Clickable electrochemiluminescence biosensor based on 3D DNA walker and its application

Abstract

The application discloses a click-type electrochemiluminescence biosensor based on a 3D DNA Walker and application of the click-type electrochemiluminescence biosensor in preparation of a detection system for detecting myocardial infarction miRNA. The 3D DNA Walker accurately starts relative motion between particles, and the application realizes signal amplification for a click reaction connector. An ECL signal of AgNCs is located at the other end of alkyne DNA constituting a click reaction, and is used for super-sensitive detection of the ECL signal. The application uses simple and low-toxicity AgNCs as an ECL emitter, avoids a labeling process of the whole experiment, and is conducive to further sensing application. The combination of click chemistry and relative motion between particles of a 3D DNA Walker ECL sensing platform fully utilizes the relative motion between particles of the 3D DNA Walker and the advantages of the click chemistry reaction, so that myocardial miRNA can be stably and accurately detected in different real samples.

Description

Technical Field

[0001] This invention relates to the field of bioanalytical detection, specifically to a click-type electrochemiluminescence biosensor based on 3D DNA Walker and its application in the preparation of a detection system for detecting myocardial infarction miRNA. Background Technology

[0002] Acute myocardial infarction (AMI) is a serious cardiovascular disease. Prolonged ischemia can lead to myocardial tissue death and even heart failure, significantly impacting health. Therefore, early diagnosis and treatment of AMI are crucial. Current research indicates the presence of miRNAs associated with AMI in the circulatory system. These miRNAs are a class of non-coding single-stranded RNAs composed of approximately 22 nucleotides. They play a central regulatory role in gene expression by inhibiting mRNA translation or promoting mRNA degradation, and are involved in various biological processes such as cell differentiation, proliferation, apoptosis, and transformation, exhibiting high specificity and stability. Mature miRNAs can reach the complex blood environment through passive release or entry into lipoprotein complexes. Studies have confirmed that the levels of these miRNAs reaching the blood are extremely low, yet they are pathogens of various diseases. Therefore, establishing a stable and efficient biosensor platform for the accurate detection of miRNAs is imperative.

[0003] Electrochemiluminescence (ECL) technology combines the advantages of electrochemical and luminescent analysis, becoming the mainstream analytical method for detecting miRNAs in myocardial infarction (AMI) due to its low background, simple equipment, high sensitivity, and high controllability. However, using only ECL technology to construct a sensing platform without nucleic acid amplification methods for signal amplification results in relatively low sensor sensitivity. Therefore, it is necessary to combine electrochemiluminescence technology with nucleic acid amplification sensing principles for accurate analysis and detection of AMI-related miRNAs. However, quantitative reverse transcription polymerase chain reaction (RT-PCR) for accurate miRNA detection requires large amounts of sample and complex instruments with time-consuming procedures. To avoid the use of complex instruments and achieve continuous signal accumulation in a short time, cyclic miRNA analysis based on rolling loop amplification (RCA) reaction was proposed. Considering that the actual detection of rolling loop amplification of miRNA involves the participation of template and enzymes, it is prone to false positive signals and high background interference. Subsequently, enzyme-free cleavage hairpin self-assembly (CHA), hybridization chain reaction (HCR), and strand displacement amplification (SDA) have been developed as easy-to-operate nucleic acid amplification methods; however, they lack the sensitivity and specificity for detecting miRNAs in complex blood environments.

[0004] DNA walkers are typical dynamic DNA devices that use DNA, ATP, and other small molecules as fuel to move mechanically at the micro or nanoscale through biased Brownian motion. Three-dimensional DNA walkers (3D DNA Walkers) are a prime example. Due to their large surface-to-volume ratio and high DNA loading density, once the 3D DNA Walker, which drives the relative motion between particles, is activated by a target, the multi-walking strands in the walking particles (WPs) and the orbital strands on the orbital particles (TPs) bind synergistically. These strands are stable and resistant to derailment. Through continuous interaction between the walking particles (WPs) and orbital particles (TPs), large signal accumulation is achieved. Due to the high chemical stability, ease of synthesis, ease of modification, and low production cost of DNA, 3D DNA Walkers have been used in various sensing platforms, showing promising and emerging applications in nucleic acid detection.

[0005] Cu(I) (Cu reduced by AA) 2+ Cu(I)-catalyzed click chemistry is widely used in biosensor platforms due to its high selectivity, efficient ligation, and mild reaction conditions. Furthermore, compared to ligases, Cu(I)-catalyzed click chemistry offers lower production costs and achieves ligation efficiencies exceeding 92%. However, in practice, click reactions are limited to ligation and cannot be used for signal amplification, thus failing to meet the practical needs for sensitive detection of low-abundance miRNAs in blood. Summary of the Invention

[0006] To improve sensor performance, fully utilize the effective connection efficiency of click reactions, and ensure excellent signal amplification performance of the constructed biosensor, this invention utilizes a sandwich biosensing platform constructed by coupling click reactions with a 3D DNA Walker based on the relative motion between particles. This platform enables accurate identification and stable, efficient detection of miRNAs. Compared with conventional biosensing methods, this biosensing scheme significantly improves the detection efficiency and stability of myocardial miRNAs, providing more reliable approaches for the precise detection of miRNAs using ECL sensing platforms.

[0007] This invention designs and synthesizes a 3D DNA Walker with relative interparticle motion to achieve cascaded amplification of connectors. The connectors can also be used in sandwich sensing platforms constructed from click chemistry reactions with excellent stability and high connection efficiency, containing AgNCs at one end. AgNCs, as luminescent agents for ECL signals, possess low toxicity, good biocompatibility, and good stability, and have been widely used for detecting ECL signals from various organisms.

[0008] An ECL biosensor of the present invention is prepared by the following method: (1) pre-treatment of the electrode; (2) modification of the electrode surface with azido DNA (Azido-S3); (3) addition of MCH (thiol-polyethylene glycol SH-PEG) to prevent non-specific binding; (4) addition of walking particles WPs composed of thiol DNA (DNA-SH), walking strand, locking strand and AuNPs, and orbital particles TPs composed of thiol DNA (DNA-SH), hairpin strand Si-Hp(rA) and AuNPs, mixing WPs and TPs and adding miRNA to construct a 3D DNA molecular machine (3D DNA Walker) for relative movement between particles to amplify the signal of the connector (S1); (5) addition of alkynyl DNA (Alkynyl-S2) and catalyst Cu. 2+ (6) A click chemical reaction with a sandwich structure occurs; (7) AgNO3 solution is dropped onto the surface of the modified electrode and then reduced with NaBH4 to obtain the modified electrode, which is then combined with other electrodes to form an ECL biosensor.

[0009] As a preferred embodiment, in the ECL biosensor described above, step (1) pretreatment includes polishing, cleaning, and drying.

[0010] As a preferred embodiment, in the ECL biosensor described above, step (2) of modifying the electrode surface refers to incubating Azido-S3 on the electrode.

[0011] As a preferred embodiment, in the ECL biosensor described above, step (2) involves first electrochemically depositing HAuCl4 onto the electrode surface and then incubating Azido-S3 on the electrode.

[0012] As a preferred embodiment, in the aforementioned ECL biosensor, in step (4), the walking particles (WPs) are formed by annealing thiol DNA, walking strand, locking strand, and AuNPs at 90°C for 10 minutes, and the orbital particles (TPs) are formed by annealing thiol DNA, Si-Hp(rA), and AuNPs at 90°C for 10 minutes. After mixing, miRNA is added, followed by Mg. 2+ After incubation for 24 hours, centrifuge and drop the supernatant onto the electrode surface. In this step, thiol DNA (DNA-SH) can be first modified onto AuNPs to form Au@DNA-SH.

[0013] As a preferred embodiment, in the ECL biosensor described above, in step (5), Alkynyl-S2 is annealed and dropped onto the electrode surface, while Cu is simultaneously added. 2+ Incubate with sodium ascorbate (AA) for 3 hours.

[0014] As a preferred embodiment, in the ECL biosensor described above, step (6) involves first dropping AgNO3 solution onto the modified electrode surface and incubating it in the dark, and then dropping freshly prepared NaBH4 solution onto the electrode surface and incubating it in the dark.

[0015] In this invention, the azide DNA (Azido-S3) sequence was designed by the author and is SEQ ID NO.1 in the sequence listing, as follows:

[0016] 5'-TTTTTCGAATAGTCACGACAG-3'.

[0017] Step (3) MCH is mercapto polyethylene glycol SH-PEG.

[0018] Preferably, the thiol DNA, walking strand, locking strand, and hairpin strand Si-Hp(rA) in step (4) are self-designed sequences, as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 in the sequence listing:

[0019] Thiol DNA (DNA-SH): 5'-CTGATAAGCTACAGGACATTTTTTTTTT-3'.

[0020] walking strand: 5'-TGTCCTGTAGCTTATCAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGAACCAAATTCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'

[0021] locking strand: 5'-AAGAGAATTTGGTTCCATTTACCAGCT-3';

[0022] Si-Hp(rA): 5'-TGTCCTGTAGCTTATCAGTCGAATAGTCTTTTTTGAGCGACACACTATrAGGAAGAGATACTGTCGTGACTATTCGA-3'

[0023] rA is the cleavage site, an adenine ribonucleotide. A sequence S1 is embedded in the hairpin Si-Hp(rA) as a specific connector for subsequent click chemistry reactions.

[0024] In step (5), the alkyne DNA (Alkynyl-S2) and the connector (S1) dropped onto the electrode surface have self-designed sequences, namely SEQ ID NO.6 and SEQ ID NO.7 in the sequence listing.

[0025] Alkynyl-S2: 5'-GTATCTCTTCCTTTTTTTTTTTTTTTGTCTCCCCCCCCCCCCCCC-3'

[0026] S1: 5'-GGAAGAGATACTGTCGTGACTATTCGA-3'

[0027] As a preferred embodiment, the ECL biosensor described above can be prepared using the following method:

[0028] (1) Polish the electrode with alumina paste, rinse it thoroughly with ultrapure water, and then sonicate it in ethanol and water and dry it at room temperature;

[0029] (2) Place the electrode in the electrolyte (25mM HAuCl4), perform electrochemical deposition for 60s, and then add 10μL of 3μM DNA with azide functional groups (Azido-S3) to the electrode and incubate for 4h-6h.

[0030] (3) Add 1 mM MCH and incubate for half an hour;

[0031] (4) Anneal thiol DNA, walking strand, locking strand and AuNPs at 90℃ for 10 minutes to form walking particles WPs, anneal thiol DNA, Si-Hp(rA) and AuNPs at 90℃ for 10 minutes to form orbital particles TPs, mix and add miRNA, then add MgAc2 and incubate for 24 hours, centrifuge, take the supernatant and drop it on the electrode surface for incubation.

[0032] (5) Add 10 μL of 3 μM DNA with alkynyl functional groups (Alkynyl-S2) and 1 μL of 20 mM Cu 2+ 2 μL of 20 mM AA was dropped onto the electrode surface and incubated for 3 hours.

[0033] (6) Drop 8 μL of 100 μM AgNO3 solution onto the modified electrode surface and incubate in the dark for 30 minutes. Then, drop 8 μL of freshly prepared 100 μM NaBH4 solution onto the electrode surface and incubate in the dark at ambient temperature for 2 hours. Finally, a modified electrode with DNA template silver nanoclusters DNA-AgNCs is obtained. This electrode can be combined with other electrodes to form a commonly used two-electrode or three-electrode system for ECL measurement.

[0034] This invention relates to the field of bioanalytical detection, specifically to the ECL biosensor and its application in the preparation of a detection system for detecting myocardial infarction miRNA.

[0035] The principle of this invention is analyzed as follows:

[0036] 1. Basis for the design of cascaded signals for the 3D DNAWalker connector (S1) based on inter-particle relative motion

[0037] In the extremely complex blood environment, circulating miRNAs exhibit low abundance but can stably exist within the blood environment with high specificity. A 3D DNA Walker was designed and synthesized to trigger relative movement between particles, thereby amplifying the cascade signal of the connector (S1). The miRNA acts as the key to triggering the relative movement of the 3D DNA Walker. First, inactive walking particles (WPs) and orbital particles (TPs) with cleavage sites were designed. Walking particles (WPs) consist of 18 nM AuNPs and a locking DNA strand and a walking DNA strand. Orbital particles (WPs) consist of 18 nM AuNPs and a hairpin DNA strand (Si-Hp(rA)) with cleavage sites. A carefully designed, locked walking machine composed of hairpin DNA strands with cleavage sites can precisely capture miRNAs. The captured miRNAs act as the key to activate the walking machine. Once the walking machine (WP) is activated, the multi-walking strands on the WP synergistically bind to the hairpin DNA (Si-Hp(rA)) with cleavage sites on the track machine. Through continuous interactions between WPs and TPs, continuous accumulation of S1 signals is achieved. Secondly, to improve the specificity of the inactive walking machine (WP), mismatched miRNAs are used as the key to activate the WP, and the sequence of the locked walking strands is adjusted to avoid binding of highly similar miRNAs to the locked strands, thus preventing false positive signals caused by the activation of walking particles. Finally, a large number of connectors (S1) are used for subsequent click chemistry reactions, forming a stable sandwich structure for a stable and efficient ECL sensing platform for the detection of miRNA (miRNA-133a).

[0038] 2. Basis for the study of 3D DNA Walker's capture of myocardial infarction miRNAs and connector signal amplification

[0039] Based on the design concept of using a specific miRNA (miRNA-133a) as the initiator and lock-on walking machine (WP) and orbital machine (TP), a connector (S1) was constructed to specifically capture myocardial infarction miRNA-133a, thereby initiating the amplification of two types of nanomachine particles (WPs and TPs) for subsequent research. Using a database (http: / / www.mirbase.org), miRNA-133a was screened, and using miRNA-133a as the analysis object, the initiation and signal amplification effects of the 3D DNA Walker involving relative motion between particles were investigated.

[0040] 3. The basis for designing a stable sandwich structure based on click chemical reactions.

[0041] Cu(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC), a revolutionary bioorthogonal click chemistry reaction, is a powerful tool for linking two ssDNAs (Azido-S3 and Alkynyl-S2) modified with azide and alkyne, respectively. However, the [3+2] cycloaddition products formed by click chemistry lack signal amplification, making it difficult to meet the practical needs of low-abundance miRNA-133a. Therefore, a 3D DNA Walker (WP, TP) with relative interparticle motion is introduced into the click chemistry reaction. In this case, a sequence (S1) is embedded in the hairpin Si-Hp(rA) with a cleavage site rA on the track machine (TP) as a specific connector for the click chemistry reaction. Multiple walking strands on the surface of WPs cleave the Si-Hp(rA) hairpin on the surface of TPs, and the cleaved Si-Hp(rA) hairpin generates two DNA strands, one of which is the connector S1.

[0042] When miRNA-133a is present, the relative motion of WPs and TPs is initiated to generate a large number of connectors S1, which are used to connect stabilizers of the [3+2] cycloaddition products of click chemistry, thereby constructing a stable sandwich sensing platform composed of click chemistry.

[0043] 4. Basis for DNA templated AgNCs design

[0044] AgNCs were synthesized by reducing AgNO3 with NaBH4 as a reducing agent, leading to the aggregation of AgNCs and the formation of large nanoparticles. These nanoparticles could then be naturally integrated into DNA through a designed synthetic sequence. Click chemistry used an alkynyl-S2 DNA with multiple C-sequences at one end to immobilize ssDNA polymers, which were then reduced in situ to generate ECL luminescent AgNCs. In the ECL detection system, the modified electrode was prepared in PBS containing 0.05 M K2S2O8 and 0.1 M KCl. K2S2O8 served as a co-reactant for the AgNCs luminescent group, and KCl enhanced the conductivity of the reaction. The ECL intensity continuously increased with increasing miRNA-133a concentration.

[0045] The continuous signal amplification performance coupling of a three-dimensional molecular machine with relative interparticle motion exhibits excellent connection efficiency, highly selective click chemistry, and low toxicity and good biocompatibility, making silver nanoclusters suitable for the efficient detection and specific capture of myocardial infarction miRNA-133a. Compared with the traditional sandwich-based ECL biosensing platform, the ECL biosensing platform based on the relative interparticle motion of a 3D DNA Walker coupled with click chemistry achieves accurate identification and stable and efficient detection of miRNA, greatly improving the stability of the ECL sensing platform and providing more reliable approaches for the accurate detection of miRNA using ECL sensing platforms. This invention achieves signal amplification for the click reaction connector by synthesizing a 3D DNA Walker with relative interparticle motion that is accurately initiated by the target miRNA, while the ECL signal of AgNCs is located at the other end of the alkyne DNA constituting the click reaction, enabling ultrasensitive detection of the ECL signal.

[0046] This invention firstly expands the contact area of ​​the biosensing platform by using an electrochemically deposited thin layer of Au as a substrate, while avoiding the use of AuNPs and simplifying the experimental steps for synthesizing AuNPs. Secondly, it synthesizes simple and low-toxicity silver nanoclusters (AgNCs) as ECL luminescent groups, avoiding the labeling process throughout the experiment and facilitating their further sensing applications (AgNCs are generally no larger than 10 nanometers, while silver nanoparticles (AgNPs) are generally tens to hundreds of nanometers in size). Finally, by coupling the relative motion of 3D DNA Waller particles with a click chemical reaction, an ECL biosensor with excellent stability and high sensitivity was successfully constructed for the detection of myocardial infarction miRNA-133a, with a wide detection range from 100 aM (angomolar concentration) to 1 nM (nanomolar concentration) and a detection limit as low as 32.34 aM. Attached Figure Description

[0047] Figure 1A schematic diagram illustrating the principle of using 3D DNA Waller coupling click chemistry to detect miRNA based on the relative motion between particles;

[0048] Figure 2 The feasibility of the 3D DNAWaller amplification and click chemistry reaction design scheme based on the relative motion between particles was verified by gel electrophoresis experiment.

[0049] Figure 3 TEM plots of WPs and TPs running at different time periods;

[0050] Figure 4 Comparison of analytical capabilities for miRNAs in serum and buffer solutions from 10% of patients with acute myocardial infarction.

[0051] Figure 5 The graph shows the relationship between miRNA-133a concentration and ECL intensity, as well as the linear relationship.

[0052] Figure 6 The ECL strength was evaluated to assess the connectivity efficiency and stability of efficient biosensing platforms consisting of and without click chemistry reactions. Detailed Implementation

[0053] Using the mirbase database, myocardial infarction-related miRNAs (miRNA-133a) were screened out as the analysis targets. These miRNAs were provided by Suzhou Gemma Gene Co., Ltd., and their sequences are SEQ NO.8 in the sequence listing, as follows:

[0054] miRNA-133a:AGCUGGUAAAAUGGAACCAAAU

[0055] Example 1

[0056] 3D DNA Walker design and synthesis.

[0057] First, 6 μL of 100 μM thiol DNA (DNA-SH), annealed at 90 °C for 10 minutes, was added to 835 μL of 1.01 nM AuNPs (18 nm particle size). Simultaneously, 100 μL of 1% Tween 20 and 80 μL of 0.025% citrate were added, and the mixture was incubated for 6 hours. (A base sequence of DNA-SH is complementary to the walking strand and Si-Hp(rA). Longer walking strands and Si-Hp(rA) can indirectly modify AuNPs, reducing experimental costs.) Then, 20 μL of 0.5 M citrate was added to the mixture, and incubation continued for 3 hours. After this series of steps, 20 μL of 0.3 M NaCl was added to the mixture every 30 minutes to prevent AuNP aggregation. A total of 72 hours was required to ensure that DNA-SH was completely modified onto AuNPs to form Au@DNA-SH. Subsequently, Au@DNA-SH was centrifuged, and the supernatant (containing successfully modified WPs and TPs) was collected. Unmodified DNA-SH was removed, and the mixture was divided into two equal portions, A and B. 1 μL of 100 μM Walking strand, 1 μL of 100 μM Locking strand, and 5 mM MgAc2 were added to solution A; 1 μL of 100 μM Si-HP(rA) was added to solution B. Solutions A and B were annealed at 90°C for 10 minutes to form walking particles (WPs) (Au@DNA-SH@Walking strand@locking strand) and orbital particles (TPs) (Au@DNA-SH@Si-HP(rA)). Finally, the annealed solutions A and B were mixed, and miRNA-133a and 5 mM MgAc2 were added (to initiate WPs and TPs). The mixture was incubated at 37°C for 24 hours to form a large number of linker S1s, constructing a 3D DNA Walker with relative interparticle movement. The mixture was then centrifuged for later use. Because the multiple walking chains of the walking particles TPs cleave the hairpin chains Si-Hp(rA) of the orbital particles, the cleavage is completed after incubation at 37°C for 24 hours, generating a large number of connectors S1 (S1 is formed by the walking chains of WPs cleaving the Si-Hp(rA) of TPs). TEM images on a 10% (w / w) polyacrylamide gel and of WPs and TPs running at different time points confirm the successful synthesis of the 3D DNA Walker with relative interparticle motion.

[0058] Click chemical reactions to design and synthesize stable sandwich structures.

[0059] First, polish the GCE electrode (glassy carbon electrode) with alumina paste. Then, thoroughly rinse the GCE with ultrapure water and sonicate it in ethanol and water, followed by drying and cleaning at room temperature. Electrochemically deposit the GCE electrode in an electrolyte solution (25 mM HAuCl4) for 60 seconds, allow it to dry, and then add 10 μL of 3 μM Azido-S3 to the electrode and incubate for 4-6 hours. Discard the solution on the electrode, add 1 mM of purchased MCH, and incubate for half an hour to prevent non-specific binding. Take the supernatant from the previous centrifugation step (containing a large amount of connector S1) and drop it onto the electrode surface for incubation. Simultaneously add 10 μL of 3 μM annealed Alkynyl-S2 and 1 μL of 20 mM Cu. 2+ 2 μL of 20 mM sodium ascorbate (AA) and 2 μL of 10 mM Mg 2+ Click reaction occurs after co-incubation for 3 hours. The solution on the electrode is discarded, and 8 μL of 100 μM AgNO3 solution is dropped onto the modified electrode surface. Incubation is performed in the dark for 30 minutes. Then, 8 μL of 100 μM freshly prepared NaBH4 solution is dropped onto the electrode surface and incubated in the dark at ambient temperature for 2 hours. Finally, a modified electrode with DNA template silver nanoclusters DNA-AgNCs is obtained, which can be used for ECL measurement. ECL measurement can be performed using three electrodes: a platinum wire electrode, an Ag / AgCl reference electrode, and a glassy carbon electrode. The modifier is applied to the glassy carbon electrode. Measurement is performed in PBS with 0.05 M K2S2O8 and 0.1 M KCl electrolyte, an 800 V ECL photomultiplier tube, and a scan rate of 0.1 s from -0.6 to 0.2.

[0060] like Figure 2 The image shows the analysis of DNA structure using polyacrylamide gel electrophoresis (PAGE).

[0061] Figure 2In section A: 6% PAGE analysis of 3D DNA Walker. Well 1, Walking strand; Well 2, Locking strand; Well 3, miRNA-133a; Well 4, Walking strand + Locking strand; Well 5, Locking strand + miRNA-133a; Well 6, Si-Hp(rA); Well 7, S1; Well 8, Walking strand + Locking strand + Si-Hp(rA); Well 9, Walking strand + Locking strand + miRNA-133a + hairpin Si-Hp(rA) with cleavage site; Well 10, Walking strand + Locking strand + miRNA-133a + hairpin Si-Hp without cleavage site; Well 11, Walking strand + Si-Hp(rA); Well 12, Walking strand + Si-Hp.

[0062] Figure 2 In section B: 10% PAGE analysis of the chemical reaction. Channels 1-8 are respectively: Alkynyl-S2, Azido-S3, S1, Alkynyl-S2+S1, Azido-S3+S1, Alkynyl-S2+Azido-S3, Alkynyl-S2+, Azido-S3+S1, Alkynyl-S2+Azido-S3+S1+ catalyst.

[0063] like Figure 2 As shown in Figure A, when miRNA-133a was introduced into lane 4, a new band was observed in lane 5, demonstrating that miRNA-133a can undergo a strand displacement reaction with the locking strand. Similarly, the new band in lane 5 also appeared in lanes 9 and 10, further indicating the feasibility of the miRNA-133a strand displacement reaction with the locking strand. Lane 7 is the location of S1. Compared to lane 10, a new band appeared in lane 9, and this new band is located in the same position as in lane 7, demonstrating that the relative movement of WPs and TPs successfully produced the connector S1. Figure 2As shown in Figure B, Alkynyl-S2 and Azido-S3, when mixed with S1, produced clear bands in channels 4 and 5, demonstrating their mutual reaction. When the catalyst, Azido-S3, Alkynyl-S2, and S1 were added to channel 8, a distinct new band appeared; this new band represents a sandwich structure formed by a click chemistry reaction. In contrast, without the catalyst (channel 7), only a sandwich structure formed by the hybridization of three DNA molecules was observed, and no new band was detected. These results indicate that the designed biosensor was successfully assembled.

[0064] like Figure 3 The image shows TEM images of WPs and TPs operating at different time points. From 0 to 15 hours, the relative movement of WPs and TPs produces dimers and trimers. From 18 to 24 hours, the cleavage reaction of WPs and TPs proceeds slowly or even stops, and WPs and TPs slowly separate.

[0065] like Figure 4 To evaluate the practical application of this method, the analytical capability of miRNA in the serum of 10% of patients with acute myocardial infarction (Ma'anshan People's Hospital) was studied. As shown in the figure, the analytical results in the patient serum sample were largely consistent with those in the Tris buffer (a buffer solution for miRNA-133a) (the left column in the figure is the buffer, and the right column is the serum), indicating that the designed sensor platform has good anti-interference performance. It also demonstrates good analytical performance for miRNA in real patient serum.

[0066] like Figure 5 The graph shows the relationship between miRNA-133a concentration and ECL intensity, as well as the linear relationship. Adding a series of miRNA-133a concentrations ranging from 100aM to 1nM can significantly enhance ECL intensity, resulting in a linear relationship between ECL intensity and the logarithm of miRNA-133a concentration.

[0067] like Figure 6 The ECL strength of the high-efficiency biosensing platforms constructed with and without click chemistry was used to evaluate the connectivity efficiency and stability. By randomly selecting 20 points for ECL strength, the RSD of the ECL sensing platform constructed with click chemistry was only 1.73%, while the relative standard deviation of the sensing platform without click chemistry was 5.53%, demonstrating the superior connectivity efficiency and stability of the click-chemistry-constructed sensing platform.

[0068] It should be noted that the above-described technical content of this invention is merely an explanation and clarification to enable those skilled in the art to understand the technical essence of this invention, and therefore is not intended to limit the scope of protection of this invention. The scope of protection of this invention should be determined by the claims. Those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made based on the essential spirit of this invention should be within the scope of protection of this invention.

Claims

1. A clickable electrochemiluminescence biosensor based on 3D DNA Walker, obtained by a method comprising the following steps: (1) Pretreatment electrode; (2) The azide DNA, namely Azido-S3, is modified onto the electrode surface; the Azido-S3 sequence is SEQ ID NO.1 in the sequence listing; (3) Add MCH to prevent nonspecific binding; (4) Mix walking particles (WPs) and orbital particles (TPs) and add miRNA to construct a 3D DNA Walker with relative interparticle motion to generate a connector S1; the walking particles (WPs) are formed by annealing thiol DNA, walking strand, locking strand and AuNPs, and the orbital particles (TPs) are formed by annealing thiol DNA, Si-Hp(rA) and AuNPs; the sequences of thiol DNA, walking strand, locking strand and Si-Hp(rA) are SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, SEQ ID NO. 5 in the sequence listing, respectively; (5) Add alkynyl DNA (Alkynyl-S2), connector S1, and catalyst Cu. 2+ Sodium ascorbate (AA) and sodium ascorbate (AA) are jointly modified on the electrode surface, resulting in a click chemical reaction with a sandwich structure; the Alkynyl-S2 and connector S1 sequences are SEQ ID NO. 6 and SEQ ID NO. 7 in the sequence listing; (6) Drop AgNO3 solution onto the surface of the modified electrode, then reduce it with NaBH4 to obtain the modified electrode, and combine it with other electrodes to form an ECL biosensor.

2. The biosensor as described in claim 1, characterized in that, Step (1) Pretreatment includes polishing, cleaning and drying.

3. The biosensor as described in claim 1, characterized in that, Step (2) Modifying the electrode surface refers to incubating Azido-S3 on the electrode.

4. The biosensor as described in claim 1, characterized in that, Step (2) First, electrochemically deposit HAuCl4 and then modify the electrode surface with Azido-S3.

5. The biosensor as described in claim 1, characterized in that, In step (4), the walking particles (WPs) are formed by annealing thiol DNA, walking strand, locking strand, and AuNPs at 90°C for 10 minutes, and the orbital particles (TPs) are formed by annealing thiol DNA, Si-Hp(rA), and AuNPs at 90°C for 10 minutes. The two types of particles formed by annealing are mixed and then miRNA is added, followed by the addition of Mg. 2+ Centrifuge after 24 hours of incubation.

6. The biosensor as described in claim 1, characterized in that, Step (5) involves annealing Alkynyl-S2, connector S1, and Cu. 2+ Sodium ascorbate AA was dropped onto the electrode surface and incubated for 3 hours.

7. The biosensor as described in claim 1, characterized in that, Step (6) First, drop AgNO3 solution onto the modified electrode surface and incubate it in the dark. Then, drop freshly prepared NaBH4 solution onto the electrode surface and incubate it in the dark.

8. The biosensor as described in claim 1, characterized in that, It was prepared using the following method: (1) Polish the electrode with alumina paste, rinse it thoroughly with ultrapure water, and sonicate it in ethanol and water, then dry it at room temperature; (2) Place the electrode in an electrolyte containing 25 mM HAuCl4 and electrochemically deposit for 60 s. Then add 10 μL of 3 μM DNA with azide functional groups, namely Azido-S3, and incubate on the electrode for 4-6 h. (3) Add 1 mM MCH and incubate for half an hour; (4) Anneal thiol DNA, walking strand, locking strand and AuNPs at 90°C for 10 minutes to form walking particles WPs, anneal thiol DNA, Si-Hp(rA) and AuNPs at 90°C for 10 minutes to form orbital particles TPs, mix and add miRNA, then add MgAc2 and incubate at 37°C for 24 hours and centrifuge to generate a large number of connectors S1, take the supernatant and drop it on the electrode surface for incubation; (5) Add 10 μL of 3 μM alkynyl-S2 DNA and 1 μL of 20 mM Cu 2+ 2 μL of 20 mM sodium ascorbate (AA) and 2 μL of 10 mM Mg 2+ Drop them onto the electrode surface and incubate for 3 hours; (6) Drop 8 μL of 100 μM AgNO3 solution onto the modified electrode surface and incubate in the dark for 30 minutes. Then drop 8 μL of 100 μM freshly prepared NaBH4 solution onto the above electrode surface and incubate in the dark for 2 hours to obtain the modified electrode. Combine it with other electrodes to form an ECL biosensor.

9. The use of the click-type electrochemiluminescence biosensor based on any one of claims 1-8 in the preparation of a detection device for detecting myocardial infarction miRNA-133a.

10. The application as described in claim 9, characterized in that, The miRNA-133a sequence is SEQ ID NO.8 in the sequence listing.

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