A sers-based three-dimensional dna walker biosensor, and a preparation method and application thereof

Abstract

The application discloses a three-dimensional DNA walker biosensor based on SERS as well as a preparation method and application thereof, relates to the technical field of biosensors, and is used for detecting target lncRNA. The biosensor comprises an MB\@DNA\@Au\@Ag compound, a 3D DNA walker and an aptamer. The nucleotide sequence of the ssDNA linker is shown in SEQ ID NO:1. The nucleotide sequence of the 3D DNA walker is SEQ ID NO:7 or SEQ ID NO:8 or a corresponding reverse complementary sequence. The biosensor realizes isothermal autonomous walking of the three-dimensional DNA walker and releases MGITC coded Au@Ag SERS nanolabels through TapSAKI triggered strand displacement and Nb.BbvCI enzymatic cleavage, thereby generating a'signal-on' type Raman output. The detection limit of the biosensor for the AKI early diagnosis potential biomarker TapSAKI is as low as 1.16 fM, and reliable quantification can be realized in the range of 4.30-850 fM. Compared with RT-qPCR (LOD is about 0.26 pM), the biosensor is improved by about 2.3 orders of magnitude in the detection limit of molar concentration.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and in particular to a three-dimensional DNA walker biosensor based on SERS, its preparation method, and its application. Background Technology

[0002] Acute kidney injury (AKI) is a rapidly progressing clinical syndrome with a high mortality rate, often accompanied by long-term renal function sequelae. Due to the narrow treatment window, early and accurate identification of AKI is crucial for improving prognosis. However, renal biopsy is invasive and has limited diagnostic value in the early stages; routine functional indicators such as serum creatinine and urine output are lagging and lack specificity, and other laboratory indicators and imaging examinations also have limited sensitivity for early functional impairment. Therefore, there is an urgent need to develop precise, minimally invasive diagnostic biomarkers and supporting detection strategies for the early diagnosis of AKI.

[0003] Long non-coding RNAs (lncRNAs) play important regulatory roles in kidney inflammation, apoptosis, and repair processes. Among them, TapSAKI is considered a potentially clinically viable biomarker for akin to kidney injury (AKI). However, existing methods such as RT-qPCR, microarrays, and high-throughput sequencing still face limitations in sensitivity, operational complexity, cost, and quantitative robustness for detecting low-abundance lncRNAs. Surface-enhanced Raman scattering (SERS) offers advantages such as ultrasensitivity, interference resistance, and applicability to complex biological samples; however, robust lncRNA detection still requires integration with efficient, programmable, and isothermal signal amplification modules. A three-dimensional DNA walker can achieve target-triggered autonomous cyclic amplification within a spatially confined orbit. Coupled with a plasmonic nanoprobe, it can convert molecular recognition into a strong and stable "signal-on" type SERS output. Summary of the Invention

[0004] In view of this, the present invention provides a SERS-based three-dimensional DNA walker biosensor, its preparation method and application.

[0005] This invention proposes a magnetic bead-assisted three-dimensional DNA walker-SERS sensor (MBDAA) for early and accurate detection of the AKI-related lncRNA biomarker TapSAKI. The sensor comprises a programmed 3D DNA walker, densely packed orbital sites, and MGITC-encoded Au@Ag SERS nanotags. The walker employs an asymmetric bipedal structure, consisting of long and short legs, with a poly(A) spacer introduced between the legs to reduce steric hindrance and facilitate continuous walking within the three-dimensional orbital network. TapSAKI first activates the walker through strand displacement, exposing the stepping domain. Subsequently, enzyme-assisted cleavage and rehybridization occur on the orbitals of the pre-encoded Nb.BbvCI cleavage site, enabling continuous walking under isothermal conditions and target recycling. Simultaneously, it programmatically releases MGITC-labeled Au@Ag nanoparticles, outputting an accumulative "signal-on" type SERS signal.

[0006] In a first aspect, the present invention provides a SERS-based three-dimensional DNA walker biosensor for detecting target lncRNA. The biosensor includes an MB@DNA@Au@Ag complex, a 3D DNA walker, and an aptamer capable of specifically recognizing the target lncRNA. The MB@DNA@Au@Ag complex consists of streptavidin magnetic beads and core-shell gold and silver nanoparticles linked by an ssDNA linker; the nucleotide sequence of the ssDNA linker is shown in SEQ ID NO:1, and the ssDNA linker is labeled with biotin; the MB@DNA@Au@Ag complex is labeled with a Raman reporter molecule. The nucleotide sequence of the 3D DNA walker is SEQ ID NO:7 or SEQ ID NO:8 or the corresponding reverse complementary sequence.

[0007] Furthermore, the Raman reporter molecule is malachite green isothiocyanate (MGITC).

[0008] Further, the target lncRNA is TapSAKI, and the nucleotide sequence of the aptamer is SEQ ID NO:16 or SEQ ID NO:17 or the corresponding reverse complementary sequence; the complementary DNA sequence of TapSAKI is shown in SEQ ID NO:21.

[0009] In a second aspect, the present invention provides a method for preparing any of the above-described biosensors, comprising the following steps: (1) Preparation of AuNPs: Take trisodium citrate solution A, heat to boiling, add chloroauric acid solution A, stir under reflux for 10-20 min, after the reaction system turns dark red, keep the system at 85-95℃, add trisodium citrate solution B and chloroauric acid solution B repeatedly at intervals to obtain gold nanoparticles, i.e., AuNPs, stir, cool and store for later use; (2) Preparation of Au@Ag NPs: The prepared gold nanoparticles were stirred with MGITC at room temperature for 20-40 min, and the gold nanoparticles that were not bound to MGITC were removed by centrifugation. Ascorbic acid and silver nitrate were added, and after reacting at room temperature for 1-2 h, MGITC was added and stirred for 20-40 min to obtain core-shell structured gold and silver nanoparticles, namely Au@Ag NPs. (3) Linking DNA to Au@Ag NPs: Take the Au@Ag NPs prepared in step (2), add thiol-modified ssDNA linker, and after the reaction is completed by microwave heating under nitrogen protection, remove the core-shell structured gold and silver nanoparticles that are not bound to ssDNA linker, and resuspend the product in PBS buffer to obtain Au@Ag NPs linked with ssDNA linker, i.e. DNA@Au@Ag, which should be stored in the dark and refrigerated. (4) Mix streptavidin magnetic beads, let stand, discard supernatant, wash, add an equal volume of biotin-labeled DNA@Au@Ag, gently shake and incubate at room temperature for 40-80 min, then perform magnetic separation, wash, and obtain MB@DNA@Au@Ag complex. (5) Construction of 3D DNA walker-SERS sensor: Mix MB@DNA@Au@Ag with 3D DNA walker and aptamer and incubate for 20-40 min.

[0010] Further, in step (1), the concentration of trisodium citrate solution A is 2.2 mM, the concentration of chloroauric acid solution A is 25 mM, and the volume ratio of trisodium citrate solution A to chloroauric acid solution A is 120-180:1; The specific details of repeatedly adding trisodium citrate solution B and chloroauric acid solution B at intervals are as follows: the number of repetitions is 10-15 times, with an interval of 2 minutes between each repetition; each time, a 60 mM trisodium citrate solution B and a 25 mM chloroauric acid solution B are added, and the volume ratio of trisodium citrate solution B to chloroauric acid solution B added each time is 1:1.

[0011] Further, in step (1), gold nanoparticles AuNPs were obtained. During stirring, the temperature was 90 ℃ and the time was 30 min. The average particle size of the prepared AuNPs was 45-55 nm.

[0012] Further, in step (2), the concentration of ascorbic acid is 10 mM, the concentration of silver nitrate is 10 mM, and the concentration of MGITC is 0.1 mM.

[0013] Furthermore, in step (3), a microwave oven with an input power of 1150 W and an output power of 700 W is used to heat the reaction on high for 3 minutes. The removal of unbound ssDNA linker core-shell structured gold and silver nanoparticles specifically involves resuspending them in PBS buffer, centrifuging, and then washing them with 0.01 M phosphate buffer containing 0.3 M NaCl. The volume ratio of Au@Ag NPs to thiol-modified ssDNA linker is 10:1; the concentration of Au@Ag NPs is 1 nM, and the concentration of ssDNA linker is 100 μM.

[0014] In a third aspect, the present invention provides the application of any of the above-described biosensors in the detection of target lncRNAs, the application including preparation of diagnostic products.

[0015] Furthermore, the target lncRNA is TapSAKI, a biomarker for early diagnosis of acute kidney injury.

[0016] The present invention also provides a method for detecting the concentration of target lncRNA using the above-described biosensor, comprising: adding a sample containing target lncRNA to the biosensor, reacting at a constant temperature for 40-80 min, resuspending after magnetic separation, adding Nb.BbvCI enzyme, buffer and water, reacting at 37°C, and after the reaction is completed, centrifuging to collect the supernatant for SERS detection.

[0017] In this invention, a sensor constructs a three-dimensional DNA walking track on the surface of streptavidin-modified magnetic beads and activates the DNA walker through tapSAKI-triggered strand displacement, achieving isothermal, autonomous, and efficient cyclic signal amplification without the need for external thermal cycling under Nb.BbvCI enzyme mediation. MGITC-encoded Au@Ag nanoparticles generate a stable and enhanced "signal-on" type SERS response during dynamic release, thereby converting target recognition into quantifiable Raman output. This sensor has a detection limit of 1.16 fM and a quantitation limit of 4.30 fM, enabling reliable quantification within the 4.30-850 fM range, representing an improvement of approximately 2.3 orders of magnitude in molar concentration detection limit compared to RT-qPCR (detection limit approximately 0.26 pM). Clinical sample validation showed that the serum TapSAKI level in 32 AKI patients was significantly higher than that in 32 healthy controls (P<0.001), with an area under the ROC curve of 0.876. Using 71.8 fM as the early warning screening threshold, the sensitivity was 100% and the specificity was 68.8%. This invention establishes a SERS platform with high sensitivity, rapid response, and clinical interpretability, providing a powerful tool for early diagnosis and risk stratification of AKI.

[0018] The beneficial effects of this invention include at least the following: (1) The biosensor provided by the present invention can detect the AKI-related lncRNA biomarker TapSAKI with ultrasensitive, rapid and highly specificity; (2) The biosensor of the present invention achieves isothermal, autonomous, and cyclic signal amplification without external thermal cycling through target-triggered chain substitution and Nb.BbvCI enzymatic cleavage.

[0019] (3) The detection limit of the biosensor of the present invention can be as low as 1.16 fM and can achieve reliable quantification in the range of 4.30-850 fM, which is suitable for quantitative analysis of low abundance TapSAKI and early warning of AKI. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of lncRNA-TapSAKI detection based on a three-dimensional DNA walker-SERS platform. (a) Serum sample processing and target molecule detection workflow; (b) Schematic diagram of the DNA walker structure, which consists of long legs, a poly(A) spacer region, and short legs; (c) The target lncRNA triggers the 3D DNA walker on the surface of the magnetic bead to cycle and release the MGITC-labeled Au@Ag SERS nanotag, thereby generating a "signal-on" type Raman readout.

[0021] Figure 2This is a validation diagram of key molecular events of the three-dimensional DNA walker. (a) Schematic diagram of the chain design of the three-dimensional DNA walker module, including ssDNA orbitals (long legs - poly(A) spacer region - short legs), DNA walking chains, and TapSAKI recognition elements; (b) PAGE analysis of the complementary length optimization of DNA walking chains and aptamers (lanes I-VIII correspond to 7-14 bp); (c) PAGE analysis of TapSAKI-triggered competitive substitution and complex formation, lanes I-IX are ssDNA orbitals, DNA walking chains, aptamers (P), targets, orbitals / walking chains, walking chains / P, orbitals / walking chains / P, P / targets, and samples after co-incubation of targets and pre-assembled complexes, respectively; (d) PAGE analysis of Nb.BbvCI digestion specificity, single-stranded substrates showed no cleavage, while double-stranded substrates showed 7-14 bp cleavage products after Nb.BbvCI treatment.

[0022] Figure 3 Characterization diagrams of functionalized Au@Ag nanoparticles. (a) Schematic diagram of the synthesis of core-shell structured Au@Ag nanoparticles (Au@M@Ag@M) with MGITC and thiol DNA (Au@Ag@DNA); (b) Au@M@Ag@M structure at 1614 cm⁻¹ under different AgNO₃ addition volumes (20, 40, 60, 80, 100 μL). -1 (c) Comparison of SERS signal intensity at 1614 cm⁻¹; (d) TEM image of Au@M@Ag@M nanoparticles under optimized conditions; (e) UV-Vis absorption spectra of Au@M, Au@M@Ag, Au@Ag@M and Au@M@Ag@M; (f) SERS signal intensity comparison of the four structures at 1614 cm⁻¹. -1 (f) Comparison of SERS spectra at different locations; (g) NanoDrop measurements of DNA concentration before and after modification with Au@M@Ag@M nanoparticles; (h) Changes in Zeta potential of Au@Ag nanoparticles at different functionalization stages: unmodified (-24 mV), after MGITC modification (+28 mV), and after DNA modification (-35 mV); (i) Schematic diagram of biotin-modified Au@Ag@DNA and streptavidin-modified magnetic beads coupling; (j) Comparison of DNA content in free magnetic beads and coupled Au@Ag@DNA@MB; (k) Photographs of the magnetic separation process at different incubation times (0, 10, 20, 30, 40, 50, 60 min); (f) Quantitative analysis of the change in DNA binding to magnetic beads with incubation time (total time 60 min).

[0023] Figure 4The images show the assembly and enzyme-triggered release characterization of the magnetic bead-based 3D DNA walker-SERS system. (a) Schematic diagram of MBDAA system assembly and target response; (b) Photograph of the Au@Ag nanotags after magnetic separation capture and enzyme-triggered release; (c) TEM images before and after assembly and after enzyme digestion and release; (d) UV-Vis absorption spectra of Au@Ag@DNA, magnetic bead-loaded complex, and supernatant after enzyme digestion; (e) Corresponding SERS spectra; (f) 1614 cm⁻¹ at different reaction times. -1 (g) SERS signal intensity change; particle size stability analysis of the complex over 72 h.

[0024] Figure 5 Figure 1 shows the clinical benchmark detection of TapSAKI and the performance evaluation of MBDAA analysis. (a) Schematic diagram of the RT-qPCR detection process for serum TapSAKI; (b) NanoDrop absorption spectra of representative cDNA samples; (c) Comparison of TapSAKI levels in serum of AKI patients and healthy controls detected by RT-qPCR; (d) Representative SERS spectra of MBDAA for different concentrations of TapSAKI; (e) 1614 cm⁻¹ -1 (f) SERS spectrum within the selected quantitative working interval; (g) Linear regression results of TapSAKI concentration and Raman intensity within the working interval.

[0025] Figure 6 This is a graph showing the detection of serum TapSAKI in clinical AKI and its clinical assessment using MBDAA. (a) Comparison of serum TapSAKI levels in 32 healthy controls and 32 AKI patients; (b) TapSAKI reading thermogram of clinical samples; (c) ROC curve of TapSAKI distinguishing AKI from healthy controls; (d) Representative renal ultrasound images; (e) Baseline and follow-up changes in serum creatinine in 4 representative cases; (f) Relative increase in serum creatinine in 48 h in 4 representative cases; (g) TapSAKI concentration in 4 representative cases at 12 h. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0028] The materials and experimental procedures used in this embodiment are as follows: 1. Experimental Materials DNA oligonucleotides, TE buffer, TBE buffer, PBS buffer, sodium chloride, 30% acrylamide, ammonium persulfate (APS), TEMED, and 10×TBE buffer were purchased from Shanghai Sangon Biotech Co., Ltd., China. Streptavidin-modified magnetic beads, BeyoRT™ Q cDNA first-strand synthesis kit, and SYBR Green real-time PCR kit (dye method) were purchased from Beyotime Biotechnology Co., Ltd., China. Nb.BbvCI single-stranded nickase and 10×NEB CutSmart buffer were purchased from New England Biolabs, USA; SYBR nucleic acid dye was purchased from BioActive Molecules, China; Mara Green Isothiocyanate (MGITC) and DNA molecular weight standards were purchased from Thermo Fisher Scientific, USA; chloroauric acid was purchased from Shanghai Bojing Chemical Co., Ltd., China; trisodium citrate dihydrate, silver nitrate, and ascorbic acid were all purchased from Xilong Scientific Co., Ltd., China; electrophoresis loading buffer was purchased from Solarbio, China; transmission electron microscope copper mesh was purchased from Zhongjing Scientific Instruments Co., Ltd.; capillary glass tubes were purchased from Mingrui Teaching Instruments Co., Ltd.; BIOG serum / plasma free RNA extraction kit was purchased from Baida Biotechnology Co., Ltd.; DEPC water was purchased from Sangon Biotech Co., Ltd.

[0029] The main instruments include: a thermostatic magnetic stirrer (Tianjin Aote Scientific Instruments), a thermostatic mixer (ThermoFisher, USA), an ultra-low temperature freezer (Haier Group), a drying cabinet (Shenzhen Saijie), a multi-functional microplate reader (BioTek Instruments, USA), a high-speed centrifuge (Hunan Tiangen), a dynamic light scattering (DLS) and zeta potential analyzer (Malvern, UK), a transmission electron microscope (Hitachi, Japan), an electrophoresis apparatus (Bio-Rad, USA), an ultrapure water system (Millipore, USA), an electronic balance (Mettler-Toledo, Switzerland), a gel imaging system (Shanghai Tianneng), a real-time fluorescence PCR instrument (Applied Biosystems, USA), a gradient PCR instrument (Bio-Rad, USA), a Raman spectrometer (Renishaw, UK), and a NanoDrop micro spectrophotometer (IMPLEN, Germany).

[0030] 2. Experimental Procedure (1) Synthesis of gold nanoparticles (AuNPs) 75 mL of 2.2 mM trisodium citrate solution was added to a 250 mL three-necked round-bottom flask and stirred under reflux for 15 minutes. After the solution boiled, 0.5 mL of 25 mM chloroauric acid solution was added dropwise. During the reaction process of 10–20 minutes, the solution color changed from light pink to purple, and finally to deep red, indicating the formation of gold nanoseeds. The system was then maintained at 90 °C, and 0.5 mL of 60 mM trisodium citrate solution and 0.5 mL of 25 mM chloroauric acid solution were added sequentially, mixing thoroughly after each addition. This process was repeated 12 times, with a 2-minute interval between each round, to gradually increase the particle size. Finally, the mixture was stirred at 90 °C for 30 minutes, cooled to room temperature, and stored at 4 °C for later use.

[0031] (2) Synthesis of core-shell structured gold and silver nanoparticles (Au@AgNPs) AuNPs preparation: AuNPs with an average particle size of approximately 50 nm were obtained by reduction with trisodium citrate. First labeling (internal labeling): AuNPs were mixed with MGITC (pre-labeling was performed by vigorous stirring at room temperature for 30 min) to allow some probe molecules to adsorb onto the surface of the gold core. After centrifugation to remove unbound molecules, pre-labeled AuNPs were obtained. Silver shell growth: Mix 5 mL of pre-labeled AuNPs with 10 mM ascorbic acid, then add 10 mM silver nitrate solution. The ascorbic acid will cause the Ag to... + The silver salt was reduced and deposited on the surface of AuNPs to form an Au@Ag core-shell structure. The thickness of the silver shell depended on the amount of silver salt added. A second labeling was performed 1 hour after the reaction. Second labeling (surface modification): After the reaction is complete, add MGITC (0.1 mM, stir for 30 min) to allow the remaining probe molecules to modify the silver shell surface, resulting in Au@Ag NPs; Storage: Store the obtained Au@AgNPs at 4 ℃.

[0032] (3) Microwave-assisted ligation of DNA to Au@AgNPs 200 μL of Au@Ag NPs (1 nM) was mixed with 20 μL of thiol-modified DNA (SH-DNA, 100 μM) in a 5 mL glass bottle and placed under nitrogen protection to prevent oxidation. The mixture was then heated in a household microwave oven (1150 W input, 700 W output) on high for 3 minutes. After heating, the nanoparticles were resuspended in PBS buffer (0.1 M NaCl, pH 7.4). The mixture was centrifuged and washed with 0.01 M phosphate buffer (pH 7.4) containing 0.3 M NaCl to remove unbound material. The final product was resuspended in PBS and stored at 4°C protected from light.

[0033] (4) Immobilization of DNA@Au@Ag on streptavidin magnetic beads (MBs) First, thoroughly mix the streptavidin magnetic beads, and add 20 μL to a 1.5 mL centrifuge tube. Place the tube on a magnetic rack and let it stand for 1 minute. Discard the supernatant and wash three times with 0.5 mL of 1×TBS buffer. Then add an equal volume of biotin-labeled DNA probe (DNA@Au@Ag) and incubate gently at room temperature for 1 hour to promote binding. Afterward, perform magnetic separation and wash once to obtain the MB@DNA@Au@Ag complex, and determine the DNA concentration after binding using NanoDrop.

[0034] (5) DNA probe quantification Nucleic acids, due to their aromatic ring structure, exhibit characteristic absorption at 260 nm. Their absorbance is calculated using the formula A = -log(sample intensity / blank intensity), and concentration is estimated using the Lambert-Beer law. Each measurement requires only 2 μL of sample and is completed within 1 second. With NanoDrop set to nucleic acid mode, after cleaning the detection stage, first measure the blank, then measure the samples sequentially.

[0035] (6) 3D DNA walker reaction system DNA strand assembly: Mix 10 μL of 1 μM single-stranded DNA, DNA walking strand, and Aptamer p, and incubate at 37°C for 1 hour. Then add the target lncRNA TapSAKI strand, and use PAGE to verify hybridization and competitive binding.

[0036] Enzyme digestion reaction: After verifying successful assembly, add 10 μL of 1 μM ssDNA, 10 μL of 1 μM DNA walker, 2 μL of (5 U) Nb.BbvCI enzyme, 10 μL of 10×CutSmart buffer and 48 μL of sterile water, keeping the total volume at 80 μL, and carry out the enzyme digestion reaction.

[0037] (7) Construction of 3D DNA Walker – SERS Sensor MB@DNA@Au@Ag was mixed with 10 μL of 1 μM DNA Walker and 10 μL of 1 μM Aptamer p and incubated at room temperature for 30 minutes. Then, the target lncRNA TapSAKI was added, and the reaction was carried out at room temperature for 1 hour. After magnetic separation and resuspending, 2 μL (20 U) Nb.BbvCI enzyme, 10 μL of 10× buffer, and 73 μL of water were added, and the reaction was carried out at 37 °C. After the reaction, the supernatant was collected by centrifugation for SERS detection. The buffer consisted of 50 mM Potassium acetate, 20 mM Tris-acetate (pH 7.9, 25 °C), 10 mM Magnesium acetate, and 100 µg / mL BSA (bovine serum albumin).

[0038] (8) Polyacrylamide gel electrophoresis (PAGE) Prepare a 20% gel by adding 30% acrylamide, 5×TBE, 10% APS, TEMED, and water, avoiding air bubbles during gel pouring. After gel polymerization, place the gel in an electrophoresis tank, add 1×TBE, remove the comb teeth, and load the sample (10 μL sample + 2 μL 6×loading buffer). Run at 80V for 30 minutes, then increase to 110V and run for 2 hours. After electrophoresis, stain with SYBR Green I for 30 minutes and take images.

[0039] (9) Characterization of sensor components The UV-Vis absorption spectra of the nanoparticles were recorded using an ELISA reader. The morphology of the nanostructures was observed using TEM (accelerating voltage 120 kV). DLS and zeta potential analysis were used to determine particle size and charge properties.

[0040] (10) SERS detection Using monocrystalline silicon at 520cm -1 The Raman spectrometer was calibrated using characteristic peaks. Excitation wavelength: 633 nm; laser power: 5 mW; objective lens: 20×; integration time: 1 second; detection range: 600–1800 cm⁻¹. -1 .

[0041] (11) Collection of clinical serum samples Serum was collected from 30 AKI patients and 30 healthy volunteers at the First Affiliated Hospital of Hainan Medical University. 3–5 mL of venous blood was collected at 7–8 AM after an 8-hour fast. Plasma was separated by centrifugation at 4000 rpm for 10 minutes, and the supernatant was stored at -80℃. Informed consent was obtained from all participants.

[0042] (12) Extraction of total RNA from serum Prepare anhydrous ethanol and RNase-free centrifuge tubes in advance. Prepare wash buffers A and B with 30% and 70% ethanol, respectively. If precipitation occurs, dissolve by preheating to 37°C. Mix 200 μL of serum with 4 μL of vector RNA, then add 200 μL of lysis buffer and 20 μL of enzyme digestion buffer, and incubate at 65°C for 10 minutes. Add 0.9 mL of anhydrous ethanol, mix well, transfer to an RNA adsorption column, centrifuge at 12000 rpm at 4°C for 1 minute, and discard the eluent. Repeat the binding and washing process according to the kit instructions, and finally elute with 30 μL of preheated RNase-free water.

[0043] (13) RT-PCR quantitative detection of lncRNA Total RNA was extracted using the BIOG kit and cDNA was synthesized via reverse transcription using the BeyoRT™ Q kit. qPCR was performed using the SYBR Green qPCR reagent on the QuantStudio™ 3 platform. The reaction mixture contained cDNA template, lncRNA-TapSAKI primers, and dyes. The cycling program included denaturation, annealing, and extension. GAPDH was used as an internal control, and a 2... -ΔΔCt The method was used for relative quantitative analysis.

[0044] (14) Construct the SERS standard curve Prepare a series of standard solutions with known concentrations and record their Raman spectral signals. Plot a standard curve with concentration on the x-axis and Raman intensity on the y-axis, and use linear regression. Quantify unknown samples using interpolation.

[0045] (15) Sensitivity and specificity calculation Serum samples from AKI and control groups were analyzed using SERS, and the concentration of lncRNA-TapSAKI was determined based on a standard curve. ROC curve analysis was performed using SPSS to evaluate diagnostic performance. Sensitivity and specificity were calculated, and the optimal cut-off value was determined using the Youden index (sensitivity + specificity - 1). AUC was used to assess overall diagnostic accuracy; an AUC closer to 1 indicates better efficacy.

[0046] (16) Statistical analysis All statistical analyses were performed using SPSS 20.0 software. Data are expressed as mean ± standard error (SEM), and one-way ANOVA was used for comparisons between groups. ROC curves were used to calculate AUC values, and P < 0.05 was considered statistically significant.

[0047] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Construction of a 3D DNA Walker – SERS Sensor The construction principle of the 3D DNA walker – SERS sensing platform is as follows: Figure 2 As shown. First ( Figure 2 a) AuNPs were synthesized via seed-mediated growth and surface-functionalized with the Raman reporter molecule MGITC. Subsequently, a silver shell was coated onto their surface to form core-shell Au@Ag nanoparticles, further enhancing SERS signaling activity. These nanoparticles bound to thiol-modified DNA strands to construct mobile Raman signaling probes. To construct a three-dimensional DNA walking track, biotin-labeled DNA sequences were immobilized on the surface of streptavidin-modified magnetic beads (…). Figure 2 (b) The DNA walking system consists of a long "leg" DNA sequence, a poly-A spacer region, and a short "leg" DNA sequence, precisely designed to achieve specific hybridization with the orbital sequence. Furthermore, the system incorporates an aptamer module that specifically recognizes the target lncRNA TapSAKI. In the presence of TapSAKI, the aptamer binds specifically to it, triggering the release of the walker. With the participation of the Nb.BbvCI restriction endonuclease, the specific site formed between the walker and the orbital sequence is precisely cleaved. Figure 2 As shown in Figure c, the walker autonomously moves along the DNA orbital through a continuous cycle of cleavage and hybridization, dynamically releasing MGITC-labeled Au@Ag nanoparticles into the solution. This cascade reaction process produces a significantly enhanced "signal-on" type Raman response. It is worth emphasizing that the entire signal amplification reaction is completed under isothermal conditions, without the need for external thermal cycling or complex enzyme cascade systems, enabling rapid, ultrasensitive, and highly specific detection of low-abundance lncRNA biomarkers in complex biological environments.

[0048] Example 2: Feasibility Verification of a Three-Dimensional DNA Walker To verify the feasibility of the three-dimensional DNA walker design, a hybridization system consisting of ssDNA, a DNA walking strand, and an aptamer (P strand) was first constructed. All relevant base sequences are shown in Table 1. The design principle is as follows: Figure 2As shown in Figure a, the device includes a walking track (ssDNA linker arm), a DNA walking strand, and a target lncRNA TapSAKI recognition element (cTapSAKI). The track design includes a long "leg" (blue and red strands, near the 5' end), a spacer region (gray strand), and a short "leg" (red strand, near the 3' end) to ensure efficient and specific activation of the walker under target excitation.

[0049] To optimize hybridization conditions, this invention systematically evaluated the impact of different numbers of base pairings between the DNA walking chain and the aptamer chain, aiming to improve subsequent target competitive binding efficiency while ensuring structural stability. Figure 2 As shown in b, combinations of 7 to 14 complementary base pairings were evaluated. The results showed that both the 11bp and 12bp pairing systems (lanes V and VI) formed new hybridization bands, while further increases in the number of bases (lanes VII and VIII) readily led to the formation of additional secondary structures. Considering both pairing affinity and structural stability, 12bp was ultimately selected as the optimal hybridization configuration for subsequent experiments.

[0050] After confirming successful hybridization of the three DNA strands, further verification was conducted to determine whether the target molecule could competitively bind to the aptamer. For ease of electrophoresis verification, a fully complementary DNA sequence (sTapSAKI) was selected as a target to mimic lncRNA for system optimization. This mimic target was added to the pre-assembled three-dimensional DNA walker complex, and after incubation at isothermal conditions for 30 minutes, new double-stranded bands corresponding to hybridization patterns 12, 23, 34, and 123 were observed in the electrophoresis pattern compared to the single-stranded control. Figure 2 (c) This demonstrates the efficient execution of the hybridization reaction. Specifically, after incubating the target with the already formed 123 complex, new bands of approximately 37 bp and 34 bp appeared near lane IV, corresponding to the reformation of the 12 and 34 double-stranded structures. This result indicates that the target sequence can specifically recognize and bind to the aptamer, thus providing preliminary validation for the functional realization of a three-dimensional DNA walker.

[0051] The recognition and cleavage capabilities of the Nb.BbvCI endonuclease were then evaluated. Figure 2 As shown in d, no cleavage was observed in single-stranded DNA (lanes II and IV), while cleavage products of approximately 7–14 bp were visible in the double-stranded hybrid structure (lane VI), demonstrating that Nb.BbvCI can achieve sequence-specific cleavage at specific sites.

[0052] Table 1. Oligonucleotide sequences and primer information used in SERS detection and qRT-PCR

[0053] Example 3: Design and Synthesis of Functionalized Au@Ag Nanoparticles To construct a robust and efficient SERS sensing platform, this invention first prepared an MGITC-encoded Au@Ag core-shell nanotag, and further functionalized it with DNA to serve as a mobile SERS reporter probe in 3D DNA walker detection, such as... Figure 3 As shown in a. The AuNPs obtained from seed-mediated growth have good monodispersity and a particle size of about 50 nm, providing a stable core for subsequent silver shell coating.

[0054] Subsequently, the SERS enhancement performance of Au@Ag nanotags was optimized by adjusting the amount of AgNO3 added. The results showed that the nanostructures prepared under 60 μL AgNO3 conditions exhibited SERS enhancement at 1614 cm⁻¹. -1 The highest Raman intensity is found at this location. Figure 3 b), therefore, was selected as the standard condition for subsequent assembly and testing.

[0055] TEM characterization of the optimized MGITC-modified Au@Ag nanotags revealed a clear core-shell structure with a silver shell thickness of approximately 3 nm. Figure 3 c). The UV-Vis absorption spectra and corresponding SERS spectra are shown below. Figure 3 d and Figure 3 e indicates that the silver shell coating was successful and that the dual-layer MGITC coding structure has a stronger signal enhancement effect.

[0056] After immobilizing thiol-based DNA onto the surface of the nanotag, NanoDrop assays showed a significant increase in nucleic acid-related signals, and the zeta potential also exhibited a phased change from negative to positive and then back to negative, demonstrating successful DNA functionalization and a stable surface modification process. Figure 3 f and Figure 3 g). Meanwhile, DNA modification did not weaken 1614 cm. -1 The SERS output at that location.

[0057] To achieve spatially confined assembly of a 3D DNA walker, Au@Ag@DNA was further immobilized on the surface of magnetic beads via biotin-streptavidin interaction, such as... Figure 3 As shown in h.

[0058] DNA-related signals were significantly enhanced in the magnetic bead system after coupling. Figure 3 i) indicates that the nanotag has been successfully loaded onto the surface of the magnetic beads.

[0059] Example 4: Capture and Characterization of DNA-Functionalized Au@Ag Nanoprobes Based on Magnetic Beads To achieve spatial constraints and modular integration of the 3D DNA walker system, this invention uses streptavidin-modified magnetic beads (MBs) to immobilize biotin-labeled DNA-functionalized Au@Ag nanoprobes. The overall assembly strategy is as follows: Figure 3 As shown in h. When MBs and Au@Ag@DNA are mixed under gentle stirring, a visible color change occurs in the solution ( Figure 3 j): After magnetic separation, the magnetic beads of the originally brownish-yellow colloidal solution aggregated on the tube wall, while the supernatant became clear, indicating that the probe was effectively captured through streptavidin-biotin interaction.

[0060] To quantitatively evaluate the immobilization efficiency of the nanoprobes, the DNA content before and after magnetic bead coupling was determined using a NanoDrop spectrophotometer. Figure 3 As shown in i, the DNA concentration increased significantly after coupling, verifying that the Au@Ag@DNA probe successfully attached to the magnetic bead surface; at the same time, the increase in hydrated particle size further indicates the formation of a magnetic bead-nano probe complex.

[0061] To determine the optimal magnetic bead-probe coupling reaction time, the DNA binding amount at different incubation times ranging from 0 to 60 minutes was evaluated. Figure 3 As shown in k, the DNA loading increased with prolonged incubation time, reaching a plateau at 30 minutes, after which no significant changes were observed. These results indicate that under these experimental conditions, 30 minutes of incubation is sufficient for efficient conjugation of the nanoprobes.

[0062] In summary, DNA-functionalized SERS nanoprobes can be stably and efficiently captured by magnetic beads, providing a solid foundation for the subsequent construction of a 3D DNA walker – SERS sensing platform.

[0063] Example 5: Construction and Enzyme Response Performance Evaluation of a 3D DNA Walker-SERS Sensor Based on Magnetic Beads To verify the effectiveness of the sensor construction and its ability to respond to enzyme digestion, a systematic evaluation of the structural features and functional performance of the MB-assisted 3D DNA walker-SERS sensing system (MBDAA) was conducted. Figure 4 a). First, the synthesized Au@Ag@DNA nanoprobes exhibit a typical reddish-brown color ( Figure 4 b, test tube 1), indicates that the probe structure is intact and the connection is stable. After magnetic separation, the supernatant is almost colorless ( Figure 4 (b, test tube 2) indicates that the Au@Ag@DNA probe has been efficiently immobilized on the magnetic bead surface. Further, after adding Nb.BbvCI enzyme and the target lncRNA TapSAKI, the supernatant regained a significant color ( Figure 4 b, test tube 3), clearly reflects the nanoparticle release process induced by enzyme digestion.

[0064] Transmission electron microscopy (TEM) images further confirmed the structural features of the sensor assembly. Figure 4c). After DNA hybridization and heat annealing (95°C denaturation, 65°C annealing), TEM images (top) show approximately 200 nm magnetic beads (blue arrows) tightly bound to 60 nm Au@Ag@DNA nanoparticles (red arrows), forming a dense composite structure. Although single DNA strands cannot be directly observed in TEM, the distribution morphology of the nanoparticles adjacent to the surface of the magnetic beads strongly supports the success of DNA-mediated hybridization. After Nb.BbvCI treatment, the nanoparticles were clearly released from the surface of the magnetic beads ( Figure 4 c (see figure below) verifies the effectiveness of the enzyme cleavage-driven release mechanism in this sensing system.

[0065] Ultraviolet-visible spectroscopy was used to further quantify the binding and release process of nanoparticles. Figure 4 d). The initial MBDAA system exhibited a typical plasmon resonance absorption peak (520 nm, curve 1), indicating that the nanoparticles were stable. After magnetic separation, the supernatant showed almost no absorption signal (curve 2), indicating that the probe was completely immobilized on the magnetic beads. After enzymatic digestion, the released nanoparticles restored a significant absorption peak in the supernatant (curve 3), further confirming the effectiveness of the release process.

[0066] The corresponding SERS detection results ( Figure 4 e) Significant changes were also observed. Before magnetic separation, the Au@Ag@DNA probe exhibited a significant Raman signal (curve 1), originating from the enhanced Raman scattering of the MGITC molecule; after magnetic bead fixation, the signal was significantly weakened (curve 3), indicating that the probe was effectively removed. Enzymatic digestion re-enhanced the signal (curve 2), demonstrating that target-triggered probe release can significantly restore the SERS response, verifying the platform's specific response capability and signal amplification characteristics to the target molecule.

[0067] To further optimize the detection conditions, the effects of different enzyme reaction times on 1614 cm⁻¹ were investigated. -1 The influence of SERS signal strength Figure 4 f). The results showed that the signal rapidly plateaued after 15 minutes of reaction, indicating rapid enzymatic digestion kinetics. A stable and maximized signal intensity was obtained after 30 minutes of incubation, suitable for routine detection procedures. Furthermore, DLS experiments showed that the assembled nanoprobe complex remained stable for 72 hours within a particle size range of approximately 300 nm. Figure 4 g), indicating its good stability and operability in actual diagnostic environments.

[0068] In summary, the above structural and functional verifications fully demonstrate that the designed MBDAA sensor can effectively achieve highly sensitive and repeatable SERS detection through the synergistic effect of target recognition, enzyme digestion response, and Raman signal amplification, providing a solid foundation for subsequent quantitative analysis of clinical biomarkers.

[0069] Example 6: LncRNA-TapSAKI Expression Analysis Based on Traditional RT-qPCR Method To use commonly used clinical methods as a reference, real-time quantitative PCR (RT-qPCR) was employed to quantify TapSAKI in serum samples from 3 AKI patients and 3 age-matched healthy controls. The procedure is as follows: Figure 5 As shown in a, the process includes steps such as blood sample processing, RNA extraction, reverse transcription, and real-time amplification.

[0070] After RNA extraction and reverse transcription, the purity and concentration of the obtained complementary DNA (cDNA) samples were evaluated using a NanoDrop spectrophotometer. Figure 5 As shown in b, all samples exhibited a consistent characteristic absorption peak at 260 nm, indicating that the quality of RNA extraction and cDNA synthesis was good and could meet the requirements of subsequent amplification analysis.

[0071] To achieve absolute quantification, a TapSAKI standard curve was constructed and a series of gradient samples were tested. The standard curve showed that RT-qPCR can be used for absolute quantification of serum TapSAKI, with a limit of detection (LOD) of approximately 0.26 pM, which can serve as a clinical reference method for evaluating the analytical performance of the SERS platform of this invention.

[0072] Furthermore, RT-qPCR results from clinical samples showed that serum TapSAKI levels in AKI patients were significantly higher than those in healthy controls. Figure 5 c, P<0.001). This result further supports the clinical application potential of TapSAKI as an AKI-related biomarker and also illustrates the need to develop detection methods with higher sensitivity and simpler procedures.

[0073] Example 7: Quantitative Analysis of LncRNA-TapSAKI and Evaluation of Standard Curve To evaluate the application potential of a 3D DNA walker-assisted SERS sensor in ultrasensitive RNA detection, quantitative experiments were conducted targeting synthetic lncRNA-TapSAKI. This detection strategy is based on the specific hybridization between TapSAKI and its aptamer, which activates the Nb.BbvCI enzyme-mediated MBDAA system and programmatically releases MGITC-encoded Au@Ag SERS nanotags; the number of nanotags released into the supernatant can serve as a positive indicator of target RNA abundance. Figure 5 d and Figure 5 As shown in e, over a wide concentration range, 1614 cm⁻¹ -1 The MGITC characteristic peak at this location increases with increasing target concentration. Considering the effects of background noise at low concentrations and signal saturation at high concentrations, [the following is selected]. Figure 5Quantitative analysis was performed on the working interval shown in f, and a linear regression relationship between Raman intensity and the logarithmic value of TapSAKI concentration was established. Figure 5 g, R 2 = 0.9931). Based on the threshold calculation of the blank sample signal, the detection limit (LOD) of this platform is 7.0 × 10⁻⁶. 2 The limit of quantitation (LOQ) was 2.6 × 10⁻⁶ copies / μL (1.16 fM). 3 copies / μL (4.30 fM); based on this, it can be obtained at 2.6 × 10 3 -5.1×10 5 Reliable quantification is achieved within the range of copies / μL (4.30-850 fM). Compared with RT-qPCR, which has a detection limit of approximately 0.26 pM, this platform improves the detection limit by approximately 2.3 orders of magnitude at molar concentrations, while maintaining the advantages of isothermal operation, signal on-state capability, and suitability for complex biological samples.

[0074] Example 8: Diagnosis and Early Warning Assessment of LncRNA-TapSAKI in Clinical Serum Samples To evaluate the clinical application value of the MBDAA sensor, serum samples from 32 healthy controls and 32 AKI patients were analyzed using TapSAKI detection. Figure 6 The results showed that the TapSAKI level in the AKI group was significantly higher than that in the healthy control group (P<0.001); the median (interquartile range) in the AKI group was 138.3 fM (125.1-160.9 fM), while that in the healthy control group was 70.0 fM (51.5-83.5 fM). Figure 6 The heatmap shown in b further indicates that the AKI sample as a whole exhibits a higher TapSAKI reading distribution.

[0075] To further evaluate its diagnostic and early warning performance, receiver operating characteristic (ROC) curves were constructed. The results showed that, considering fluctuations in concentration-related analysis, the area under the curve (AUC) for TapSAKI in distinguishing AKI from healthy controls was 0.876 (…). Figure 6 c). To address the need for prioritizing the avoidance of missed diagnoses in early clinical warning scenarios, using 71.8 fM as the exclusionary screening threshold yielded a sensitivity of 100% (32 / 32) and a specificity of 68.8% (22 / 32). Samples exceeding this threshold suggest the need for further monitoring and confirmation using routine clinical indicators such as changes in creatinine within 48 hours.

[0076] Four representative cases were further selected, and the dynamic changes in renal ultrasound, 12-hour TapSAKI level, and 48-hour serum creatinine were analyzed in combination. Figure 6(dg). In healthy controls G1, renal ultrasound showed no significant abnormalities, with SCr decreasing from 59 μmol / L to 54 μmol / L and TapSAKI at 45.2 fM. In non-AKI case G2, although SCr only increased from 110 μmol / L to 148 μmol / L within 48 hours (an increase of 34.55%), failing to reach the 50% threshold, TapSAKI had already risen to 185.6 fM at 12 hours, exceeding the screening threshold. In AKI cases G3 and G4, SCr increased by 85.47% and 141.32% respectively within 48 hours, corresponding to TapSAKI levels of 340.5 fM and 510.8 fM. These results indicate that the MBDAA readout of TapSAKI can serve as an early warning indicator within 12 hours and complements functional assessments based on creatinine within 48 hours.

[0077] This invention constructs a magnetic bead-assisted three-dimensional DNA walker-SERS sensor (MBDAA) that achieves ultrasensitive, rapid, and highly specific detection of TapSAKI, an AKI-related biomarker. The platform utilizes TapSAKI-triggered strand displacement and Nb.BbvCI-mediated autonomous walking to achieve isothermal cycling signal amplification without external thermal cycling, converting target recognition into a "recognition-signal" output released from MGITC-labeled Au@Ag nanotags. The platform has a detection limit of 1.16 fM and can reliably quantify within the range of 4.30-850 fM. Clinical serum sample validation showed a significant difference in TapSAKI levels between 32 AKI patients and 32 healthy controls (P<0.001), with an area under the ROC curve of 0.876; using 71.8 fM as the early warning screening threshold, the sensitivity was 100% and the specificity was 68.8%. Therefore, the MBDAA platform can provide an effective tool for early risk stratification and accurate diagnosis of AKI, and can serve as a useful supplement to 12-hour molecular early warning and 48-hour dynamic creatinine assessment.

[0078] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0079] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A three-dimensional DNA walker biosensor based on SERS, characterized in that, The biosensor is used to detect target lncRNA, and the biosensor includes an MB@DNA@Au@Ag complex, a 3D DNA walker, an aptamer capable of specifically recognizing the target lncRNA, and an Nb.BbvCI enzyme; The MB@DNA@Au@Ag complex consists of streptavidin magnetic beads and core-shell gold and silver nanoparticles linked by an ssDNA linker; the nucleotide sequence of the ssDNA linker is shown in SEQ ID NO:1, and the ssDNA linker is labeled with biotin; the MB@DNA@Au@Ag complex is labeled with a Raman reporter molecule. The nucleotide sequence of the 3D DNA walker is SEQ ID NO:7 or SEQ ID NO:8 or the corresponding reverse complementary sequence.

2. The biosensor according to claim 1, characterized in that, The Raman reporter molecule is malachite green isothiocyanate.

3. The biosensor according to claim 1, characterized in that, The target lncRNA is TapSAKI, and the nucleotide sequence of the aptamer is SEQ ID NO:16 or SEQ ID NO:17 or the corresponding reverse complementary sequence.

4. The method for preparing the biosensor according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Preparation of AuNPs: Take trisodium citrate solution A, heat to boiling, add chloroauric acid solution A, stir under reflux for 10-20 min, after the reaction system turns dark red, keep the system at 85-95℃, add trisodium citrate solution B and chloroauric acid solution B repeatedly at intervals to obtain gold nanoparticles AuNPs, stir, cool and store for later use. (2) Preparation of Au@Ag NPs: The prepared gold nanoparticles were stirred with MGITC at room temperature for 20-40 min, and the gold nanoparticles that were not bound to MGITC were removed by centrifugation. Ascorbic acid and silver nitrate were added, and after reacting at room temperature for 1-2 h, MGITC was added and stirred for 20-40 min to obtain core-shell structured gold and silver nanoparticles Au@Ag NPs. (3) Linking DNA to Au@Ag NPs: Take the Au@Ag NPs prepared in step (2), add thiol-modified ssDNA linker, and after the reaction is completed by microwave heating under nitrogen protection, remove the Au@Ag NPs that have not been bound to the ssDNA linker, resuspend the product in PBS buffer to obtain DNA@Au@Ag, and store it in the dark and cold. (4) Mix streptavidin magnetic beads, let stand, discard supernatant, wash, add an equal volume of biotin-labeled DNA@Au@Ag, gently shake and incubate at room temperature for 40-80 min, then perform magnetic separation, wash, and obtain MB@DNA@Au@Ag complex. (5) Construction of 3D DNA walker-SERS sensor: Mix MB@DNA@Au@Ag with 3D DNA walker and aptamer and incubate for 20-40 min.

5. The preparation method according to claim 4, characterized in that, In step (1), the concentration of trisodium citrate solution A is 2.2 mM, the concentration of chloroauric acid solution A is 25 mM, and the volume ratio of trisodium citrate solution A to chloroauric acid solution A is 120-180:1; The specific details of repeatedly adding trisodium citrate solution B and chloroauric acid solution B at intervals are as follows: the number of repetitions is 10-15 times, with an interval of 2 minutes between each repetition; each time, a 60 mM trisodium citrate solution B and a 25 mM chloroauric acid solution B are added, and the volume ratio of trisodium citrate solution B to chloroauric acid solution B added each time is 1:

1.

6. The preparation method according to claim 4, characterized in that, In step (1), gold nanoparticles (AuNPs) were obtained. During stirring, the temperature was 90 °C and the time was 30 min. The average particle size of the prepared AuNPs was 45-55 nm.

7. The preparation method according to claim 4, characterized in that, In step (2), the concentration of ascorbic acid is 10 mM, the concentration of silver nitrate is 10 mM, and the concentration of MGITC is 0.1 mM.

8. The preparation method according to claim 4, characterized in that, In step (3), a microwave oven with an input power of 1150 W and an output power of 700 W is used to heat the reaction on high for 3 minutes. The removal of unbound ssDNA linker core-shell structured gold and silver nanoparticles specifically involves resuspending them in PBS buffer, centrifuging, and then washing them with 0.01 M phosphate buffer containing 0.3 M NaCl. The volume ratio of Au@Ag NPs to thiol-modified ssDNA linker is 10:1; the concentration of Au@Ag NPs is 1 nM, and the concentration of ssDNA linker is 100 μM.

9. The application of the biosensor according to any one of claims 1-3 in detecting a target lncRNA, wherein the target lncRNA is TapSAKI, an early diagnostic biomarker for acute kidney injury, and the application includes preparation of a diagnostic product.

10. A method for detecting the concentration of a target lncRNA using the biosensor according to any one of claims 1-3, characterized in that, include: A sample containing the target lncRNA was added to the biosensor and reacted at an incubator for 40-80 minutes. After magnetic separation, the sample was resuspended, and Nb.BbvCI enzyme, buffer, and water were added. The reaction was carried out at 37°C. After the reaction was completed, the supernatant was collected by centrifugation for SERS detection.

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