Electrochemical aptamer sensor based on single-molecule scale proximity binding induced hybridization of tri-block probe and its application

Abstract

The application provides an electrochemical aptamer sensor based on single-molecule scale proximity binding of a tri-block probe to induce hybridization and an application thereof, the electrochemical aptamer sensor comprising a tri-block polyadenine DNA probe self-assembled on a gold electrode, a target protein proximity probe and a biotin-labeled antibody; the tri-block polyadenine DNA probe comprises a polyA sequence and flanking sequences connected on both sides of the polyA sequence, comprising a first flanking sequence and a second flanking sequence; the target protein proximity probe comprises a target protein proximity probe A and a target protein proximity probe B; and the biotin-labeled antibody is an antibody for recognizing the target protein. The sensor has extremely high sensitivity and specificity, and the concentration of the target protein is indicated by detecting the change of a catalytic oxidation-reduction current.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular detection technology, specifically relating to an electrochemical aptamer sensor based on single-molecule-scale proximity binding induced hybridization of a triblock probe and its application. Background Technology

[0002] The concept of proximity analysis was first proposed by Fredriksson et al. in an article published in *Nature Biotechnology*. It allows for the in vitro analysis of proteins and other macromolecules. The principle involves two affinity ligands simultaneously binding to the same target molecule. The two ligands are spatially brought closer, resulting in a sharp increase in the local concentration of the resulting pair of neighboring probes, thus promoting subsequent reactions. The requirement for simultaneous binding of two affinity ligands is what gives this method its high specificity. Scientists have combined proximity analysis with PCR signal amplification detection methods to develop a series of sensors with extremely high sensitivity and specificity, which are widely used in the detection of cytokines, tumor markers, enzymes, and pathogens.

[0003] Electrochemical biosensors are a class of sensors that use electrodes as signal transducers within a system, biological active units (e.g., antigens, antibodies, enzymes, microorganisms, or whole cells) as analytes, and current, conductance, or potential changes as characteristic detection signals. They utilize the principles of chemical reactions to convert the concentration and composition of organic or inorganic substances into electrical signals. Electrochemical biosensors possess advantages such as high sensitivity, good specificity, and ease of operation, and have received widespread attention and research in areas such as pathogenic bacteria, heavy metals, and pesticide / veterinary drug residues. They also demonstrate great application potential in food safety testing, medicine, and biology. However, the existing types of electrochemical biosensors are limited; therefore, developing a novel electrochemical biosensor for protein detection has significant application value. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an electrochemical aptamer sensor based on single-molecule-scale proximity binding induced hybridization using a triblock probe and its applications. The sensor described in this invention features a high signal-to-noise ratio, accurately detects proteins at both low and high concentrations, has wide applications, a simple preparation method, and significant application value.

[0005] To achieve this objective, the present invention employs the following technical solution:

[0006] In a first aspect, the present invention provides an electrochemical aptamer sensor based on single-molecule-scale proximity binding induced hybridization of a triblock probe, the electrochemical aptamer sensor comprising a triblock polyadenine DNA probe self-assembled on a gold electrode, a target protein proximity probe, and a biotin-labeled antibody;

[0007] The triblock polyadenine DNA probe includes a polyA sequence and flanking sequences connected to both sides of the polyA sequence, including a first flanking sequence and a second flanking sequence.

[0008] The target protein proximity probe includes target protein proximity probe A and target protein proximity probe B;

[0009] The biotin-labeled antibody is an antibody that recognizes the target protein.

[0010] This invention designs a triblock polyadenine DNA probe (triblock polyA DNA probe): without special chemical modification, the central polyA sequence covalently binds to gold, enabling the capture probe to assemble compactly and orderly on the gold electrode surface. The flanking sequences at both ends are connected by the same polyA sequence, forming a monomolecular 1:1 ratio probe electrochemical self-assembly interface (SAM) with controllable spacing. Employing a proximity-induced hybridization strategy, the flanking sequences at both ends are designed as short-chain probes. The tail portions of the target protein's neighboring probes (Aptamer A and Aptamer B) are designed with complementary extension sequences to the flanking sequences of the triblock polyA DNA probe. Furthermore, the melting temperature of a single extension sequence with a single flanking sequence of the polyA DNA probe is relatively low. Only when the target protein is present do Aptamer A and Aptamer B bind simultaneously to the target protein. The proximity effect promotes the hybridization of the tail extension sequences of Aptamer A and Aptamer B with the short-chain probes at both ends, forming a stable composite structure. Biotinylated antibodies targeting the target protein can specifically bind to the target protein and capture horseradish peroxidase onto the gold electrode surface through the strong binding force between biotin and streptomycin. Horseradish peroxidase reacts with the substrate to generate an electrochemical signal, and the concentration of the target protein is indicated by detecting changes in the catalytic redox current.

[0011] Preferably, the flanking sequence consists of a flanking connecting sequence and a flanking complementary sequence.

[0012] Preferably, the flanking connection sequence is (T)n, where n is an integer selected from 8 to 12, for example, it can be 8, 9, 10, 11 or 12.

[0013] Preferably, the number of bases in the flanking complementary sequence is 6-10, for example, 6, 7, 8, 9 or 10.

[0014] Preferably, the target protein proximity probe consists of an extended linker sequence, a protein recognition sequence, and an extended complementary sequence.

[0015] Preferably, the extended connection sequence is (T)n, where n is an integer selected from 35-45, such as 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45, etc.

[0016] Preferably, the extended complementary sequence has 6-10 bases, for example, 6, 7, 8, 9 or 10.

[0017] Preferably, the first flanking sequence consists of a flanking linker sequence and a flanking complementary sequence 1; the target protein proximity probe A consists of an extension linker sequence, a protein recognition sequence A, and an extension complementary sequence 1; wherein, the flanking complementary sequence 1 is complementary to the extension complementary sequence 1 of the target protein proximity probe A.

[0018] Preferably, the second flanking sequence consists of a flanking linker sequence and a flanking complementary sequence 2; the target protein proximity probe B consists of an extension linker sequence, a protein recognition sequence B, and an extension complementary sequence 2; wherein the flanking complementary sequence 2 is complementary to the extension complementary sequence 2 of the target protein proximity probe B.

[0019] Preferably, the polyA sequence has 8-40 bases, more preferably 10-20.

[0020] Preferably, the electrochemical aptamer sensor further includes streptomycin-modified horseradish peroxidase.

[0021] Secondly, the present invention provides a detection method based on single-molecule-scale proximity binding induced hybridization using a triblock probe, the detection method comprising:

[0022] Construct the electrochemical aptamer sensor based on single-molecule-scale proximity binding induced hybridization of the triblock probe as described in the first aspect; establish a linear regression equation between the electrochemical current signal and the concentration of the target protein; and indicate the concentration of the target protein in the test sample by detecting the electrochemical current signal of the test sample.

[0023] Preferably, in step (1), the sensor is constructed using a method comprising the following steps:

[0024] (1) Assembly of triblock polyA capture probe on gold electrode surface: The gold electrode, which has been physically polished, is electrochemically cleaned, and the triblock polyA capture probe solution is dropped onto the gold electrode surface for isothermal reaction assembly.

[0025] (2) Gold electrode surface sealing: The assembled gold electrode was sealed with a sealing reagent;

[0026] (3) Mix the target protein, the adjacent probe, and the test solution and add them to the sealed gold electrode and incubate at a constant temperature; then add biotin-labeled antibody and streptomycin-modified horseradish peroxidase in sequence.

[0027] Preferably, in step (1), the electrochemical cleaning is carried out in a sulfuric acid solution with a concentration of 0.02-1 μM, such as 0.02 μM, 0.08 μM, 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM or 1 μM.

[0028] Preferably, in step (1), the concentration of the triblock polyA capture probe in the triblock polyA capture probe solution is 0.01-3 μM, for example, it can be 0.01 μM, 0.1 μM, 0.5 μM, 0.8 μM, 1 μM, 1.5 μM, 2 μM or 3 μM, etc., and the solvent is TE assembly buffer with pH 6.8-7.2, for example, it can be 6.8, 7.0 or 7.2.

[0029] Preferably, in step (1), the reaction assembly time is 8-12 hours, for example, 8, 10 or 12 hours; the reaction assembly temperature is 20-25°C, for example, 20°C, 22°C or 25°C.

[0030] Preferably, in step (2), the blocking reagent is a casein solution with a mass concentration of 0.5-1.5%, such as 0.5%, 1%, or 1.5%; the solvent of the casein solution is PBS buffer with a pH of 7.0-7.4, such as 7.0, 7.2, or 7.4.

[0031] Preferably, in step (2), the sealing treatment time is 25-35 minutes, for example, 25, 30 or 35 minutes; the sealing treatment temperature is 20-25°C, for example, 20°C, 22°C or 25°C.

[0032] Preferably, in step (3), the target protein adjacent probe is placed in PBS buffer and denatured at 89-95℃ (e.g., 89℃, 90℃, 91℃, 92℃, 93℃, 94℃ or 95℃, etc.) for 4-5 minutes (e.g., 4 or 5 minutes, etc.) and then cooled to 20-25℃ (e.g., 20℃, 22℃ or 25℃, etc.).

[0033] Preferably, in step (3), when the target protein proximity probe is incubated with the test solution, the final concentration of the target protein proximity probe is 20-80 nM, for example, it can be 20 nM, 50 nM or 80 nM.

[0034] Preferably, in step (3), the temperature of the constant temperature incubation is 35-39°C, for example, 35°C, 36°C, 37°C, 38°C or 39°C; the time of the constant temperature incubation is 1.5-2.5 hours, for example, 1.5, 2 or 2.5 hours.

[0035] Preferably, in step (3), the concentration of the biotin-labeled antibody is 0.5-2 μg / mL, for example, it can be 0.5 μg / mL, 1 μg / mL or 2 μg / mL.

[0036] Preferably, in step (3), after adding the biotin-labeled antibody, the reaction is carried out at 20-25°C (e.g., 20°C, 22°C, or 25°C) for 0.5-1.5 hours (e.g., 0.5, 1, or 1.5 hours); after adding the streptomycin-modified horseradish peroxidase, the reaction is carried out at 20-25°C (e.g., 20°C, 22°C, or 25°C) for 10-15 minutes (e.g., 10, 13, or 15 minutes).

[0037] Thirdly, the present invention provides a detection kit based on single-molecule-scale proximity binding induced hybridization of a triblock probe, wherein the detection kit includes the electrochemical aptamer sensor based on single-molecule-scale proximity binding induced hybridization of a triblock probe as described in the first aspect.

[0038] Fourthly, the present invention provides the application of the electrochemical aptamer sensor based on single-molecule-scale proximity binding induced hybridization of the triblock probe described in the first aspect and / or the detection kit based on single-molecule-scale proximity binding induced hybridization of the triblock probe described in the third aspect in protein quantification.

[0039] 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.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] Adding a polyA block to the middle of a triblock polyadenine DNA probe plays an important role in the measurement of proximity binding-induced hybridization.

[0042] (1) This invention can avoid the common problem of difficult spacing control of traditional end-modified DNA probes. By adjusting the number of polyA bases, the spacing of adjacent reactions can be controlled, ensuring high sensitivity detection of the target PDGF-BB.

[0043] (2) The two flanking probes are connected by polyA blocks, which can maintain a strict 1:1 relationship at the molecular level, which is conducive to the generation of hybridization reaction induced by adjacent binding.

[0044] (3) This invention has high specificity. Although the homologous PDGF-AB and PDGF-BB have the same target, since this invention cannot effectively capture PDGF-AB through a nearby probe, it can effectively distinguish the homologous PDGF-AB and PDGF-BB. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of an electrochemical aptamer sensor based on single-molecule-scale close-range induced hybridization using a triblock probe.

[0046] Figure 2 It is a cyclic voltammogram (CV) of the sensor detecting 0.5 μg / mL PDGF-BB and blank samples.

[0047] Figure 3 This is a chronoamperometry (It) curve of the sensor detecting 0.5 μg / mL PDGF-BB and a blank sample.

[0048] Figure 4 It is an electrochemical impedance spectroscopy diagram of electrodes at different stages of sensor construction.

[0049] Figure 5 This is an SPR characterization diagram of the binding status of the triblock polyA DNA probe, the neighboring probe, PDGF-BB, and the biotin-modified PDGF-BB antibody during sensor detection.

[0050] Figure 6 This is an experimental diagram for studying the sensor detection mechanism.

[0051] Figure 7 This is a selection diagram of molecules that are closed at vacant sites on the electrode surface.

[0052] Figure 8 It is an optimization of the number of polyA molecules in a triblock polyA DNA probe.

[0053] Figure 9 It is an optimization of the assembly concentration of triblock polyA DNA probes.

[0054] Figure 10 It optimizes the use of probe concentrations near the target protein during the detection process.

[0055] Figure 11 It optimizes the incubation time of the sensor with the target protein proximity probe A, the target protein proximity probe B, and PDGF-BB.

[0056] Figure 12 It is about optimizing the concentration of biotin-modified PDGF-B antibody.

[0057] Figure 13 The incubation time for biotin-modified PDGF-BB antibodies has been optimized.

[0058] Figure 14 This is a graph showing the relationship between the electrochemical current signal and the concentration of PDGF-BB at different concentrations.

[0059] Figure 15 This is a curve showing the fitting relationship between the electrochemical current signal and the concentration of high-concentration PDGF-BB.

[0060] Figure 16 This is a fitted curve of the electrochemical current signal of low-concentration PDGF-BB versus the logarithm of the concentration.

[0061] Figure 17 This is a diagram from a specific experiment. Detailed Implementation

[0062] 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.

[0063] 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.

[0064] Example 1

[0065] The DNA probe sequences in this embodiment are shown in Table 1.

[0066] Table 1

[0067]

[0068] SEQ ID NO:1:

[0069] CACACAGATTTTTTTTTTAAAAAAAAAATTTTTTTTTTAGACACAC;

[0070] SEQ ID NO:2:

[0071] CACACAGATTTTTTTTTAAAAAAAAAAAAAAAAAAAATTTTTTTTTT AGACACAC;

[0072] SEQ ID NO:3:

[0073] CACACAGATTTTTTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAA AATTTTTTTTTTAGACACAC;

[0074] SEQ ID NO: 4:

[0075] CACACAGATTTTTTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTTTTTTTTTAGACACAC;

[0076] SEQ ID NO: 5:

[0077] TCTGTGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAC TCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTA;

[0078] SEQ ID NO: 6:

[0079] TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTATTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTGTGTCT;

[0080] SEQ ID NO: 7:

[0081] TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTATTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTGTGTCT;

[0082] SEQ ID NO: 8:

[0083] CACACAGATTTTTTTTTTAAAAAAAAAAAAAAAAAAAA;

[0084] SEQ ID NO: 9:

[0085] AAAAAAAAAAAAAAAAAAAATTTTTTTTTTAGACACAC.

[0086] Construction of Sensor

[0087] (1) Assembly of Triblock polyA Capture Probe on Gold Disk Electrode Surface

[0088] First, the gold electrode was polished with alumina powder, then electrochemically cleaned in water and sulfuric acid (1 μM), and finally rinsed thoroughly with Milli-Q water. Any remaining water droplets were then dried with nitrogen (N2). Next, 5 μL of a 1.0 μM triblock polyA capture probe (prepared using TE assembly buffer: pH 7.0, 10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl) was added to the cleaned gold electrode surface and assembled overnight at 25°C.

[0089] (2) Blocking of gold electrode surface: 1% casein (prepared with PBS buffer, PBS buffer: pH 7.4, 10mM PB, 140mM, 5mM KCl, 1mM MgCl2, 1mM CaCl2) was used to block the vacancies on the gold electrode surface at room temperature for 30 minutes.

[0090] Electrochemical detection

[0091] (1) Pretreatment of neighboring probes: Neighboring probe A and neighboring probe B (both 250 nM) were denatured in PBS buffer (pH 7.4, 10 mM PB, 140 mM, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2) at 90 °C for 5 minutes, and then cooled to room temperature for 20 minutes.

[0092] (2) Incubation: Add a certain amount of PDGF-BB protein to the adjacent probe solution pretreated in step (1), and then drop 5 μL of the above solution onto the prepared sensor. Incubate at 37°C for 2 hours to capture the target PDGF-BB protein.

[0093] (3) Enzyme-labeled specific antibody reaction: Add 5 μL of Anti-Bio-PDGF-B antibody solution of a certain concentration and react at room temperature for 1 hour.

[0094] (4) Labeling: Add 3 μL of Avidin-HRP diluted 1000 times and react at room temperature for 15 minutes.

[0095] (5) Detection: All electrochemical detections were performed on an electrochemical workstation (Autolab III type) using a three-electrode system (gold disk electrode as working electrode, platinum wire as counter electrode, and Ag / AgCl as reference electrode). Cyclic voltammetry (CV) and chronoamperometry (IT) were used for electrochemical detection and analysis on TMB, a reaction substrate that can catalyze Avidin-HRP enzyme.

[0096] Results and Discussion

[0097] (1) The principle of electrochemical aptamer sensors based on single-molecule-scale close-range induced hybridization of triblock probes is as follows: Figure 1 As shown.

[0098] This invention designs a triblock polyadenine DNA probe (triblock polyA DNA probe): without special chemical modification, the central polyA sequence covalently binds to gold, enabling the capture probe to assemble compactly and orderly on the gold electrode surface. The flanking sequences at both ends are connected by the same polyA sequence, forming a monomolecular 1:1 ratio probe electrochemical self-assembly interface (SAM) with controllable spacing. A proximity-induced hybridization strategy is employed, with the flanking sequences designed as short-chain probes. The tail portions of the target protein proximity probes (Aptamer A and Aptamer B) are designed as complementary extension sequences to the flanking sequences of the triblock polyA DNA probe. The melting temperature of a single extension sequence with a single flanking sequence of the polyA DNA probe is relatively low. Only when the target protein is present do the target protein proximity probes A and B bind simultaneously to the target protein. The proximity effect promotes the hybridization of the tail extension sequences of the target protein proximity probes A and B with the short-chain probes at both ends, forming a stable composite structure. Biotinylated antibodies targeting the target protein can specifically bind to the target protein and capture horseradish peroxidase onto the gold electrode surface through the strong binding force between biotin and streptomycin. Horseradish peroxidase reacts with the substrate to generate an electrochemical signal, and the concentration of the target protein is indicated by detecting changes in the catalytic redox current.

[0099] (2) Electrochemical response of the sensor

[0100] In this embodiment, platelet-derived growth factor PDGF-BB was selected as the research object. The electrochemical response of the sensor was studied, and the feasibility of using the sensor to detect PDGF-BB was evaluated.

[0101] The electrochemical response diagrams of the sensor detecting 0.5 μg / mL PDGF-BB and blank samples are shown below. Figure 2 and Figure 3 As shown, Figure 2 The cyclic voltammogram (CV) shows two clear redox couples, indicating good electron transfer between TMB and the gold electrode. Due to the electrocatalytic reaction of horseradish peroxidase, when 0.5 μg / mL of PDGF-BB is present in the sample (solid line), the target protein neighbor probes A and B simultaneously bind to PDGF-BB, forming neighbor probes. This promotes the hybridization of their tail extension sequences with the short-chain probes on both sides of the triblock polyA DNA probe assembled on the electrode surface, forming a stable complex structure. The reduction peak at 0.3 V (relative to the Ag / AgCl electrode) is significantly increased. Figure 3The current curve (It) shows a very strong current signal, which is 10 times stronger than the blank (dashed line) under the same conditions.

[0102] (3) Sensor characterization

[0103] Two methods were used to characterize the sensor:

[0104] (1) The condition of the gold electrode surface during the sensor construction process was characterized by AC impedance technology.

[0105] (2) The binding of the triblock polyA DNA probe, the neighboring probe, PDGF-BB and the biotin-modified PDGF-BB antibody during the sensor detection process was characterized by surface plasmon resonance (SPR) and other techniques.

[0106] Figure 4 Electrochemical impedance spectroscopy (EIS) maps of electrodes at different stages were constructed for the sensor in K3[Fe(CN)6] / K4[Fe(CN)6]. The bare gold electrode (curve a) showed a very small semicircular region, indicating a relatively low electron transfer resistance value. After assembling the triblock polyA DNA probe (curve b), due to the inherent affinity between the adenine (A) bases in the polyA block and the gold electrode surface, a dense SAM layer was formed on the gold electrode surface, hindering electron transfer and causing an increase in the electrochemical impedance signal. When the vacancies on the gold electrode surface were blocked with 1% casein at room temperature for 30 minutes (curve c), although the triblock polyA DNA probe on the electrode surface became ordered, which was conducive to electron transfer, the casein on the gold electrode surface greatly hindered electron transfer, resulting in an overall increase in the electrochemical impedance signal. When both target protein proximity probe A and target protein proximity probe B simultaneously recognize PDGF-BB and hybridize with the flanking ends of the triblock polyADNA probe on the gold electrode surface (curve d), the electrochemical impedance signal continues to increase significantly due to the introduction of PDGF-BB. After adding biotin-modified PDGF-BB antibody (curve e), the biotin-modified PDGF-BB antibody specifically binds to PDGF-BB on the gold electrode surface, further hindering electron transfer, and the electrochemical impedance signal continues to increase. After adding streptomycin-modified horseradish peroxidase (Avidin-HRP) (curve f), the strong binding force between biotin and streptomycin introduces horseradish peroxidase to the electrode surface. Due to the gradual increase in negative charge density, the electrochemical impedance signal continues to gradually increase, indicating that the sensor has been successfully prepared and can be used to detect target proteins.

[0107] Surface plasmon resonance (SPR) technology utilizes traditional optical phenomena, specifically the resonance between evanescent waves generated by light in different media and plasma waves, to construct a biosensor analysis technique for biomolecular interactions. By real-time monitoring of refractive index changes caused by the binding or dissociation of biomolecules on the chip surface, the entire process of biomolecular binding and dissociation can be tracked and detected in real time, making it an ideal tool for studying biomolecular interactions. We assembled a triblock polyA DNA probe onto an SPR bare gold chip and used 1% casein to block vacant sites to prepare an SPR sensor chip. We monitored the entire detection process of an electrochemical aptamer sensor based on single-molecule-scale close-range induced hybridization using triblock probes, such as... Figure 5 As shown, after injecting the target protein proximity probe and PDGF-BB mixed solution (solid line), the target protein proximity probe, PDGF-BB and the triblock polyA probe on the chip surface bind together, and the signal increases significantly compared to the sample with only the target protein proximity probe (dashed line). Especially after rinsing the SPR chip surface with PBS buffer to remove non-specific adsorption, the signal in the absence of PDGF-BB (dashed line) decreases to the initial signal, while the signal in the presence of PDGF-BB (solid line) decreases less. This effectively proves that the target protein proximity probe can only bind to the triblock polyA DNA probe assembled on the chip surface when PDGF-BB is present. When biotin-modified PDGF-BB antibody (Anti-Bio-PDGF-B) was injected, the signal of the presence of PDGF-BB (solid line) increased significantly. This is because Anti-Bio-PDGF-B specifically binds to PDGF-BB on the chip surface, causing the signal increase. After washing the SPR chip surface with PBS buffer to remove non-specific adsorption, the signal of the absence of PDGF-BB (dashed line) decreased to even smaller than the initial signal. This may be because the chip was washed three times with PBS, resulting in a more thorough surface wash and the removal of some loosely bound triblock polyADNA probes and casein. The signal of the presence of PDGF-BB (solid line) decreased less, and compared with the previous stage (after binding to PDGF-BB and adjacent probes of the target protein), the signal increased significantly, proving that Anti-Bio-PDGF-B can specifically bind to PDGF-BB on the chip surface. SPR plots effectively reflect the entire detection process of our constructed sensor, and monitor the interactions between the target protein PDGF-BB, the target protein neighboring probes, and the triblock polyA DNA probes in real time, which further proves the feasibility of the constructed sensor.

[0108] (4) Research on sensor detection mechanism

[0109] The proximity binding-induced hybridization strategy is key to this experiment. To explore the detection mechanism of our constructed sensor, we compared the signal-to-noise ratio of different types of capture probes for detecting a certain concentration of the target protein PDGF-BB and a blank sample. We also designed two biblock polyA-DNA capture probes (capture probe A and capture probe B in Table 1) to simulate the left and right wings of the triblock polyA-DNA capture probe, respectively, and compared them to the triblock polyA-DNA capture probe to produce the effect of proximity binding-induced hybridization. In the design of the triblock polyA-DNA capture probe, the target protein proximity probes A and B bind simultaneously to the target protein. The proximity effect promotes the hybridization of the tail extension sequences of target protein proximity probes A and B with the short-chain probes on both flanks, forming a stable complex structure. Figure 6 Column 1). In the design of biblock polyA-DNA capture probes (taking capture probe A in Table 1 as an example), the target protein's neighboring probe A binds to the target protein. The proximity effect promotes hybridization of the tail extension sequence of the target protein's neighboring probe A with the two adjacent biblock polyA-DNA capture probes, such as... Figure 6 As shown in columns 2 (capture probe A) and 3 (capture probe B), the S / N ratio of the biblock polyA-DNA capture probe was significantly reduced (P values ​​were all less than 0.05). A possible reason for this phenomenon is that the spacing between the biblock polyA-DNA capture probes is not as accurate and controllable as that of the triblock polyA-DNA capture probes. For comparison, this experiment also simultaneously assembled two biblock polyA-DNA capture probes (capture probe A and capture probe B in Table 1) onto the gold electrode surface, as shown... Figure 6 As shown in column 4, the signal-to-noise ratio of the detection results decreased significantly, which may be due to the uncontrollable assembly spacing between the two diblock polyA-DNA capture probes.

[0110] Meanwhile, to verify the proximity binding induced hybridization strategy, we also separately incubated target protein proximity probe A and target protein proximity probe B with PDGF-BB protein with the triblock polyA-DNA capture probe. The experimental results are as follows: Figure 6 As shown in column 5, the S / N ratio of the detection result drops sharply. This is because, under these conditions, the target protein neighbor probes A and B cannot hybridize with the triblock polyA-DNA capture probe. Target protein neighbor probes A and B, along with PDGF-BB protein, must be present simultaneously for them to induce proximity binding hybridization with the triblock polyA-DNA capture probe. Experimental results demonstrate that the triblock polyA-DNA capture probe has the highest signal-to-noise ratio and is more advantageous, allowing for better control of the spatial arrangement of the assembly interface. This also fully proves that the sensor operates using a proximity binding induced hybridization strategy.

[0111] Example 2

[0112] Optimization of sensor construction conditions

[0113] The key conditions for sensor construction are the vacant sites on the electrode surface, the number of polyA molecules in the triblock polyA DNA probe, and the assembly concentration.

[0114] This embodiment first optimizes the blocking molecules for vacant sites on the electrode surface. Commonly used blocking molecules in electrochemical biosensor construction are employed: mercaptohexanol (MCH), mercaptohexanol and bovine serum albumin (MCH+BSA), bovine serum albumin (BSA), and casein to block vacant sites on the gold electrode surface after assembly of the triblock polyA DNA probe. Figure 7 As shown, when MCH was used for blocking, the blank signal was high, and the signal for detecting 1 μg / mL PDGF-BB was also low, resulting in a very low signal-to-noise ratio (SNR). When MCH and BSA were used sequentially for blocking, the SNR improved slightly, but it still did not meet the requirements for low-concentration detection. When BSA was used for blocking, the signal for detecting 1 μg / mL PDGF-BB decreased slightly, and the blank signal also decreased, while the SNR improved significantly. This may be because BSA blocks the vacant sites on the gold electrode surface, which not only makes the two ends of the triblock polyA DNA probe stand upright, promoting hybridization between the two ends and the target protein's neighboring probes (target protein neighboring probe A and target protein neighboring probe B), but also reduces the non-specific adsorption of PDGF-BB and Anti-Bio-PDGF-B to the gold electrode. PDGF-BB can participate better in the proximity binding-induced hybridization reaction with the neighboring probes. When casein, with a smaller molecular weight, was used for blocking, the signal for detecting 1 μg / mL PDGF-BB increased slightly, and the blank signal decreased rapidly, resulting in a significantly improved SNR. This indicates that casein, with a smaller molecular weight, can achieve a better blocking effect than BSA. Therefore, a gold electrode assembled with a casein-blocked triblock polyA probe was chosen for the electrochemical biosensor to detect PDGF-BB protein.

[0115] In triblock polyA DNA probes, the length of the polyA segments controls the spacing between the two flanking segments, which is a crucial condition for proximity-induced hybridization. The sensor detection mechanism study indicates that only an appropriate spacing can promote proximity-induced hybridization. In our experiments, we compared the detection results of 1 μg / mL PDGF-BB and blank samples using triblock polyA DNA probes with different polyA lengths (10, 20, 30, and 40 bases). Figure 8As shown, the current signal for detecting 1 μg / mL PDGF-BB gradually decreased as the polyA fragment length increased from 10 to 40. The highest signal-to-noise ratio (S / N = 16.07) was achieved when the polyA sequence had 20 bases, reaching the highest conformational stability for ortho-binding induced hybridization at the electrochemical interface. When the polyA sequence had 30 and 40 bases, the electrochemical current signal for detecting 1 μg / mL PDGF-BB decreased sharply. This may be due to two reasons: firstly, the excessive number of polyA bases in the triblock polyA DNA probe occupied too much electrode surface, reducing the total amount of triblock polyA DNA probe assembled on the electrode surface; secondly, the excessively long flanking distance of the triblock polyA DNA probe with 30 and 40 bases was unfavorable for ortho-binding induced hybridization. Therefore, a triblock polyA DNA probe with a polyA length of 20 bases was selected as the capture probe for the sensor.

[0116] After determining that the polyA length in the triblock polyA DNA probe is 20 bases, the assembly concentration of the triblock polyA DNA probe (triblock polyA20 DNA probe) was further optimized. Figure 9 The concentration of the triblock polyA20 DNA probe directly determines the assembly density of the capture probe on the electrode surface. We assembled gold electrodes using triblock polyA20 DNA probe concentrations of 0.01 μM, 0.1 μM, 0.5 μM, 1 μM, and 3 μM, respectively. Multiple experiments showed that the 1 μM triblock polyA20 DNA probe exhibited the highest signal-to-noise ratio (S / N = 18.82).

[0117] Example 3

[0118] Optimization of sensor detection conditions

[0119] The key conditions for sensor construction were determined, and the key detection conditions affecting the sensor detection performance were optimized. During the detection process, the concentrations of the target protein proximity probes (target protein proximity probe A and target protein proximity probe B), the incubation time of the sensor with target protein proximity probe A, target protein proximity probe B and PDGF-BB, the concentration and incubation time of biotin-modified PDGF-B antibody were used.

[0120] First, the concentration of the probe near the target protein was optimized. Figure 10 ) and the incubation time of the sensor with the target protein's neighboring probe A, target protein's neighboring probe B, and PDGF-BB ( Figure 11 After multiple experiments, the optimal signal-to-noise ratio was finally achieved when 50 nM target protein proximity probe A and 50 nM target protein proximity probe B were selected and the incubation time was 2 h.

[0121] Secondly, the concentration of biotin-modified PDGF-B antibody was optimized, such as... Figure 12 As shown, although the signal of the biotin-modified PDGF-B antibody at a concentration of 2 μg / mL was twice that of the 1 μg / mL sample when detecting high-concentration PDGF-BB, the 2 μg / mL biotin-modified PDGF-B antibody could not effectively detect low-concentration PDGF-BB (S / N = 0.97), showing no difference from the background signal. The 1 μg / mL biotin-modified PDGF-B antibody, however, performed significantly better (S / N = 2.42). We speculate that the high concentration of the 2 μg / mL biotin-modified PDGF-BB antibody prevented complete removal by rinsing, resulting in a high background signal. Therefore, we ultimately used the 1 μg / mL biotin-modified PDGF-B antibody. Finally, we optimized the incubation time. The incubation times were 30 minutes, 1 hour, and 2 hours. After multiple experiments, a 1-hour incubation time was selected as the optimal condition. Figure 13 As shown.

[0122] Example 4

[0123] Sensor analytical performance

[0124] (1) Operating linearity and detection limit

[0125] Under optimal experimental conditions, we used our constructed sensor to detect a range of concentrations of the target protein PDGF-BB. For example... Figure 14 As shown, the electrochemical current signal gradually increased as the PDGF-BB concentration increased from 0.1 pg / mL to 1 μg / mL (eight orders of magnitude). When the PDGF-BB concentration was in the range of 0.1 μg / mL to 1 μg / mL, as... Figure 15 As shown, the electrochemical current signal and PDGF-BB concentration exhibit a good linear relationship. The linear regression equation is: Y = 995X + 34, with a linear correlation coefficient r of 0.994, where X is the PDGF-BB concentration and Y is the electrochemical current signal. When the PDGF-BB concentration is in the range of 0.1 pg / mL to 0.1 μg / mL, as... Figure 16 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 = 11.68x + 145.51, with a linear correlation coefficient r of 0.997, where x is the logarithm of the PDGF-BB concentration and y is the electrochemical current signal. When y = S0 + 3δ (S0 = 44 nA is the background signal, and δ = 1 nA is the standard deviation of S0), the detection limit is calculated to be 4 fg / mL. These results indicate that the sensor can be used for sensitive quantitative detection of PDGF-BB.

[0126] (2) Specificity

[0127] To evaluate the specificity of this sensor, the PDGF dimer isomers PDGF-AB and PDGF-AA, as well as bovine serum albumin (BSA) and thrombin, were used as negative controls. The results are as follows: Figure 17 As shown, even when the concentrations of PDGF-AA, BSA, and thrombin are ten times that of PDGF-BB, the resulting electrochemical current signals are very small, similar to the blank signal values. Although the homologous protein PDGF-AB has the same target site as PDGF-BB, at the same concentration, the electrochemical current signal is also very small, with no significant difference from the blank signal value. This indicates that the constructed sensor cannot effectively capture PDGF-AB using only one proximity probe, and that the proximity probe has no specific interaction with other proteins, failing to generate a proximity-induced hybridization reaction. This is attributed to the fact that simultaneous recognition of the target protein by two proximity probes is a prerequisite for generating a proximity-induced hybridization reaction. The results demonstrate that the established sensor has good specificity, providing the possibility for highly selective detection of target proteins.

[0128] Example 5

[0129] Simulated biological sample analysis

[0130] To further evaluate the practicality of the sensor, we 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 low, medium, and high spiked levels, the spiked recoveries of PDGF-BB ranged from 96.8% to 115.54%. The experimental results indicate that the constructed sensor has good accuracy and precision and can meet the requirements of complex biological sample analysis.

[0131] Table 2

[0132]

[0133] 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. A detection method based on single-molecule-scale proximity binding induced hybridization using a triblock probe, characterized in that, The detection method includes: An electrochemical aptamer sensor based on single-molecule-scale proximity binding induced hybridization using a triblock probe was constructed; a linear regression equation between the electrochemical current signal and the concentration of the target protein was established; and the concentration of the target protein in the test sample was indicated by detecting the electrochemical current signal of the test sample. The sensor is constructed using a method comprising the following steps: (1) Assembly of triblock polyA capture probe on gold electrode surface: The gold electrode, which has been physically polished, is electrochemically cleaned, and the triblock polyA capture probe solution is dropped onto the gold electrode surface for isothermal reaction assembly. (2) Sealing of gold electrode surface: The assembled gold electrode is sealed with a sealing reagent; (3) Mix the target protein and the adjacent probe and the test solution and add them to the blocked gold electrode and incubate at a constant temperature; then add biotin-labeled antibody and streptomycin-modified horseradish peroxidase in sequence; The electrochemical aptamer sensor includes a triblock polyadenine DNA probe, a target protein proximity probe, and a biotin-labeled antibody, all self-assembled on a gold electrode. The triblock polyadenine DNA probe includes a polyA sequence and flanking sequences connected to both sides of the polyA sequence, including a first flanking sequence and a second flanking sequence. The target protein proximity probe includes target protein proximity probe A and target protein proximity probe B; The biotin-labeled antibody is an antibody that recognizes the target protein; The flanking sequence consists of a flanking connecting sequence and a flanking complementary sequence; The flanking connection sequence is (T)n, where n is an integer selected from 8-12; The number of bases in the flanking complementary sequence is 6-10 bases; The target protein proximity probe consists of an extension linker sequence, a protein recognition sequence, and an extension complementary sequence. The extended connection sequence is (T)n, where n is an integer selected from 35-45; The extended complementary sequence has 6-10 bases. The first flanking sequence consists of a flanking linker sequence and a flanking complementary sequence 1; the target protein proximity probe A consists of an extension linker sequence, a protein recognition sequence A, and an extension complementary sequence 1; wherein, the flanking complementary sequence 1 is complementary to the extension complementary sequence 1 of the target protein proximity probe A. The second flanking sequence consists of a flanking linker sequence and a flanking complementary sequence 2; the target protein neighbor probe B consists of an extension linker sequence, a protein recognition sequence B, and an extension complementary sequence 2; wherein, the flanking complementary sequence 2 is complementary to the extension complementary sequence 2 of the target protein neighbor probe B. The polyA sequence has 10-20 bases.

2. The detection method according to claim 1, characterized in that, The electrochemical aptamer sensor also includes streptomycin-modified horseradish peroxidase.

3. The detection method according to claim 1, characterized in that, In step (1), the electrochemical cleaning is carried out in a sulfuric acid solution with a concentration of 0.02-1 μM.

4. The detection method according to claim 1, characterized in that, In step (1), the concentration of the triblock polyA capture probe in the triblock polyA capture probe solution is 0.01-3 μM, the solvent is TE assembly buffer, and the pH is 6.8-7.

2.

5. The detection method according to claim 1, characterized in that, In step (1), the reaction assembly time is 8-12 hours; the reaction assembly temperature is 20-25℃.

6. The detection method according to claim 1, characterized in that, In step (2), the blocking reagent is a casein solution with a mass concentration of 0.5-1.5%; the solvent of the casein solution is PBS buffer with a pH of 7.0-7.

4.

7. The detection method according to claim 1, characterized in that, In step (2), the sealing process takes 25-35 minutes and the sealing temperature is 20-25°C.

8. The detection method according to claim 1, characterized in that, In step (3), the target protein adjacent probe is placed in PBS buffer, denatured at 89-95℃ for 4-5 minutes and then cooled to 20-25℃.

9. The detection method according to claim 1, characterized in that, In step (3), when the target protein proximity probe is incubated with the test solution, the final concentration of the target protein proximity probe is 20-80 nM.

10. The detection method according to claim 1, characterized in that, In step (3), the temperature of the constant temperature incubation is 35-39℃; the time of the constant temperature incubation is 1.5-2.5 hours.

11. The detection method according to claim 1, characterized in that, In step (3), the concentration of the biotin-labeled antibody is 0.5-2 μg / mL.

12. The detection method according to claim 1, characterized in that, In step (3), after adding biotin-labeled antibody, the reaction is carried out at 20-25℃ for 0.5-1.5 hours; after adding streptomycin-modified horseradish peroxidase, the reaction is carried out at 20-25℃ for 10-15 minutes.

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