Preparation method and application method of DNA chimera

Abstract

The invention relates to the technical field of biochemistry, and discloses a DNA chimera preparation and application method, which comprises the following steps: synthesizing phosphorylated linear annular L-rTDNA and template DNA, mixing in a 1 * T4 DNA ligase buffer solution according to an equal molar concentration of 10 [mu] M, heating at 95 DEG C for 5 minutes, and then carrying out gradient cooling to room temperature for renaturation for 120 minutes; adding T4DNA ligase to close a tail end notch, and carrying out enzyme digestion treatment to obtain annular rTDNA; the method comprises the following steps: synthesizing hairpin DNAHP (deoxyribonucleic acid), mixing rTDNA and HP according to an equal molar concentration of 2 mu mol / L, denaturating at 95 DEG C for 5 minutes, slowly cooling to 25 DEG C within 2 hours, and self-assembling to obtain the DNA topology limited chimera. According to the invention, a DNA chimera with dynamic response capability is constructed, an integrated detection process of enzyme-free assembly-target triggering-signal cascade amplification is realized, and meanwhile, the problems of anti-interference and ultra-sensitive detection in a complex matrix are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemical technology, in particular to a DNA chimera preparation and application method thereof. BACKGROUND

[0002] MicroRNAs (miRNAs) are key molecules in gene expression regulation, and their abnormal expression is closely related to the occurrence and development of various diseases (such as cancer, sepsis). miRNAs encapsulated in exosomes are highly stable due to the protection of lipid bilayers and can reflect the state of the source cells, making them extremely potential non-invasive diagnostic biomarkers. However, the trace amount of exosomal miRNAs (usually as low as fM level) and the complex matrix of clinical samples (such as proteases, RNases and non-specific nucleic acids in serum) put extremely high requirements on the sensitivity, specificity and anti-interference ability of detection techniques.

[0003] Current DNA chimera preparation and application methods mainly focus on nucleic acid dynamic assembly and signal amplification, and representative technologies include: 1. Strand displacement reaction (SDR) system: by designing multiple sets of DNA probes (such as probes A-E in patent CN113293199A), cascade strand displacement reaction is used to realize signal amplification without the participation of enzymes, and detection can be completed under isothermal conditions; 2. Rolling circle amplification (RCA) technology: based on the circularization of padlock probes to start RCA, combined with secondary amplification (such as loop-mediated isothermal amplification in patent CN105463110A) to improve signal strength, but it needs to rely on restriction enzyme treatment of amplification products; 3. Fluorescent labeling detection: Toehold-mediated strand displacement reaction triggers RCA, combined with fluorescence resonance energy transfer (FRET) to realize visual detection (such as patent CN110004213A), relying on fluorescent group labeling and optical detection equipment; 4. Reverse transcription-padlock probe technology: miRNA is first reverse transcribed into cDNA, and then RCA is started by padlock probe circularization (such as patent CN117925788A), which needs multiple steps of enzymatic reaction to complete detection.

[0004] Although the above methods have promoted the development of miRNA detection technology to some extent, there are still the following outstanding problems: 1. Complexity of the process and enzyme dependence: for example, patents CN105463110A and CN117925788A require reverse transcription, enzyme cutting or ligation steps, which are cumbersome to operate and enzyme activity is easily inhibited by sample matrix (such as proteases in serum), resulting in long detection time (> 6 hours) and poor repeatability; 2. Static structure and specificity bottlenecks: For example, patents CN113293199A and CN110004213A use linear or static hairpin structures, lacking the ability to undergo dynamic conformational changes triggered by the target. Nonspecific binding leads to high background signals (e.g., the cascade reaction in patent CN113293199A has a nonspecific signal of 15%-20%), and the ability to recognize single-base mismatches is insufficient (e.g., the inhibition rate in patent CN110004213A is less than 50%). 3. Limited sensitivity and insufficient clinical adaptability: The detection limit of existing technologies is mostly at the pM level (e.g., 1 pM in patent CN117925788A), which is difficult to meet the ultra-trace detection requirements of exosomal miRNA. In addition, RNA purification is required, and it is impossible to directly detect miRNA in intact exosomes. The results are easily biased due to RNase degradation or operational loss.

[0005] Although patent CN113293199A simplifies the enzyme dependency problem through entropy-driven strand displacement reaction, the fixed sequence design of pre-assembled multi-probes limits its adaptability to different miRNAs and has limited signal amplification efficiency (detection limit of 10 pM); Patent CN110004213A introduces a Toehold dynamic recognition element, which improves the target binding rate. However, the static hairpin structure lacks conformational rigidity and cannot effectively inhibit nonspecific RCA initiation. In addition, the fluorescence detection cost is high. Patent CN117925788A achieves multiplex detection through high-throughput lock probes, but the risk of enzyme contamination introduced by the reverse transcription step and the pM-level detection limit still cannot meet the needs of accurate quantification of trace exosomal miRNA in clinical samples.

[0006] How to break through the functional limitations of static nucleic acid structures, construct DNA chimeras with dynamic response capabilities, realize the integrated detection process of "enzyme-free assembly-target triggering-signal cascade amplification", and at the same time solve the problems of anti-interference and ultra-sensitive detection in complex matrices has become a technical problem that urgently needs to be overcome. Summary of the Invention

[0007] The present invention aims to provide a method for preparing DNA chimeras to solve the problem of how to design dynamically responsive DNA chimeras to achieve enzyme-free ligation, highly specific recognition, and ultrasensitive signal amplification, while being suitable for direct detection of complex clinical samples (such as serum exosomes).

[0008] In order to solve the above problems, the present invention adopts the following technical solutions: Scheme 1: A method for preparing a DNA chimera, comprising the following steps: Step 1: Synthesize phosphorylated linear circular L-rTDNA and template DNA, mix them in 1×T4 DNA ligase buffer at an equimolar concentration of 10 μM, heat at 95°C for 5 minutes, and then cool down to room temperature for 120 minutes for annealing; Step 2: T4 DNA ligase is added to seal the terminal gap, and circular rT DNA is obtained after enzyme digestion; Step 3: Synthesize hairpin DNAHP. Mix rTDNA and HP at an equimolar concentration of 2 μmol / L, denature at 95°C for 5 minutes, and then slowly cool to 25°C for 2 hours to self-assemble into a DNA topology-constrained chimera.

[0009] Beneficial effects: Gradient annealing and enzyme digestion purification ensure the integrity of the circular structure of rTDNA (circle closure rate > 95%), avoiding nonspecific amplification caused by linear DNA residues; equimolar self-assembly combined with topologically constrained conformation improves the stability of the chimera (Tm=65±2℃), and its resistance to denaturation is enhanced by 30% compared with traditional linear probes.

[0010] Preferably, the enzyme digestion step in step 2 comprises: heating at 70°C for 5 minutes to inactivate the ligase, adding exonuclease III and exonuclease I, reacting at 37°C for 60 minutes to degrade the unclosed linear DNA, and then heating at 80°C for 15 minutes to inactivate the exonuclease.

[0011] Beneficial effects: The double enzyme digestion system completely removes linear DNA impurities (residual amount <1%), ensures the purity of rTDNA, and avoids background signal interference in subsequent detection.

[0012] Preferably, the structural parameter optimization of the DNA chimera includes: designing a series of chimeras with a base number of 5-24 nt in the loop-hairpin junction region and a base number of 5-30 nt in the miRNA recognition region foothold, and screening the target product with stable conformation by 12% non-denaturing PAGE electrophoresis.

[0013] Beneficial effects: Multi-dimensional structural optimization (such as a 20nt linker region + a 10nt foothold) increases the efficiency of chimera RCA by 4 times, and the non-specific signal suppression rate reaches over 95% (the existing technology, such as patent CN110004213A, is only 70%).

[0014] Preferably, the screening conditions for conformational stability are: a single clear band in electrophoretic mobility, and fluorescence detection showing a ratio of rolling circle amplification specific signal to background signal > 10:1.

[0015] Beneficial effects: Combining PAGE and fluorescence dual verification ensures the structural homogeneity (purity > 98%) and functional effectiveness of the chimera, eliminating the interference of heterozygotes.

[0016] Preferably, the slow cooling program of the self-assembly process in step 3 is: after denaturation at 95°C, first cool to 65°C within 30 minutes, and then cool to 25°C within 40 minutes.

[0017] Beneficial effects: Segmented gradient cooling simulates the thermodynamic equilibrium process, promoting precise hybridization between the HP probe and rTDNA (hybridization efficiency > 92%) and avoiding structural disorder caused by random folding.

[0018] Preferably, the method further comprises a chimera purification step: cutting out the target band by 12% non-denaturing PAGE, extracting the target band using a UNIQ-10 centrifugal column DNA gel recovery kit, and quantifying the concentration by UV spectrophotometer to ≥10 μM.

[0019] Beneficial effects: Gel recovery and purification remove unassembled monomers (purity > 95%), and UV quantification ensures dosage accuracy for subsequent testing, with a concentration error of <5%.

[0020] Preferably, the base sequences of the rTDNA and HP are designed to satisfy the following requirements: the 5'-end toehold sequence of HP is complementary to the target miRNA, and the rTDNA contains a barcode sequence that hybridizes with the biotinylated signal probe.

[0021] Beneficial effects: Modular sequence design enables programmable adaptation of the chimera to any miRNA (such as miR-125b, miR-21, etc.), and combined with the biotin-streptavidin system, provides an interface for electrochemical signal amplification.

[0022] Solution 2: The present invention also provides a method for using a DNA chimera, wherein the DNA chimera obtained by the DNA chimera preparation method described above is used to detect miRNA, comprising the following steps: Step 1: Isolate exosomes from serum using ultrafiltration to extract total RNA or directly obtain exosomal miRNA; Step 2: Mix the exosomal miRNA with the DNA chimera, add ɸ29 DNA polymerase and dNTPs, and initiate the rolling circle amplification (RCA) reaction at 37°C for 2 hours. In step 3, the RCA product was hybridized with the biotinylated signal probe through foot-site-mediated strand displacement reaction (TSDR). A three-electrode system was used: a glassy carbon electrode modified with AuAgNPs / Ti3C2Tx-MXene, combined with differential pulse voltammetry (DPV) to detect the electrochemical signal.

[0023] Beneficial effects: The direct exosome detection process omits the RNA extraction step (shortening the time to within 4 hours). Combined with the high conductivity of the MXene / AuAgNPs composite interface (increasing the electron transfer rate by 3 times), the detection limit is as low as 0.15fM, which is 2 orders of magnitude higher than the sensitivity of traditional methods.

[0024] Preferably, the ultrafiltration method in step 1 is performed by centrifugation at 300 × g and 4°C for 10 minutes to remove cell debris, followed by filtration through a 0.22 μM Steriflip® membrane. The clarified supernatant is transferred to a 100 kDa Amicon® ultrafiltration unit, concentrated by centrifugation at 4000 × g for 10 minutes, resuspended in PBS, and stored at -80°C.

[0025] Beneficial effects: Multi-stage centrifugation combined with PBS washing ensures the purity of exosomes (Calnexin negative expression), with a recovery rate of 95.2±3.1%, which is better than the traditional kit method (85±5%).

[0026] Preferably, in step 3, the electrochemical detection parameters for detecting the electrochemical signal are: DPV voltage scanning range -0.05-0.55V, modulation amplitude 70mV, pulse width 50ms, sensitivity 1×10⁻ 6 A / V.

[0027] Beneficial effects: The optimized electrochemical parameters significantly improved the signal-to-noise ratio (SNR>20:1). Combined with the biotin-alkaline phosphatase (SA-ALP) catalytic system, secondary signal amplification was achieved, and the detection linear range reached 2fM-200nM (R²=0.982).

[0028] The advantages of the present invention are: The present invention breaks through the functional limitations of static nucleic acid structures by designing topologically constrained DNA chimeras and combining them with nanomaterial interface optimization. It constructs DNA chimeras with dynamic response capabilities, realizes an integrated detection process of "enzyme-free assembly-target triggering-signal cascade amplification", and simultaneously solves the problems of anti-interference and ultra-sensitive detection in complex matrices.

[0029] Structural innovation: This is the first "programmable topologically constrained DNA chimera" proposed, which achieves target-triggered RCA primer release through a dynamic lock-loop / hairpin conformation without the need for enzyme ligation, and its adaptability is significantly better than the static probe combination in D1. Simplified process: No reverse transcription or enzyme ligation is required, shortening the detection process to 4 hours, a 33% increase in efficiency compared to the two-step method (>6 hours) in Reference 2; Performance breakthrough: Detection limit of 0.15 fM, linear range spanning 8 orders of magnitude, and single-base mismatch signal suppression rate >85% (<50% in Document 3), achieving enhanced specificity and sensitivity. Clinical adaptability: Direct detection of serum exosomal miRNA, with spike recovery of 90-110% (RSD < 6%), which is superior to the process in comparative document 5 that requires RNA purification (recovery of 75-90%).

[0030] Unexpectedness of dynamic conformation design: the prior art (such as patent CN110004213A) adopts a static hairpin structure, while the present patent controls the conformation by topological restriction (20 nt connection region + 10 nt foothold), and unexpectedly finds that it can simultaneously optimize stability and specificity (traditional cognition that long-chain structure easily leads to increased diffusion resistance); Synergistic effect of material combination: the composite interface of AuAgNPs and Ti3C2Tx-MXene (20 nm particles uniformly loaded on 5 nm sheets) significantly increases the electrochemical active area (specific surface area increases by 8 times), which cannot be predicted by stacking a single material, and the prior art does not involve two-dimensional material and noble metal composite; Invention of enzyme-free process: in the context of the comparison file generally relying on enzyme ligation (such as the restriction endonuclease of patent CN105463110A and the reverse transcriptase of patent CN117925788A), the present patent triggers RCA by gradient annealing self-assembly and target, breaking through the technical prejudice that "enzymatic reaction is a necessary condition for signal amplification". BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 A is the HRTEM image of Ti3C2Tx-MXene sheet, B is the HRTEM image of MXene-AuAgNPs, C is the SEM image of Ti3C2Tx-MXene sheet, and D is the SEM image of MXene-AuAgNPs.

[0032] Figure 2 A is the synthesis of DNA ring / hairpin structure and signal amplification mechanism diagram; B is the DNA ring / hairpin structure lock configuration molecular dynamics simulation; C is the PAGE electrophoresis analysis of DNA assembly (lane 1: Maker (20 bp DNA ladder); Lane 2: hairpin structure DNA (HP); Lane 3: miRNA-125b / HP hybrid complex; Lane 4: linear circular DNA (L-rTDNA); Lane 5: circular DNA (rTDNA); Lane 6: DNA ring / hairpin chimera; Lane 7: miRNA-125b triggered rolling circle amplification product); D is the atomic force microscopy (AFM) morphology characterization of DNA ring / hairpin nanostructure.

[0033] Figure 3: This is a concentration-time relationship diagram of the miRNA-125b after fluorescent labeling in the present invention, wherein a is the amplification system of DNA loop / hairpin nanostructure and 200nM miRNA-125b; b is the amplification system of DNA loop / hairpin nanostructure and 200pM miRNA-125b; c is the amplification system of DNA loop / hairpin nanostructure and 200fM miRNA-125b; d is the amplification system of HP and 200nM miRNA-125b; e is the amplification system of HP and 200pM miRNA-125b; f is the amplification system of HP and 200fM miRNA-125b.

[0034] Figure 4 Schematic diagram of the structural optimization and specificity analysis of DNA self-assembly in the present invention, wherein A is the PAGE analysis of DNA loop / hairpin assembly with different toe lengths in the hairpin probe (HP). Lane 1: 20 bp DNA molecular weight standard; Lane 2: circular DNA template (rTDNA); Lane 3: HP with 5 bp toe; Lane 4: DNA loop / hairpin (5 bp toe); Lane 5: HP with 10 bp toe; Lane 6: DNA loop / hairpin (10 bp toe); Lane 7: HP with 15 bp toe; Lane 8: DNA loop / hairpin (15 bp toe); Lane 9: HP with 20 bp toe; Lane 10: DNA loop / hairpin (20 bp toe). B is PAGE analysis of DNA loop / hairpin assembly with different hybridization region lengths in rTDNA-HP complexes. Lane 1: 20 bp DNA molecular weight standard; Lane 2: rTDNA (30 bp hybridization region); Lane 3: DNA loop / hairpin (30 bp hybridization region); Lane 4: HP (30 bp hybridization region); Lane 5: DNA loop / hairpin (24 bp hybridization region); Lane 6: HP (24 bp hybridization region); Lane 7: DNA loop / hairpin (20 bp hybridization region); Lane 8: HP (20 bp hybridization region); Lane 9: DNA loop / hairpin (15 bp hybridization region); Lane 10: HP (15 bp hybridization region); Lane 11: rTDNA (24 bp hybridization region). C is the fluorescence reaction of the DNA loop / hairpin structure triggered by target miRNA-125b. In the presence of target miRNA-125b, the fluorescence intensities of 7 DNA loop / hairpin variants and negative controls were measured by a fluorescence spectrophotometer: a. 5bp hairpin footpoint DNA loop / hairpin; b. 10bp hairpin footpoint DNA loop / hairpin; c. 15bp hairpin footpoint DNA loop / hairpin; d. 20bp hairpin footpoint DNA loop / hairpin; e. 15bp hybridization region DNA loop / hairpin; f. 24bp hybridization region DNA loop / hairpin; g. 30bp hybridization region DNA loop / hairpin.

[0035] Figure 5The images of the exosomes in the present invention are shown, where A is a TEM image of exosomes; B is an NTA analysis of exosomes; and C is a WB analysis of exosomes.

[0036] Figure 6 The successful stepwise preparation of the working electrodes in this invention is demonstrated by measuring EIS patterns after assembly of the GCE (a), Ti3C2Tx-MXene-modified electrode (b), AuAgNPs-modified electrode (c), DNA topology-constrained chimera-modified electrode (d), MCH-modified electrode (e), target miRNA-125b-modified electrode (f), biotin signal probe-modified electrode (g), and streptavidin-alkaline phosphatase-modified electrode (h). Inset: Enlarged EIS patterns of the GCE, Ti3C2Tx-MXene-modified electrode, AuAgNPs-modified electrode, and DNA topology-constrained chimera-modified electrode.

[0037] Figure 7 This is a comparison chart of laboratory condition optimization used in the present invention. In Panel A, circles represent AuAgNPs concentration, squares represent Ti3C2Tx-MXene concentration, Panel B shows the optimal reaction temperature for φ29 polymerase, Panel C shows the φ29 polymerase concentration, Panel D shows the concentration of the signaling probe, Panel E shows the pH of the DEA buffer, and Panel F shows the DNA topology-constrained chimera concentration and signal-to-noise ratio. Error bars represent the standard deviation of three replicate measurements.

[0038] Figure 8 Figure 1 is a performance analysis comparison chart of the electrochemical biosensor of the present invention, wherein: A represents the stability assessment of the biosensor; B represents the DPV response of the electrochemical biosensor to different oligonucleotides (200 nM): (a) single-base mismatch strand (B1); (b) two-base mismatch strand (B2); (c) three-base mismatch strand (B3); (d) non-complementary strand; (e) blank; (f) target miRNA-125b; C represents the reproducibility analysis: the current response of five independent measurements at 200 fM, 200 pM, and 200 nM target miRNA-125b; D is the DPV curve of a miRNA-125b concentration gradient (from bottom to bottom: 0 fM, 2 fM, 20 fM, 200 fM, 2 pM, 20 pM, 200 pM, 2 nM, 20 nM, 200 nM); E is the linear correlation between the DPV signal and the logarithmic target miRNA-125b concentration (range: 2 fM–2 uM). Error bars represent the standard deviation of three replicate measurements.

[0039] Figure 9 Figure 2 is a heat map of the data of sepsis (SPs), inflammation (GIPs), and healthy subjects (HDs) groups detected using electrochemical sensors in the present invention. DETAILED DESCRIPTION

[0040] The following is further described in detail through specific implementation methods: (1) DNA chimera preparation method Example 1: Preparation of rT DNA Mixing and renaturation: Add 10 μL each of phosphorylated L-rTDNA and template DNA (sequence complementary) (10 μM) to 80 μL of 1× T4 ligase buffer, heat at 95°C for 5 minutes, and then cool to room temperature at a rate of 1°C / minute (total time: 120 minutes); Ligation and enzyme digestion: Add 5μL T4 ligase (5U / μL) and incubate at room temperature for 8 hours; heat at 70℃ for 5 minutes to inactivate the enzyme, then add 2μL exonuclease III (10U / μL) and 2μL exonuclease I (10U / μL) and react at 37℃ for 60 minutes; heat at 80℃ for 15 minutes to inactivate the exonuclease to obtain rTDNA solution (concentration ≈ 8μM).

[0041] Example 2: Chimera Self-Assembly and Optimization Assembly conditions: 10 μL of rT DNA and 10 μL of HP (20 μM) were added to 80 μL of reaction buffer (10 mM Tris-HCl, pH 7.5), denatured at 95°C for 5 min, cooled to 65°C at 1°C / min (30 min), and then cooled to 25°C at 0.5°C / min (40 min); Structural screening: Take 10 μL of the assembled product and run it on 12% non-denaturing PAGE (120V, 45 minutes). Cut out the band with a slower migration rate than the linear DNA (such as Figure 2 Lane C 6), UV quantification to 10 μM after gel recovery; Parameter optimization: By designing a series of chimeras with 5-24nt linker and 5-30nt toehold, the optimal parameter was determined to be 20nt linker + 10nt toehold (e.g. Figure 4 Lane B 7, Figure 4 C Bar graph b), at this time, the fluorescence intensity of the RCA product is 12 times the background value.

[0042] (II) Application methods of DNA chimeras Example 3: Detection of exosomal miRNA Exosome isolation: 2 mL of serum was centrifuged at 300 × g at 4°C for 10 minutes to remove cellular debris, followed by filtration through a 0.22 μM Steriflip® membrane. The clarified supernatant was transferred to a 100 kDa Amicon® ultrafiltration unit and concentrated by centrifugation at 4000 × g for 10 minutes. The supernatant was washed twice with 2 mL of PBS to remove impurities. The precipitate was resuspended in PBS, gently pipetted, and centrifuged at 4000 × g for 30 minutes. The resulting pellet was the exosomes.

[0043] RCA reaction: 20 μL of exosome suspension (containing miRNA) was mixed with 20 μL of chimera (10 μM), 8 μL of dNTPs (2.5 mM), 1.6 μL of ɸ29 polymerase (10 U / μL), and 20 μL of 10× reaction buffer were added, and incubated at 37°C for 2 h. Electrochemical detection: Electrode modification: The glassy carbon electrode was polished with 300 nm and 50 nm alumina, and then ultrasonically cleaned and drop-coated with 10 μL Ti3C2Tx-MXene (0.1 mg / mL) and 10 μL AuAgNPs (20 nM). After drying, the chimera probe was drop-coated and incubated at 4°C overnight. Signal reading: After hybridization of the RCA product with the biotinylated probe, the SA-ALP complex was added and DPV detection was performed in DEA buffer containing 1-NPP. The miRNA concentration was calculated based on the peak current.

[0044] Comparative Example: Traditional Lock Probe Method (Patent CN117925788A) Process: serum RNA extraction → reverse transcription into cDNA → padlock probe circularization → RCA → fluorescence detection; Performance comparison: Time consumption: 6 hours (4 hours for the present invention); Detection limit: 1 pM (0.15 fM in the present invention); Serum recovery rate: 85±5% (98.1±5.6% in the present invention).

[0045] Conclusion: The present invention significantly improves the efficiency, sensitivity and clinical applicability of miRNA detection through dynamic chimera design and nanomaterial combination, and has outstanding substantive characteristics and significant progress.

[0046] The specific implementation process is as follows: This study innovatively constructed a programmable topologically constrained DNA chimera. By specifically recognizing a target miRNA, it triggers a conformational transition in the hairpin structure, releasing the topological constraints of the DNA nanostructure, enabling autonomous assembly of the circular template and primer, and initiating RCA. The amplified product hybridizes with a biotinylated signal probe via TSDR, generating a cascade-amplified electrochemical response signal. Compared with traditional detection methods, this strategy eliminates the need for miRNA reverse transcription and padlock probe ligation, significantly simplifying the operational process. Addressing theoretical gaps in existing DNA chimera research regarding spatial conformational regulation, entropy-driven mechanisms, and signal leakage control, this study systematically analyzed its molecular assembly mechanism through a multidimensional experimental system. By fine-tuning the number of base pairs between the circular structure and the hairpin structure, the study elucidated the dynamic regulation of the system's thermodynamic stability and reaction kinetics by topological constraints. Based on orthogonal optimization experiments on toehold sequence length and composition, a structure-activity relationship model was established between the specificity of the molecular recognition interface and the efficiency of probe hybridization, identifying the optimal combination of structural parameters. Ultimately, a synergistically enhanced cascade catalytic interface of the AuAgaNPs / Ti3C2Tx-MXene layered composite was constructed, achieving an exponential increase in electrochemical signal amplification efficiency. The developed sensor exhibited excellent linear response over a concentration range of 2fM to 200nM, with a detection limit as low as 0.15fM, successfully enabling precise quantitative analysis of exosomal miR-125b in clinical serum samples. This study not only provides an innovative technological platform for the precise diagnosis and treatment of sepsis but also, through the establishment of a DNA self-assembly strategy, opens new avenues for ultrasensitive detection of clinical diagnostic biomarkers, significantly promoting the translational application of functionalized DNA nanodevices in precision medicine.

[0047] 1. Experimental methods 1.1 Chemical reagents Ti3C2Tx-MXene dispersion (lateral size 2-5 μm), Fe3O4 nanoparticles, TiO2 nanoparticles, Ti3AlC2, and WS2 nanosheets (diameter 2-5 μm) were obtained from Jiangsu Xianfeng Nanomaterials Co., Ltd. (Nanjing, China, www.chinaxfnano.com). MoS2 nanosheets (diameter 20-500 nm) were provided by Nanjing Jicang Nanotechnology Co., Ltd. (Nanjing, China, www.jcnano.com). NH2-Fe3O4, Nafion solution, gold chloride trihydrate (HAuC l4 3H2O, 99.9%), silver nitrate (AgNO3, 99%), and ascorbic acid (AA, 99.0%) were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China, www.aladdin-e.com). Octadecyltrimethylammonium chloride (OTAC, 90%) was obtained from Lusen Chemical (Linyi, China). Gold chloride (HAuC l4), sodium citrate, 6-mercaptohexanol (MCH), 1-naphthol phosphate (1-NPP), 1-naphthol, streptavidin-alkaline phosphatase (SA-ALP), 4-nitrophenol, and diethanolamine (DEA) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA; www.sigmaaldrich.com). Bovine serum albumin (BSA) was purchased from Sigma (St. Louis, MO, USA). Exonuclease III was purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Tris(2-carboxyethyl)phosphine (TCEP), T4 DNA ligase, deoxyribonucleotide mix (dNTPs), polymerase (ɸ29 DP), and exonuclease I were all purchased from Bioengineering Technology Service Co., Ltd. (Shanghai, China). All DNA oligonucleotides were purchased from Bioengineering Technology Service Co., Ltd. (Shanghai, China). The sequences of the DNA oligonucleotides used in this experiment are shown in Table 1. Clinical serum samples were obtained from the University Town Hospital Affiliated to Chongqing Medical University (Chongqing, China). The following buffers and solutions were used in this experiment: 20 mM Tris-HCl, 5 mM potassium chloride (KCl), 140 mM sodium chloride (NaCl), 1 mM magnesium chloride (MgCl2), and 1 mM calcium chloride (CaCl2), pH 7.4, for DNA preparation. 140 mM NaCl, 5 mM KCl, 20 mM Tris-HCl, and 10 mM TCEP, pH 7.4, for solubilizing the capture probe. Tris buffer: 20 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, pH 7.4. Tris-T buffer: 20 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, 0.05% Tween-20, pH 7.4, for the wash buffer. Diethanolamine (DEA) buffer: 0.1 M DEA, 100 mM KCl, and 1 mM MgCl2, pH 9.6. The working buffer for electrochemical measurements consisted of 5 mL of 0.1 M DEA buffer containing 1 mg / mL 1-NP, pH 9.6. The buffer for cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) consisted of 0.1 M KCl and 5 mM [Fe(CN)6]3- / 4-, pH 7.4. Ultrapure distilled and deionized water (18.2 MΩ) obtained from an Arium® pro (Sartorius, Germany) purification system was used for all solution preparations throughout the experiments.

[0048] Table 1

[0049] B1, B2, B3, and B4 are single-base mismatched strands, double-base mismatched strands, triple-base mismatched strands, and non-complementary strands, respectively.

[0050] 1.2 Instruments and Equipment All electrochemical measurements in this experiment, including differential pulse voltammetry (DPV), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS), were performed on a DH7003 electrochemical workstation (Jiangsu, China). The three-electrode system consisted of a saturated calomel electrode (reference electrode), a platinum wire (auxiliary electrode), and a 3 mm diameter glassy carbon electrode (working electrode). Gel electrophoresis was performed using a Bio-Rad electrophoresis analyzer (USA), and gel images were acquired using a Quantum CX5 (Vilber, France) gel imaging system. UV-visible absorption spectra (UV-vis) of the samples were measured using a Cary Eclipse (Agilent, USA). Transmission electron microscopy (TEM) images were acquired using a Bruker Dimension Icon microscope (Bruker, USA). Energy-dispersed photoelectron spectroscopy (EDS) was collected using a JEOL JSM-6700F microscope (Japan). All electrochemical measurements were performed at room temperature (25 ± 1°C).

[0051] 1.3 Preparation of AuAgaNPs The synthesis of gold-silver nanoparticles (AuAgaNPs) was performed as follows: Octadecyltrimethylammonium chloride (OTAC, 200 mM, 0.25 mL), ultrapure water (4.75 mL), chloroauric acid (HAuCl4, 0.5 mM, 3 mL), and silver nitrate (AgNO3, 0.5 mM, 3 mL) were added sequentially to a 20 mL glass reaction vial. The mixed solution was allowed to stand in an ice-water bath for 10 minutes. Ascorbic acid (AA, 10 mM, 1 mL) was then rapidly added to initiate a reduction reaction, and the reaction was stirred continuously for 1 hour. After the reaction, the product was collected by centrifugation (12,000 rpm, 10 minutes) and washed once with ultrapure water to remove unreacted reagents. The resulting nanoparticles were stored at 4°C until use.

[0052] 1.4 Synthesis of DNA topology-constrained chimeras Preparation of circular DNA: Phosphorylated linear circular DNA (L-rTDNA) and template DNA were mixed at equimolar concentrations (10 μM) in 1× T4 DNA ligase buffer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, pH 7.8). The mixture was heated at 95°C for 5 minutes, followed by a temperature ramp (cooling to room temperature over 120 minutes) to allow DNA strands to anneal. 5 μL of T4 DNA ligase was added, and the reaction was incubated at room temperature for 8 hours to close the terminal nicks. After inactivating the ligase by heating at 70°C for 5 minutes, exonuclease III and exonuclease I were added and reacted at 37°C for 60 minutes to completely degrade any unclosed linear DNA. The reaction system was heated at 80°C for 15 minutes to completely inactivate the exonucleases. The resulting circular DNA (rTDNA) solution was stored at 4°C until further use.

[0053] Assembly and purification of DNA topology-constrained chimeras: Equimolar concentrations of rT DNA and hairpin DNA (HP) were mixed in reaction buffer to a final concentration of 2 μmol / L. The mixture was denatured at 95°C for 5 minutes and then induced to self-assemble using a slow cooling procedure (approximately 2 hours to 25°C). The DNA nanostructures were characterized by 12% native polyacrylamide gel electrophoresis (PAGE). The target band (DNA loop / hairpin complex) was excised and enriched from the gel slice using the UNIQ-10 spin column DNA gel extraction kit. Quantitative analysis was performed by UV spectrophotometry.

[0054] 1.5 Optimization and specificity verification of DNA topology-constrained chimeras We designed and synthesized DNA chimera with different structural parameters, including DNA chimera with 5, 10, 15 and 24 nt in the loop-hairpin linker region, and DNA chimera with 5, 10, 15, 20 and 30 nt in the miR-125b recognition region. All the synthesized products were characterized by 12% polyacrylamide gel electrophoresis (PAGE), and the chimera with better conformational stability was selected for subsequent experiments according to the electrophoresis results. The selected chimera was purified and concentrated using the previous method for subsequent research. Then the rolling circle amplification reaction system was constructed, 20 uL of 500 nM DNA chimera solution was mixed with 8.0 uL of 2.5 mM dNTP mixture, 1.6 uL of Phi29 DNA polymerase (10 U / uL), 20 uL of 10x reaction buffer and 4.0 uL of 200 nM miR-125b standard, and incubated at 37℃ for 2 hours to complete the amplification reaction. The fluorescence detection system was configured in parallel, 4 uL of 200 nM Cy5 labeled molecular beacon probe and 4 uL of 200 nM BHQ2 quenched probe were added to 138.4 uL of PBS buffer containing 2 mmol / L MgCl2 (pH 7.4), and incubated at room temperature for 10 minutes to allow the probe to fully anneal. Finally, 200 uL of amplification product was mixed with fluorescence quenched DNA double-stranded (dsDNA) signal probe, and real-time fluorescence detection was immediately performed using a fluorescence spectrophotometer at an excitation wavelength of 650 nm and an emission wavelength of 670 nm to evaluate the rolling circle amplification specificity of different structural chimeras.

[0055] 1.6 Extraction of miRNA in exosomes Exosomes were isolated from serum by ultracentrifugation, and the whole process was carried out at 4℃. First, the serum sample was centrifuged at 2000 x g for 30 minutes to remove apoptotic cells and cell debris, and then centrifuged at 10000 x g for 30 minutes to further remove impurities. The exosomes were preliminarily precipitated by ultracentrifugation at 110000 x g for 80 minutes, resuspended with phosphate buffered saline (PBS) to remove soluble protein contaminants, and then centrifuged at 110000 x g for 80 minutes to obtain high-purity exosomes. The final precipitate was resuspended in PBS solution (100 uL PBS for every 1 mL of original serum) and stored in a ultra-low temperature freezer at -80℃. The total RNA was extracted from the exosome suspension using TRI Reagent BD lysis buffer (Molecular Research Center, USA), and the manufacturer's operation manual was strictly followed. The concentration and purity of the total RNA were determined using a NanoDrop 2000 ultramicro spectrophotometer (Thermo Fisher Scientific, USA).

[0056] 1.7 Construction of electrochemical biosensor Glassy Carbon Electrode Pretreatment: Bare glassy carbon electrodes (GCEs) were polished to a mirror-like finish using 300nm and then 50nm alumina polishing slurries. Ultrasonic cleaning was then performed in ultrapure water, anhydrous ethanol, and ultrapure water for 5 minutes each to remove residual surface particles. After cleaning, the electrodes were dried with nitrogen at room temperature and then used.

[0057] Preparation of nanomaterial dispersion: Ti3C2Tx-MXene (0.1 mg / mL) was dispersed in ultrapure water containing 0.1% Nafion solution and ultrasonicated for 60 min to ensure uniform dispersion. The product was stored in the dark at 4 °C until use.

[0058] Layer-by-layer electrode modification: 10 μL of a Ti3C2Tx-MXene suspension (0.10 mg / mL) was drop-coated on the GCE surface and slowly dried at room temperature to form a uniform coating. Then, 10 μL of an AuAgNPs solution was drop-coated and dried at room temperature to prepare an AuAgNPs / Ti3C2Tx-MXene / GCE composite electrode. 10 μL of a 200 nmol / L thiolated DNA loop and hairpin nanoprobe pretreated with tris(2-carboxyethyl)phosphine (TCEP) was drop-coated on the modified electrode surface and incubated overnight at 4°C. After rinsing with wash buffer, the electrode was immersed in a 1.0 mM 6-mercaptohexanol (MCH) solution and blocked at room temperature for 1 hour to block nonspecific sites. This resulted in the MCH / DNA loop / hairpin / AuAgNPs / Ti3C2Tx-MXene / GCE electrochemical biosensing platform. A 10 μL mixture containing 1× ɸ29DP reaction buffer (50 mmol L⁻¹ Tris-HCl, 10 mmol L⁻¹ MgCl₂, 10 mmol L⁻¹ (NH₄)₂SO₄, 4 mmol L⁻¹ DTT, pH 7.5), 500 μmol L⁻¹ dNTPs, 0.08 U L⁻¹ phi29DP, and the target miRNA was added to the electrochemical biosensor platform. After a 2-hour incubation at 37°C, the RCA reaction was terminated by heating to 65°C for 10 minutes. Finally, the working electrode was incubated with 10 μL of 2.5 μM biotinylated detection probe at 37°C for 1 hour. After each step, the electrode was rinsed with wash buffer. Next, the prepared electrode was treated with 10 μL of DEA buffer containing 1.25 μg / mL SA-ALP and 8 mg / mL BSA at 37°C for 30 minutes to block nonspecific binding sites and ensure specific binding of SA-ALP to biotin. Finally, the sensor was thoroughly washed with DEA ​​buffer, and the electrochemical signal was detected by DPV method in DEA buffer containing 1 mg / mL 1-NPP.

[0059] 1.8 Electrochemical measurement parameters All electrochemical tests were performed using a three-electrode system (working electrode: modified glassy carbon electrode; counter electrode: platinum wire electrode; reference electrode: calomel electrode with saturated KCl solution) on an electrochemical workstation. The entire experiment was carried out in a constant temperature environment (25±1°C). The specific test conditions are as follows: Cyclic voltammetry (CV): scanning potential range is -0.2 to 0.6 V, scanning rate is 100 mV / s, and it is stabilized at the open circuit potential for 300 s before testing. Electrochemical impedance spectroscopy (EIS): applied AC amplitude of 10 mV, frequency scanning range of 0.1 Hz–100 kHz, and DC bias is the open circuit potential of the system. Differential pulse voltammetry (DPV): voltage scanning range is -0.05–0.55 V, modulation amplitude is 70 mV, pulse width is 50 ms, sampling interval is 200 ms, and sensitivity is set to 1×10⁻ 6 A / V.

[0060] Characterization of AuAgNPs / Ti3C2Tx materials: Transmission electron microscopy (TEM) was used to characterize the morphology of the Ti3C2Tx-MXene and MXene-AuAgNPs samples. Ti3C2Tx-MXene showed very large flakes and some stacked small fragments ( Figure 1 A), the MXene-AuAgNPs sample appears as ultrathin nanosheets with wavy morphology aggregated on large flakes, which makes it contain more interfaces and chemically active sites ( Figure 1 B). The morphology and structure of the prepared MXene and MXene-AuAgNPs were analyzed by SEM. Figure 1 As shown in Figure C, the layered structure of MXene is composed of thin and transparent nanosheets stacked on top of each other. Figure 1 D shows the SEM image of MXene-AuAgNPs, where AuAgNPs are uniformly distributed on the surface and between the layers of MXene.

[0061] Characteristics of DNA topology-constrained chimeras: like Figure 2 Figure A shows the synthesis and preparation route of the DNA topology-constrained chimera, as well as the mechanism by which the target miRNA initiates in situ RCA to achieve signal amplification and detection. The DNA topology-constrained chimera is constructed through precise molecular design. It integrates the HP probe and circular template DNA (rTDNA), and exhibits a locked configuration ( Figure 2B). This chimeric probe exhibits dual functionality: HP not only serves as a recognition element for the target miRNA but also as a primer for RCA, while the rTDNA carries a specific barcode sequence that can be used to capture the signal probe. During RCA, the rTDNA template is continuously amplified, generating an ultra-long single-stranded DNA product. This product then undergoes multi-site hybridization with a biotinylated electrochemical probe or a double-stranded fluorescent probe, achieving cascade signal amplification. During preparation, HP and rTDNA are mixed in equimolar amounts and then self-assembled using a gradient annealing protocol (95°C for 5 minutes, followed by cooling to 65°C over 30 minutes, and then further cooling to 25°C over 40 minutes). Thermodynamic analysis shows that decreasing the temperature from 95°C to 65°C promotes HP unfolding and hybridization with rTDNA, while subsequent cooling promotes self-folding of the HP ends, forming a stable chimera. This stable topology effectively inhibits nonspecific RCA activation.

[0062] To verify the effect of chimera construction, we used atomic force microscopy (AFM) tapping mode to characterize the typical lock-ring morphology ( Figure 2 D), which is highly consistent with the simulated conformation. At the same time, 12% PAGE was used to verify the preparation process of DNA topology-restricted chimeras and the RCA reaction triggered by miRNA. Figure 3 As shown in the gel electrophoresis image in Figure c, after the addition of target miRNA-125b to HP, a band with significantly slower electrophoretic migration appeared in lane 2, indicating that HP binds to the target miRNA-125b. Although linear rTDNA (L-rTDNA) and rTDNA have the same number of bases, the electrophoretic mobility of rTDNA is significantly slower than that of L-rTDNA (compare lanes 4 and 3). This is due to the different electrophoretic hindrances generated by the circular structure of L-rTDNA. Subsequently, rTDNA and HP were annealed and assembled, resulting in the appearance of multiple bands with significantly slower electrophoretic migration in lane 5, indicating the successful assembly of the larger molecular weight DNA loop / hairpin nanostructure. After the introduction of target miRNA-125b and ɸ29 DNA polymerase (lane 6), the complex band disappeared and a high-molecular-weight RCA product band appeared, confirming the specific amplification reaction triggered by the target.

[0063] The experiment further verified the feasibility of using DNA chimeras to detect miRNA-125b. Figure 3As shown in Figure (b), during the 2-hour RCA process, the fluorescence signal gradually increased over time, confirming that the RCA reaction was successfully activated. Notably, when the DNA loop / hairpin nanostructure was present in the system, the concentration of the target miRNA-125b showed a significant positive correlation with the Cy5 fluorescence signal (curves ac). In sharp contrast, although the system containing only the HP probe also detected an increase in fluorescence signal (curve df), its intensity was completely unrelated to the target concentration. These data strongly demonstrate that the DNA loop / hairpin nanostructure can specifically recognize the miRNA target and initiate the RCA reaction through precise molecular recognition, ultimately generating a large number of long DNA products.

[0064] DNA topology is related to specificity: In the chimera structure optimization experiment, we systematically adjusted the number of bases (5-20bp) of the miRNA binding site at the 5' end of the HP probe. Through PAGE analysis, we found that although the 5bp binding site can form a chimera ( Figure 4 A, lane 4), but induces nonspecific RCA activation ( Figure 4 C Bar graph a). When the binding site reaches 10 bp or more, the system shows strict target dependence ( Figure 4 Bar graphs (b, d). Further detection using a fluorescence quenching probe revealed that chimeras with a 15-20 bp binding site not only maintained high synthesis efficiency but also exhibited excellent detection specificity. Through cross-analysis of thermodynamic simulations and experimental verification, the optimal parameter range for the miRNA recognition domain was determined to be 10-20 bp. This optimization scheme effectively maintained structural and functional stability while ensuring molecular recognition accuracy.

[0065] Under the premise of keeping the hairpin structure miRNA foothold 10bp unchanged, we systematically adjusted the length of the rTDNA-HP hybridization region (15-30bp). When the hybridization region was 15bp, PAGE analysis showed obvious band tailing phenomenon ( Figure 4 Lane B 9) indicates that the chimera structure is not stable enough; when extended to 20 bp, the electrophoresis shows a clear single band ( Figure 4 Lane B 7) shows that the structural stability is significantly improved. When the number of bp is increased to 24 bp, a miscellaneous band appears ( Figure 4 Lane B 5), presumably due to the formation of rTDNA dimers, but the main product still maintains the correct configuration; while in the 30bp system, although there are a few miscellaneous bands ( Figure 4However, the structure of the 30-bp hybridization region was disordered, which was significantly different from the ideal configuration. The length of the hybridization region also had a significant impact on the specificity of RCA. Experimental data showed that the 15-24 bp system could achieve specific amplification, but when the hybridization region was extended to 30 bp, the specificity of amplification decreased significantly (possibly related to structural disorder). Comprehensive analysis showed that the 20-bp hybridization system not only had the best structural stability, but also exhibited the highest RCA amplification efficiency.

[0066] Characterization of exosomes: Before carrying out the detection of exosomal miRNA (Exo-miR), we first successfully isolated exosomes from the serum of sepsis patients and extracted total RNA. Transmission electron microscopy (TEM) images clearly showed that these exosome particles were uniformly dispersed, exhibiting a typical cup-shaped morphology and an intact membrane structure (Fig. 1A). Figure 5 A). Further confirmation by nanoparticle tracking analysis (NTA) showed that the concentration of exosomes after purification reached 2.3 x 10 8 particles / mL after 35-fold dilution, with a particle size mainly distributed in the range of 100-250 nm (Fig. 1B). Figure 5

[0067] To strictly verify the identity and purity of exosomes, we used Western Blot (WB) to detect key molecular markers. CD9 is a member of the TM4SF / Tetraspanins family, a membrane protein with four transmembrane structures, which can aggregate on the cell membrane surface and is a surface marker of exosomes. TSG101 was initially discovered as a tumor suppressor and negative growth regulator, and is a key component of the endosomal sorting complex required for transport, involved in the biogenesis of multivesicular bodies and the secretion of exosomes. Therefore, TSG101 is considered a hallmark protein of exosomes. The detection of TSG101 by WB can further verify whether the extracted or isolated material is an exosome. ALIX is also a marker of exosomes and is closely related to the biogenesis and secretion process of exosomes. Calnexin is a type I integral endoplasmic reticulum membrane protein, usually located between the endoplasmic reticulum and the cytosol. In the WB experiment, the negative expression of Calnexin is usually considered as one of the conditions for obtaining pure exosomes. In other words, if the expression of Calnexin is detected in the exosome sample, it may mean that there is some endoplasmic reticulum or other organelle contamination in the sample. When we used WB to characterize the extracted exosomes, CD9, TSG101, and ALIX were specifically expressed in the sample; while the endoplasmic reticulum marker Calnexin was not detected, effectively excluding organelle contamination (Fig. 1C). Figure 5 ​C). These results fully verified the successful isolation of exosomes from three dimensions: morphological characteristics, particle size distribution, and molecular markers, laying a solid foundation for subsequent miRNA detection.

[0068] Characterization of electrochemical sensors: To verify the successful fabrication of the biosensor, we characterized the sensor assembly process step by step using electrochemical impedance spectroscopy (EIS). Figure 6 As shown, analysis based on the Randle equivalent circuit model reveals that, based on a bare glassy carbon electrode (curve a), Ti3C2Tx-MXene modification significantly reduces the semicircular diameter in the high-frequency region (curve b), thanks to the material's excellent conductivity, effectively reducing the interfacial resistance. Subsequent introduction of AuAg nanoparticles (curve c) further minimizes the electron transfer resistance. When loaded with a DNA topology-constrained chimera (curve d), the interfacial resistance significantly increases due to the insulating properties of the structure. The subsequent steric hindrance created by the immobilization of MCH molecules (curve e) further increases the resistance, successfully blocking electron penetration to the electrode surface. After specific recognition of the target miRNA-125b (curve f), the step-wise increase in impedance confirms the precise capture of the target by the DNA chimera. When assembling the biotinylated signal probe (curve g), the negatively charged DNA backbone enhances electrostatic repulsion, significantly increasing the electron transfer barrier. Finally, the introduction of the SA-ALP complex (curve h) leads to a peak interfacial resistance. The continuous evolution of the EIS impedance spectrum systematically verifies the successful step-by-step construction of each functional layer of the biosensor.

[0069] Optimization of experimental conditions: In order to optimize the analytical performance of biosensors, Figure 7 As can be seen, we systematically screened seven key parameters. The results showed that the lowest signal-to-noise ratio was achieved when the AuAgNPs concentration was 20 nM, the Ti3C2Tx-MXene material concentration was 3 mg / mL, the φ29 polymerase optimal reaction temperature was 37°C, the φ29 polymerase concentration was 0.08 U / uL, the signal probe concentration was 500 nM, the DEA buffer pH was 9.0, and the DNA topology-constrained chimera concentration was 700 nM. These optimized conditions synergistically improved the sensitivity and specificity of the detection system.

[0070] Performance analysis of electrochemical biosensors: In stability testing, Figure 8 As shown in Figure A, the signal attenuation of the modified electrode stored at 4°C was less than 6.64% within the first 5 days, and 85.90% of the initial response value was still retained after 20 days of storage, demonstrating that the sensor has good long-term stability.

[0071] To verify the specificity of the sensor, we introduced single-base mismatch chains, double-base mismatch chains, triple-base mismatch chains, and non-complementary sequences at a target concentration of 200 nM. Figure 8 As shown in Figure B, the signal response for a single-base mismatch strand was only 13.3% of that for a perfectly matched strand, while for double-base and triple-base mismatch strands it dropped to 13.5% and 9.3%, respectively, demonstrating the sensor's ability to discriminate between base mismatches. The signal response for the non-complementary strand (8.9%) was not significantly different from that for the blank control (5.4%) (p>0.05), further confirming the high selectivity of the detection system.

[0072] Repeatability evaluation showed that ( Figure 8 C) Five independent tests were performed for miRNA-125b at three concentration gradients of 200 fM, 200 pM, and 200 nM. The relative standard deviations (RSDs) of the current responses were 5.5%, 3.2%, and 4.2%, respectively, indicating that the sensor has excellent batch-to-batch consistency.

[0073] To systematically evaluate the analytical performance of the biosensor, we used differential pulse voltammetry (DPV) to detect the current response of miRNA-125b at different concentrations under optimized conditions. Figure 8 As shown, the peak current exhibited an excellent linear relationship with the logarithmic concentration of miRNA-125b over the target concentration range of 2 fM to 200 nM (R=0.982). The linear regression equation was I=1.81048×1g(c / pm)+7.47117 (I: peak current; c: target concentration). The detection limit, calculated by 3 times the standard deviation of the blank signal, was as low as 0.15 fM, representing two orders of magnitude improvement in sensitivity compared to traditional ELISA methods, confirming the ultrasensitive detection capabilities of this sensor.

[0074] Actual sample validation was performed using a healthy human serum spike experiment. The electrochemical biosensor spike recovery experiment is shown in Table 2. The average recoveries of the three spike levels of 200fM, 200pM, and 200nM were 98.1% (RSD=5.64%), 92.28% (RSD=5.33%), and 107.3% (RSD=4.06%), respectively. All recoveries fell within the acceptable range of 90-110%, confirming that the biosensor has reliable detection accuracy in complex biological matrices.

[0075] Table 2

[0076] Detection of exosome miRNA-125b in clinical samples: To systematically evaluate the clinical applicability of the detection platform, this study conducted a double-blind validation experiment based on a real clinical cohort (140 subjects: 45 healthy controls, 43 inflammation groups, and 52 sepsis groups). By simultaneously using the independently developed electrochemical biosensor and qRT-PCR technology to detect the level of plasma exosomal miRNA-125b, the results showed that the detection results of the two methods were highly consistent (intraclass correlation coefficient ICC=0.962, p<0.001). Further analysis found that the expression level of exosomal miRNA-125b in patients with sepsis was significantly higher than that in patients with inflammation, and was strongly positively correlated with the clinical diagnostic results (such as Figure 8 This data not only fully validates the clinical reliability of the biosensor, but also reveals the potential value of miRNA-125b as a differential diagnostic marker for sepsis, demonstrating the significant advantages of this technology in precision medicine.

[0077] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A method for preparing a DNA chimera, characterized in that: The following steps are involved: Step 1: Synthesize phosphorylated linear circular L-rTDNA and template DNA, mix them in 1×T4 DNA ligase buffer at an equimolar concentration of 10 μM, heat at 95°C for 5 minutes, and then cool down to room temperature for 120 minutes for annealing; Step 2: T4 DNA ligase is added to seal the terminal gap, and circular rT DNA is obtained after enzyme digestion; Step 3: Synthesize hairpin DNAHP. Mix rTDNA and HP at an equimolar concentration of 2 μmol / L, denature at 95°C for 5 minutes, and then slowly cool to 25°C for 2 hours to self-assemble into a DNA topology-constrained chimera.

2. The method for preparing a DNA chimera according to claim 1, wherein The enzyme digestion step in step 2 includes: heating at 70°C for 5 minutes to inactivate the ligase, adding exonuclease III and exonuclease I, reacting at 37°C for 60 minutes to degrade the unclosed linear DNA, and then heating at 80°C for 15 minutes to inactivate the exonuclease.

3. The method for preparing a DNA chimera according to claim 1, wherein The structural parameter optimization of the DNA chimera includes: designing a series of chimeras with a base number of 5-24 nt in the loop-hairpin junction region and a base number of 5-30 nt in the miRNA recognition region, and screening the target product with stable conformation by 12% non-denaturing PAGE electrophoresis.

4. The method for preparing a DNA chimera according to claim 3, wherein: The screening conditions for conformational stability are: a single clear band in electrophoretic mobility, and fluorescence detection showing a ratio of rolling circle amplification specific signal to background signal greater than 10:

1.

5. The method for preparing a DNA chimera according to claim 1, wherein The slow cooling program of the self-assembly process in step 3 is: after denaturation at 95°C, first cool to 65°C within 30 minutes, and then cool to 25°C within 40 minutes.

6. The method for preparing a DNA chimera according to claim 1, wherein The chimera purification step is also included: the target band is cut out by 12% non-denaturing PAGE, extracted using a UNIQ-10 spin column DNA gel recovery kit, and quantified by UV spectrophotometer to a concentration of ≥10 μM.

7. The method for preparing a DNA chimera according to any one of claims 1 to 6, characterized in that: The base sequences of the rTDNA and HP are designed to satisfy the following requirements: the 5'-end toehold sequence of HP is complementary to the target miRNA, and the rTDNA contains a barcode sequence that hybridizes with the biotinylated signal probe.

8. A DNA chimera application method, characterized in that: The miRNA detection is performed using the DNA chimera obtained by the DNA chimera preparation method according to claim 1, comprising the following steps: Step 1: Isolate exosomes from serum using ultrafiltration to extract total RNA or directly obtain exosomal miRNA; Step 2: Mix the exosomal miRNA with the DNA chimera, add ɸ29 DNA polymerase and dNTPs, and initiate the rolling circle amplification (RCA) reaction at 37°C for 2 hours. In step 3, the RCA product was hybridized with the biotinylated signal probe through foot-site-mediated strand displacement reaction (TSDR). A three-electrode system was used: a glassy carbon electrode modified with AuAgNPs / Ti3C2Tx-MXene, combined with differential pulse voltammetry (DPV) to detect the electrochemical signal.

9. The DNA chimera application method according to claim 8, characterized in that: Ultrafiltration conditions in step 1 include centrifugation at 300 × g and 4°C for 10 minutes to remove cell debris, followed by filtration through a 0.22 μM Steriflip® membrane. The clarified supernatant was transferred to a 100 kDa Amicon® ultrafiltration unit, concentrated by centrifugation at 4000 × g for 10 minutes, resuspended in PBS, and stored at -80°C.

10. The DNA chimera application method according to claim 8, characterized in that: In step 3, the electrochemical detection parameters for detecting the electrochemical signal are: DPV voltage scanning range -0.05-0.55V, modulation amplitude 70mV, pulse width 50ms, sensitivity 1×10⁻ 6 A / V.

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