An electrochemical sensor for detecting PDGF-BB using a poly A-DNA tetrahedral probe and its application.

Abstract

This invention provides an electrochemical sensor for detecting PDGF-BB using a poly A-DNA tetrahedral probe and its application. The electrochemical sensor is a base-stack-dependent electrochemical sensor, comprising a poly A-tetrahedral probe self-assembled on a gold electrode, aptamer DNA, horseradish peroxidase, and a chromogenic substrate. The aptamer DNA is biotin-modified at its tail, and the horseradish peroxidase is avidin-modified. The electrochemical sensor detects PDGF-BB. The aptamer DNA is triggered by PDGF-BB to transform into a stem-loop structure and stacks with the poly A-tetrahedral probe. Based on the base stacking effect, the aptamer DNA stably hybridizes with the target complementary sequence of the poly A-tetrahedral probe immobilized on the gold electrode. The aptamer DNA and horseradish peroxidase bind via biotin-avidin. The chromogenic substrate is catalyzed to generate an electrochemical redox catalytic signal, which is then output.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular detection technology, specifically relating to an electrochemical sensor for detecting PDGF-BB using a poly A-DNA tetrahedral probe and its application. Background Technology

[0002] DNA tetrahedra are rigid three-dimensional DNA structures with numerous advantages, including high stability, abundant structural sites, and ease of modification. DNA possesses a specific AT / GC base pairing rule, allowing for the synthesis of DNA tetrahedra through simple self-assembly. Furthermore, the size of the nucleic acid framework structure, the introduction of functionalized sequences, and the position of vertices can be precisely controlled. Polyadenine (poly A) has been developed for assembling oligonucleotides on gold surfaces, exhibiting affinity comparable to Au-S bonds. Various biosensors based on poly A-based two-dimensional capture probes immobilized on gold electrodes have been developed. Label-free poly A probes are covalently attached to the gold electrode surface via adenine, requiring no chemical modification. The Turberfield group successfully constructed framework nucleic acid (FNA) structures using four single-stranded DNA strands, which are among the most widely used DNA nanostructures.

[0003] The thermal stability of the DNA double helix is ​​affected by factors such as temperature, base composition, chain length, secondary structure, and ionic strength. Base pairing between complementary strands and stacking between adjacent bases are the main factors contributing to the stability of the DNA double helix. Base stacking is a dipole-induced dipole interaction between planar aromatic bases in two adjacent nucleotides. When two consecutive tandem sequences are annealed into a longer chain, the coaxial base stacking at the nick site provides additional stability for hybridization and facilitates primary stacking interactions between adjacent base pairs. Numerous theoretical studies have confirmed that stacking and base pairing are beneficial to the stability of the DNA double helix, and this research has been successfully applied to the chemiluminescence detection of proteins.

[0004] Platelet-derived growth factor (PDGF) is an important serum cytokine, classified into three subtypes: PDGF-AA, PDGF-AB, and PDGF-BB. PDGF-BB is a cancer-associated protein involved in cell transformation, tumor growth, and progression, and is a crucial biomarker for cancer diagnosis and identification. Traditional PDGF-BB detection methods are antibody-based immunoassays, such as enzyme-linked immunosorbent assay (ELISA). While these methods are highly practical, they still require improvement due to their long detection time, high cost, and complex operation. Therefore, it is necessary to establish a rapid, sensitive, accurate, and economical method for measuring PDGF-BB for disease diagnosis and treatment.

[0005] Electrochemical detection is a promising technology due to its simplicity, high sensitivity, low detection limit, low cost, and miniaturization capabilities. Typical electrochemical biosensors usually consist of single-stranded DNA capture probes immobilized on an electrode. However, controlling the orientation and density of conventional capture probes on the electrode surface is difficult. Therefore, developing a method that facilitates control over the orientation and density of capture probes on the electrode surface, and establishing an efficient and sensitive electrochemical detection method for PDGF-BB, has significant application value. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an electrochemical sensor for detecting PDGF-BB using a poly A-DNA tetrahedral probe and its applications. This electrochemical biosensor detects the protein PDGF-BB. In the presence of the target protein, the aptamer DNA is triggered to undergo structural changes, forming a stem-loop. Subsequently, under the influence of base stacking, it stably hybridizes with the extended end of a poly A-DNA tetrahedral capture probe immobilized on a gold electrode surface. The biotin-labeled end binds to streptavidin, generating a signal output in the presence of the TMB substrate. The proposed method can detect PDGF-BB concentrations as low as 3 pg / mL, with a dynamic range of four orders of magnitude, comparable to traditional detection methods such as enzyme-linked immunosorbent assay (ELISA).

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an electrochemical sensor for detecting PDGF-BB using a poly A-DNA tetrahedral probe. The electrochemical sensor is a base stacking-dependent electrochemical sensor, comprising a poly A-tetrahedral probe self-assembled on a gold electrode, aptamer DNA, horseradish peroxidase, and a chromogenic substrate.

[0009] The poly A-tetrahedral probe is formed by the self-assembly of four single-stranded DNA strands onto a gold electrode to obtain a tetrahedral structure; the tail of the aptamer DNA is modified with biotin, and the horseradish peroxidase is modified with avidin.

[0010] The electrochemical sensor is used to detect PDGF-BB. The aptamer DNA is triggered by PDGF-BB to transform into a stem-loop structure and stacks with a poly A-tetrahedral probe. Based on the base stacking effect, the aptamer DNA and the target complementary sequence of the poly A-tetrahedral probe immobilized on the gold electrode stably hybridize. The aptamer DNA and horseradish peroxidase bind through biotin-avidin. The catalytic chromogenic substrate generates an electrochemical redox catalytic signal, producing a signal output.

[0011] This invention combines base stacking and DNA hybridization methods with electrochemical technology to design an aptamer biosensor based on a polyadenine three-dimensional DNA tetrahedral capture probe, which, combined with base stacking, is used to detect PDGF-BB. The biosensor requires no chemical modification; the distance between tetrahedra can be controlled by changing the number of tail A atoms on the probe on the electrode surface, forming a tightly ordered biosensing interface. Only in the presence of platelet-derived factor PDGF-BB is the aptamer triggered to transform into a stem-loop structure and stack with the capture probe. Due to the enhanced base stacking, the aptamer DNA can stably hybridize with the poly A-DNA tetrahedral capture probe immobilized on the gold electrode, forming a stem-loop structure. The aptamer tail is designed to be biotin-modified, binding to avidin-polyHRP80, generating an electrochemical redox catalytic signal in the TMB-H2O2 substrate, producing a signal output.

[0012] Preferably, the poly A-tetrahedral probe is formed by self-assembling single-chain A, single-chain B-poly A, single-chain C-poly A and single-chain D-poly A on a gold electrode to obtain a tetrahedral structure.

[0013] Preferably, the single-stranded A consists of a target complementary sequence, a poly T sequence, and an extension sequence.

[0014] Preferably, the single-stranded B-poly A, single-stranded C-poly A, and single-stranded D-poly A are each composed of a poly A sequence and an extended sequence.

[0015] Preferably, the poly A sequence has 5-30 bases, such as 5, 10, 15, 20, 25 or 30, and more preferably 10-20, such as 10, 12, 14, 15, 16, 18 or 20.

[0016] Preferably, the target complementary sequence is complementary to the aptamer DNA.

[0017] Preferably, the number of bases in the target complementary sequence that is complementary to the aptamer DNA is 7-9, for example, 7, 8 or 9, preferably 8.

[0018] Preferably, the single-stranded A consists of a target complementary sequence, a poly T sequence, and an extended sequence A, wherein the extended sequence A consists of extended sequences A1, A2, and A3.

[0019] Preferably, the extended sequences A1 and A2 are connected by 1-3 (e.g., 1, 2 or 3) bases, and the extended sequences A2 and A3 are connected by 1-3 (e.g., 1, 2 or 3) bases.

[0020] Preferably, the single-chain B-poly A consists of a poly A sequence and an extended sequence B, wherein the extended sequence B consists of extended sequences B1, B2 and B3.

[0021] Preferably, the extended sequences B1 and B2 are connected by 1-3 (e.g., 1, 2 or 3) bases, and the extended sequences B2 and B3 are connected by 1-3 (e.g., 1, 2 or 3) bases.

[0022] Preferably, the single-stranded C-poly A consists of a poly A sequence and an extended sequence C, wherein the extended sequence C consists of extended sequences C1, C2 and C3.

[0023] Preferably, the extended sequences C1 and C2 are connected by 1-3 (e.g., 1, 2 or 3) bases, and the extended sequences C2 and C3 are connected by 1-3 (e.g., 1, 2 or 3) bases.

[0024] Preferably, the single-stranded D-poly A consists of a poly A sequence and an extended sequence D, wherein the extended sequence D consists of extended sequences D1, D2 and D3.

[0025] Preferably, the extended sequences D1 and D2 are connected by 1-3 (e.g., 1, 2 or 3) bases, and the extended sequences D2 and D3 are connected by 1-3 (e.g., 1, 2 or 3) bases.

[0026] Preferably, the extended sequence A1 is complementary to the extended sequence D1; the extended sequence A2 is complementary to the extended sequence B2; the extended sequence A3 is complementary to the extended sequence C3; the extended sequence B1 is complementary to the extended sequence C1; the extended sequence B3 is complementary to the extended sequence D3; and the extended sequence C2 is complementary to the extended sequence D2.

[0027] In this invention, the number of bases in the extended sequences A1-A3, B1-B3, C1-C3 or D1-D3 is 15-20, for example, 15, 16, 17, 18, 19 or 20.

[0028] Secondly, this invention provides a method for detecting PDGF-BB based on a poly A-DNA tetrahedral probe electrochemical sensor, the detection method comprising:

[0029] Construct the poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB as described in the first aspect, and establish a linear regression equation between the electrochemical current signal and the concentration of PDGF-BB; the concentration of PDGF-BB in the test sample is indicated by detecting the electrochemical current signal of the test sample.

[0030] Preferably, the detection method includes the following steps:

[0031] (1) Preparation of poly A-tetrahedron

[0032] A buffer solution containing single-chain A, single-chain B-poly A, single-chain C-poly A and single-chain D-poly A was heated and kept at a constant temperature, and then cooled to obtain poly A-tetrahedron;

[0033] (2) Construction of electrochemical biosensors

[0034] Poly A-tetrahedrons self-assemble on the surface of the gold electrode, and the surface of the gold electrode is sealed with a sealing agent.

[0035] (3) Electrochemical detection

[0036] The electrochemical biosensor prepared in step (2) is co-incubated with the sample to be tested and aptamer DNA. Based on the base stacking effect, the aptamer DNA and the target complementary sequence of the poly A-tetrahedral probe fixed on the gold electrode are stably hybridized. The aptamer DNA and horseradish peroxidase are bound through biotin-avidin. The catalytic chromogenic substrate generates an electrochemical redox catalytic signal, and a signal output is generated.

[0037] Preferably, in step (1), the concentration of the single-chain A, single-chain B-poly A, single-chain C-poly A and single-chain D-poly A in the buffer solution is 5-15 μM, for example, it can be 5 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM or 15 μM.

[0038] Preferably, in step (1), the buffer solution is a TE buffer solution.

[0039] Preferably, in step (1), the heating temperature is 90-95℃, for example, it can be 90℃, 91℃, 92℃, 93℃, 94℃ or 95℃, etc.

[0040] Preferably, in step (1), the heat preservation time is 8-12 minutes, for example, it can be 8, 9, 10, 11 or 12 minutes.

[0041] Preferably, in step (2), the blocking agent is a 0.8-1.2% casein solution, for example, it can be 0.8%, 0.9%, 1%, 1.1% or 1.2%, etc.

[0042] Preferably, in step (3), the concentration of the aptamer DNA is 5-5000 nM, for example, it can be 5 nM, 50 nM, 100 nM, 1000 nM, 2000 nM, 3000 nM, 4000 nM or 5000 nM, etc., more preferably 50-500 nM, for example, it can be 50 nM, 100 nM, 200 nM, 300 nM, 400 nM or 500 nM, etc., more preferably 50-100 nM, for example, it can be 50 nM, 60 nM, 70 nM, 80 nM, 90 nM or 100 nM, etc.

[0043] Preferably, in step (3), the co-incubation temperature is 25-37℃, for example, it can be 25℃, 27℃, 29℃, 30℃, 31℃, 33℃, 35℃ or 37℃, etc.

[0044] Preferably, in step (3), the co-incubation time is 1-3 hours, for example, it can be 1, 2 or 3, preferably 2-3 hours.

[0045] Thirdly, the present invention provides a kit for detecting PDGF-BB based on an electrochemical biosensor, the kit comprising the poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB described in the first aspect.

[0046] Fourthly, the present invention provides the application of the poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB as described in the first aspect in the preparation of products for detecting PDGF-BB.

[0047] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] This invention provides a base stacking-dependent poly A-DNA tetrahedral probe electrochemical sensing platform for detecting PDGF-BB. The biosensor's capture probe is designed as a poly A-DNA tetrahedron. Polyadenine has a similar affinity to Au-S, enabling the tetrahedron to be immobilized on a gold electrode without chemical modification. Furthermore, utilizing the base stacking effect, in the presence of the target protein, the aptamer is triggered to transform into a stem-loop structure and stacks with the capture probe. Due to the enhanced base stacking, the aptamer DNA can stably hybridize with the poly A-DNA tetrahedral capture probe immobilized on the gold electrode, generating a signal output. This sensor can detect proteins down to 3.0 pg / mL, with a linear dynamic range of four orders of magnitude. Moreover, it performs well in real serum sample assays and has significant application potential. Attached Figure Description

[0050] Figure 1 This is a schematic diagram of a poly A-DNA tetrahedral electrochemical aptamer sensor based on the base superposition effect used to detect PDGF-BB.

[0051] Figure 2 It is a cyclic voltammogram (CV) of the sensor detecting 0.5 μg / mL PDGF-BB and blank samples.

[0052] Figure 3 This is a chronoamperometry (It) curve of the sensor detecting 0.5 μg / mL PDGF-BB and a blank sample.

[0053] Figure 4 It is an electrochemical cyclic voltammogram constructed by the sensor at different stages.

[0054] Figure 5 These are AC impedance diagrams from different stages of sensor construction.

[0055] Figure 6 This is a polyacrylamide gel electrophoresis image of a poly A-DNA tetrahedron.

[0056] Figure 7 These are experimental results from optimizing the number of bases in the capture probe.

[0057] Figure 8 These are experimental results from the optimization of the capture probe type.

[0058] Figure 9 These are the experimental results of optimizing the number of poly A atoms in the poly A-DNA tetrahedral probe.

[0059] Figure 10 This is the result of optimizing the assembly concentration of poly A-DNA tetrahedral probes.

[0060] Figure 11 This is the result of optimizing the aptamer concentration.

[0061] Figure 12 This is the result of optimizing the incubation time.

[0062] Figure 13 This is a curve showing the fitting relationship between the electrochemical current signal and the concentration of high-concentration PDGF-BB.

[0063] Figure 14 This is a curve showing the fitting of the electrochemical current signal of low-concentration PDGF-BB to the logarithm of the concentration.

[0064] Figure 15 This is the result of a specificity verification experiment. Detailed Implementation

[0065] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0066] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0067] The DNA probe sequences required in the following examples are shown in Table 1.

[0068] Table 1

[0069]

[0070]

[0071] This section only uses single-chain A and single-chain B-poly A (poly A) 10 -B), single-chain C-poly A (poly A) 10 -C) and single-chain D-poly A (poly A) 10 Taking -D) as an example, the connection situation of the poly A-tetrahedron is described in detail.

[0072] The single-stranded A: target complementary sequence (CACACAGA) - poly T sequence (TTTTTTTTTT) - extension sequence A1 ( ACATTCCTAAGTCTGAA )-AC-Extended Sequence A2 -GA-Extended sequence A3 Specifically as follows:

[0073]

[0074] Single-stranded B-poly A: poly A sequence (AAAAAAAAAAAAAAAAAAAAAAA) - extended sequence C1 -CA-Extended sequence C2 -AG-Extended sequence C3 Specifically as follows:

[0075]

[0076] Single-stranded C-poly A: poly A sequence (AAAAAAAAAAAAAAAAAAAAAAA) - extended sequence C1 -AA-Extended sequence C2 -TC-Extended sequence C3 Specifically as follows:

[0077]

[0078] Single-stranded D-poly A: poly A sequence (AAAAAAAAAAAAAAAAAAAA) - extended sequence D1 ( TTCAGACTTAGGAATGT )-GC-Extended Sequence D1 -TT-Extended Sequence D1 Specifically as follows:

[0079]

[0080] The extended sequence A1 is complementary to the extended sequence D1; the extended sequence A2 is complementary to the extended sequence B2; the extended sequence A3 is complementary to the extended sequence C3; the extended sequence B1 is complementary to the extended sequence C1; the extended sequence B3 is complementary to the extended sequence D3; and the extended sequence C2 is complementary to the extended sequence D2, forming a polyA-tetrahedron. The aptamer DNA is triggered by PDGF-BB to transform into a stem-loop structure and stacks with the polyA-tetrahedron probe. Based on base stacking, the aptamer DNA stably hybridizes with the target complementary sequence of the polyA-tetrahedron probe immobilized on the gold electrode. The aptamer DNA and horseradish peroxidase bind via biotin-avidin. This catalyzes the generation of an electrochemical redox catalytic signal from the chromogenic substrate, resulting in signal output.

[0081] Example 1

[0082] 1. Sensor fabrication and testing

[0083] (1) Preparation of poly A tetrahedron

[0084] Single-stranded A, single-stranded B-poly A, single-stranded C-poly A, and single-stranded D-poly A (final concentration 10 μM) were dispersed in TM buffer. The resulting mixture was placed in a PCR instrument at 95°C for 10 minutes, then rapidly cooled to 4°C to form stable poly A tetrahedra. Finally, the poly A tetrahedra were stored at 4°C for later use.

[0085] (2) Assembly of poly A tetrahedra on the surface of gold electrodes

[0086] First, the gold electrode was physically polished on a cloth for 5 minutes using 0.05 μm alumina powder, followed by ultrasonication with anhydrous ethanol and Milli-Q water. Then, it was electrochemically cleaned with 0.5 M sulfuric acid, rinsed with Milli-Q water, and dried with nitrogen (N2) to remove any remaining water droplets. 5 μL of the prepared capture probe poly A tetrahedron was added and incubated overnight for later use.

[0087] (3) Electrode surface sealing

[0088] A self-assembled layer of capture probe / casein was constructed by using 1% casein for interface sealing to prevent subsequent non-specific adsorption.

[0089] (4) Incubation

[0090] The pre-treated PDGF-BB and aptamer DNA solution was then added dropwise to the sealed electrode interface and incubated at 37°C for 2 hours. Finally, the electrode was rinsed with rinsing buffer and dried with nitrogen.

[0091] (5) Mark

[0092] After incubation, the electrode was rinsed with PBS buffer, and then 5 μL of avidin-PolyHRP80 (diluted 1000 times) was added. The reaction was carried out at room temperature for 15 minutes.

[0093] (6) Detection

[0094] All electrochemical detections were performed on an electrochemical workstation (Autolab III type). A three-electrode system was used in the experiments, with a gold electrode (2 mm in diameter) as the working electrode, an Ag / AgCl electrode (saturated KCl) as the reference electrode, and a platinum wire electrode as the counter electrode for electrochemical detection. Cyclic voltammetry (CV) and chronoamperometry (IT) electrochemical detection and analysis were performed on TMB, a reaction substrate that catalyzes avidin-HRP enzyme.

[0095] The buffer solutions used in the experiment were as follows: washing buffer (10 mM Na₂HPO₄, 10 mM KH₂PO₄, 2.7 mM KCl, 137 mM NaCl, pH = 7.4), assembly buffer (pH 8.0, 20 mM Tris·HCl, 50 mM MgCl₂), and hybridization buffer (pH 7.4, 10 mM phosphate buffer, 1 mM NaCl, 20 mM MgCl₂). All solutions were prepared using Milli-Q ultrapure water (18 MΩ·cm resistance) from the Millipore system.

[0096] 2. Principle of using a poly A-DNA tetrahedral electrochemical aptamer sensor based on base superposition effect for the detection of PDGF-BB.

[0097] This embodiment designs a poly A-DNA electrochemical biosensor based on base stacking interactions to achieve simple and stable detection of platelet-derived factor PDGF-BB. The electrochemical biosensor's capture probe consists of four DNA strands, three of which are modified with polyadenine. Figure 1 This is a schematic diagram of a poly A-DNA tetrahedral electrochemical aptamer sensor based on the base stacking effect for detecting PDGF-BB. Unlike traditional thiol-modified DNA probes (SH-B, SH-C, and SH-D), the label-free poly A-tetrahedron is covalently bound to the gold electrode surface via adenine, requiring no chemical modification. The distance between the tetrahedra can be modulated by changing the number of tail A atoms on the electrode surface, thus forming a tightly ordered biosensing interface. Utilizing the base stacking effect, the biosensor is triggered to transform into a stem-loop structure and stack with the capture probe only in the presence of platelet-derived factor PDGF-BB. Due to the enhanced base stacking, the aptamer DNA can stably hybridize with the poly A-DNA tetrahedral capture probe immobilized on the gold electrode. The aptamer tail is designed to be biotin-modified, binding to avidin-polyHRP80, generating an electrochemical redox catalytic signal in the TMB-H2O2 substrate, producing a signal output. In the absence of the target protein, the complementary fragment is too short to promote effective annealing, so the aptamer DNA does not bind to the capture strand immobilized on the gold electrode, and no signal is generated.

[0098] 3. Electrochemical response of the sensor

[0099] (1) Cyclic voltammetry

[0100] The electrochemical behavior of polyadenine tetrahedral probes assembled in an electrochemical DNA biosensor was analyzed using cyclic voltammetry. Figure 2It is a cyclic voltammogram (CV) of the sensor detecting 0.5 μg / mL PDGF-BB and a blank sample, such as... Figure 2 As shown, the cyclic voltammetry results reveal two clear redox peaks, indicating good electron transfer between the TMB and the gold electrode. Steady-state current signals were collected using chronoamperometry. Figure 3 This is a chronoamperometry (It) curve of the sensor detecting 0.5 μg / mL PDGF-BB and a blank sample, as shown. Figure 3 As shown, the magnitude of this signal value depends on the concentration of the target protein. In this embodiment, platelet-derived growth factor PDGF-BB was selected as the research object to study the electrochemical response of the sensor. When 0.5 μg / mL of PDGF-BB was present in the sample (red), the binding of the aptamer to PDGF-BB increased the base stacking force, enabling stable hybridization with the poly A-DNA tetrahedral capture probe immobilized on the gold electrode, generating a signal output. In the chronoamperometry curve, a very strong current signal was collected, which was 20 times stronger than the blank (dashed line) under the same conditions.

[0101] (2) Electrochemical impedance spectroscopy (EIS)

[0102] Meanwhile, the fabrication of the sensor was characterized using electrochemical impedance spectroscopy (EIS). Figure 4 The sensor constructs electrochemical cyclic voltammograms at different stages, such as... Figure 4 As shown, the bare gold electrode exhibits a very small charge transfer resistance, indicating that the electrode surface is clean. Figure 5 It is the AC impedance diagram constructed by the sensor at different stages, such as Figure 5 (Black) As shown, when the poly A-DNA tetrahedral capture probe is modified onto the gold electrode, the increased electrostatic repulsion between the probe and ferricyanide / ferrous ferrocyanide anions leads to an increase in negative charge density, resulting in a significant increase in the electrochemical impedance signal value. Figure 5 As shown in blue, therefore, as casein is gradually added to the surface of the gold electrode, the negative charge density gradually increases, and the electrochemical impedance increases, as... Figure 5 (As shown in orange), when the target protein and aptamer are added, such as Figure 5 As shown in (green), the electrochemical impedance signal continues to gradually increase due to the gradual increase in negative charge density.

[0103] 4. Polyacrylamide gel electrophoresis to verify the formation of poly A-DNA tetrahedra.

[0104] The formation of the capture probe poly A-DNA tetrahedron is one of the key aspects of this experiment, and the formation of the poly A tetrahedron was verified by polyacrylamide gel electrophoresis. Figure 6This is a polyacrylamide gel electrophoresis image of poly A-DNA tetrahedra. As can be seen from the image, the A+B+C+D band has the lowest migration rate, proving that it has the largest molecular weight, that is, the poly A-DNA tetrahedra were successfully synthesized.

[0105] Example 2

[0106] Base number optimization of capture probe

[0107] Base stacking is key to this experiment. To investigate the detection mechanism of the biosensor, a series of control experiments were conducted to compare the detection performance of different types of capture probes on platelet-derived factor PDGF-BB. First, the chain lengths between capture probes and aptamer probes with different base numbers were designed as 4bp, 6bp, 8bp, and 10bp, respectively. Figure 7 The results of the optimization experiment on the base number of the capture probe show that the relatively short base numbers of 4 bp and 6 bp did not produce signal output even in the presence of protein, while the 10 bp capture probe could capture the aptamer and produce a signal output even without protein stacking. Compared to the above three capture strands, when the strand length between the capture probe and the aptamer probe is 8 base pairs, the DNA double helix is ​​unstable due to its short length and no signal output is produced. However, after the introduction of protein, a strong signal output is produced. This is because the aptamer is triggered to transform into a stem-loop structure and stack with the capture probe. Due to increased binding and reduced dissociation, hybridization is brought additional stability and efficiency. Therefore, it is demonstrated that the cumulative base pairing and stacking between adjacent bases stabilizes the hybridization between the capture probe and the aptamer probe, which is also the key to this experiment.

[0108] Example 3

[0109] Optimization of capture probe type

[0110] To verify the design advantages of the poly A-DNA tetrahedral capture probe, this embodiment also designed a biblock single-stranded DNA probe and a traditional thiol tetrahedral probe as capture probes for comparison, which were used to capture PDGF-BB. Figure 8The results of this study are from an experiment optimizing the type of capture probe. The results showed that the biblock DNA single-strand capture probe generated almost no signal. This is likely due to two factors: firstly, the single strand lacks a stable tetrahedral rigid structure, failing to adequately support PDGF-BB; and secondly, steric hindrance prevents the biotin modified at the aptamer tail from binding to avidin-PolyHRP80, resulting in no signal output. In contrast to the traditional, well-established thiol tetrahedron, the poly A-DNA tetrahedron of this invention, due to its unique structure, provides a relatively large vertical space between the probe and the interface, offering a recognition microenvironment. This not only achieves the desired signal value but also simplifies the operation and is more cost-effective. The experimental results demonstrate the feasibility of the poly A-DNA tetrahedron capture probe and its highest signal-to-noise ratio. It allows for better adjustment of the spatial arrangement of the assembly interface and fully proves that the base-stacking hybridization strategy employed by the sensor is feasible and advantageous.

[0111] Example 4

[0112] Optimization of sensor construction conditions

[0113] (1) Optimization of poly A length

[0114] The length of the a atom in the poly A-DNA tetrahedron is crucial in determining whether the capture probe can be immobilized on the gold electrode. In this embodiment, capture probes with different atom lengths were designed: 5 nt, 10 nt, 20 nt, and 30 nt. The experimental results for optimizing the number of poly A atoms in the poly A-DNA tetrahedron probe are shown below. Figure 9 As shown in the experimental results, the 5nt capture probe produced the lowest signal-to-noise ratio, which may be due to the short A base length, which is insufficient to effectively anchor the DNA tetrahedron to the electrode. When the A length increased to 10nt, the highest signal value and the highest signal-to-noise ratio were produced, indicating that poly A... 10 DNA tetrahedra not only anchor well to the electrode but also enable highly efficient detection. While 20nt and 30nt DNA tetrahedra can firmly immobilize DNA tetrahedra compared to 10nt tetrahedra, a gradual decrease in PDGF-BB signal and signal-to-noise ratio was observed. This is presumably due to the increased length of the a-molecules, leading to greater spacing between the DNA tetrahedra. Excessive a-molecules occupying the electrode surface reduce the number of DNA tetrahedra that can be immobilized. Therefore, 10nt poly A-DNA tetrahedra were chosen as the capture probe to construct the biosensor.

[0115] (2) Optimization of capture probe concentration, aptamer concentration and incubation time

[0116] After determining the poly A length, this embodiment further optimized a series of conditions, including the selection of capture probe concentration, aptamer concentration, and incubation time.

[0117] The concentration of the capture probe directly determines the assembly density on the electrode surface and affects subsequent hybridization experiments, making it a crucial condition. The optimized assembly concentration of the poly A-DNA tetrahedral probe is shown below. Figure 10 As shown, after multiple experiments, a concentration of 1 μM was finally selected for overnight assembly.

[0118] Subsequently, this embodiment further optimized the aptamer concentration and incubation time. The optimization results of the aptamer concentration are as follows: Figure 11 As shown, the optimization results of the incubation time are as follows: Figure 12 As shown, after multiple experiments, it was found that the highest signal-to-noise ratio was achieved when the aptamer concentration was 100 nM and the incubation time was 2 h. Therefore, all subsequent experiments were conducted under these conditions.

[0119] Example 5

[0120] Sensor analysis performance test

[0121] (1) Operating linearity and detection limit

[0122] Under optimal conditions, a series of concentration gradients of target proteins were detected, such as Figure 13-14 As shown, with the PDGF-BB concentration increasing from 10 pg / mL to 1 μg / mL (four orders of magnitude), the electrochemical current signal increased with the increase of the target protein concentration. This demonstrates that the constructed sensor has good sensitivity. When the PDGF-BB concentration is in the range of 0.1 μg / mL to 1 μg / mL, the electrochemical current signal increases accordingly. Figure 13 As shown, the electrochemical current signal and PDGF-BB concentration exhibit a good linear relationship. The linear regression equation is: Y = 1874.65X + 16.85, with a linear correlation coefficient r of 0.997, where X is the PDGF-BB concentration and Y is the electrochemical current signal. When the PDGF-BB concentration is in the range of 10 pg / mL to 0.1 μg / mL, such as... Figure 14 As shown, the electrochemical current signal exhibits a good linear relationship with the logarithm of the target protein concentration. The linear regression equation is: Y = 27.97X + 205.4, with a linear correlation coefficient r of 0.996, where x is the logarithm of the PDGF-BB concentration and y is the electrochemical current signal. When y = S0 + 3δ (S0 = 48 nA is the background signal, and δ = 1 nA is the standard deviation of S0), the detection limit is calculated to be 3 pg / mL. The results indicate that this sensor can be used for sensitive quantitative detection of PDGF-BB.

[0123] (2) Specificity verification

[0124] To evaluate the specificity of this sensor, PDGF-AB, PDGF-AA, bovine serum albumin (BSA), and thrombin were used as negative controls to verify the sensor's specificity. The results of the specificity verification experiment are shown in the figure below. Figure 15 As shown, the protein concentrations used in the experiment were: PDGF-BB (0.5 μg / mL), PDGF-AB (0.5 μg / mL), PDGF-AA (5 μg / mL), thrombin (5 μg / mL), and BSA (5 μg / mL). The results showed that even when using PDGF-AA at ten times the concentration of PDGF-BB, bovine serum albumin (BSA) and thrombin, the generated electrochemical current signals were still very small, almost blank. This indicates that the aptamer has no specific interaction with other proteins and cannot produce a base-stacking hybridization reaction. Furthermore, although PDGF-AB shares the same target as PDGF-BB, at the same concentration, the aptamer's capture efficiency for PDGF-AB is much lower than that of PDGF-BB, resulting in a very small electrochemical signal, almost background, indicating that the biosensor cannot effectively capture PDGF-AB. The results demonstrate that the constructed biosensor has good specificity and can specifically detect PDGF-BB.

[0125] Example 6

[0126] Simulated biological sample analysis

[0127] To further evaluate the practicality of the sensor, this embodiment added different concentrations of PDGF-BB to human serum for spiked recovery experiments. All measurements were performed three times, and the results are shown in Table 2. At the three spiked levels (high, medium, and low), the spiked recovery rate of PDGF-BB was 96%-112.8%. The experimental results show that the constructed sensor has good accuracy and precision and can meet the requirements of complex biological sample analysis.

[0128] Table 2

[0129]

[0130] In summary, this invention designs a base-stack-dependent poly A-DNA tetrahedral probe electrochemical sensing platform for the detection of PDGF-BB. The capture probe in this biosensor is designed as a poly A-DNA tetrahedron. Polyadenine has a similar affinity to Au-S, enabling the tetrahedron to be immobilized on a gold electrode without chemical modification. Furthermore, utilizing the base stacking effect, in the presence of the target protein, the aptamer is triggered to transform into a stem-loop structure and stacks with the capture probe. Due to the enhanced base stacking, the aptamer DNA can stably hybridize with the poly A-DNA tetrahedral capture probe immobilized on the gold electrode, generating a signal output. The sensor can detect proteins down to 3.0 pg / mL, with a linear dynamic range of four orders of magnitude. Moreover, it performs well in real serum sample assays.

[0131] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An electrochemical sensor for detecting PDGF-BB using a poly A-DNA tetrahedral probe, characterized in that, The electrochemical sensor is a base stacking-dependent electrochemical sensor, comprising a poly A-tetrahedral probe, aptamer DNA, horseradish peroxidase, and chromogenic substrate self-assembled on a gold electrode. The poly A-tetrahedral probe is formed by the self-assembly of four single-stranded DNA strands onto a gold electrode to obtain a tetrahedral structure; the tail of the aptamer DNA is modified with biotin, and the horseradish peroxidase is modified with avidin. The electrochemical sensor is used to detect PDGF-BB. The aptamer DNA is triggered by PDGF-BB to transform into a stem-loop structure and stacks with a poly A-tetrahedral probe. Based on the base stacking effect, the aptamer DNA stably hybridizes with the target complementary sequence of the poly A-tetrahedral probe immobilized on the gold electrode. The aptamer DNA and horseradish peroxidase bind through biotin-avidin. The catalytic reaction of the chromogenic substrate generates an electrochemical redox catalytic signal, producing a signal output. The poly A-tetrahedral probe is obtained by self-assembling single-chain A, single-chain B-poly A, single-chain C-poly A and single-chain D-poly A on a gold electrode to obtain a tetrahedral structure. The single-stranded A consists of a target complementary sequence, a poly T sequence, and an extension sequence; The single-stranded B-poly A, single-stranded C-poly A, and single-stranded D-poly A are respectively composed of a poly A sequence and an extended sequence; The poly A sequence has 10-20 bases; The target complementary sequence is complementary to the aptamer DNA; The number of bases in the target complementary sequence that is complementary to the aptamer DNA is 8. The sequence of the aptamer DNA is shown in SEQ ID NO:18, wherein the sequence of the specific region that hybridizes with the target complementary sequence is biotin-TCTGTGTG. The target complementary sequence is CACACAGA.

2. The poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB according to claim 1, characterized in that, The single-stranded A consists of a target complementary sequence, a poly T sequence, and an extension sequence A, wherein the extension sequence A consists of extension sequences A1, A2, and A3.

3. The poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB according to claim 2, characterized in that, The extended sequences A1 and A2 are connected by 1-3 bases, and the extended sequences A2 and A3 are connected by 1-3 bases.

4. The poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB according to claim 1, characterized in that, The single-chain B-poly A consists of a poly A sequence and an extended sequence B, wherein the extended sequence B consists of extended sequences B1, B2 and B3.

5. The poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB according to claim 4, characterized in that, The extended sequences B1 and B2 are connected by 1-3 bases, and the extended sequences B2 and B3 are connected by 1-3 bases.

6. The poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB according to claim 1, characterized in that, The single-stranded C-poly A consists of a poly A sequence and an extended sequence C, wherein the extended sequence C consists of extended sequences C1, C2 and C3.

7. The poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB according to claim 6, characterized in that, The extended sequences C1 and C2 are connected by 1-3 bases, and the extended sequences C2 and C3 are connected by 1-3 bases.

8. The poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB according to claim 1, characterized in that, The single-chain D-poly A consists of a poly A sequence and an extended sequence D, wherein the extended sequence D consists of extended sequences D1, D2 and D3.

9. The poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB according to claim 8, characterized in that, The extended sequences D1 and D2 are connected by 1-3 bases, and the extended sequences D2 and D3 are connected by 1-3 bases.

10. The poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB according to claim 9, characterized in that, The extended sequence A1 is complementary to the extended sequence D1; the extended sequence A2 is complementary to the extended sequence B2; the extended sequence A3 is complementary to the extended sequence C3; the extended sequence B1 is complementary to the extended sequence C1; the extended sequence B3 is complementary to the extended sequence D3; and the extended sequence C2 is complementary to the extended sequence D2.

11. A method for detecting PDGF-BB based on a polyA-DNA tetrahedral probe electrochemical sensor, characterized in that, The method includes: Construct a poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB as described in any one of claims 1-10, establish a linear regression equation between the electrochemical current signal and the concentration of PDGF-BB, and indicate the concentration of PDGF-BB in the test sample by detecting the electrochemical current signal of the test sample.

12. The method for detecting PDGF-BB based on a poly A-DNA tetrahedral probe electrochemical sensor according to claim 11, characterized in that, The method includes the following steps: (1) Preparation of poly A-tetrahedron A buffer solution containing single-chain A, single-chain B-poly A, single-chain C-poly A and single-chain D-poly A was heated and kept at a constant temperature, and then cooled to obtain poly A-tetrahedron; (2) Construction of electrochemical biosensors Poly A-tetrahedrons self-assemble on the surface of the gold electrode, and the surface of the gold electrode is sealed with a sealing agent. (3) Electrochemical detection The electrochemical biosensor prepared in step (2) is co-incubated with the sample to be tested and aptamer DNA. Based on the base stacking effect, the aptamer DNA and the target complementary sequence of the polyA-tetrahedral probe fixed on the gold electrode are stably hybridized. The aptamer DNA and horseradish peroxidase are bound through biotin-avidin. The catalytic chromogenic substrate generates an electrochemical redox catalytic signal, and a signal output is generated.

13. The method for detecting PDGF-BB based on a poly A-DNA tetrahedral probe electrochemical sensor according to claim 12, characterized in that, In step (1), the concentrations of single-chain A, single-chain B-poly A, single-chain C-poly A and single-chain D-poly A in the buffer solution are 5-15 μM.

14. The method for detecting PDGF-BB based on a poly A-DNA tetrahedral probe electrochemical sensor according to claim 12, characterized in that, In step (1), the buffer solution is a TE buffer solution.

15. The method for detecting PDGF-BB based on a poly A-DNA tetrahedral probe electrochemical sensor according to claim 12, characterized in that, In step (1), the heating temperature is 90-95℃.

16. The method for detecting PDGF-BB based on a poly A-DNA tetrahedral probe electrochemical sensor according to claim 12, characterized in that, In step (1), the heat preservation time is 8-12 minutes.

17. The method for detecting PDGF-BB based on a poly A-DNA tetrahedral probe electrochemical sensor according to claim 12, characterized in that, In step (2), the sealing agent is a 0.8-1.2% casein solution.

18. The method for detecting PDGF-BB based on a poly A-DNA tetrahedral probe electrochemical sensor according to claim 12, characterized in that, In step (3), the concentration of the aptamer DNA is 50-100 nM.

19. The method for detecting PDGF-BB based on a poly A-DNA tetrahedral probe electrochemical sensor according to claim 12, characterized in that, In step (3), the co-incubation temperature is 25-37℃.

20. The method for detecting PDGF-BB based on a poly A-DNA tetrahedral probe electrochemical sensor according to claim 12, characterized in that, In step (3), the co-incubation time is 2-3 hours.

21. A kit for detecting PDGF-BB based on an electrochemical biosensor, characterized in that, The kit includes the poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB according to any one of claims 1-10.

22. The use of the poly A-DNA tetrahedral probe electrochemical sensor for detecting PDGF-BB according to any one of claims 1-10 in the preparation of products for detecting PDGF-BB.

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