Electrochemical sensor based on functionalized DNA cube, construction method of electrochemical sensor and application of electrochemical sensor in exosome miRNA detection

Abstract

The invention discloses an electrochemical sensor based on a CHA functionalized DNA cube in the technical field of electrochemical sensor detection, the electrochemical sensor comprises a signal probe Pt (at) AuNPs (at) HRP (at) DNA and the functionalized DNA cube which are combined on a glassy carbon electrode, and the signal probe is obtained by synthesizing Pt (at) AuNPs by adopting gold nanoparticles, then performing SA functionalization, and reacting with Bio-HRP and Bio-DNA. The functionalized DNA cube is obtained by self-assembling four ssDNAs into a wireframe DNA cube and incubating the wireframe DNA cube with a capture probe and a hairpin probe. When the target exosome miRNA exists, an efficient CHA reaction is generated, target circulation is rapidly transferred among cubic nodes, a large number of H1H2 double-stranded structures are formed, free single-stranded sequences are exposed to be combined with a signal probe, H2O2 is catalyzed to generate a remarkable electrochemical signal, and the probe is used for rapid and stable detection of the exosome miRNA.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical sensor detection, and particularly relates to an electrochemical sensor based on functionalized DNA cubes, a construction method thereof, and an application thereof in exosome miRNA detection. Background Art

[0002] Cervical cancer (CC) is the fourth most common cancer in women worldwide. Late-stage cervical cancer is one of the leading causes of cancer death in women. Early screening of CC is important for identifying cervical intraepithelial neoplasias (CIN) to prevent it from developing into invasive cancer. Papanicolaou test, Thinprep cytologic test (TCT), and human papillomavirus (HPV) infection screening have significantly reduced the incidence of cervical cancer. In recent years, exosomal miRNAs have been found to be of great significance in the diagnosis, treatment, and prognosis monitoring of cervical cancer and are considered to be potential biomarkers. Exosomal miRNAs are one of the main components of exosomes. Compared with the complex mechanism of long non-coding RNA (lncRNA) and the unstable characteristics of mRNA, the content of miRNAs in exosomes is high and relatively stable, making them ideal biomarkers for the diagnosis and monitoring of cervical cancer. The latest research shows that exosomal miRNA-30d-5p and let-7d-3p are valuable diagnostic biomarkers for cervical cancer and precancerous lesions, and the accurate and rapid detection of these two exosomal miRNAs is of great significance. However, due to the short sequence, high homology and low abundance of miRNAs, there are still some challenges in the accurate quantitative detection of miRNAs.

[0003] At present, a variety of detection methods have been successfully applied to the detection of exosomal miRNAs, including RT-qPCR, fluorescent sensors, colorimetry, SERS, etc. The RT-qPCR method is the gold standard for miRNA detection, but it requires cumbersome primer design and sample processing procedures, strict environmental and professional personnel requirements, which limits its usability. Fluorescent sensors are simple to operate, but the additional fluorescent labeling is costly and has low sensitivity, making it difficult to detect low levels of exosomal miRNAs. SERS is prone to cross-interference between signals, resulting in high background signals. Therefore, it is of great clinical significance to develop a simple detection strategy with high specificity and sensitivity to detect exosomal miRNAs.

[0004] Isothermal amplification is a new type of in vitro nucleic acid amplification technology developed after PCR. It can achieve target amplification and detection at a constant temperature, simple conditions, or even in complex biological environments. Thanks to the high programmability of nucleic acids and the high specificity of base complementary pairing, researchers have designed various DNA isothermal amplification technologies, including CHA, HCR, RCA, SDA, LAMP, etc. In recent years, catalytic hairpin self-assembly (CHA) has been widely studied because dsDNA products containing hanging free single-stranded parts are easier to capture and generate enhanced electrochemical signals, low cost, high specificity, and simple operation. CHA uses an enzyme-free isothermal signal amplification strategy to produce hundreds of times of amplification products. By converting analyte recognition events into electrochemical signal detection modes, a variety of miRNAs detection strategies have been established. However, CHA has the following shortcomings for electrochemical detection: (1) The CHA reaction relies on the random collision of free hairpins, with low reaction efficiency and long reaction time, which brings certain difficulties to detection; (2) In the electrode reaction with ssDNA as the capture probe, the nucleic acid chains are entangled and crowded with each other, and the reaction efficiency is low.

[0005] Combining the excellent performance of FNAs with the advantages of electrochemical technology has opened up a new path for the detection of exosomal miRNAs. Various electrochemical biosensors have been developed based on FNAs electrochemical probes on the interface for high-sensitivity and specific detection of nucleic acids, small molecules, proteins and cells, which has significantly enhanced the molecular recognition on the electrochemical biosensing interface. DNA tetrahedrons can be firmly anchored on the electrode to avoid entanglement between probes, and the local aggregation of self-assembled structures can resist nonspecific adsorption. Combined with electrochemical methods, DNA tetrahedrons have been used for the detection of DNA, miRNAs, proteins and CTCs. Recently, researchers have proposed a localized DNA tetrahedron-assisted CHA strategy, which uses DNA nanowires to confine tetrahedron-CHA in a compact space, accelerates CHA through DNA tetrahedrons, and creates a precise control space for each DNA tetrahedron-CHA to reduce the leakage of local reactions. It is used for exosomal miRNA detection and shows excellent sensitivity. Some researchers have also designed a dual-engine-triggered ECL detection strategy using a DNA cube as a scaffold, a double swing arm fixed on it as an engine, and a track as a signal switch, which effectively improved the operational efficiency and controllability of the DNA walker. Although these detection strategies have achieved efficient detection of exosomal miRNAs, there are still the following shortcomings that need to be improved: (1) There are many reaction steps and the operation is cumbersome; (2) Electrochemical biosensors for multiple detections should be further explored; (3) The time-consuming process of multiple metastable hairpin-driven amplification limits their practical application. Summary of the invention

[0006] The present invention aims to provide an electrochemical sensor based on functionalized DNA cubes and a construction method thereof, and the electrochemical sensor is used for the detection of exosome miRNA to solve the problems of the existing exosome miRNA detection technology, which generally has many reaction steps, cumbersome operation and long time consumption.

[0007] In a first aspect, a method for constructing an electrochemical sensor in the present application comprises the following steps:

[0008] S1. Preparation of signal probe Pt@AuNPs@HRP@DNA

[0009] S1-1, using gold nanoparticles AuNPs and H2PtCl6 as the main components to synthesize platinum nanoparticles Pt@AuNPs;

[0010] S1-2, after the Pt@AuNPs are SA-functionalized, they are mixed with Bio-HRP and Bio-DNA to react at room temperature, the reaction product is washed, and resuspended with PBST buffer to obtain the signal probe Pt@AuNPs@HRP@DNA;

[0011] S2. Preparation of CHA-functionalized DNA cubes

[0012] S2-1, using 4 ssDNA strands as the main skeleton to self-assemble into a wireframe DNA cube;

[0013] S2-2, mixing the wireframe DNA cube, the capture probe CP, the hairpin probe H1 and the hairpin probe H2 and incubating them to obtain a functionalized DNA cube combined with the capture probe CP;

[0014] S3. Construction of electrochemical sensor

[0015] The functionalized DNA cube solution described in S2-2 is dripped onto the purified glassy carbon electrode GE for incubation, and then washed and reacted with BSA buffer at room temperature to block the nonspecific sites on the GE surface, and then the target let-7d-3p or miRNA-30d-5p is added for reaction, and then the signal probe Pt@AuNPs@HRP@DNA solution is dripped onto the GE surface, and dried after the reaction to obtain the electrochemical sensor;

[0016] Among them, the nucleotide sequences of the four ssDNAs are shown as SEQ ID NO.1 to SEQ ID NO.4 respectively; the nucleotide sequence of the CP is shown as SEQ ID NO.5; the nucleotide sequence of let-7d-3p is shown as SEQ ID NO.6; and the nucleotide sequence of miRNA-30d-5p is shown as SEQ ID NO.7.

[0017] Furthermore, the concentrations of the Bio-HRP and Bio-DNA are: Bio-HRP 1 mg / mL, Bio-DNA 100 μM.

[0018] Furthermore, the capture probe CP is first used to activate the thiol group with tris(2-carboxyethyl)phosphine hydrochloride.

[0019] Furthermore, the reaction time of the target in S3 is 60 min, and the reaction time of the signal probe is 30 min.

[0020] In a second aspect, the present application also provides a material for improving the detection sensitivity of an electrochemical sensor, wherein the material comprises the signal probe Pt@AuNPs@HRP@DNA or / and the functionalized DNA cube as described in the first aspect.

[0021] Thirdly, the electrochemical sensor constructed using the above method showed excellent sensitivity for exosomal miRNA detection.

[0022] Electrochemical biosensors have become a promising area of ​​research and development for the detection of exosomal miRNAs due to their high sensitivity, rapid and simple detection, instrument miniaturization, and cost-effectiveness. Compared with other methods, electrochemical methods are easier to set up integrated systems, making them more suitable for POCT.

[0023] Working principle of the present invention: The present invention uses CHA-functionalized DNA cubes combined with Pt@AuNPs@HRP nanozyme labels to research and develop a highly sensitive electrochemical sensor for the detection of exosomal miRNA. During the assembly process of the CHA-functionalized DNA cube, the first prepared wireframe DNA cube is assembled from 4 ssDNA single strands in one step, with 8 single-stranded domains that can specifically bind to ssDNA. The single-stranded domains on the upper surface of the wireframe DNA cube are combined with the CHA response element H1 and H2 hairpin probes to assemble into a functionalized DNA cube. And it is fixed to the surface of the gold electrode (GE) through the thiol modified at the bottom node.

[0024] Electrochemical detection principle: Pt@AuNPs are synthesized on the basis of AuNPs, and then ssDNA and HRP are connected through SA functionalization and biotin to form Pt@AuNPs@HRP@DNA as a signal probe. The DNA in the signal probe binds to the free single-stranded sequence exposed by the CHA reaction product on the functionalized DNA cube. Pt@AuNPs@HRP has efficient peroxidase activity and can reduce H2O2 to produce a strong electrochemical signal. The functionalized DNA cube is fixed on the GE through a gold-thiol bond. The presence of exosomal miRNA can activate the efficient CHA reaction within the confinement of the functionalized DNA cube framework, and quickly transfer between cube nodes through target circulation to form a large number of H1H2 double-stranded structures, exposing free single-stranded sequences, and combining with the signal probe Pt@AuNPs@HRP@DNA, thereby catalyzing H2O2 to produce a significant electrochemical signal for the rapid and stable detection of exosomal miRNA.

[0025] Beneficial technical effects of the present invention:

[0026] 1. The present invention integrates CHA amplification elements on the basis of the wireframe DNA cube framework and combines Pt@AuNPs@HRP@DNA signal probe to construct an electrochemical biosensor for exosomal miRNA detection. By utilizing confined space to greatly improve the amplification efficiency of CHA and synthesize complex nanozyme probes with efficient catalytic performance, the detection sensitivity of the sensor is jointly improved.

[0027] 2. The presence of DNA cubes not only improves the reaction rate by localizing the CHA reaction substrate, but also improves the operational efficiency and controllability of CHA based on the DNA framework structure on the interface, thus achieving efficient detection of exosomal miRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Assembly process of functionalized DNA cubes.

[0029] Figure 2 Schematic diagram of the electrochemical sensor based on functionalized DNA cubes for rapid and sensitive detection of exosomal miRNA. (A) Preparation process of Pt@AuNPs@HRP@DNA signal probe. (B) Detection process of biosensor and generation of electrochemical signal. (C) Reaction process of functionalized DNA cubes.

[0030] Figure 3 Characterization of Pt@AuNPs (A) Low-magnification TEM image of Pt@AuNPs; (B) High-magnification TEM image of Pt@AuNPs; (C) UV absorption spectra of AuNPs and Pt@AuNPs; (D) STEM-EDS spectrum of Pt@AuNPs.

[0031] Figure 4 Characterization of Pt@AuNPs@HPR@DNA. TEM (A) and DLS (D) characterization of Pt@AuNPs; TEM (B) and DLS (E) characterization of Pt@AuNPs@SA; TEM (C) and DLS (F) characterization of Pt@AuNPs@HRP@DNA.

[0032] Figure 5 UV characterization of Pt@AuNPs@HRP@DNA. (A) UV-visible absorption spectra of the supernatant before and after DNA was linked to Pt@AuNPs; (B) UV absorption spectra of oxTMB of Pt@AuNPs@DNA and Pt@AuNPs@HRP@DNA.

[0033] Figure 6 Characterization of functionalized DNA cubes. (A) PAGE analysis of wireframe DNA cube self-assembly; (B) PAGE analysis of CHA; (C) PAGE analysis of functionalized DNA cubes.

[0034] Figure 7 Wireframe DNA cube characterization. (A) AFM characterization of wireframe DNA cube; (B) TEM characterization of wireframe DNA cube.

[0035] Figure 8 Figure 2 shows fluorescence detection based on functionalized DNA cubes. (A) Schematic diagram of traditional CHA reaction; (B) Schematic diagram of reaction based on functionalized DNA cubes; (C) Real-time fluorescence signals of functionalized DNA cubes and traditional CHA; (D) Corresponding fluorescence intensity when the reaction time is 2 hours.

[0036] Fig. 9 Feasibility verification of biosensor. (A) Time-dependent UV absorption spectra of oxTMB after adding TMB and H2O2 to Pt@AuNPs@DNA and Pt@AuNPs@HRP@DNA; (B) DPV signal response of Pt@AuNPs@DNA and Pt@AuNPs@HRP@DNA as signal probes, with miRNA concentration of 100pM; (C) EIS and (D) CV curves characterizing the step-by-step preparation process of the electrode; (a) GE, (b) functionalized DNA cube / GE, (c) BSA / functionalized DNA cube / GE, (d) miRNA / BSA / functionalized DNA cube / GE, (e) signal probe / miRNA / BSA / functionalized DNA cube / GE.

[0037] Fig.10 Optimization of experimental parameters. (A) Number of bases of H1 sequence bound to the signal probe; (B) Target reaction time; (C) Signal probe reaction time.

[0038] Fig.11 Sensitivity evaluation of the designed electrochemical biosensor. (A) DPV response signal of different concentrations of miRNA-30d-5p; (B) DPV signal and lgC miRNA-30d-5p (C) Detection of DPV response signals of different concentrations of let-7d-3p; (D) DPV signal and lgC let-7d-3p linear relationship.

[0039] Fig.12 Performance evaluation of electrochemical sensors. (A) DPV responses of different miRNAs: blank control, miRNA-122, miRNA-141, miRNA-155, miRNA-373, miRNA-30d-5p at 1pM concentration, and mixture (1pM miRNA-30d-5p + 1pM / interferor) (B) DPV signal response of electrochemical sensor to 10pM miRNA-30d-5p within 7 days; (C) DPV signal response of 1pM, 100pM, and miRNA-30d-5p in buffer and exosome extracts; (D) DPV signal response of 1pM, 100pM, and 1nM let-7d-3p in buffer and exosome extracts. DETAILED DESCRIPTION

[0040] The following is further described in detail through specific implementation methods:

[0041] 1. Materials and Reagents

[0042] Table 1: Chinese-English comparison table of this application

[0043]

[0044] Table 1 continued:

[0045]

[0046] The nucleotide sequences used in the present invention were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), and the nucleic acid sequences are shown in Table 2. Chloroplatinic acid (H2PtCl6), chloroauric acid (HAuCl4), sodium citrate, polyvinylpyrrolidone (PVP), L-ascorbic acid (L-AA), bovine serum albumin (BSA), Tween-20 (Polyethylene glycol sorbitan monolaurate), Tris (2-carboxyethyl)-phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (St. Louis, USA), and 30% hydrogen peroxide (H2O2) and anhydrous ethanol (Ethanol) were from Chuandong Chemical Co., Ltd. (Chongqing, China). DNAMarker and RNase-free water were purchased from Bao Biotech (Dalian, China). The universal miRNA purification kit was provided by EZBioscience (Roseville, USA). Streptavidin (SA), GoldView I, and PBS were purchased from Solebio Biotech (Beijing, China). Biotin labeled horseradish peroxidase (Bio-HRP) was from Bio-Osen Biotech (Beijing, China). The ultrapure water used in the experiments was prepared by Millipore water purification system (≥18 MΩ Millipore-Q) (Millipore, USA).

[0047] Table 2 Nucleotide sequences used in the present invention

[0048]

[0049]

[0050] 2. Instruments and Equipment

[0051] Differential pulse voltammetry (DPV) was performed on a Chenhua CHI 660E electrochemical workstation (Shanghai, China). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed on a Donghua DH7000 electrochemical workstation. Electrochemical detection used a conventional three-electrode system: (1) GE (3 mm in diameter) as the working electrode; (2) a platinum wire electrode as the counter electrode; and (3) a silver / AgCl electrode (immersed in saturated KCl solution) as the reference electrode. Fluorescence kinetics was measured using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, California, USA). Nucleic acid concentrations were determined using a Thermo Fisher Nano Drop 1000 spectrophotometer (Wilmington, DE, USA). A Bio-Rad electrophoresis instrument was used for PAGE analysis, and images were acquired using a ChemiDoc-XRS system (California, USA). Dynamic light scattering (DLS) (Holtzville, USA) was used to analyze the particle size of nanoparticles. The morphology of nanomaterials was characterized using a Bruker Dimension icon atomic force microscope (AFM) (Massachusetts, Germany) and a transmission electron microscopy (TEM) JEM-1200EX (Tokyo, Japan). Elemental analysis of nanomaterials was performed using an energy dispersive spectrometer (EDS, Oxford X-MaxN, Britain).

[0052] 3. Construction of electrochemical sensors

[0053] 1. Synthesis of AuNPs and Pt@AuNPs

[0054] The synthesis of gold nanoparticles (AuNPs) adopts the method of reducing HAuCl4 with sodium citrate, and the particle size of synthesized AuNPs is about 14nm. The specific steps are as follows: add 1mL of HAuCl4 (20mM) to 18mL of deionized water and heat to 100℃. Quickly add 1mL of sodium citrate (68mM), stir the reaction for 10min, and stop heating when the solution turns wine red. After the product is cooled to room temperature, centrifuge the product and redisperse it with deionized water. The specific steps for the synthesis of Pt@AuNPs are as follows: add 155μlAuNPs to 4.845mL of deionized water, add 100μL of 20wt% PVP solution, stir and incubate for 5min to allow the polymer to coat and stabilize the AuNPs. 1 mL H2PtCl6 (20 mM) and 200 μL L-AA (100 mg / mL) were added to the mixture and stirred well. The mixture was immediately incubated at 65°C for 30 min until the solution color changed to brown / black, indicating the successful deposition of platinum (Pt). The product was cooled to room temperature and centrifuged at 1250 rcf for 12 min, washed 3 times, and resuspended in 3 mL deionized water.

[0055] 2. Preparation of Pt@AuNPs@HRP@DNA

[0056] Pt@AuNPs were SA functionalized by mixing 1 mL Pt@AuNPs, 100 μl PBS buffer (50 mM, pH 6.4) and 100 μl SA (1 mg / mL) and stirring at room temperature for 3 h. 100 μL PBST buffer (0.01 M PBS, 0.05% (w / v) Tween-20, pH 7.4) was added and the reaction continued for 1 h. The reaction product was centrifuged at 7000 g for 5 min, washed with PBST for 3 consecutive times, and the excess reagent was removed and resuspended in 1 mL PBST to obtain SA-functionalized Pt@AuNPs. The preparation method of Pt@AuNPs@HRP@DNA is as follows: 200 μl SA-functionalized Pt@AuNPs, 3 μl Bio-HRP (1 mg / mL), and 10 μl Bio-DNA (100 μM) were mixed and reacted at room temperature for 1 h. The reaction product was centrifuged at 7000 g for 5 min, washed three times with PBST, excess reagents were removed, and resuspended in 600 μL PBST to obtain the Pt@AuNPs@HRP@DNA signal probe.

[0057] 3. Preparation of wireframe DNA cubes and functionalized DNA cubes

[0058] Self-assembly of wireframe DNA cubes: Four ssDNAs (ClipA, Clip B, Clip C, Clip D) were mixed at equimolar concentrations in TA buffer (40mM Tris-Ac, 10mM MgAc2, pH 8.0). The mixture was heated to 95°C for 5min and slowly cooled to room temperature to form a wireframe DNA cube. At the same time, probes H1 and H2 were heated to 95°C for 5min and slowly cooled to room temperature to form a hairpin structure. The capture probes (CP) were activated with TCEP for thiol groups in advance. The wireframe DNA cubes (500nM) and CP (2μM) were mixed, and the hairpin probes H1 (1μM) and H2 (1μM) were mixed and incubated at 37°C for 30 minutes to obtain functionalized DNA cubes combined with CP, which were stored at 4°C for later use.

[0059] 4. Construction of electrochemical sensor

[0060] GE was polished with 0.05 μm alumina for 5 min, treated with piranha solution (H2SO4:H2O2=3:1) for 5 min, repeated 3 times, and ultrasonicated with deionized water, repeated 3 times to obtain a clean GE, which was blown dry with nitrogen for later use. 10 μl of the functionalized DNA cube solution bound to CP was added to the GE and incubated at 4 °C for 8 h. After gently rinsing with PBST, 10 μL of BSA (1 wt%) was used to react at room temperature for 1 h to block the nonspecific sites on the GE surface, and the prepared electrochemical sensor was gently rinsed with PBST for later use.

[0061] 4. Application of electrochemical biosensors in the detection of exosomal miRNA

[0062] 1. Electrochemical determination of exosomal miRNA

[0063] A certain concentration of exosome miRNA was added to the electrode, reacted at 37℃ for 1h, and the electrode was gently rinsed with PBST. Then, the signal probe was added and reacted at 37℃ for 0.5h. The electrode was gently rinsed with PBST, and DPV detection was performed in PBS working solution containing 2mM H2O2 (0.01M, pH 7.4). Parameter setting: Scanning at 1.0-1.0V potential, amplitude 70mV, pulse width 50ms, pulse period 200ms. In a solution containing 5mM Fe(CN)6 3- / 4- CV and EIS measurements were performed in 0.1M KCl solution. The frequency range of EIS detection was 0.01Hz-10kHz, the amplitude was 10mV, the CV scan range was -0.2V-0.6V, and the scan rate was 0.15V / S

[0064] 2. PAGE electrophoresis

[0065] 5% non-denaturing PAGE was used to verify the assembly of wireframe DNA cubes and functionalized DNA cubes. Reaction solutions with different components were prepared. After a certain reaction time, 1 μM samples (10 μL) containing different reaction solutions were added to a 5% non-denaturing polyacrylamide gel tank, and the gel was immersed in 1×TBE buffer (89 mM boric acid, 2 mM EDTA, 89 mM Tris, PH 8.3) and electrophoresed at 70 V for at least 2 h at 4 ° C. The obtained gel was stained with GoldView I for 30 min and imaged on a gel imaging system.

[0066] 3. Fluorescence kinetic detection of exosomal miRNA

[0067] Fluorescence kinetics detection was used to verify the feasibility of functionalized DNA cubes. Functionalized DNA cubes (125nM wireframe DNA cubes, 250nM hairpin probe H1, 250nM hairpin probe H2) and traditional CHA reaction system (250nM hairpin probe H1 and 250nM hairpin probe H2) were introduced to detect 20nM exosomal miRNA-30d-5p. The fluorescence kinetics detection conditions were set as follows: excitation wavelength of 490nm, emission wavelength of 518nm, excitation and emission slit width of 5nm, fluorescence collection period of 0.1min, and detection time of 2h.

[0068] 4. Results and Discussion

[0069] Electrochemical biosensor detection principle

[0070] Based on CHA-functionalized DNA cubes and Pt@AuNPs@HRP nanozyme tags, this study developed an electrochemical sensor for highly sensitive exosomal miRNA detection. Figure 1 As shown: The wireframe DNA cube is assembled from 4 single strands in one step and has 8 single strand domains that can specifically bind to ssDNA. The single strand domains on the upper surface of the DNA cube are combined with the CHA response element H1 and H2 hairpin probes to assemble into a functionalized DNA cube. The single strand domains on the lower surface are combined with the CP modified with thiol groups and fixed to the GE through gold-thiol bonds. The electrochemical detection principle is shown in Figure 2 As shown: Pt@AuNPs were synthesized on the basis of AuNPs, and then ssDNA and HRP were connected through SA and biotin to form Pt@AuNPs@HRP@DNA as a signal probe (as shown in Figure 2 A), the DNA in the signal probe binds to the free single-stranded sequence exposed by the CHA reaction product on the functionalized DNA cube, and Pt@AuNPs@HRP has efficient peroxidase activity and can reduce H2O2 to produce a strong electrochemical signal. Figure 2As shown in Figure B, the functionalized DNA cube is fixed to the GE through a gold-thiol bond. The presence of exosomal miRNA can activate the efficient CHA reaction within the confinement of the cube framework, and quickly transfer between cube nodes through target circulation to form a large number of H1H2 double-stranded structures, exposing free single-stranded sequences, and combining with the signal probe Pt@AuNPs@HRP@DNA, thereby catalyzing H2O2 to produce a significant electrochemical signal. In this study, an electrochemical biosensor for exosomal miRNA detection was constructed by integrating CHA amplification elements on the basis of the DNA cube framework and combining Pt@AuNPs@HRP nanozyme tags. By utilizing the confined space to greatly improve the amplification efficiency of CHA and synthesizing complex nanozyme probes with efficient catalytic performance, the detection sensitivity of the sensor was jointly improved.

[0071] 5. Characterization of Pt@AuNPs@HPR@DNA

[0072] In this study, TEM and STEM-EDS were used to characterize the size, morphology and elemental composition of the synthesized Pt@AuNPs. Figure 3 A and Figure 3 As shown in Figure B, TEM characterization shows that Pt@AuNPs are spherical nanoparticles with a particle size of about 80nm and 1-2nm protrusions on the surface. The nanoscale protrusions can contact small molecules (such as H2O2) and significantly increase the catalytic amplification surface area. The UV absorption spectrum shows ( Figure 3 C) AuNPs have an obvious absorption peak at 520nm. After the synthesis of Pt@AuNPs, the ultraviolet absorption peak disappears. STEM-EDS( Figure 3 D) confirmed that Pt@AuNPs had a core-shell structure and was composed of two elements, Au and Pt, in which AuNPs were confined in the Pt shell structure. The above results confirmed the successful synthesis of Pt@AuNPs.

[0073] Subsequently, TEM and DLS were used to characterize the assembly process of Pt@AuNPs@HPR@DNA. Figure 4 A, 4B, 4D, 4F), although the particle size of Pt@AuNPs did not change significantly, a thin layer of amorphous material was formed on its surface, indicating that the surface of Pt@AuNPs nanoparticles was coated with SA. After adding Bio-HRP and Bio-ssDNA (see Figure 4 C and 4F), a more obvious translucent film was formed on the surface of Pt@AuNPs, and the particle size was significantly increased, indicating the successful preparation of Pt@AuNPs@HPR@DNA.

[0074] In order to further prove that Bio-ssDNA and Bio-HRP were successfully connected to Pt@AuNPs, the present invention detected:

[0075] (1) UV-visible absorption spectra of the supernatant before and after Bio-ssDNA was connected to Pt@AuNPs;

[0076] (2) UV-visible absorption spectra and UV-time-dependent absorption spectra of oxTMB before and after Bio-HRP was linked to Pt@AuNPs, and after the addition of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 to Pt@AuNPs and Pt@AuNPs@HRP. Figure 5 After the Bio-DNA was connected to the nanomaterials as shown in A, the absorbance value of the supernatant at 260nm was significantly lower than that before the connection, proving that the Bio-DNA was successfully bound to Pt@AuNPs@HRP. After adding TMB and H2O2, the absorbance value of Pt@AuNPs@HRP at 652nm was significantly higher than that of Pt@AuNPs( Figure 5 B). Time-dependent UV absorption spectra show ( Fig. 9 A), the absorbance slope of Pt@AuNPs@HRP at 652 nm is about 2.5 times that of Pt@AuNPs. These results fully demonstrate that HRP is successfully bound to Pt@AuNPs and significantly enhances its peroxidase catalytic activity.

[0077] VI. Characterization of Functionalized DNA Cubes

[0078] PAGE was used to verify the self-assembly process of wireframe DNA cubes, such as Figure 6 As shown in A, lanes 1-5 represent DNAmaker, ClipA, ClipA+Clip B, ClipA+Clip B+Clip C, ClipA+Clip B+Clip C+Clip D, respectively. As the amount of ssDNA increases, DNA bands with gradually decreasing migration speed and increasing molecular weight appear in the lanes. A DNA band with the lowest migration speed and the largest molecular weight appears in lane 5, representing the wireframe DNA cube. In addition, the structure and size of the synthesized wireframe DNA cube were clearly observed by TEM and AFM images ( Figure 7 A and Figure 7 B), these results demonstrate the successful assembly of wireframe DNA cubes. The feasibility of individual CHAs was subsequently verified, e.g. Figure 6 As shown in B, in the absence of the target, the hairpin probes H1 and H2 are two bands with different molecular weights (lane 3). After adding the target, the DNA band becomes one, and its migration rate is significantly reduced, indicating that CHA is successfully activated and a large number of H1H2 double strands are produced (lane 2). Finally, the assembly of the functionalized DNA cube was verified, as shown in Figure 6 As shown in C, lanes 1-6 represent DNAmaker, wireframe DNA cube, wireframe DNA cube + H1, wireframe DNA cube + H2, functionalized DNA cube, and functionalized DNA cube + miRNA-30d-5p, respectively. After adding hairpin probes H1 and H2, the electrophoretic migration of the wireframe DNA cube slowed down, proving the successful assembly of the functionalized DNA cube. After adding the target (lane 6), the position of the DNA band is basically the same as that of the functionalized DNA cube (lane 5). This is because although the CHA reaction changes the conformation of the functionalized DNA cube, its base number does not change.

[53] , so the electrophoretic migration speeds of the two are similar.

[0079] VII. Feasibility analysis of CHA fluorescence detection based on functionalized DNA cubes

[0080] In order to explore the reaction speed of functionalized DNA cubes, this study used fluorescence kinetic experiments to monitor and compare the reaction speed of the developed functionalized DNA cubes (125nM functionalized DNA cubes, including 125nM wireframe DNA cubes, 250nM H1 and 250nM H2) and traditional CHA (250nM H1 and 250nM H2) to 20nM exosomal miRNA-30d-5p. Figure 8 As shown in Figure C, in the absence of the target, no enhanced fluorescence signal was detected in the two reaction systems, indicating that H1 and H2 alone cannot trigger CHA. After adding the target, the fluorescence signal of the functionalized DNA cube increased rapidly and reached a plateau at about 60 minutes, while the fluorescence signal intensity of the traditional CHA increased slowly and did not reach the maximum value within 2 hours of reaction. The reaction speed of the functionalized DNA cube was 3.12 times that of the traditional CHA. When the reaction time was 2 hours (such as Figure 8 D), the signal-to-noise ratio of the functionalized DNA cube is as high as 8.81, while the S / B of the traditional CHA is only 5.18. The reason for the accelerated CHA reaction kinetics and S / B is that the spatial confinement of CHA on the wireframe DNA cube increases the local concentration of the reaction substrate hairpins H1 and H2, greatly improving the reaction efficiency and reaction rate.

[0081] 8. Feasibility Verification of Biosensors

[0082] Pt@AuNPs@DNA and Pt@AuNPs@HRP@DNA were used as signal probes to construct electrochemical sensors. The electrochemical sensor (BSA / functionalized DNA cube / GE) was reacted with 100 pM miRNA-30d-5p and then co-incubated with Pt@AuNPs@DNA and Pt@AuNPs@HRP@DNA, respectively. Fig. 9As shown in Figure B, the DPV signal of Pt@AuNPs@HRP@DNA as a signal probe is significantly higher than that of Pt@AuNPs@DNA. This further proves that Pt@AuNPs@HRP@DNA has excellent catalytic performance for H2O2 as a signal probe and can be successfully applied on an electrochemical sensing platform. Subsequently, EIS and CV tests were used to verify the successful construction of the electrochemical sensor. The semicircle diameter in EIS represents the electron transfer resistance (Electron transfer resistance, Ret). Fig. 9 As shown in Figure C, after the functionalized DNA cube was fixed to the GE (curve b), a significantly increased semicircle diameter was observed compared to the bare GE (curve a). This is because the steric hindrance of the DNA 3D structure is greatly increased compared to the bare GE. After the unbound sites were blocked with BSA, BSA hindered the electron transfer on the electrode surface, so an increase in Ret was observed (curve c). After the target reaction was added, there was no significant change in the semicircle diameter (curve d). This is because although the conformation of the functionalized DNA cube changed, its base number did not change.

[53] , which has little effect on the electron transfer on the electrode surface. After adding the signal probe, the signal probe hybridizes with the free ends of the H1 H2 double strands on the functionalized DNA cube, and Ret increases further (curve e), indicating that Pt@AuNPs@HRP@DNA binds to the DNA cube framework on the electrode. At the same time, CV is used to detect the changes in the redox peak current after electrode modification to verify the assembly process of the biosensor ( Fig. 9 D). When GE (curve a) was gradually modified by functionalized DNA cubes and BSA, the redox peak current decreased successively (curves b, c), and after the addition of the target, the peak current did not change significantly (curve d). Finally, the peak current decreased significantly after the addition of the signal probe (curve e). The CV results were consistent with those of the EIS, indicating the successful construction of the electrochemical biosensor.

[0083] 9. Optimization of experimental conditions

[0084] In order to obtain the best experimental results, key experimental conditions were optimized, including the number of bases that the H1 probe binds to the reporter probe, the target reaction time, and the signal probe reaction time. First, the number of bases that the free end of the H1 probe binds to the signal probe was explored (8nt, 9nt, 10nt, 11nt, 12nt). As the number of bases increased, the DPV signal gradually increased, and the DPV signal was strongest when the number of bound bases was 10nt ( Fig.10 A). Target reaction time is an important reaction condition of electrochemical sensors. This study detected the DPV signals when the target reaction time was 30min, 60min, 90min, 120min, and 150min. The electrochemical signal was optimal at 60min ( Fig.10B) Finally, the reaction time between the signal probe and the functionalized DNA cube was optimized. Fig.10 As shown in Figure C, the DPV signal was strongest when the reaction time was 30 min. Therefore, the reaction conditions for the subsequent experiments were selected as follows: the number of bases bound between the free end of the H1 probe and the signal probe was 10 nt, the target reaction time was 60 min, and the signal probe reaction time was 30 min.

[0085] 10. Sensitivity evaluation of electrochemical sensors

[0086] Under the optimal experimental conditions, the newly constructed electrochemical sensor was used to detect the DPV signals of exosomes let-7d-3p and miRNA-30d-5p at different concentrations. Fig.11 A and Fig.11 As shown in C, with the gradual increase of miRNA-30d-5p and let-7d-3p concentrations (1fM-10nM), the DPV signal increases successively, and the logarithm of miRNA concentration shows a good linear correlation with DPV ( Fig.11 B and Fig.11 D). The linear equations of the fitting are I=2.56lgC miRNA-30d-5p +17.15(R 2 =0.994), I = 2.211gC let-7d-3p +18.12(R 2 =0.997) where I is the current intensity and C is the concentration of miRNA-30d-5p and let-7d-3p. The LODs of miRNA-30d-5p and let-7d-3p were calculated to be 0.55fM and 0.45fM, respectively, based on the standard deviation of the 3 DPV signals of the blank control group. Compared with the previously reported miRNA electrochemical detection methods, as shown in Table 3, the electrochemical sensor proposed in the present invention exhibits better performance, which is attributed to the efficient catalytic performance of the Pt@AuNPs@HRP nanozyme probe and the spatial confinement effect of the DNA cube framework, which greatly improves the amplification efficiency of CHA. The biosensor not only has better analytical performance, but also has wide versatility.

[0087] Table 3 Comparison of analytical performance of different electrochemical biosensors in miRNA detection

[0088]

[0089] MOF: Metal-Organic Framework

[0090] 11. Specificity and stability analysis of electrochemical sensors

[0091] miRNAs have high homology, and this study compared the DPV signals generated by similar miRNAs (miRNA-122, miRNA-141, miRNA-155, and miRNA-373) to evaluate the specificity of the constructed electrochemical sensor. Fig.12 As shown in A, the DPV current value will increase significantly only when miRNA-30d-5p or its mixture is present. Compared with the blank control group, the presence of other miRNAs will not produce obvious changes in the DPV signal. This shows that the electrochemical sensor has good specificity for the detection of miRNA. In order to ensure the long-term stability of the developed electrochemical sensor, this study recorded the DPV response performance of the electrode stored at 4°C for 7 days. Fig.12 As shown in B, after being stored at 4°C for 7 days, the DPV signal gradually decreased, but still retained 95.57% of the original DPV signal. The good long-term stability of the developed electrochemical sensor is attributed to the excellent biological stability of the framework structure of the DNA cube.

[0092] The accuracy of the developed electrochemical sensor was evaluated by adding different concentrations of target let-7d-3p (1pM, 100pM, 1nM) and miRNA-30d-5p (1pM, 100pM, 1nM) to the exosome miRNA matrix to prepare the test samples. Fig.12 As shown in Figures C and 12D, the DPV signal remained stable even when the target was added to the exosomal miRNA mixture compared with PBS buffer. As shown in Tables 4 and 5, the recovery of let-7d-3p was 96.47% to 102.12%, with a relative standard deviation of 1.43% to 3.92%, and the recovery of miRNA-30d-5p was 100.0% to 105.34%, with a relative standard deviation of 0.54% to 5.24%. These results show that the electrochemical sensor has potential application prospects in the detection of complex clinical samples.

[0093] Table 4 Electrochemical sensors for detecting different concentrations of miRNA-30d-5p in exosomes

[0094]

[0095] Table 5 Electrochemical sensors for detection of different concentrations of let-7d-3p in exosomes

[0096]

[0097] In summary, this paper proposes an electrochemical sensing strategy for quantitative detection of exosomal miRNA. The functionalized DNA cube is fixed on the GE surface through the thiol modified by the bottom node. The wireframe DNA cube is used as a scaffold, and the single-stranded domain is combined with the CHA reaction element to construct a CHA functionalized DNA cube. Pt@Au@HRP@DNA is used as a signal probe for the first time to construct an electrochemical sensor to output an electrochemical signal for sensitive and rapid detection of exosomal miRNA. This strategy greatly improves the amplification efficiency of CHA by using confined space, and designs and synthesizes nanozyme probes with high catalytic performance, which together improve the detection sensitivity and reaction efficiency of the sensor. The development of this study provides a new technical platform for the highly sensitive detection of exosomal miRNA, showing excellent analytical detection performance, and has important clinical significance and translational value.

Claims

1. A method for constructing an electrochemical sensor, characterized in that: The following steps are involved: S1. Preparation of signal probe Pt@AuNPs@HRP@DNA S1-1, using gold nanoparticles AuNPs and H2PtCl6 as the main components to synthesize platinum nanoparticles Pt@AuNPs; S1-2, after the Pt@AuNPs are SA-functionalized, they are mixed with Bio-HRP and Bio-DNA to react at room temperature, the reaction product is washed, and resuspended with PBST buffer to obtain the signal probe Pt@AuNPs@HRP@DNA; S2. Preparation of CHA-functionalized DNA cubes S2-1, using 4 ssDNA strands as the main skeleton to self-assemble into a wireframe DNA cube; S2-2, mixing the wireframe DNA cube, the capture probe CP, the hairpin probe H1 and the hairpin probe H2 and incubating them to obtain a functionalized DNA cube combined with the capture probe CP; S3. Construction of electrochemical sensor The functionalized DNA cube solution described in S2-2 is dripped onto the purified glassy carbon electrode GE for incubation, and then washed and reacted with BSA buffer at room temperature to block the nonspecific sites on the GE surface, and then the target let-7d-3p or miRNA-30d-5p is added for reaction, and then the signal probe Pt@AuNPs@HRP@DNA solution is dripped onto the GE surface, and dried after the reaction to obtain the electrochemical sensor; Among them, the nucleotide sequences of the four ssDNAs are shown as SEQ ID NO.1 to SEQ ID NO.4 respectively; the nucleotide sequence of the CP is shown as SEQ ID NO.5; the nucleotide sequence of let-7d-3p is shown as SEQ ID NO.6; and the nucleotide sequence of miRNA-30d-5p is shown as SEQ ID NO.

7.

2. The method for constructing an electrochemical sensor according to claim 1, characterized in that: The concentrations of the Bio-HRP and Bio-DNA are: Bio-HRP 1 mg / mL, Bio-DNA 100 μM.

3. The method for constructing an electrochemical sensor according to claim 1, characterized in that: The capture probe CP is used after the thiol group is activated by tris(2-carboxyethyl)phosphine hydrochloride.

4. The method for constructing an electrochemical sensor according to claim 1, characterized in that: The reaction time of the target in S3 is 60 min, and the reaction time of the signal probe is 30 min.

5. An electrochemical sensor, characterized in that: The electrochemical sensor is constructed according to the method according to claim 1 or 2.

6. Use of the electrochemical sensor according to claim 5 in detecting exosomal miRNA.

7. A material for improving the detection sensitivity of an electrochemical sensor, characterized in that: The material includes the signal probe Pt@AuNPs@HRP@DNA as described in claim 1 or / and the functionalized DNA cube as described in claim 1.

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