SPCE / ZrGA / mDNA-t biosensing platform, application and detection method

Abstract

The application discloses an SPCE / ZrGA / mDNA-T biosensing platform and application and a detection method. + The specific DNAzyme trigger reaction converts the exosome-derived miRNA analysis into DNAzyme catalyzed signal amplification. Through the high signal amplification ability of HCR and the high catalysis of DNAzyme, the biosensor can sensitively determine the exosome-derived miRNA, and the detection limit is as low as 34.6 aM. In addition, the biosensor has the characteristics of low price, simple operation and high sensitivity, and becomes a convenient and user-friendly tool for POC diagnosis.

Description

Technical Field

[0001] This invention relates to the field of detection, specifically to the SPCE / ZrGA / mDNA-T biosensor platform, and also to the application of the SPCE / ZrGA / mDNA-T biosensor platform and detection methods. Background Technology

[0002] microRNA (miRNA) is an endogenous, non-protein-coding single-stranded RNA that plays an important role in physiology and pathology. MiRNA in peripheral circulating body fluids is abnormally expressed in cancer and has advantages over miRNA in tissues: it has a longer half-life, is more resistant to RNase degradation, does not require extraction of total cellular RNA, is convenient to sample, and can continuously monitor disease development. Therefore, it is more suitable as a non-invasive biomarker for early cancer diagnosis and prognosis.

[0003] In recent years, miRNA detection technologies have mainly included microarray analysis, RNA sequencing, Northern blotting, and real-time quantitative PCR, but these methods suffer from low sensitivity, insufficient detection efficiency, large equipment size, and time-consuming operation. The development of biosensing technology has provided new opportunities for miRNA detection. Electrochemical biosensing technology, with its advantages of high sensitivity, ease of operation, fast response, low cost, and ease of miniaturization, has been widely used in the field of nucleic acid molecular detection.

[0004] DNA possesses advantages such as programmability and high precision, and can be assembled into various shapes through bottom-up construction. Hairpin DNA is a well-known structure used in biosensors, enhancing specificity and sensitivity. Amplification strategies based on DNA-hairpin structures (such as hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA)) have been used for miRNA detection and imaging, improving detection sensitivity. However, DNA amplification strategies may require enzymes, suffer from high background noise, side reactions, and false positive signals, resulting in high costs and limited sensitivity, specificity, or reliability. Besides two-dimensional hairpin DNA, three-dimensional tetrahedral DNA (THD) has attracted chemists' attention due to its favorable regulation of DNA hybridization, supramolecular density, cell permeability, and high resistance. THD possesses mechanical rigidity, addressing capabilities, and multifunctionality, making it suitable for various molecular recognition applications and greatly enhancing the sensitivity and specificity of biosensors. THD-assisted biosensors can improve sensitivity by more than two orders of magnitude. Therefore, THD-based biosensors coupled with various signal expressions, fluorescence colorimetry, and electrochemical detection methods have been proposed. However, on the one hand, double-stranded specific nucleases are needed for signal amplification; on the other hand, THD is only used as a nanocarrier, ignoring its significant advantages in recognition and hybridization. Therefore, truly enzyme-free and hairpin-free high-performance electrochemical biosensors have enormous potential for miRNA measurement in practical applications.

[0005] Metal-organic frameworks (MOFs) are novel porous crystalline materials synthesized by assembling metal ions and organic ligands. They possess advantages such as large specific surface area, high porosity, and tunable pore structure and morphology, and are widely used in catalysis, capacitors, adsorption, and sensors. Biofunctional MOFs can achieve signal transduction and selective recognition, and can be used to build biosensors that simultaneously detect multiple targets. UiO-66 is a typical MOF with good porosity and high surface area, capable of encapsulating a large number of signal molecules and responding to external stimuli. However, UiO-66 is often limited by low loading capacity and slow response processes, affecting its applications. In in-depth research on composite materials, the idea of ​​assembling MOFs from different materials has been proposed to utilize synergistic effects and maximize the advantages of the constituent materials. MOFs containing redox-active metals (such as zinc, iron, nickel, cobalt, and manganese) and functional organic ligands (such as trimellitic acid and terephthalic acid) are suitable for designing composite materials, such as the MIL series, ZIF series, UiO series, and HKUST-1 series, because the structure and properties of the parent material MOF are the main considerations in composite material design.

[0006] In recent years, many reports have confirmed that graphene-based materials with excellent electrochemical and mechanical properties can be combined with MOFs, which can not only improve their conductivity and stability, but also avoid the aggregation and re-stacking phenomenon between graphene sheets, and have broad application potential, especially in the field of electrochemistry. Its advantages include: (1) the functional groups on the graphene surface guide the growth of MOFs; (2) the combination reduces the limitation of the conductivity of MOFs; (3) the unique structure of graphene can solve the coordination bond problem in MOFs; (4) prevent the process from degrading the performance of MOFs. In-situ growth is one of the most widely used methods for preparing MOF / graphene nanocomposites. Since GO and RGO surfaces contain abundant oxygen-containing functional groups, uniform in-situ MOF growth can be achieved. The metal clusters of MOFs combine with the oxygen-containing functional groups on the GO surface, and ligands are added to promote the nucleation and growth of MOFs. This in-situ method not only saves time, but also enhances the adhesion between MOFs and graphene. Therefore, MOFs grown on graphene substrates will be more uniform and have a stronger interaction with graphene. Summary of the Invention

[0007] In view of this, one objective of the present invention is to provide an SPCE / ZrGA / mDNA-T biosensing platform; a second objective of the present invention is to provide the application of the SPCE / ZrGA / mDNA-T biosensing platform in the detection of miRNA; and a third objective of the present invention is to provide a method for detecting miRNA using the SPCE / ZrGA / mDNA-T biosensing platform.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] 1. SPCE / ZrGA / mDNA-T biosensing platform, wherein the SPCE / ZrGA / mDNA-T biosensing platform includes SPCE / ZrGA sensing electrodes and hairpin chains;

[0010] The SPCE / ZrGA / mDNA-T sensing electrode is obtained by modifying the SPCE graphite working electrode surface with Zr-MOF-rGO, electrodepositing Au nanoparticles to obtain SPCE / ZrGA, and fixing multi-arm DNA tetrahedral mDNA-T onto SPCE / ZrGA through Au-S bonds to obtain the SPCE / ZrGA / mDNA-T sensing electrode.

[0011] The hairpin chain includes hairpin chain I and hairpin chain II, which are modified with stem-loop structures and have electrical signal tags at their ends. The hairpin chains I and hairpin chain II are capable of complementary pairing and binding with sequences remaining after multi-arm DNA tetrahedral cleavage.

[0012] The Zr-MOF-rGO is prepared by hydrothermal method using ZrCl4 and 2-aminoterephthalic acid as raw materials to obtain octahedral Zr MOF, and then the obtained octahedral Zr MOF is physically mixed with graphene powder to obtain Zr-MOF-rGO composite material.

[0013] The multi-armed DNA tetrahedron is a tetrahedral structure assembled from 5 DNA single strands and 3 DNA single strands modified with thiol groups. It also combines with a LOCK chain to temporarily block the target miRNA recognition and binding site. The tetrahedral structure has a swing arm chain at its apex, and the three multi-arm chains adjacent to this swing arm chain contain adenosine ribonucleotides as substrate chains for DNAzyme cleavage sites. The 3' end of each swing arm chain contains Na... + The activated DNAzyme structure's enzyme chain binds to the LOCK chain, the 3' end of which is complementary to the target sequence.

[0014] After activation, the DNAzyme sequentially cleaves adenosine ribonucleotide sites on the substrate strand. The remaining single-stranded nucleic acid residues after cleavage can complementarily bind with hairpin chain I to form a double strand with sticky ends. The formed sticky ends open hairpin chain II to form sticky ends, which triggers a hybridization chain reaction and releases an electrical signal.

[0015] Preferably, the mass ratio of the octahedral Zr MOF to the graphene powder is 1:0.5.

[0016] Preferably, the method for electrodepositing Au nanoparticles in this invention involves deposition at -0.2V for 200s in a mixed solution of 0.01M Na2SO4 and H2SO4 containing 5mM HAuCl4.

[0017] Preferably, the nucleotide sequence of the assembly strand of the multi-armed DNA tetrahedron is shown in SEQ ID NO.1 to 8.

[0018] Preferably, the nucleotide sequence of the LOCK chain is shown in SEQ ID NO.9.

[0019] Preferably, the sequences of the hairpin chain I and hairpin chain II are as shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0020] 2. Application of the SPCE / ZrGA / mDNA-T biosensing platform in the detection of miRNA.

[0021] Preferably, the miRNA in this invention is miRNA-21.

[0022] 3. The method for detecting miRNA using the SPCE / ZrGA / mDNA-T biosensor platform is as follows:

[0023] Using the SPCE / ZrGA / mDNA-T sensing electrode as the working electrode, carbon as the auxiliary electrode, and a silver paste electrode as the reference electrode, the SPCE / ZrGA / mDNA-T sensing electrode was immersed in a solution containing Na. + The sample was incubated in a solution containing hairpin chains I, II, and the target miRNA, and the electrochemical signal was detected.

[0024] Preferably, the incubation time is 30 min to 150 min.

[0025] The beneficial effects of this invention are as follows: This invention discloses an SPCE / ZrGA / mDNA-T biosensing platform. Utilizing the tightly encapsulated Zr MOF octahedral composite material, electron transport is effectively facilitated, and graphene is used to maximally suppress electron-hole pair formation. This results in a SPCE / ZrGA electrode with high conductivity, providing a large specific surface area and loading a large number of AuNPs, offering numerous immobilization sites for mDNA-Ts. Furthermore, the precisely controllable three-dimensional mDNA-T probes ensure a rigid spatial conformation of the probe array at the sensing interface, effectively reducing steric hindrance and maintaining probe spatial orientation. The design of three "robotic arms" improves molecular hybridization and detection efficiency. In addition, the electrode developed in this invention can be fabricated into a disposable portable electrode combined with a wireless USB-based electrochemical analyzer (plug-and-play). The three-dimensional electrode structure generates local molecular constraints, increasing the collision probability of trace target molecules in the micro-reaction system (10 μL), enabling convenient, rapid, and sensitive detection of exosome-derived miRNAs, greatly improving detection efficiency.

[0026] This invention utilizes the recognition of exosome-derived miRNAs by mDNA-T on the SPCE / ZrGA sensing electrode, triggering HCR to form a DNAzyme, thus transforming miRNA analysis into signal amplification catalyzed by the DNAzyme. Leveraging the high signal amplification capability of HCR and the strong catalytic effect of the DNAzyme, this paper-based electrochemical biosensor can highly sensitively measure exosome-derived miRNAs, with a detection limit as low as 34.6 aM.

[0027] The paper-based sensing electrode developed using this invention, combined with a portable USB-type radiochemical analyzer, is characterized by its low cost, simple operation, and high sensitivity, making it a convenient and user-friendly tool for POC diagnosis. Attached Figure Description

[0028] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0029] Figure 1 Flowchart for detecting miRNAs in exosomes using the SPCE / ZrGA / mDNA-T portable biosensor platform;

[0030] Figure 2 The entire flowchart for mDNA-T detection of miRNA-21;

[0031] Figure 3Fabrication and characterization of SPCE / Zr-MOF-GO-Au sensing electrodes (A: Assembly procedure of Zr MOF-rGO-Au; B: SEM image of Zr MOF; CD: TEM images of Zr MOF, EF: Zr MOF-rGO and GH: Zr MOF-rGO-Au; I: EDS elemental mapping).

[0032] Figure 4 XRD full spectrum and XPS spectrum of Zr MOF, Zr MOF-rGO and Zr MOF-rGO-Au (A: XRD full spectrum; B: XPS spectrum);

[0033] Figure 5 XPS wide-scan measurement spectra of SPCE / ZrGA and high-resolution XPS spectra of C1s (B), Zr 3d (C), and Au 4f (D) of SPCE / ZrGA (A: XPS wide-scan measurement spectra; B: C1s high-resolution XPS spectrum of SPCE / ZrGA; C: Zr 3d high-resolution XPS spectrum; D: Au 4f high-resolution XPS spectrum).

[0034] Figure 6 Characterization of a multi-arm DNA tetrahedral nanomachine (mDNA-T) component (A: PAGE electrophoresis image of mDNA-T, M: 20bp ladder, lane 1: S4, lane 2: S) 1-a Lane 3:S 1-r-b Lane 4:S W Lane 5: Lock, Lane 6: S4+S 1-a Lane 7:S4+S 1-a +S 2-a Lane 8:S4+S 1-a +S 2-a +S 3-a Lane 9:S4+S 1-a +S 2-a +S 3-a +S 1-r-b Lane 10:S4+S 1-a +S 2-a +S 3-a +S 1-r-b +S 2-r-b Lane 11: S4+S 1-a +S 2-a +S 3-a +S 1-r-b +S 2-r-b +S 3-r-b Lane 12:S4+S 1-a +S 2-a +S 3-a +S1-r-b +S 2-r-b +S 3-r-b +S W Lane 13:S4+S 1-a +S 2-a +S 3-a +S 1-r-b +S 2-r-b +S 3-r-b +S W +Lock; B: Characterization of mDNA-T using atomic force microscopy (AFM); C: Functional domains of the designed mDNA-T).

[0035] Figure 7 Electrochemical performance tests of SPCE / ZrGA sensing electrodes (A: CV curves of SPCE / ZrGA in 5mM [Fe(CN)6]3- / 4- containing 0.1M KCl at different scan rates; B: Linear relationship between peak current and scan rate at the anode and cathode; C: Linear curve of fitted lgv versus potential).

[0036] Figure 8 Electrochemical performance study of the SPCE / ZrGA / mDNA-T biosensor platform (A: Flowchart of miRNA detection using the SPCE / ZrGA / mDNA-T portable biosensor platform; B: Electrochemical performance study using 5mM [Fe(CN)6] containing 0.1M KCl) 3- / 4- CV curves of naked SPCE, SPCE / Zr-MOF-rGO, SPCE / Zr-MOF-rGO-Au (ZrGA), SPCE / ZrGA / mDNA-T, and SPCE / ZrGA / mDNA-T / miRNA / H1-H2; Feasibility study: C: Procedure for mDNA-T detection of miRNA; D: CV curves of 200 nM miRNA-21 and 60 mM Na + (SWV response of the electrode prepared under the condition of...)

[0037] Figure 9 To optimize the results (A: the response of SPCE / ZrGA to different mDNA-T concentrations; B: the response of SPCE / ZrGA surface immobilized with mDNA-T to different incubation times; C: the response of H1 and H2 HCR reaction time on the SPCE / ZrGA / mDNA-T / miRNA sensing surface).

[0038] Figure 10(a) Schematic diagram of the SPCE / ZrGA / mDNA-T portable biological platform for detecting miRNA-21; (b) SWV reactions for different concentrations of miRNA-21 (from a to h: 0, 100aM, 1fM, 100fM, 10pM, 1nM, 100nM and 200nM); (c) Calibration diagram corresponding to (b).

[0039] Figure 11 To enhance the selectivity and reproducibility of the SPCE / ZrGA / mDNA-T biosensing platform (A: SPCE / ZrGA / mDNA-T electrodes targeting (miR-21), interfering miRNAs (miR-155, miR-26a, miR-192, miR-10b, single-base mismatch (SM) miR-21, triple-base mismatch (TM) miR-21), and different cofactors (K) + ,Zn 2+ Mg 2+ ,Fe 3+ A: Current response of the SPCE / ZrGA / mDNA-T biosensor; B: Reproducibility of the recommended SPCE / ZrGA / mDNA-T biosensor.

[0040] Figure 12 Comparison of clinical sample detection with qRT-PCR (A: TEM image of isolated exosomes from clinical blood samples; B: Immunoblot bands of CD63 and CD81 on exosome membranes; C: Nanoparticle tracking analysis (NTA) of exosomes; D: Validation of clinical identification of exosomal miRNA-21 in clinical samples from healthy individuals (10 samples as controls) and NSCLC patients at tumor stages (16 samples); ***: p<0.001, ****: p<0.0001; (E)(F) Correlation between exosomal miRNA-21 detection results measured by the proposed sensor and real-time quantitative PCR (qRT-PCR). Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0042] Example 1: Design of SPCE / ZrGA / mDNA-T Biosensing Platform

[0043] Based on a multi-arm DNA tetrahedron (mDNA-T) robotic arm to bind Na + The experimental principle of detecting exosome-derived miRNAs using a specific DNAzyme strand displacement reaction, specifically by triggering an HCR signal amplification strategy to detect miRNA-21 in exosomes, is as follows:

[0044] Zr MOFs tightly encapsulated in GO were prepared using a physical mixing and stirring method. The synthesized graphene contacted all faces of the MOF octahedron to form a Zr-MOF-rGO composite material. This material can effectively facilitate electron transport and maximize the utilization of graphene to effectively suppress the formation of electron-hole pairs. Zr-MOF-rGO was then modified onto the surface of the SPCE graphite working electrode, i.e., SPCE / ZrGA. After electrodeposition of Au nanoparticles, mDNA-T cells with "robotic arms" were immobilized on the SPCE / ZrGA via Au-S bonds.

[0045] mDNA-T is a tetrahedral structure assembled from five DNA single strands and three DNA single strands modified with thiol groups. It also temporarily blocks the target miRNA recognition and binding site via a lock strand. The tetrahedral structure has a swing arm chain at its apex, with three strands adjacent to this swing arm chain containing adenosine ribonucleotides (rA) as substrate strands for DNAzyme cleavage. The 3' end of the swing arm chain contains Na+. + The activated DNAzyme's enzyme chain structure binds to the LOCK chain, and the 3' end of the LOCK chain is complementary to the target sequence. The thiol groups modified on the three DNA single strands are firmly fixed to SPCE / ZrGA.

[0046] During the detection process, when the target molecule is present, it can directly displace the lock chain on the swing arm chain, and the enzyme chain on the swing arm chain combines with the substrate chain to form a DNAzyme. In Na… + In the presence of ions, a robotic arm is activated to cleave the "rA" sites on the mDNA-T side waist substrate chain one by one. The cleaved residues serve as the "toehold" for the hybridization chain reaction (HCR), forming a stable dsDNA copolymer in situ until the supply of hairpin chain 1 (H1) or hairpin chain 2 (H2) modified with the "Fc" electroactive substance is exhausted. After all reactions are completed, the target concentration value can be obtained directly on a mobile APP using a portable USB radiochemical analyzer.

[0047] miRNA-21 was selected as a model to validate the proposed SPCE / ZrGA / mDNA-T biosensing platform. Primers were designed as shown in Table 1.

[0048] Table 1. Detection of miRNA-21 mDNA-T

[0049]

[0050]

[0051] The single-stranded DNA sequences SEQ ID NO.1–9 in Table 1 were diluted to 100 μM with TE Buffer and stored. They were then diluted to appropriate concentrations with TM buffer (20 mM Tris, 50 mM MgCl2, 3 mM TCEP, pH 8.0), heated to 95 °C for 10 min in a PCR instrument, and then gradually cooled to 4 °C (each 4 min at -1 °C) and held for 30 min to form multi-armed DNA tetrahedral nanoprobes (mDNA-T).

[0052] The detection principle of miRNA-21 using assembled mDNA-T is as follows: Figure 1 and Figure 2 As shown. During the detection process, the exosomal target miRNA-21, acting as a "target," initiates interaction with the blue LOCK domain. Subsequently, a complementary double strand formed by a portion of the enzyme chain and a portion of LOCK (ΔG enzyme chain: LOCK = -26.62 kcal mol) -1 The miRNA-21:LOCK complex is opened, forming a miRNA-21:LOCK complex (ΔG miRNA-21:LOCK = -33.35 kcal / mol). -1 The red domain of the enzyme strand is no longer blocked and can bind to the red domain of the hairpin substrate strand, then open the hairpin substrate strand to form Na+. + -Specific DNAzyme (ΔG enzyme chain: substrate chain = -34.06 kcal mol) -1 With Na + With the addition of [a specific ingredient], the DNAzyme is activated to specifically cleave the substrate chain on the tetrahedral side waist. The sticky ends of the cleaved residues will open the hairpin H1 (ΔG H1 = -3.75 kcal mol). -1 And form viscous ends: H1 double chain (ΔG viscous ends: H1=-25.10kcal mol) -1 In the viscous end: H1 double chain, the new viscous end will continue to open the hairpin H2 (ΔG H2=-6.19kcal mol). -1 And form a new viscous end: H2 double-chain structure (ΔG new viscous end H1:H2=-43.97kcal mol) -1 Due to the continuous presence of sticky ends, a series of hybridization chain reactions (HCR) are triggered. Fc electroactive markers are modified on the H1 and H2 hairpin probes, thereby enabling the detection of miRNA.

[0053] Example 2: Fabrication and characterization of SPCE / Zr-MOF-GO-Au sensing electrode

[0054] The preparation process of SPCE / Zr-MOF-GO-Au is as follows: Figure 3As shown in Figure A, firstly, tightly packed Zr MOFs with rGO were prepared using a physical mixing method. Then, rGO and Zr MOFs were mixed to improve the conductivity and stability of the MOFs and avoid aggregation and recombination between graphene sheets. The specific steps are as follows:

[0055] Preparation of Octahedral Zr MOFs: Octahedral Zr MOFs were prepared via a typical hydrothermal method. The specific procedure is as follows: First, ZrCl4 (0.233 g) and 2-aminoterephthalic acid (BDC-NH2, 0.181 g) were dissolved in 102.1 mL of N,N-dimethylformamide (DMF). After sonication for 20 min, 22.9 mL of acetic acid was added to the solution. After thorough mixing, the solution was poured into a 200 mL Teflon-lined stainless steel autoclave and reacted at 120 °C for 16 h. After cooling to room temperature, the product was collected by centrifugation, washed three times with DMF to remove excess reactants, then washed three times with methanol to remove DMF, and finally dried under vacuum for at least 12 h.

[0056] Preparation of Zr-MOF-GO composite material: 0.1 g of octahedral Zr MOF was mixed thoroughly with 30 mL of deionized water and 1 mL of HCl in a beaker and sonicated for 10 min. Then, 0.05 g of GO powder was added to the mixture, sonicated for 10 min, and stirred for 2 h. After the reaction was complete, the product was collected by centrifugation at 5000 rpm, washed three times with primary water, and then vacuum dried at 80 °C for 24 h.

[0057] Preparation of SPCE / Zr-MOF-GO-Au (SPCE / ZrGA) sensing electrode: 2.0 mg of Zr-MOF-GO nanocomposite material was weighed and dispersed in 1 mL of primary water, and ultrasonicated for more than 2 h to obtain a homogeneous dispersion. 10 μL of the mixed solution was added dropwise to a clean SPCE electrode and dried at room temperature. The prepared electrode was washed with deionized water, and then deposited for 200 s at -0.2 V in a 0.01 M Na2SO4 and H2SO4 mixed solution containing 5 mM HAuCl4 using chronoamperometry (it) to obtain the SPCE / ZrGA biosensing electrode.

[0058] The morphology and structure of Zr-MOF, Zr-MOF-GO, and Zr-MOF-GO-Au were characterized using field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). Elemental composition was analyzed using energy-dispersive X-ray spectroscopy (EDS) surface scanning results, and changes in the materials were compared and analyzed. The results are as follows: Figure 3 As shown in Figures B through H. Figure 3Figure B shows that we synthesized Zr-MOFs with uniform particle size (400±50 nm). Furthermore, transmission electron microscopy (TEM) revealed that the Zr-MOFs exhibit a typical octahedral structure. Figure 3 (C, D) Figure 3 In images E and F, the graphene layer encapsulating the Zr-MOF octahedron is clearly visible. (High-resolution transmission electron microscopy) Figure 3 In the middle F, the edges of Zr-MOF and rGO are clearly visible, and the rGO lattice structure is clear with a crystal interplanar spacing of 0.34 nm. Figure 3 China G and Figure 3 The image shows the successful deposition of Au NPs on the Zr MOF-rGO surface. The gold nanoparticles have a size of 15 ± 5 nm and a row spacing of 0.238 nm. Figure 3 (H). In addition, EDS ( Figure 3 Analysis showed that elements C, N, O, Zr, and Au were uniformly distributed on the Zr MOF-rGO-Au surface.

[0059] X-ray diffraction (XRD) is used to characterize the crystal structure of the prepared electrodes and obtain diffraction patterns, as shown in the following figures. Figure 4 As shown in Figure A. The results show that the synthesized Zr MOF, Zr MOF-rGO, and Zr MOF-rGO-Au exhibit sharp diffraction peaks in the XRD patterns, which are highly consistent with the single-crystal simulation data, indicating good purity and high crystallinity.

[0060] The chemical composition of the prepared samples was determined using X-ray photoelectron spectroscopy (XPS), and the results are as follows: Figure 4 As shown in B. Figure 4 Figure B shows the full X-ray photoelectron spectroscopy (XPS) of Zr MOF, Zr MOF-rGO, and Zr MOF-rGO-Au. Among them, Zr MOF-rGO-Au has six distinct characteristic peaks: O 1s, N 1s, C 1s, Zr 3p, Zr 3d, and Au 4f. Figure 5 Image A clearly shows the high-resolution XPS spectrum of C1s, where the strong peak at 284.99 eV is attributed to the sp2 hybridization of the CC / CH bond, the binding energy peak at 286.40 eV is attributed to the CN bond, and the weak binding energy peak at 289.00 eV corresponds to the OC=O bond. Figure 5 The data shows the presence of Zr3d in the Zr3d spectrum. 3 / 2 (183.33eV) and Zr 3d 5 / 2 (185.69eV) Two peaks. Figure 5 The binding energies of 84.54 eV and 88.20 eV shown in the C-value are attributed to Au 4f, respectively. 7 / 2 and Au 4f 5 / 2The chemical binding state indicates that AuNPs were successfully bound to Zr MOF-rGO.

[0061] Example 3: Assembly and Characterization of the mDNA-T Robotic Arm

[0062] Single-stranded DNA was dissolved in TE buffer and diluted to a suitable concentration with TM buffer (20 mM Tris, 50 mM MgCl2, 3 mM TCEP, pH 8.0). The mixture was heated to 95 °C for 10 min in a PCR instrument, then gradually cooled to 4 °C (1 °C every 4 min) and held for 30 min to form multi-armed DNA tetrahedral nanoprobes (mDNA-T). The formed DNA tetrahedra (mDNA-T) were verified by 8% polypropylene gel electrophoresis (PAGE) and atomic force microscopy (AFM). Polypropylene gel electrophoresis (PAGE) experiment: 0.5 μM single-stranded (S4, S1-a, S1-rb*, SW, LOCK), double-stranded, triple-stranded, quadruple-stranded, penta-stranded, hexa-stranded, heptapride, octa-stranded, and intact mDNA-T were mixed with 2 μL of 6× loading buffer and loaded into pre-prepared 8% PAGE wells. The gels were run at 80 V for 10 min, then at 100 V for 60 min. Afterward, the gels were stained in 50 mL of 1% gel-red aqueous solution for 10 min. The images were then captured using a Bio-Rad ChemDoc XRS (BioRad, USA). The results are shown below. Figure 6 As shown. From Figure 6 As can be seen from lanes 1-13 of the A-band, with the addition of a new chain, the migration distance decreases due to the increase in molecular weight and more complex spatial structure, while the single-chain Sw migrates even slower due to its longer sequence. Figure 6 (Middle A, Lane 4). Any other assembly structure of mDNA-T, built from sequences of fewer than 9 strands, migrates more slowly, and the presence of clear bright bands on the gel ensures successful assembly and high yield of mDNA-T. Figure 6 Lane A, lane 13). Atomic force microscopy (AFM) was used to further verify the tetrahedral structure of the DNA. The AFM images clearly showed that the prepared mDNA-T possessed a typical tetrahedral structure with a diameter ≈ 5.8 nm. Figure 6 (B)

[0063] like Figure 6 As shown in C, Na +The catalytically active core of a specific DNAzyme is split in two by a lock, inhibiting its catalytic function. Considering that ribonucleotides are approximately 100,000 times more sensitive to hydrolysis than their corresponding deoxyribonucleotides, a DNA-RNA chimeric sequence composed of adenosine mononucleotide (rA) flanked by two DNA domains of a hairpin substrate chain that can complementaryly pair with the two arms of the enzyme chain. To avoid spatial interference of mDNA-T with DNA hybridization and increase the accessibility of the hairpin substrate chain to the walking enzyme chain, a multi-T spacer was designed between the mDNA-T and the enzyme chain. The mDNA-T can be anchored to the SPCE / ZrGA electrode in a highly oriented manner via an Au-S chemical conjugate. This unique pyramidal structure composed of three hairpin substrate chains ensures that all fixed hairpin substrate chains are distributed at fixed distances on the DNA tetrahedrons, thus maintaining efficient assembly of Na+. + Spatial orientation of specific DNAzymes. DNA tetrahedrons are used as a basis because this size of DNA tetrahedron can be defined at nanometer-scale distances, effectively reducing steric hindrance and maintaining spatial orientation, thereby improving miRNA recognition rates.

[0064] Example 4: Electrochemical performance testing of SPCE / ZrGA sensing electrode

[0065] All electrochemical performance test data in this embodiment were acquired using an ECWP100 single electrochemical analyzer. A Zr-MOF-rGO-Au sensing electrode was used as the working electrode, carbon as the auxiliary electrode, and a silver paste electrode as the reference electrode. Cyclic voltammetry (CV) was employed in 10 mL of 5 mM [Fe(CN)6] containing 0.1 M KCl. 3- / 4- Electrochemical performance tests of the sensing electrodes were conducted in the electrolyte system. When using SPCE / ZrGA electrodes to modify multi-arm DNA tetrahedral nanoprobes (mDNA-T) for microRNA sensing, square wave voltammetry (SWV) experiments were performed in 0.01M pH 7.4 PBS buffer. SWV test parameters: operating voltage range +0.2–+0.7V, amplitude 0.025V. All experiments were performed at room temperature.

[0066] The SPCE / ZrGA electrode modification process was characterized using CV. The CV test parameters were: operating potential -0.4V to 0.6V, at different scan rates (10–295 mV s). -1 CV tests were performed on SPCE / ZrGA; the results are as follows: Figure 7 As shown in Figure A. Figure 7 Figure B shows the linear relationship between peak current and the square of the scan rate, where anode: I pa (μA)=293.34v1 / 2 (Vs -1 ) 1 / 2 +9.97(R 2 =0.997) and cathode: I pc (μA)=-262.69v 1 / 2 (V s -1 ) 1 / 2 -12.26(R 2 =0.998). This indicates that the redox reaction of SPCE / ZrGA is a diffusion-controlled process.

[0067] Figure 7 C shows the linear relationship between the peak potential of the anode-cathode pair and the logarithm of the scan rate, where anode: E pa (V)=0.0582lgv(V s -1 )+0.277(R 2 =0.988), Cathode: E pc (V)=-0.0698lgv(V s -1 )+0.011(R 2 =0.881). Based on Laviaron theory:

[0068]

[0069]

[0070] In the formula, k a and k c Let be the slopes of the lines from Epa to lg(v) and from Epc to lg(v), respectively, in terms of 8.314 J·(mol·K). -1 , T=298k, F=96,493C·mol -1 n = 1. According to formulas (I) and (II), α = 0.455 (charge transfer coefficient), k s =1.243s -1 (Apparent electron transfer rate constant). The current k s The electron transfer rate was higher or at least comparable to that reported in the literature (Table 2), which indicates that Zr-MOF-rGO-Au accelerated electron transfer.

[0071] Table 2. Comparison of electron transfer rate constant (ks) with existing reported sensors

[0072]

[0073] Square wave voltammetry (SWV) was used to characterize the changes in the sensing electrode before and after attachment. The previously prepared SPCE / ZrGA electrode was used to immobilize the mDNA-T array onto the electrode surface via Au-S bonds at room temperature. The modified electrode was then washed three times with 0.01M PBS. The cleaned electrode was then immersed in 60mM Na... + 200 nM miRNA-21 and 60 mM Na + Incubate in a mixture of +200 nM miRNA-21 H1 and H2 at room temperature and wash.

[0074] Furthermore, the electroactive surface area (A) of the three electrodes was calculated according to the Randles-Sevcik equation:

[0075]

[0076] Where Ip = peak current (A), n = 1 (number of electron transfers), D = (6.7 ± 0.02) × 10 –6 cm 2 s -1 (Diffusion coefficient), υ = 0.05V s -1 C0 = 5 × 10 -6 mol cm -3 ([Fe(CN)6) 3- / 4- Concentration). According to equation (III), SPCE / ZrGA provides 0.191 cm. 2 The electroactive surface areas are SPCE / ZrMOF-rGO (0.148 cm²). 2 1.3 times that of SPCE (0.076cm) 2 2.5 times that of ).

[0077] Figure 8 Figure A illustrates the assembly process of the recommended SPCE / ZrGA / mDNA-T portable bio-platform, and the electrochemical behavior of the electrodes during modification on a radiochemical analyzer was characterized using cyclic voltammetry (CV). Different modified electrodes were tested in 5 mM [Fe(CN)6] containing 0.1 M KCl. 3- / 4- The CV curve for detecting the solution is shown below. Figure 8In Figure B, after modifying the SPCE electrode surface with ZrMOF-rGO composite nanomaterials (red curve), the current response value was significantly higher than that of the bare SPCE electrode (black curve). This is mainly because the highly conductive ZrMOF-rGO composite nanomaterials greatly increased the specific surface area of ​​the electrode. Notably, the current signal response value of ZrMOF-rGO-AuNPs (ZrGA, blue curve) is approximately three times that of ZrMOF-rGO, demonstrating that ZrMOF-rGO provides a large specific surface area to support the attachment of many AuNPs and can immobilize a large number of capture probes. When the tentacles of mDNA-T are immobilized on the electrode surface through Au-S (SPCE / ZrGA / mDNA-T, purple curve), the current response value decreases significantly. This is due to the interaction between the negatively charged phosphate backbone and Fe in the solution. 2+ / 3+ The electrostatic repulsion between them hinders [Fe(CN)6] 3- / 4- Current diffusion occurs between the electrode surface and the electrode, thus reducing the redox peak current. The current response value further decreases after the HCR reaction due to the hybridization of more non-electroactive DNA strands (SPCE / ZrGA / mDNA-T / miRNA / H1-H2, green curve). This is because the hybrid double strands of DNA formed on the electrode surface further hinder the [Fe(CN)6] reaction. 3- / 4- As the current diffuses across the electrode surface, the current response signal gradually decreases.

[0078] Figure 8 Figure C shows the presence of 200 nM miRNA-21 (target) and 60 mM Na in 0.01 M PBS solution. + A feasibility study was conducted by measuring changes in electrochemical signals induced by the target miRNA, along with the corresponding detection procedures. Figure 8 The SWV curves of the SPCE / ZrGA / mDNA-T biological platform under different detection conditions are shown in Figure D. As can be seen from the figure, the SWV curves of the SPCE / ZrGA / mDNA-T biological platform under only 60mM Na... + In the case of (black curve) or miRNA-21 (target, blue curve), the SPCE / ZrGA / mDNA-T biosensor platform showed almost no response, indicating that the HCR response was not triggered. We can detect miRNA-21 and Na... + A significant SWV response peak (Fc, 0.52V, red curve) was observed in the presence of HCR, which was attributed to the activation of DNA hairpins with Fc (H1-Fc and H2-Fc) to hybridize with each other.

[0079] Example 5: Optimization of experimental conditions for the SPCE / ZrGA / mDNA-T biosensing platform

[0080] Optimizing the junction concentration of the mDNA-T robotic arm: Prepare 5 prepared SPCE / ZrGA electrodes. Immerse the electrodes in mixed solutions containing 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 μM mDNA-T, respectively, and incubate at room temperature for 2 h. Block the cleaned electrodes with MCH solution (1.0 mM) for 15 min. Immerse the cleaned SPCE / ZrGA / mDNA-T in 60 mM Na... + In a mixed solution of miRNA-21 and 4 μM hairpin H1 + H2, the target miRNA-21 initiates a series of strand displacement reactions. After the reaction, the SWV electrochemical signal was measured in 10 mL PBS (0.01 M, pH 7.4). The results are as follows: Figure 9 As shown in Figure A. The results showed that the SWV current signal detected by 0.2 μM miRNA-21 in the mDNA-T concentration range of 0.2 μM to 1.2 μM increased rapidly at the beginning, reached a maximum response at 1.0 μM, and then the current signal tended to stabilize.

[0081] Optimizing the connection time of the mDNA-T robotic arm: The experimental procedure was the same as above. The electrodes were incubated in a 1.0 μM mDNA-T mixed solution for 30, 60, 90, 120, 150, and 180 min, respectively. After washing, the next step of the experiment was performed. SWV electrochemical measurements were performed after all reactions were complete. The results are as follows: Figure 9 As shown in Figure B. The results show that when the incubation time increased from 30 min to 150 min, the generated current signal increased almost linearly in the first 120 min, and then tended to stabilize after 120 min.

[0082] Optimize the coupling time of hairpin H1 and H2 hybridization chain reaction (HCR): The experimental procedure is the same as above. After mDNA-T incubation is completed and the electrodes are blocked with MCH solution, immerse them in 60mM Na. + The mixture of miRNA-21 and 4 μM hairpin H1 + H2 was incubated for 30, 60, 90, 120, 150, and 180 min, respectively. After the reaction was complete, the SWV signal was measured and recorded. The results are as follows: Figure 9 As shown in Figure C. The results showed that within the time range of 30 min to 150 min, the SWV response signal began to increase almost linearly within 60 min and then tended to stabilize, indicating that the reaction had reached equilibrium. Therefore, an mDNA-T concentration of 1.0 μM, an mDNA-T incubation time of 150 min, and an HCR reaction time of 60 min were selected as the experimental parameters for subsequent experiments.

[0083] All of the above optimization experiments were conducted in triplicate.

[0084] Example 6: Optimization of SPCE / ZrGA / mDNA-T Biosensor Platform and Linearity Study of miRNA-21

[0085] Linearity detection was performed under the optimized experimental conditions described above, and the SPCE / ZrGA electrode was freshly prepared in advance for each detection.

[0086] Prepare 8 prepared SPCE / ZrGA electrodes. Immerse the electrodes in a 1.0 μM mDNA-T mixture and incubate at room temperature for 120 min. After washing, block the electrodes with 1.0 mM MCH solution (15 min). Then, immerse the washed SPCE / ZrGA / mDNA-T electrodes in a solution containing 60 mM Na... + In a mixed solution of 4 μM hairpin H1+H2 and 0, 100 aM, 1 fM, 100 fM, 10 pM, 1 nM, 100 nM and 200 nM miRNA-21, the target will initiate a series of strand displacement reactions. After 60 min of reaction, the solution is washed and the SWV electrochemical signal is measured. The results are as follows: Figure 10 As shown. The results indicated the presence of 200 nM miRNA-21 (target) and 60 mM Na. + The SPCE / ZrGA / mDNA-T portable biosensor platform was used to detect the response of miRNA-21. Under optimal experimental conditions, the current response of the constructed ZrMOF-rGO-AuNPs biosensor platform to a series of target concentrations (from a to g: 0, 100 aM, 1 fM, 100 fM, 10 pM, 1 nM, 100 nM, and 0.2 μM) was examined using the SWV method. Figure 10 (B). Therefore, as the concentration of target miRNA-21 increases, the Fc signal gradually increases. Linear fitting was performed based on the relationship between concentration and current signal ( Figure 10 (C). Within the range of 100 aM to 0.2 μM, the linear fitting equation is as follows: miRNA-21: y = 5.84 logC miRNA-21 +95.54(R 2 =0.997)C corresponds to the miRNA-21 concentration. The calculated LOD is miRNA-21: 34.6 aM (S / N = 3). The proposed SPCE / ZrGA / mDNA-T portable biological platform has advantages in analytical performance in terms of detection range and limit of detection compared to previous work.

[0087] All of the above linear experiments were conducted in triplicate.

[0088] Example 7: Selectivity and reproducibility study of the SPCE / ZrGA / mDNA-T biosensing platform

[0089] The selectivity and reproducibility of SPCE / ZrGA / mDNA-T in detecting miRNA-21 were investigated. The experimental conditions and SWV scanning parameters were the same as in Example 6.

[0090] Selectivity experiment: Prepare 11 SPCE / ZrGA / mDNA-T electrodes. After blocking with MCH solution, immerse the washed SPCE / ZrGA / mDNA-T electrodes in a solution containing miRNA-21+Na. + miRNA-155+Na + miRNA-26a+Na + miRNA-192+Na + miRNA-10b+Na + SM miRNA-21+Na + TM miRNA-21+Na + miRNA-21+K + miRNA-21+Zn 2+ miRNA-21+Mg 2+ and miRNA-21+Fe 3+ The solution was reacted with a 4 μM hairpin H1+H2 mixture. After reacting for 60 min, the solution was washed and the SWV electrochemical signal was measured. The results are as follows: Figure 11 As shown in Figure A. The results show that only with 60 mM Na + The peak current change (ΔI) after hybridization with the complementary target (miRNA-21) was the highest, while any other interfering miRNAs and different cofactors produced only very weak current signals, which is attributed to the high specificity of the chain substitution reaction.

[0091] Reproducibility test: Prepare 8 SPCE / ZrGA / mDNA-T electrodes, and scan the 8 parallel electrodes connected to 60mM Na. + The SWV curves of a mixed solution of 0.2 μM miRNA-21 and 4 μM hairpin H1 + H2 were used to examine the reproducibility of the SPCE / ZrGA / mDNA-T sensing platform. The results are as follows: Figure 11 As shown in Figure B. The results showed that the average standard deviation (RSD) between different replicates was 2.86% (miRNA-21), indicating that the prepared biosensor has satisfactory reproducibility.

[0092] All of the above experiments were conducted in triplicate.

[0093] Example 8: Comparison of Clinical Sample Detection and qRT-PCR Method

[0094] Twenty-six clinical blood samples, including 16 non-small cell lung cancer (NSCLC) patients and 10 healthy individuals as controls, were analyzed using the recommended SPCE / ZrGA biosensor and classic qRT-PCR. This study was approved by the ethics committee, and all human blood samples used in this study were obtained from Southwest Hospital (Chongqing). Serum was centrifuged at 3000 rpm for 10 min to remove blood cells and large vesicles. The supernatant was then used as the experimental part, and exosomes were extracted using the exoRNeasy Midi / Maxi Kit. The extracted exosomes were characterized by morphology, characteristic proteins, and particle size distribution. (See TEM images). Figure 12 (A) The exosomes exhibited the same goblet membrane structure as previously reported. For protein expression assessment, Western blot (WB) experiments confirmed the presence of characteristic protein markers CD63, CD81, and CD9 on the exosome membrane, corresponding to 32, 20, and 23 kDa bands, respectively, consistent with previous reports. Figure 12 (B). Meanwhile, nanoparticle tracking analysis (NTA) showed that approximately 98% of the exosomes from serum samples were in the size range of 30–250 nm, with an average size of 105.2 nm. Figure 12 (C). Therefore, the extracted exosomes maintained good membrane structure and obvious specific protein distribution, proving the effective extraction of exosomes from serum samples.

[0095] Then, total RNA derived from exosomes was extracted as the experimental part. Using the recommended SPCE / ZrGA platform and qRT-PCR, the total RNA from each clinical sample was divided into two equal detection fractions. qRT-PCR was used as the standard method for analyzing the concentration of exosome-derived miRNAs. The concentration of isolated miRNAs was quantified using a BioMate 3S spectrophotometer (Thermo Scientific). The miRNAs were reverse transcribed into cDNA using a reverse transcription kit (Sangon, China) via a stem cell loop-mediated method. The transcribed cDNA was then used as a qPCR template using a 2×SG rapid qPCR Master Mix kit (Sangon, China). The results showed that there was no significant difference between the two methods. Furthermore, the results analysis revealed that... Figure 12 (D and E) The SPCE / ZrGA / mDNA-T portable bio-platform signal proposed in the clinical sample text increased proportionally with the stage of NSCLC patients. The miRNA-21 expression level in stage IV patients was significantly higher than in these healthy individuals, comparable to levels reported in the literature. Furthermore, the proposed bio-platform and classic qRT-PCR were observed to have the same assay capability in differentiating non-small cell lung cancer patient samples (R... 2=0.988), indicating that the proposed method and qRT-PCR have good consistency. Figure 12 In summary, the above results clearly demonstrate that our portable biological platform has high accuracy and applicability, and has great potential in accurately reflecting the staging of non-small cell lung cancer in clinical samples.

[0096] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The SPCE / ZrGA / mDNA-T biosensing platform, characterized in that: The SPCE / ZrGA / mDNA-T biosensing platform includes SPCE / ZrGA sensing electrodes and hairpin chains. The SPCE / ZrGA sensing electrode is obtained by modifying the SPCE graphite working electrode surface with Zr-MOF-rGO, electrodepositing Au nanoparticles, and then fixing multi-armed DNA tetrahedral mDNA-T onto SPCE / ZrGA through Au-S bonds to obtain the SPCE / ZrGA / mDNA-T sensing electrode. The hairpin chain includes hairpin chain I and hairpin chain II, which are modified with stem-loop structures and have electrical signal tags at their ends. The hairpin chains I and hairpin chain II are capable of complementary pairing and binding with sequences remaining after multi-arm DNA tetrahedral cleavage. The Zr-MOF-rGO is prepared by hydrothermal method using ZrCl4 and 2-aminoterephthalic acid as raw materials to obtain octahedral ZrMOF. Then, the obtained octahedral ZrMOF is physically mixed with graphene powder to obtain Zr-MOF-rGO composite material. The multi-armed DNA tetrahedron is a tetrahedral structure assembled from 5 DNA single strands and 3 DNA single strands modified with thiol groups. It also combines with a LOCK chain to temporarily block the target miRNA recognition and binding site. The tetrahedral structure has a swing arm chain at its apex, and the three multi-arm chains adjacent to this swing arm chain contain adenosine ribonucleotides as substrate chains for DNAzyme cleavage sites. The 3' end of each swing arm chain contains Na... + The activated DNAzyme structure's enzyme chain binds to the LOCK chain, the 3' end of which is complementary to the target sequence. After activation, the DNAzyme sequentially cleaves adenosine ribonucleotide sites on the substrate strand. The remaining single-stranded nucleic acid residues after cleavage can complementarily bind with hairpin chain I to form a double strand with sticky ends. The formed sticky ends open hairpin chain II to form sticky ends, and the generated sticky ends trigger a hybridization chain reaction and release electrical signals.

2. The SPCE / ZrGA / mDNA-T biosensing platform according to claim 1, characterized in that: The mass ratio of the octahedral ZrMOF to the graphene powder is 1:0.

5.

3. The SPCE / ZrGA / mDNA-T biosensing platform according to claim 1, characterized in that: The method for electrodepositing Au nanoparticles involves deposition at -0.2 V for 200 s in a mixed solution of 0.01 M Na2SO4 and H2SO4 containing 5 mM HAuCl4.

4. The SPCE / ZrGA / mDNA-T biosensing platform according to claim 1, characterized in that: The nucleotide sequences of the assembly strand of the multi-armed DNA tetrahedron are shown in SEQ ID NO.1~8.

5. The SPCE / ZrGA / mDNA-T biosensing platform according to claim 1, characterized in that: The nucleotide sequence of the LOCK chain is shown in SEQ ID NO.

9.

6. The SPCE / ZrGA / mDNA-T biosensing platform according to claim 1, characterized in that: The sequences of hairpin chain I and hairpin chain II are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively.

7. The application of the SPCE / ZrGA / mDNA-T biosensing platform according to any one of claims 1 to 6 in the detection of miRNA.

8. The application according to claim 7, characterized in that: The miRNA is miRNA-21.

9. A method for detecting miRNA using the SPCE / ZrGA / mDNA-T biosensor platform according to any one of claims 1 to 6, characterized in that, The specific steps are as follows: Using the SPCE / ZrGA / mDNA-T sensing electrode as the working electrode, carbon as the auxiliary electrode, and a silver paste electrode as the reference electrode, the SPCE / ZrGA / mDNA-T sensing electrode was immersed in a solution containing Na. + The sample was incubated in a solution containing hairpin chains I, II, and the target miRNA, and the electrochemical signal was detected.

10. The method according to claim 9, characterized in that: The incubation time is 30 min to 150 min.

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