Nucleic acid aptamer of heat shock protein 70 and application thereof

Abstract

The present application relates to a kind of nucleic acid aptamer of heat shock protein 70 and its application, the nucleic acid aptamer is at least one of HS-1, HS-2, its sequence is as shown in SEQ ID NO.1-2.The nucleic acid aptamer HS-1, HS-2 screened by the present application has good affinity to HSP70, and the dissociation constant of HSP70 protein and HS-1, HS-2 is in the order of μM by SPR detection. a ) reflects molecular binding efficiency, and the K a of the above two aptamers and HSP70 binding is in e 4 M ‑1 s ‑1 , indicating that it is rapid to combine, and it is easy to form complex between molecules.The nucleic acid aptamer HS-1, HS-2 screened by the present application has good specificity.Nucleic acid aptamer HS-1 and HS-2 can be used for the construction of magnetic microparticle double-aptamer sandwich chemiluminescence detection method.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a nucleic acid aptamer for heat shock protein 70 and its applications. Background Technology

[0002] HSP70 (heat shock protein 70) is a key stress protein and molecular chaperone. It has a molecular weight of approximately 70 kDa and an isoelectric point between 5.0 and 6.5. Structurally, HSP70 consists of an N-terminal ATPase domain and a C-terminal peptide-binding domain.

[0003] Multiple studies have shown that HSP70 can stabilize protein structure, preventing protein denaturation or aggregation under high temperatures, thereby protecting cells from further damage. Secondly, HSP70 also participates in the repair and refolding of damaged proteins, helping to restore intracellular protein homeostasis. Furthermore, HSP70 plays a crucial role in maintaining cell survival and function by regulating apoptosis and autophagy. Given the central role of HSP70 in cellular homeostasis, changes in its serum levels have significance for early warning, diagnosis, prognostic assessment, and treatment monitoring in heatstroke, cancer, and inflammation.

[0004] In various cancers, elevated serum HSP70 levels typically predict greater invasiveness and a poorer prognosis. HSP70, CAP2, mannose protein 3, and glutamine synthase can serve as molecular markers for early-stage hepatocellular carcinoma (HCC) in routine histopathological diagnosis. Furthermore, serum HSP70 shows significant elevations in the early stages of HCC, exhibiting superior sensitivity compared to the traditional marker AFP, making it a complementary indicator for liver cancer screening. Studies have also shown that the intensity of membrane-expressed heat shock protein 70 (mHsp70) is associated with lung cancer progression and treatment resistance. mHSP70 is released into the bloodstream in the form of exosomes, and the levels of these exosomes are correlated with lung cancer lymph node metastasis and early recurrence.

[0005] In conclusion, developing efficient methods for HSP70 detection and analysis is crucial. Currently, the commonly used HSP70 enzyme-linked immunosorbent assay (ELISA) detects HSP70 levels in the serum of cancer patients, while Hsp70-exo ELISA is used to detect microvesicle-related Hsp70. Both methods utilize antigen-antibody reactions, where enzyme-labeled antibodies or antigens react with the analyte, ultimately generating a measurable signal through an enzymatic substrate reaction to quantify the target substance. However, these methods have high reagent preparation costs. Furthermore, ELISA results typically take 4-5 hours to produce, and its sensitivity is two orders of magnitude lower than that of chemiluminescence methods.

[0006] To address the issues of low sensitivity and low detection efficiency in existing detection kits, this invention screened and obtained two high-affinity nucleic acid aptamers and verified that they can realize the detection of HSP70 by magnetic microparticle chemiluminescence using a dual-aptamer sandwich strategy. The constructed dual-aptamer sandwich magnetic microparticle chemiluminescence kit can rapidly and effectively detect HSP70 concentration, reduce detection costs, and improve detection sensitivity, accuracy, and efficiency. Summary of the Invention

[0007] Given that existing methods for detecting HSP70 levels in the serum of tumor patients using enzyme-linked immunosorbent assay (ELISA) or Hsp70-exo ELISA for detecting microvesicle-related Hsp70 suffer from high reagent preparation costs, low sensitivity, and low detection efficiency, this invention provides a nucleic acid aptamer for heat shock protein 70 and its application. The nucleic acid aptamer for heat shock protein 70 comprises two high-affinity nucleic acid aptamers, and its ability to achieve HSP70 detection using a dual-aptamer sandwich strategy with magnetic microparticle chemiluminescence is verified. The constructed dual-aptamer sandwich magnetic microparticle chemiluminescence kit can rapidly and effectively detect HSP70 concentration, reducing detection costs and improving detection sensitivity, accuracy, and efficiency.

[0008] The objective of this invention is achieved through the following technical solution: A nucleic acid aptamer for heat shock protein 70, wherein the nucleic acid aptamer is at least one of HS-1 and HS-2, and their sequences are as follows: HS-1: 5'- TCAAG TCACA GGTTC CAGGT AGTGA CGAGT CATGG TCCAT CATGA TCCACTCTTC ATAGG CACTG ACACG ACACT-3' (SEQ ID NO.1), HS-2: 5'-TCAAG TCACA GGTTC CAGGT CCCGG GTACC AAGAA CATGA TAACA ACGTGCGATC ATAGG CACTG ACACG ACACT-3' (SEQ ID NO. 2).

[0009] The nucleic acid aptamers were fitted at 37°C and 0.15M salt ion concentration. The spatial structures of the nucleic acid aptamers HS-1 and HS-2 are as follows:

[0010] HS-1 HS-2.

[0011] The nucleic acid aptamer wherein at least one of the nucleic acid aptamers HS-1 and HS-2 has its 5' or 3' end chemically modified with a fluorescent group, an amino group, biotin, or polyethylene glycol.

[0012] The application of the nucleic acid aptamer of heat shock protein 70 in the preparation of reagents, kits, sensors, and molecular probes for detecting heat shock protein 70.

[0013] The application of the nucleic acid aptamer of heat shock protein 70 in the chemiluminescence detection of heat shock protein 70.

[0014] A kit for specifically recognizing heat shock protein 70, i.e., a kit for detecting heat shock protein 70, is prepared from the aforementioned nucleic acid adaptor.

[0015] The kit includes reagent R1, reagent R2, reagent R3, reagent R4 and reagent R5; The reagent R1 comprises a suspension containing magnetic microparticles of the surface-coupled amino-modified nucleic acid aptamer HS-1. The reagent R2 includes an HS2-pHRP solution; the HS2-pHRP solution is prepared by mixing and incubating 10 µL of 10 µM HS2-Biotin with 12.5 µL of 0.5 µg / mL streptavidin polyhorseradish peroxidase, and then diluting with 1×PBS to 500 µL to obtain a 0.2 µM HS2-pHRP solution; the HS2-Biotin is the biotinylated nucleic acid aptamer HS-2.

[0016] The reagent R3 includes an analysis buffer, which is 1×PBS containing the following components: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4. The reagent R4 is ECL ultrasensitive luminescent solution (Dalian Meilun Biotechnology, catalog number: MA0187, including luminescent solution A and luminescent solution B).

[0017] The magnetic microparticles are carboxyl magnetic beads; 2 µL of carboxyl magnetic beads are coupled with 4 µL of 10 µM amino-modified nucleic acid aptamer HS-1 to obtain magnetic microparticles with surface-coupled amino-modified nucleic acid aptamer HS-1 and a particle size of approximately 300 nm.

[0018] A molecular probe comprising the aforementioned nucleic acid aptamer.

[0019] Compared with the prior art, the advantages of the present invention are as follows: 1. The nucleic acid aptamers HS-1 and HS-2 obtained by screening in this invention both exhibit good affinity for HSP70. SPR analysis showed that the affinity dissociation constants of HSP70 protein with HS-1 and HS-2 were both in the µM range. Binding rate constant (Kb) a The Kc of the two aptamers mentioned above binding to HSP70 reflects the molecular binding efficiency. a All in e 4 M -1 s -1 This indicates rapid binding and easy formation of complexes between molecules.

[0020] 2. The nucleic acid aptamers HS-1 and HS-2 obtained by screening in this invention have good specificity.

[0021] 3. Nucleic acid aptamers HS-1 and HS-2 can be used to construct a magnetic microparticle dual-aptamer sandwich chemiluminescence detection method. HS-1 and HS-2 can be used to prepare a kit specifically recognizing heat shock protein 70, i.e., a heat shock protein 70 detection kit. The detection method established using this kit involves immobilizing HS-1 and the HSP70 target on the surface of magnetic beads, and linking streptavidin-polyhortradiction peroxidase to biotinylated HS-2. These three reagents form a sandwich structure in 1×PBS buffer. After adding low-femto-sensitive ECL chemiluminescence solution, chemiluminescence imaging is performed. The intensity of the chemiluminescence is detected using SpectraMax iD5 to detect heat shock protein 70. Furthermore, using this kit, only the HSP70 detection group showed a strong signal, while homologs HSP47 and HSP27 showed weak enhancement. The other groups showed weak signals, indicating that the chemiluminescence method established by this kit has good specificity and can specifically recognize HSP70, making it suitable for the detection of HSP70 in clinical serum. Attached Figure Description

[0022] Figure 1 This invention relates to the screening process for detecting library recovery rate and determining the enrichment degree of binding bands in each secondary library.

[0023] Figure 2 This is a homology analysis of the first 30 enriched sequences in the HSP70 aptamer library of this invention.

[0024] Figure 3 This invention provides predictions of the secondary and three-dimensional structures of three candidate aptamers HS-1, HS-2, and HS-3 for HSP70.

[0025] Figure 4 It is a single-concentration SPR test signal of three DNA aptamers and HSP70 protein.

[0026] Figure 5The results are from the cross-competition study of aptamer affinity and recognition sites, including (A) the affinity of aptamers HS-1, HS-2 and HS-3 to HSP70; (B) the cross-competition study between aptamers HS-1 and HS-2; (C) the cross-competition study between aptamers HS-1 and HS-3; and (D) the cross-competition study between aptamers HS-2 and HS-3.

[0027] Figure 6 It is a flow cytometry assay used to detect the fluorescence signals of different concentrations of FAM fluorescently labeled HS-1 (A, B) and HS-2 (C, D) aptamers bound to magnetic beads coupled with HSP70.

[0028] Figure 7 These are SPR-based sensor maps of the interaction between HSP70 protein and DNA aptamers HS-1 and HS-2. (A) Sensor map of the interaction between HSP70 protein and DNA aptamer HS-1; (B) Sensor map of the interaction between HSP70 protein and DNA aptamer HS-2.

[0029] Figure 8 It is a flow cytometry assay used to determine the specificity of HS-1 and HS-2 binding to the target.

[0030] Figure 9 This is a schematic diagram of the overall structure of HSP70 docking with HS-1, HS-2, and HS-3 molecules and their hydrogen bond interactions.

[0031] Figure 10 The efficiency of coupling between different amounts of fluorescent amino-modified HS-1 and carboxyl magnetic beads in EDC solutions of different concentrations was determined by flow cytometry.

[0032] Figure 11 These are PAGE gel images showing the binding of different concentrations of streptavidin horseradish peroxidase to HS2-Biotin.

[0033] Figure 12 This study explores the feasibility of the sandwich method for detecting HSP70 and presents the chemiluminescence imaging results of the optimized dosage.

[0034] Figure 13 This is an optimization of the reaction time of the ECL luminescent solution. (A) Chemiluminescence imaging results; (B) chemiluminescence quantitative results from the microplate reader.

[0035] Figure 14 This is the standard curve for the detection of HSP70 by magnetic particle chemiluminescence.

[0036] Figure 15 It is a specific detection. Detailed Implementation

[0037] The present invention will now be described in detail with reference to the accompanying drawings and embodiments: Experimental steps: The MB-SELEX screening process for HSP70 aptamers includes (1) library renaturation; (2) coupling of the target protein HSP70 with Ni-NTA magnetic beads; (3) coupling of serum protein with carboxyl magnetic beads; (4) forward and reverse screening; (5) preparation of secondary libraries; and (6) purification of secondary libraries. Repeating steps 1-6 constitutes one round of screening. A total of eight rounds of screening are performed, with reverse screening added in rounds 3 and 7, while forward screening is used in the remaining rounds. Library renaturation: To unlock potential secondary structures in the library, the initial 5OD library diluted to 10 μM is denatured at 95°C for 10 min. After denaturation, the library is slowly cooled to room temperature to ensure structural stability.

[0038] Ni-NTA magnetic beads were used to conjugate the target protein HSP70: For HSP70 conjugation with magnetic beads, 50 µL of Ni-NTA magnetic beads were first washed with 200 µL of ddH2O. The beads were then resuspended in 500 µL of PBSM buffer, and 50 µg of recombinant human HSP70 protein was added. This protein has a His-tag (6 consecutive histidine residues) at its N-terminus. The mixture of magnetic beads and protein was incubated on a shaker at 25 °C for 1 h to immobilize the HSP70 protein onto the beads. After incubation, the HSP70-bound beads were separated using a magnetic rack, and the supernatant was discarded. The beads were washed twice with PBS solution to remove unbound protein and other impurities. The beads treated in the above steps were labeled MB-HSP70 for subsequent experiments.

[0039] Carboxyl magnetic beads coupled with serum proteins: Before use, the carboxyl magnetic beads were activated. 100 µL of carboxyl magnetic beads were taken and washed with 200 µL of ddH₂O, and the supernatant was removed. Next, 50 µL each of 0.1 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.4 M N-hydroxysuccinimide (NHS) were mixed in equal volumes and added to the washed magnetic beads. The mixture was incubated at room temperature for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads. The supernatant was removed, and the beads were immediately washed with ddH₂O. 80 µL of serum and 80 µL of pH 4.0 sodium acetate solution were added. The mixture was incubated in a shaker at 25 °C for 60 minutes to covalently couple the serum proteins to the magnetic beads. Subsequently, the magnetic beads were adsorbed using a magnetic rack, and the supernatant was removed. 100 µL of 1M ethanolamine (pH 8.5) was added, and the mixture was incubated with shaking at room temperature for 10 minutes to block unreacted activation sites on the magnetic bead surface. Finally, the beads were washed with 200 µL of PBS to remove unbound proteins. The resulting serum protein-coupled magnetic beads, labeled MB-S, were then used for reverse screening.

[0040] Forward and reverse screening: In the total of 8 rounds of screening, the 3rd and 7th rounds add a reverse screening process for serum proteins, while the remaining rounds directly use forward screening.

[0041] Positive screening process: First, 400 μL of the library (initial library or secondary library) was added to the positive screening magnetic beads (MB-HSP70), mixed thoroughly, and incubated on a shaker at room temperature for 1 hour. Next, the magnetic beads were captured using a magnetic rack, and the supernatant was aspirated and labeled as S. Then, the library-bound magnetic beads were washed twice with PBS, resuspended in 200 μL of ddH2O, and incubated at 95°C for 10 min. While still hot, the magnetic beads were precipitated with a magnet, and the supernatant was labeled as E. This eluted bound fragment is used for amplifying the next round of secondary libraries.

[0042] Reverse screening process: The secondary libraries obtained from rounds 3 and 6 are mixed with reverse screening magnetic beads (coupled with serum proteins) and incubated on a shaker at room temperature for 1 hour. Then, the magnetic beads are captured, the supernatant is aspirated, and the supernatant without serum protein binding is used as the forward screening library.

[0043] Preparation and purification of the secondary library: Biotin-modified reverse amplification primers (Rp-Bio) and forward primers were used. PCR amplification was performed using E as a template in each round to generate biotin-labeled double-stranded products. The amplification conditions were as follows: Pre-denaturation was performed at 95℃ for 3 minutes, followed by cycling. Each cycle consisted of denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 40 seconds, and a final extension at 72℃ for 5 minutes, followed by maintenance at 4℃.

[0044] Double-stranded PCR products were incubated with streptavidin-coated magnetic beads at room temperature for 2 hours. The magnetic beads bound to the PCR products were then recovered, washed twice with PBS, and resuspended in PBS. The mixture was then heated at 95°C for 10 minutes to separate the magnetic beads. The recovered supernatant was the single-stranded secondary library, denoted as P. The nucleic acid concentration was determined using a micro-ultraviolet spectrophotometer to facilitate subsequent screening cycles.

[0045] 1. Screening process control Flow cytometry was used to monitor the dynamic changes in the recognition ability of single-stranded DNA libraries for HSP70 protein. Forward primers with FAM fluorescent labeling and reverse primers modified with biotin were used to amplify HSP70-binding DNA single-stranded recovery buffer E at each round. The amplified fluorescent single-stranded libraries were then recovered and purified using streptavidin magnetic beads. To ensure accuracy, the purified libraries were quantitatively analyzed using an ultra-micro UV spectrophotometer. Finally, 4 µL of magnetic beads and 4 µg of the HSP70 binding complex were incubated with 200 µg of each round of the library at 25 °C for one hour. The affinity of the sequences for HSP70 protein was assessed by comparing the fluorescence shift results at each round.

[0046] The random library and primers used for HSP70 aptamer screening are shown in the table below:

[0047] Screening process, including library recovery rate detection and determination of binding band enrichment in each secondary library, such as... Figure 1 As shown, it was observed that with the increase of screening rounds, the sequence retention rate of each round gradually increased, while the number of fluorescent sequences binding to the target gradually increased, and the shift phenomenon became more obvious. This reflects that the increase in screening rounds enriched the oligonucleotide chains in the library that can bind to MB-HSP70.

[0048] 2. Sequencing and homology analysis HSP70 aptamers were obtained through screening using MB-SELEX technology with the addition of a high-pressure serum matrix. Single-stranded DNA samples recovered in rounds 2, 3, 5, 6, 7, and 8 were selected and high-throughput sequencing was performed by Anhui Angpu Tuomai Biotechnology Co., Ltd. After obtaining the sequencing results, the top 30 most abundant sequences were analyzed and compared using ClustalX and MEGA software to understand the evolutionary relationships and enrichment characteristics among these core random sequences.

[0049] Figure 2 As shown, the sequences enriched by constructing a phylogenetic tree can be divided into 7 families. Using MEME software, conserved regions were identified in the first 30 sequences. For example, HS-1, HS-7, and HS-8 all contain the "RRTCCNKCATCAGTCVCT" sequence region; HS-2, HS-5, HS-9, and HS-20 all contain the "CGGNANTDACNTGTWBTTKGT" sequence region; and HS-3, HS-21, and HS-26 all contain the "CCNRDTGGTACTG" sequence region.

[0050] 3. Computer simulation of the secondary and three-dimensional structures of three candidate aptamers The secondary structures of the DNA of three candidate aptamers were simulated using the Matthews model (2004) program in the RNAfold software at 37°C and a salt ion concentration of 0.15 M. The secondary structures were then used to predict the 3D structures via the RNA Composer system, and the U-mutation was performed to T using Pymol software.

[0051] 4. SPR detection of the interaction between HSP70 protein and HS-1, HS-2, and HS-3 The samples were sent to the eTesting Lab for surface plasmon resonance (SPR) assay to determine the affinity of the HSP70 protein for candidate aptamers. The protein was immobilized on a CM5 chip using EDC / NHS coupling. A single concentration of candidate aptamers was diluted to 10 µM in a 96-well plate and flowed at 10 μL / min for 60 s during each run. At the end of each flow, the chip was regenerated for 5 minutes with 10 mM glycine hydrochloride (pH 2.0) solution. This process was repeated until all three DNA candidate aptamers were assayed.

[0052] Results: All three candidate aptamers showed affinity, with HS-1 and HS-2 exhibiting better binding ability after binding to the target protein HSP70, with a signal greater than 20 RU.

[0053] 5. Investigation of aptamer affinity for HSP70 and cross-competitiveness in biaptamer recognition. The three most abundant DNA strands (HS-1, HS-2, and HS-3) belong to different families and have significant structural differences; therefore, we selected these three for affinity analysis. Target proteins were immobilized using magnetic beads and incubated with fluorescein-modified HS-1F, HS-2F, and HS-3F at room temperature for 1 hour. Flow cytometry was used to monitor their binding affinity. Simultaneously, to develop a subsequent dual-aptamer sandwich detection method, we investigated whether the addition of dual aptamers would cause cross-competition.

[0054] Figure 5 A shows that HS-1, HS-2, and HS-3 all exhibit good affinity for HSP70. This may be because HSP70 has a large molecular weight and complex three-dimensional structure, providing a sufficient number of binding sites and DNA strands with different structures. Meanwhile, as... Figure 5 Cross-reactivity analysis of the addition of dual aptamers in B, C, and D showed that HS-2 had lower non-specific binding to magnetic beads. The combined application of HS-1 and HS-2, as well as the combined application of HS-2 and HS-3, did not result in competitive cross-reactivity. However, the combined application of HS-1 and HS-3 would somewhat affect the design of the sandwich method. Considering both affinity and complementary interference of dual recognition, we ultimately chose HS-1 and HS-2 for the construction of the subsequent magnetic microparticle dual aptamer sandwich chemiluminescence detection method.

[0055] 6. Flow cytometry was used to detect the dissociation constants of the two candidate aptamers on the HSP70 protein. 3 μL of Ni-NTA magnetic beads were conjugated to HSP70 protein. 2 μL of candidate aptamers labeled with FAM fluorescein at concentrations of 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, and 0.078125 μM were added, and the mixtures were incubated with the magnetic bead-HSP70 complex for 15 minutes. The mixture was washed three times with deionized water to remove free or unstable aptamers. Simple magnetic beads (MB) and the magnetic bead-HSP70 complex (MB-HSP70) were used as control groups. Fluorescence intensity at 525 nm was detected by flow cytometry. Nonlinear fitting was performed using GraghPad Prism 9.5, and the equation Y=B was used to calculate the fluorescence intensity. max X / (K d The +X) equation was used to evaluate the binding affinity of different concentrations of candidate aptamers to the HSP70 protein and estimate K. d Value, Y is fluorescence intensity, B max X represents the maximum binding site between the aptamer and the target, and X represents the concentration of the aptamer.

[0056] The binding and dissociation properties of the two aptamers were investigated using flow cytometry. Figure 6As shown, with increasing aptamer concentration, the fluorescence intensity shifts significantly to the right, and at a certain concentration, the fluorescence signals tend to gradually stack. The HS-1 flow cytometry fluorescence shift is as follows: Figure 6 As shown in Figure A and Figure 6C, aptamers HS-1 and HS-2 both exhibit affinity for HSP70, and their fluorescence shift reaches a plateau phase with increasing aptamer concentration. The fluorescence intensity trends of different aptamer concentrations were nonlinearly fitted, and the Kd value of candidate aptamer HS-1 was estimated to be 1.058 µM and that of HS-2 to be 0.5527 µM using the equation Y = BmaxX / (Kd +X).

[0057] 7. SPR detection of affinity dissociation constants of HSP70 protein with HS-1 and HS-2 The samples were sent to the e-testing laboratory, where surface plasmon resonance (SPR) technology was used to determine the binding and dissociation constants of HSP70 protein with candidate aptamers. The basic method was as follows: an interaction model was established using SPR to measure the interaction between HSP70 protein (ligand) and compounds HS-1 and HS-2 (analytes). Protein immobilization was performed using the amino-coupling method; protein coupling buffer: 1.0×PBS-P+ (pH 7.4); interaction buffer: 1.0×PBS-P+ (pH 7.4). Each compound was diluted to several concentrations in a 96-well plate, and the interaction buffer was run, coupling the target protein from low to high concentrations through the microarray. The flow rate was 30 μL / min, and the duration was 60 s. After each concentration point, the microarray was regenerated for 5 min with 10 mM glycine hydrochloride (pH 2.0) solution. This process was repeated until all corresponding concentrations of HS-1 and HS-2 were obtained.

[0058] The data were globally fitted to a 1:1 Langmuir binding model using Biacore Insight evaluation software (Cytiva, V6.0 Marlborough, MA, USA) to obtain binding and dissociation constants.

[0059] Table 1. Binding and dissociation constants of HSP70 with HS-1 and HS-2, respectively

[0060] like Figure 7As shown, the affinity between compounds and HSP70 protein was determined using Biacore, employing a CM5 chip-coupled protein assay. The assay primarily consisted of four parts: baseline, binding phase, equilibrium phase, and dissociation phase. Due to the presence of a biological matrix on the biosensor chip surface, a background signal was generated when buffer solution flowed through it. During the binding phase, HS-1 or HS-2 analyte flows through the HSP70 immobilized on the chip surface, generating a binding signal. Before the injection phase ended, the curve exhibited a certain curvature; the dynamic equilibrium state was not obvious. The dissociation phase curve also conformed to a single exponential characteristic before reaching a certain signal.

[0061] The binding and dissociation constants of molecular interactions are shown in Table 1: Affinity constant K D The smaller the value, the stronger the affinity. The binding KD values ​​of the two aptamers mentioned above with HSP70 are both in the µM range, indicating good intermolecular affinity.

[0062] Binding rate constant (K) a K reflects the molecular binding efficiency. a The higher the value, the faster the binding. The two aptamers mentioned above bind to HSP70 at a Kc value. a All in e 4 M -1 s -1 This indicates rapid binding and easy formation of complexes between molecules.

[0063] Dissociation rate constant (K) d () represents the rate of dissociation of the complex, measured in seconds. -1 This reflects the stability of the complex. The Kc of the complexes of the two aptamers and HSP70 mentioned above reflects this stability. d In 10 - 2 s -1 The range and stability of the bound complex can be further optimized.

[0064] 8. Specificity of aptamer recognition of HSP70 Because HSP60 and HSP90b1 are structurally similar to HSP70, they were used to verify the target recognition capabilities specific to HS-1 and HS-2. 3 μL of Ni-NTA magnetic beads were conjugated with 3 μg of each protein, followed by binding with 2 μL of a 10 μM candidate aptamer labeled with FAM fluorescein, providing a fluorescence signal intensity at an emission wavelength of 525 nm that could be detected by flow cytometry.

[0065] The specificity of the two aptamers for binding to HSP70 and its homologs was investigated by flow cytometry. Figure 8The results showed that the candidate aptamers HS-1 and HS-2, labeled with FAM fluorescein, exhibited significant fluorescence shifts when interacting with HSP70 coupled to MB, while the two candidate aptamers showed no significant binding effect with HSP60 and HSP90b1 immobilized by magnetic beads. This indicates that candidate aptamers HS-1 and HS-2 exhibited good specificity.

[0066] 9. Molecular docking simulation of HSP70 with HS-1, HS-2, and HS-3 First, a search was performed using UniProt (Universal Protein Resource), an authoritative bioinformatics database. After entering the Accession number of HSP70 (P0DMV8), the corresponding PDB number (7KW7_2) was retrieved. Then, the required target PDB file was downloaded. The PDB file of the DNA aptamer's three-dimensional conformation predicted in Part 3 above, along with the PDB file of HSP70, were imported into HDock software for molecular docking simulation. Molecular visualization was then performed in Pymol, and the interdependent hydrogen bond binding sites were analyzed.

[0067] Molecular docking analysis, such as Figure 9 As shown, HS-1 has 7 key bases that interact with HSP70, HS-2 has 6 key bases that interact with HSP70, and HS-3 has 5 key bases that interact with HSP70. By simulating the physical binding process between proteins and aptamers, the interaction patterns and potential binding sites between them were revealed.

[0068] 10. Investigation into the coupling conditions between aptamer HS1-NH2 (i.e., amino-modified HS-1) and carboxyl magnetic beads Carboxylated magnetic beads (300nm, Xianfeng Nano, catalog number: 104354) are formed by combining the carboxyl groups on the surface of magnetic microparticles with amino-modified HS-1 via EDC reagent.

[0069] Figure 10 The results showed that 5 µL of 10 µM HS1-NH2 could be coupled with 2 µL of Xianfeng nanocarboxylated magnetic beads via a reaction in 100 µL of 500 mM EDC for 2 h. Further increasing the aptamer concentration did not affect the coupling efficiency. To allow some space for the aptamer HS-1 on the surface of the magnetic beads to reduce steric hindrance for protein binding, 2 µL of carboxylated magnetic beads will be coupled with 4 µL of 10 µM amino-modified aptamer HS-1 in subsequent reactions.

[0070] 11. Optimization of the ratio of streptavidin-horseradish peroxidase to HS2-Biotin (Biotin-modified HS-2) Streptomycin-avidin polyhortradiction peroxidase stock solution (Thermo Fisher Scientific, catalog number: 21140) was dissolved in 1×PBS solution and diluted 4, 10, 40, and 100 times, respectively. 10 µL of each solution was added to 2 µL of HS2-Biotin and mixed well. Then, 1×PBS solution was added to a final volume of 30 µL. After binding at room temperature for 2 h, 8 µL of the sample was taken for PAGE gel electrophoresis.

[0071] The results are as follows Figure 11 As shown, 2 µL of 10 µM HS-2 (Biotin) can be completely linked with 10 µL of SA-pHRP diluted 4-fold, which is equivalent to 2.5 µL of streptavidin poly-HRP stock solution. The disappearance of the aptamer band indicates that it is completely bound to polyhortradiction peroxidase through streptavidin-biotin interaction to form the complex HS2-pHRP. Since this complex has a large molecular weight, it remains on the sample well.

[0072] 12. Feasibility of chemiluminescence detection of HSP70 based on aptamers and magnetic microparticles To investigate the immobilization of HS-1, HSP70 target, and biotinylated HS-2 linked to streptavidin-polyhortradic peroxidase on magnetic beads, a sandwich structure was formed using three reagents in 1×PBS buffer. Different amounts of MB-HS1 (HS-1 immobilized on magnetic beads), 10 µL of biotinylated HS-2 (10 µM), and 12.5 µL of a 0.5 µg / mL polyHRP complex (streptavidin-polyhortradic peroxidase) were added. This was diluted to 500 µL with 1×PBS for sample loading, i.e., 0.2 µM HS2-pHRP. After reacting with the target HSP70 (50 ng / mL, 10 µL) in the sandwich structure for 1 h, the mixture was washed 6 times with 100 µL of 1×PBS. Then, 100 µL of low-femtochromic ultrasensitive ECL chemiluminescence solution (Meilun, catalog number: MA0187-2) was added via a multi-channel pipette, and chemiluminescence imaging was performed after 1 min.

[0073] Figure 12 The results showed that chemiluminescence detection of HSP70 based on aptamers using magnetic microparticles is feasible. Specifically, there was no significant non-specific adsorption between the magnetic beads and HS-2-pHRP; furthermore, in the absence of a target, MB-HS1 and HS2-pHRP could not form a sandwich structure, thus failing to induce a strong chemiluminescence signal. However, with the addition of a target, the optimal chemiluminescence intensity was achieved when 4 µL of 10 µM HS-1 and 10 µL of 0.2 µM HS2-pHRP were immobilized in 2 µL of magnetic beads.

[0074] 13. Optimization of reaction time after adding ECL luminescent liquid For sample preparation, refer to Part 12 of the procedure. After sandwich reaction for 1 h, wash 6 times with 100 µL 1×PBS, then add 100 µL of low-femtosensitive ECL chemiluminescence solution (the reaction solution after mixing solutions A and B according to the instructions) using a multi-channel pipette. Set the chemiluminescence reaction time to 30 s, 1 min, 2 min, 4 min, and 5 min. Use chemiluminescence imaging to capture the changes in the imaging light intensity, and use SpectraMax iD5 to detect the chemiluminescence intensity.

[0075] Figure 13 As shown: the luminescence intensity reached its peak at 2 min when detected by a chemiluminescence analyzer. However, when detected by an enzyme-linked immunosorbent assay (ELISA) reader, after the same sample was repeatedly tested twice, the luminescence quenched strongly on the third test at 2 min. The luminescence intensity showed an upward trend in both imaging and quantitative results between 30 s and 1 min. Therefore, 1 min was subsequently used as the reaction time for the ECL luminescence solution.

[0076] 14. Detection sensitivity Since the amount of enzyme captured is directly proportional to the target concentration, it catalyzes the luminescent substrate, causing a chemical reaction that releases a large amount of energy. When the excited-state intermediate returns to the stable ground state, it can simultaneously emit photons. The photon yield is directly proportional to the amount of the analyte in the sample. The following steps involve adding 10 µL of HSP70 at gradient concentrations of 80 ng / mL, 60 ng / mL, 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, 1 pg / mL, and a blank concentration to the above reaction system. Refer to step 12 for specific steps. After adding reagent R4 and reacting the ECL luminescent solution for 1 min, the chemiluminescence intensity was detected using a SpectraMax iD5, and imaging was performed using a Bio-Rad ChemiDoc XRS+.

[0077] Figure 14 The results showed that the signal response of HSP70 in the range of 1 pg / mL to 80 ng / mL was investigated, and the results showed a biexponential curve relationship in the range of 1 pg / mL to 40 ng / mL: Y = 1210995 + 3871814.08 × (1 - e -96.10X )+ 3223318.92×(1-e -0.1125X ), R 2 =0.9877.

[0078] 15. Detection Specificity The above method was used to detect HSP47, HSP27, SAA1, IL-6, TnI, MPO, and CHI3L1 at concentrations of 100 ng / mL HSP70 and 500 ng / mL, and the chemiluminescence intensity was detected using SpectraMax iD5.

[0079] Figure 15 The results showed that chemiluminescence signal intensity was measured using five-fold concentrations of heat shock protein homologs, inflammation-related recombinant proteins, cardiac injury markers, and liver fibrosis markers to explore the specificity of the HSP70 magnetic microparticle chemiluminescence method. The results indicated that only the HSP70 detection group showed a strong signal, while homologs HSP47 and HSP27 showed weak enhancement. The other groups showed weak signals, indicating that the nucleic acid aptamer magnetic microparticle chemiluminescence method has good specificity and can specifically identify HSP70, making it suitable for the detection of HSP70 in clinical serum.

[0080] The nucleic acid aptamers HS-1 and HS-2 obtained by the present invention are not only applicable to the establishment of a detection method for nucleic acid aptamer magnetic microparticle chemiluminescence, but can also be used alone to establish colorimetric analysis methods, etc. Since colorimetric analysis is a commonly used method in this field, this method will not be described in detail here.

[0081] The above description is a general account, specific implementation method and test. However, the present invention is not limited to the above embodiments. Any modifications or improvements based on the present invention, as well as equivalent substitutions or changes to the concept of the present invention, should be covered within the protection scope of the present invention.

Claims

1. A nucleic acid aptamer for heat shock protein 70, characterized in that: The nucleic acid aptamer is at least one of HS-1 and HS-2, and their sequences are as follows: HS-1: 5'- TCAAG TCACA GGTTC CAGGT AGTGA CGAGT CATGG TCCAT CATGA TCCACTCTTC ATAGG CACTG ACACG ACACT-3' HS-2: 5'-TCAAG TCACA GGTTC CAGGT CCCGG GTACC AAGAA CATGA TAACA ACGTG CGATCATAGG CACTG ACACG ACACT-3'.

2. The nucleic acid aptamer according to claim 1, characterized in that: Fitting was performed at 37℃ and 0.15M salt ion concentration. The spatial structures of the nucleic acid aptamers HS-1 and HS-2 are as follows: 。 3. The nucleic acid aptamer according to claim 1, characterized in that: The 5' or 3' end of at least one of the nucleic acid aptamers HS-1 and HS-2 is chemically modified with a fluorescent group, amino group, biotin, or polyethylene glycol.

4. The use of the nucleic acid aptamer of heat shock protein 70 as described in any one of claims 1-3 in the preparation of reagents, kits, sensors, and molecular probes for detecting heat shock protein 70.

5. The application of the nucleic acid aptamer of heat shock protein 70 as described in any one of claims 1-3 in the chemiluminescence detection of heat shock protein 70.

6. A kit for specifically recognizing heat shock protein 70, characterized in that: It is obtained from the nucleic acid adaptor system described in any one of claims 1-3.

7. The reagent kit according to claim 6, characterized in that: The kit includes reagent R1, reagent R2, reagent R3, reagent R4 and reagent R5; The reagent R1 comprises a suspension containing magnetic microparticles of the surface-coupled amino-modified nucleic acid aptamer HS-1. The reagent R2 comprises an HS2-pHRP solution; the HS2-pHRP solution is prepared by mixing and incubating 10 µL of 10 µM HS2-Biotin with 12.5 µL of 0.5 µg / mL streptavidin-polyhortradiction peroxidase, and then diluting with 1×PBS to 500 µL to obtain a 0.2 µM HS2-pHRP solution; the HS2-Biotin is the biotinylated nucleic acid aptamer HS-2; The reagent R3 includes an analysis buffer, which is 1×PBS containing the following components: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4. The reagent R4 is an ECL hypersensitive luminescent liquid.

8. The reagent kit according to claim 7, characterized in that: The magnetic microparticles are carboxyl magnetic beads; 2 µL of carboxyl magnetic beads are coupled with 4 µL of 10 µM amino-modified nucleic acid aptamer HS-1 to obtain magnetic microparticles with surface-coupled amino-modified nucleic acid aptamer HS-1.

9. A molecular probe, characterized in that: It comprises the nucleic acid aptamer as described in any one of claims 1-3.

Images