFU / mL range were incubated with APT, T1, and T2 at 37°C for 1 h. The solution after displacement centrifugation was added dropwise to the electrode surface and incubated at 37°C for 2 h. (5) 10 μL of a mixed solution containing two DNA circles with a final concentration of 1 μM, 1 mM dNTPs, and 0.1 U / μL phi29 polymerase was added to the electrode surface and incubated at 37°C for 2 h. 2+ The final concentration was 10 mM; (6) 10 μL of DNA-encapsulated AuNCs solution was dropped onto the electrode surface and incubated at 37°C in the dark for 2 h to maintain its Mg 2+ The final concentration was 10 mM. 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-1.2 V.

[0014] 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.

[0015] The principle of the present invention is analyzed as follows: Basis of Tetrahedral Framework Nucleic Acid Design 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 tetrahedron 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 sequence bases are placed at the inflection points of the tetrahedron. The 5' end of the DNA is modified with a thiol group to form an Au-S bond on the gold electrode surface. Similarly, the 55 bases of TND-1 contribute to the construction of the tetrahedral framework's base, while the remaining bases serve to capture the auxiliary strand S1, which then pairs complementary with the hairpins H1 and H2. At this point, TND-1, TND-2, TND-3, TND-4, S1, H1 and H2 formed a nucleic acid tetrahedral framework for modification on the surface of the gold electrode. The 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 the modification of signal materials.

[0016] 2. Basis for the design of the double helix incubation process between MRSA and aptamer (APT) In nucleic acid sensing strategies, aptamers are widely used by researchers due to their strong and specific recognition ability with their targets. We first designed complementary strands T1 and T2 of the aptamer and annealed them to form a double helix structure. When this double helix structure is incubated with MRSA, the MRSA binds to the aptamer, releasing two trigger strands, T1 and T2.

[0017] 3. Basis for the design of RCA nucleic acid amplification process To achieve RCA, this experiment requires the synthesis of two DNA circles. First, the primer chain brings the 5' and 3' ends of the phosphate-modified padlock chain closer together (the primer chain is complementary to the 5' and 3' ends of the circle, bringing the two ends closer together). The lysine active site of T4 ligase then nucleophilically attacks the α-phosphate group of ATP, releasing PPi and generating a T4 DNA ligase-AMP intermediate. The phosphate group at the 5' end of the padlock chain attacks the ligase-AMP intermediate, transferring AMP to the 5' phosphate group of the padlock chain, generating a DNA-adenylate intermediate. Finally, the phosphate group at the 5' end of the padlock chain combines with the hydroxyl group at the 3' end to form a phosphodiester bond, simultaneously releasing AMP. This results in a double helix structure consisting of the DNA circle and primer. When EXO III and EXO I are added to the solution, EXO III progressively removes single nucleotides from the 3' end of the double helix, cleaving the primer chain, while EXO I removes excess single-stranded DNA from the mixed solution. Under the action of multiple enzymes, we obtained two types of DNA rings. After the DNA rings combined with the hairpins on the electrode, under the action of phi29 polymerase, they continuously consumed the dNTPs in the solution to generate long chains with repetitive DNA sequences for capturing signal materials.

[0018] Design basis for DNA functionalization on AuNCs L-methionine-functionalized AuNCs were synthesized using L-methionine. Carboxyl-modified DNA chains were then enriched on the AuNC surface via amide bonds. DNA was enriched on the AuNC surface through an amidation reaction between the amino-bearing AuNCs synthesized with methionine and the carboxyl-modified DNA. The numerous repetitive DNA fragments generated during RCA paired complementary bases with the long DNA chains modified on the AuNC surface, thereby being immobilized on the nucleic acid framework on the electrode surface.

[0019] In this work, we combined RCA nucleic acid amplification with a DNA tetrahedron framework to design an ECL platform for MRSA detection. First, by fixing the tetrahedron framework on the surface of the gold electrode, a stable basic condition for the occurrence of RCA was provided. Secondly, through the binding and displacement of the MRSA aptamer, the two DNA hairpins on the DNA tetrahedron framework were opened. This step fully utilized the upper space of the tetrahedron framework and improved the ability of the sensor to capture gold nanoclusters. In the detection of MRSA, the minimum detection concentration of the platform was 1 CFU / mL, and it could specifically distinguish different bacteria. These excellent data show that the sensor has great potential for bacterial detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the RCA strategy sensor for MRSA detection; 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; Figure 3 Transmission image of MRSA and AuNCs; Figure 4 shows the relationship between MRSA concentration and ECL intensity; Figure 5 The selectivity of the biosensor for different bacteria. DETAILED DESCRIPTION

[0021] The following embodiments are further descriptions of the present invention to illustrate 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. Example 1

[0022] 1. Preparation of tetrahedral framework nucleic acid: 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.

[0023] 2. Preparation of DNA circles: Take 10 µL of 100 µM padlock DNA strand and 10 µL of 100 µM primer DNA strand, add them to 58 µL of TAE buffer solution (40 mM TAE, pH = 8.0), and then add 10 µL of 100 mM Mg 2+ The solution was thoroughly vortexed and centrifuged, then annealed at 90°C for 10 minutes (this procedure was repeated for both padlocks and primers). After the solution cooled to room temperature, 2 µL of 5 weissU / µL T4 ligase was added and the mixture was incubated at 37°C for 2 hours. 1 µL of 10 U / µL EXO III and 1 µL of 10 U / µL EXO I were added to the solution and the mixture was incubated at 37°C for 2 hours. DNA circle 1 was generated using padlock 1 and primer 1 through the action of T4 ligase, while DNA circle 2 was generated using padlock 2 and primer 2 through the action of T4 ligase.

[0024] 3. Synthesis and modification of AuNCs materials: A solution of HAuCl₄ (0.8 mL, 20 mg / mL) and NaOH (1.2 mL, 0.5 mol / mL) was added to an L-methionine (L-met) solution (8 mL, 0.5 mol / mL) and stirred continuously at 37°C in the dark for 6 h. Subsequently, H₂SO₄ (1 mL, 1 mol / mL) was added to the solution to precipitate AuNCs. The AuNCs were then rinsed and collected with H₂SO₄ (12 mL, 0.1 mol / mL). The collected AuNCs were then evenly dispersed in aqueous ammonia (4 mL, 1.4%) and incubated at 70°C for 10 min. The incubated AuNCs solution was purified with isopropanol, removed, and allowed to dry naturally at room temperature. The resulting solid AuNCs were dispersed in water to form a 100 µg / mL solution, which was then stored in the dark at 4°C to yield amino-functionalized AuNCs.

[0025] Next, carboxyl-modified DNA1 (Au-DNA1) and DNA2 (Au-DNA2) were subjected to an amidation reaction with L-methionine-functionalized AuNCs to enrich the DNA on the AuNCs surface, yielding an AuNCs-DNA solution. Specifically, EDC, NHS, and TAE buffer were added to carboxyl-modified DNA1 and DNA2 to a final concentration of 3 μM, 20 mM EDC, and 10 mM NHS, respectively. The solution was incubated at 37°C for 2 hours to activate the -COOH groups. This solution was then added to amino-functionalized AuNCs to enrich the DNA on the AuNCs surface via an amidation reaction.

[0026] 4. Establishment of HCR nucleic acid amplification process on the electrode.

[0027] Polish the glassy carbon electrode (GCE) with Al2O3 polishing powder and rinse the electrode with ultrapure water for use. Use an electrochemical workstation to coat the GCE electrode with a gold film using a three-electrode electrolytic cell, and remove excess hexahydrate chloroauric acid solution on the gold electrode surface with ultrapure water. Treat thiol DNA (TND-2, TND-3, TND-4) with 10 mM TCEP solution and react at room temperature for 1 h. Then, TND-1, TND-2, TND-3, TND-4, and auxiliary DNA (S1, H1, and H2) were annealed together at 90°C for 10 minutes and naturally cooled to room temperature. 10 µL of the above solution was added to the gold electrode and incubated at 4°C overnight. Wash the electrode surface with PBS solution to remove excess nucleic acid frameworks. 10 µL of different bacterial concentrations (10 0 -10 7CFU / mL) to displace the T1 and T2 solutions and incubate at 37°C for 2 h. Similarly, the surface was washed with PBS to remove excess replacement solution. A 10 µL mixture of the two synthesized DNA circles 1 and 2 (1 µM each), phi29 polymerase (0.1 U / µL), and dNTPs (1 mM) was added to the electrode and allowed to react at 37°C for 1.5 h. The excess DNA circle mixture was removed with PBS, and 10 µL of the AuNCs-DNA solution was added dropwise, followed by incubation at 37°C for 1.5 h. The electrode system at the end of the incubation period was monitored for ECL signals using a photomultiplier tube at 800 V, a scan rate of 0.1 V / s, and a scan range of 0 V–1.2 V.

[0028] like Figure 2 Shown is the use of polyacrylamide gel electrophoresis (PAGE) to analyze DNA structure alone.

[0029] (A) 10% PAGE image. Lane 1-17: TND1; TND2; TND3; TND4; S1; H1; H2; T1; T2; 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; TND(1-4)+S1+H1+H2+T1+T2; (B) 10% PAGE imaging. Lanes 1-18: S1; H1; H2; T1; T2; padlock1; primer1+padlock1; DNA circle 1 (primer1+padlock1+EXO III+EXO I); padlock2; primer2+padlock2; DNA circle 2 (primer2+padlock2+EXO III+EXO I); S1+H1; S1+H1+H2; S1+H1+H2+T1; S1+H1+H2+T1+T2; S1+H1+H2+T1+T2+DNA circle 1; S1+H1+H2+T1+T2+DNA circle 1+DNA circle 2; S1+H1+H2+T1+T2+DNA circle 1+DNA circle 2+Phi29+dNTPs.

[0030] exist Figure 2In (A), lanes 1-7 are DNA single strands that form the tetrahedral base framework structure, and lanes 8-9 are the trigger strands T1 and T2 of the two MRSA displacement fluids. Lanes 10-15 are the assembly process of the tetrahedral framework. It can be found that starting from lane 10, the migration rate of the structure band formed is much lower than that of the nucleic acid single strand. These indicate that base complementary pairing has been successfully carried out between different DNA structures. Lanes 16-17 are the process of T1 and T2 opening the hairpins H1 and H2 on the tetrahedral framework respectively. In gel electrophoresis Figure 1 In this work, we verified the successful construction of the tetrahedral framework, which laid the foundation for the subsequent RCA process. Figure 2In this study, we demonstrated the RCA process. Lanes 1-3 depict auxiliary strands S1, H1, and H2 on the tetrahedral framework. The purpose of this demonstration is to connect the tetrahedral framework with RCA and demonstrate the feasibility of the entire sensor. Lanes 4-5 depict priming strands T1 and T2. Lanes 6-8 depict the formation of DNA circle 1, and Lanes 9-11 depict the formation of DNA circle 2. Lanes 6 and 9 depict the padlock strand, which has not yet formed a DNA circle. Lanes 7 and 10 depict the product of the ligation of the padlock strand with the primer strand. Due to its relatively long length, the primer strand can complementarily pair with the 5' and 3' ends of the padlock strand, thereby bringing the 5' and 3' ends of the padlock strand closer together. Lanes 8 and 11 depict the DNA circle generated by the combined action of T4 ligase, EXO III, and EXO I. When the primer brings the two ends of the padlock chain together, the lysine active site of T4 ligase nucleophilically attacks the α-phosphate group of ATP, releasing PPi and forming a T4 DNA ligase-AMP intermediate. The phosphate group at the 5' end of the padlock chain attacks the ligase-AMP intermediate, and APM is transferred to the 5' phosphate group of the padlock chain, forming a DNA-adenylate intermediate. Finally, the phosphate group at the 5' end of the padlock chain combines with the hydroxyl group at the 3' end to form a phosphodiester bond, simultaneously releasing AMP. This results in a double helix structure consisting of a DNA ring and primer. When EXO III and EXO I are added to the solution, EXO III gradually removes single nucleotides from the 3' end of the double helix, thereby cleaving the primer chain, while EXO I removes excess single-stranded DNA from the mixed solution. In Lanes 8 and 11, we observed that the padlock DNA circular structure (of the same base number) migrated faster than the padlock DNA single strand (Lanes 6 and 9). This result confirms that circular DNA migrates faster than linear DNA, confirming our successful formation of a DNA circular structure. Lanes 12-18 depict the opening of hairpins H1 and H2 on auxiliary strand S1 and the onset of RCA. In Lane 18, the RCA process generates two long DNA strands, resulting in an overly large nucleic acid structure and a low migration rate, leading to pore blockage. In summary, we successfully constructed the nucleic acid framework and initiated RCA.

[0031] like Figure 3In Figures A and B, TEM images show that individual AuNCs are less than 10 nm in size and exhibit corresponding lattice fringes, demonstrating the successful synthesis of the AuNC material. Figures C and D show transmission electron microscopy and scanning electron microscopy images of MRSA, respectively. The successful cultivation of MRSA provides material support for the detection of MRSA using our ECL sensor.

[0032] As shown in Figure 4, we obtained ECL signals related to bacterial concentrations by looking at the performance of bacteria at different concentrations in the sensor. Figure 4A As shown in Figure 2, with the continuous increase of bacterial concentration (10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 CFU / mL), the ECL signal intensity also continued to increase. Figure 4B The bacterial concentration (10 0 -10 7 CFU / mL) and ECL signal intensity showed a good linear relationship, with a correlation coefficient of R 2 The linear relationship between the two groups was 0.996, and the detection limit (LOD) reached 1 CFU / mL. The good linear relationship provides a basis for the quantitative detection of MRSA and fully demonstrates that the sensor has relatively excellent detection performance.

[0033] like Figure 5 Several other bacteria showed only low ECL signals, but the sensor generated a strong ECL signal when detecting MRSA and its bacterial mixture. This phenomenon indicates that the presence of MRSA in the sensor determines whether a strong signal is generated. 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.

[0034] 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 RCA nucleic acid amplification for MRSA detection was prepared by the following method: (1) Pretreatment of GCE electrodes; (2) Plating a gold film on the surface of the GCE electrode; (3) Modifying the nucleic acid tetrahedral framework onto the gold electrode surface; (4) The replacement solution of aptamer APT and MRSA is added to the surface of the gold electrode. The trigger chain T1 and trigger chain T2 in the solution respectively open the hairpin H1 and hairpin H2 on the nucleic acid tetrahedron framework; (5) Two pre-synthesized DNA circles, Phi29 polymerase, and dNTPs are added to the electrode surface. H1 and H2, with their sticky ends exposed, bind to the two DNA circles respectively and continuously consume dNTPs under the action of Phi29 polymerase, forming a long DNA chain with a repetitive sequence. (6) DNA-modified AuNCs are dropped onto the electrode surface.

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

3. The ECL sensor according to claim 1, wherein: Step (2) plating a gold film on the electrode surface refers to depositing gold on the GCE electrode surface by electro-reducing HAuCl4 solution.

4. The ECL sensor according to claim 1, wherein: Step (3) is to mix and anneal TND-1, TND-2, TND-3, TND-4, S1, hairpin H1 and hairpin H2 to form a nucleic acid tetrahedron framework, and then drop the nucleic acid tetrahedron framework onto the gold electrode, and the nucleic acid tetrahedron framework and the gold electrode are connected to each other through Au-S bonds; the sequences of TND-1, TND-2, TND-3, TND-4, S1, hairpin H1 and hairpin 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 ECL sensor according to claim 1, wherein: In step (4), the replacement solution of the aptamer APT and MRSA 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 concentration of the MRSA bacterial solution is 10 0 -10 7 CFU / mL; through the binding of MRSA to the aptamer APT, trigger chains T1 and T2 that are complementary to the base pairing of the aptamer APT are released, and a mixed solution containing T1 and T2 is obtained by centrifugation. The mixed solution is dropwise added to the electrode surface, and T1 and T2 respectively open the hairpins H1 and H2 on the tetrahedral framework; the sequences of the aptamer APT, trigger chain T1, and trigger chain T2 are SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 in the sequence listing, respectively.

6. The ECL sensor according to claim 1, wherein: Step (5) The synthesis method of the two DNA circles is as follows: Primer 1 and padlock 1 are mixed and annealed, T4 ligase is added and incubated, and then exonuclease EXO I and exonuclease EXO III are added to remove excess DNA structure, thereby generating DNA circle 1; Primer2 and padlock2 were mixed and annealed, and then T4 ligase was added and incubated. Then, exonuclease EXO Ⅰ and exonuclease EXO Ⅲ were added to remove excess DNA structure to obtain DNA circle 2; The hairpin H1 opened by T1 binds to the DNA circle 1 and continuously consumes dNTPs under the action of Phi29 polymerase to generate a long DNA chain 1 with a repetitive sequence; similarly, the hairpin H2 opened by T2 undergoes the same process with the DNA circle 2 to obtain a long DNA chain 2 with a repetitive sequence; the sequences of primer 1, primer 2, padlock 1 and padlock 2 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 ECL sensor according to claim 1, wherein: Step (6) First, amino-functionalized AuNCs are synthesized, and DNA chains Au-DNA1 and Au-DNA2 are wrapped on their surfaces to obtain DNA-modified AuNCs. The DNA-modified AuNCs are added dropwise to the electrode surface, and the electrode is placed in the dark for incubation. The sequences of Au-DNA1 and Au-DNA2 are SEQ ID NO. 15 and SEQ ID NO. 16 in the sequence listing, respectively.

8. The ECL sensor according to any one of claims 1 to 7, wherein: Prepared by the following method: (1) Polish the electrode surface with Al2O3 powder and clean the electrode surface by ultrasonic treatment in ultrapure water and ethanol; (2) Place the GCE electrode in a 25 mM HAuCl4 solution and electrochemically deposit for 60 s; (3) The thiol-modified TND-2, TND-3, and TND-4 were pretreated with 10 mM TCEP for 1 h, and then TND-1, TND-2, TND-3, TND-4, S1, H1, and H2 were mixed and annealed at 90°C to form a tetrahedral framework mixed solution; the mixed solution was fully mixed with 1 M NaCl solution, and 10 μL of the solution was pipetted and dropped onto the gold electrode surface, and incubated at 4°C overnight; the final concentration of the nucleic acid tetrahedral framework in the mixed solution was 1 μM, and Mg was 2. 2+ The final concentration was 10 mM; (4) 10 0 -10 7 CFU / mL of MRSA was incubated with the double helix structure formed by APT, T1, and T2 at 37°C for 1 h. The solution after displacement centrifugation was added dropwise to the electrode surface and incubated at 37°C for 2 h. (5) 10 μL of a mixed solution containing 1 μM DNA circle 1, 1 μM DNA circle 2, 1 mM dNTPs, and 0.1 U / μL phi29 polymerase was added to the electrode surface and incubated at 37°C for 2 h. 2+ The final concentration was 10 mM; (6) 10 μL of DNA-modified AuNCs was added to the electrode surface and incubated at 37°C in the dark for 2 h to maintain its Mg 2+ The final concentration is 10 mM.

9. Use of the ECL sensor based on RCA nucleic acid amplification according to any one of claims 1 to 8 in MRSA detection.

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