Multi-channel electrochemical aptamer sensor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of electrochemical sensing, in particular to a multi-channel electrochemical aptamer sensor and a preparation method and application thereof.The multi-channel electrochemical aptamer sensor adopts a commercial silk-screen printing electrode and is combined with a polymer substrate material for amplifying electrochemical signals, and the electrochemical aptamer sensor is prepared. According to the present invention, the corresponding aptamer is fixed by using the gold nanoparticle through the chemical bond so as to specifically detect the three hemorrhage conversion biomarkers after acute cerebral infarction, such as human matrix metalloproteinase (MMP-9), fibrinogen (FN) and plasminogen activation inhibitor (PAI-1); wherein the FN and the PAI-1 are detected through an electrochemical aptamer sensor method for the first time, and simultaneous detection of the three biomarkers is realized through integration with commercial multi-channel micro electrochemical equipment.

Description

Technical Field

[0001] This application relates to the field of electrochemical sensing technology, and in particular to a multichannel electrochemical aptamer sensor, its preparation method, and its application. Background Technology

[0002] Post-stroke hemorrhage (hemorrhagic transformation) is both a natural part of the disease's course and a common complication of blood flow-improving therapies such as thrombolysis and anticoagulation. It not only leads to poor patient prognosis but also results in insufficient clinical use of these effective treatments.

[0003] Currently, Western consensus only provides recommendations for managing symptomatic bleeding transformation after thrombolysis, failing to cover a wide range of clinical issues such as bleeding transformation from causes other than intravenous thrombolysis, spontaneous and asymptomatic bleeding transformation. Compared to Western populations, Asian populations have a higher risk of bleeding transformation after receiving antithrombotic and thrombolytic therapy, and there is a lack of unified standards both domestically and internationally regarding how to restart antithrombotic therapy after bleeding transformation. Therefore, a scientific understanding and appropriate management of bleeding transformation is crucial. Notably, the consensus of the Chinese Society of Neurology points out that biomarkers such as matrix metalloproteinase-9 (MMP-9), fibrinogen (FN), and plasminogen activator inhibitor-1 (PAI-1) may be closely related to the occurrence of bleeding transformation. This suggests that simultaneous and rapid detection of MMP-9, FN, and PAI-1 is of great value for early assessment of bleeding transformation risk and guiding the development of individualized treatment plans in clinical practice.

[0004] Currently, commonly used clinical methods for detecting MMP-9, FN, and PAI-1 biomarkers include enzyme-linked immunosorbent assay (ELISA), gelatin zymography, immunoturbidimetry, and activity assays. These methods detect only a single biomarker and lack high specificity or sensitivity. To avoid these problems, multiple biomarker analyses need to be performed on each sample. Currently, multi-biomarker analysis is typically performed in clinical research laboratories, relying on sophisticated experimental procedures and large-scale testing equipment. Commercial point-of-care testing equipment remains bulky, and only a limited number of protein multi-biomarker assays have been validated in point-of-care devices. Therefore, developing a rapid, simple, economical, highly sensitive, specific, and portable method for the early diagnosis, disease monitoring, and prognostic assessment of hemorrhagic transformation after acute ischemic stroke (AIS) is of significant clinical value and practical importance. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a multi-channel electrochemical aptamer sensor, its preparation method, and its application. The multi-channel electrochemical aptamer sensor provided in this application, as a portable integrated micro-electrochemical sensor, facilitates the rapid and simultaneous on-site detection of multiple related biomarkers after acute cerebral infarction hemorrhage, providing a new theoretical basis for the development of electrochemical aptamer sensors and for comprehensive personalized diagnosis and treatment of diseases through biomarkers.

[0006] Therefore, the following technical solutions are adopted in the embodiments of this application:

[0007] In a first aspect, this application provides a multi-channel electrochemical aptamer sensor, including a screen-printed electrode; the surface of the screen-printed electrode is modified with a polymer substrate material that amplifies electrochemical signals, the polymer substrate material is loaded with gold nanoparticles and a specific aptamer is fixed by chemical bonds; wherein the specific aptamer specifically recognizes three biomarkers: human matrix metalloproteinase, fibrinogen, and plasminogen activator inhibitor, and the multi-channel electrochemical aptamer sensor is integrated with a multi-channel electrochemical detection device.

[0008] As one possible implementation, the polymer substrate material is a composite material of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid), polyvinyl alcohol, and polypyrrole, PPy / DPH.

[0009] In one possible implementation, the gold nanoparticles are loaded onto the surface of the polymer substrate material by electrochemical deposition, and the specific aptamer is fixed to the surface of the gold nanoparticles by Au-S bonds; the non-specific binding sites on the surface of the screen-printed electrode are sealed with 1% BSA solution.

[0010] Secondly, embodiments of this application also provide a method for preparing the above-mentioned multichannel electrochemical aptamer sensor, comprising the following steps:

[0011] Step (1): Design and prepare polymer substrate materials to amplify electrochemical signals;

[0012] Step (2) involves modifying a specific aptamer on the polymer substrate material-modified electrode to prepare a multichannel electrochemical aptamer sensor.

[0013] As one possible implementation, the method includes:

[0014] (1) Preparation of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) (PEDOT:PSS) crosslinked polyvinyl alcohol (PVA): Take 0.05 g of 3% polyvinyl alcohol (PVA) aqueous solution melted at 95 °C, add 1 g of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) (PEDOT:PSS) solution, stir at 200 rpm / s for more than 6 h, and store at room temperature (24 °C) for later use;

[0015] (2) Preparation of polypyrrole (PPy) electrolyte: Mix 0.1M sodium carbonate and 0.1M p-toluenesulfonic acid in water until homogeneous, add 1% volume fraction of pyrrole monomer solution and mix until homogeneous, and store at 4℃ for later use;

[0016] (3) Preparation of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol (PEDOT:PSS / PVA) hydrogel (DPH) modified electrode: The mixed solution in step (1) was drop-coated onto the surface of the working electrode, dried at room temperature (24°C) and swollen to obtain a poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol (PEDOT:PSS / PVA) hydrogel (DPH) substrate modified electrode;

[0017] (4) Preparation of polypyrrole / poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol (PPy / DPH) modified electrode: using Ag / AgCl as the reference electrode, platinum wire as the counter electrode, and the poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol (PEDOT:PSS / PVA) hydrogel (DPH) modified electrode of step (3) as the working electrode, and using the polypyrrole (PPy) electrolyte of step (2) as the electrolyte, the electrode was deposited for 700s by time-amperometry at a potential of 1.2V to obtain the polypyrrole / poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol (PPy / DPH) modified electrode;

[0018] (5) Preparation of gold / polypyrrole / poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol (Au / PPy / DPH) modified electrode: with 0.1 mM gold ions Au + The solution is an electrolyte, and the electrode in step (4) is deposited by chronoamperometry at a potential of -0.2V to obtain a gold / polypyrrole / poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol (Au / PPy / DPH) modified electrode;

[0019] (6) Preparation of specific detection electrode: Incubate the electrode from step (5) with the corresponding specific aptamer at 4°C;

[0020] (7) Blocking treatment: The electrode from step (6) is incubated with 1% bovine serum albumin (BSA) solution at 4°C to block non-specific binding sites and obtain the sensor.

[0021] As one possible implementation, the deposition time of the chronoamperometry in step (5) is 40 s; the aptamer incubation time in step (6) is 2 h and the aptamer concentration is 1 μg / mL; and the BSA blocking time in step (7) is 1 h.

[0022] Thirdly, this application also provides the use of a multi-channel electrochemical aptamer sensor in the combined and simultaneous detection of hemorrhage transformation biomarkers after acute cerebral infarction. The use is to simultaneously detect the biomarkers human matrix metalloproteinase MMP, fibrinogen FN, and plasminogen activator inhibitor PAI-1, with detection concentration ranges of 1 fg / mL to 1 ng / mL, 1 fg / mL to 10 ng / mL, and 10 fg / mL to 10 ng / mL, respectively.

[0023] As one possible implementation method, the detection method employs differential pulse voltammetry (DPV) with a three-electrode system and an electrolyte of 5 mM [Fe(CN)6] containing 0.1 M KCl. 4- / 3- Solution.

[0024] As one possible implementation, the three-electrode system uses Ag / AgCl as the reference electrode, platinum wire as the counter electrode, and the modified electrode of the multichannel electrochemical aptamer sensor according to any one of claims 1-3 as the working electrode.

[0025] In one possible implementation, the detection voltage of the differential pulse voltammetry (DPV) is 0–0.5 V, and the sample incubation time is 20 min.

[0026] The sensor in this embodiment exhibits excellent sensitivity, specificity, and reproducibility for the three biomarkers MMP-9, FN, and PAI-1 across a wide detection range of six orders of magnitude from 10 fg / mL to 1 ng / mL. Furthermore, the detection results for real samples show a high degree of consistency with ELISA results. When detecting multiple biomarkers simultaneously, no additional operational steps are required, and the time and labor costs for detecting MMP-9, FN, and PAI-1 are the same as for detecting a single biomarker.

[0027] Fourthly, this application also provides a method for the combined and simultaneous detection of biomarkers of hemorrhage transformation after acute cerebral infarction, characterized in that a multi-channel electrochemical aptamer sensor as described in any one of claims 1-3 is used, and detection is performed by a portable multi-channel electrochemical detection device or a Chenhua CHI660e electrochemical workstation, with 1 to 3 detection channels, and the detection data is displayed and stored by a mobile terminal or the cloud.

[0028] In summary, the multi-channel electrochemical aptamer sensor provided in this application employs an integrated structure of screen-printed electrodes and functionalized modifications. Specifically, this application modifies the electrode substrate with PPy / DPH composite material (PEDOT:PSS and PVA crosslinked to form a three-dimensional network, then polypyrrole is deposited), while simultaneously loading gold nanoparticles. Then, specific aptamers corresponding to MMP-9, FN, and PAI-1 are immobilized via Au-S bonds, and non-specific binding sites are blocked with 1% BSA solution. Finally, the sensor is integrated with a portable multi-channel electrochemical device, achieving a single device design corresponding to three biomarker detection channels. Thus, this application achieves efficient simultaneous detection, wide-range quantification, and high-precision analysis of biomarkers for transformation after acute ischemic stroke, combining clinical practicality with technological innovation. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 Scanning electron microscope (SEM) images of DPH, PPy, PPy / DPH, and Au / PPy / DPH.

[0031] Figure 2 This is the energy scattering spectrum (EDS) of Au / PPy / DPH.

[0032] Figure 3 X-ray photoelectron spectroscopy (XPS) spectra of DPH, PPy, and PPy / DPH.

[0033] Figure 4 Cyclic voltammetry (CV) plots for GCE, DPH / GCE, PPy / GCE, and PPy / DPH / GCE.

[0034] Figure 5 Electrochemical impedance spectroscopy (EIS) for GCE, DPH / GCE, PPy / GCE, and PPy / DPH / GCE.

[0035] Figure 6 Cyclic voltammetry comparison after 100 scans for the PPy / DPH modified electrode.

[0036] Figure 7 This is a schematic diagram showing the results of condition optimization for an electrochemical sensor.

[0037] Figure 8 This is a comparison chart of detection results for different modified electrodes.

[0038] Figure 9 A schematic diagram showing the DPV curves and results analysis for MMP-9, FN, and PAI-1 detection.

[0039] Figure 10 This is a schematic diagram of the anti-interference detection results of the electrochemical sensor.

[0040] Figure 11 This is a schematic diagram of the three-dimensional image of the DPV curve simultaneously detected by the electrochemical sensor and the analysis of the results.

[0041] Figure 12 This is a schematic diagram of the reproducibility test results of the electrochemical sensor.

[0042] Figure 13 This study compares the detection results of a multi-channel electrochemical sensor on real blood samples with those of ELISA.

[0043] Figure 14 This is a schematic diagram of a multichannel electrochemical aptamer sensor and the sensor's detection results for three biomarkers (MMP-9, FN, and PAI-1) in the transformation of hemorrhage after acute ischemic stroke. Detailed Implementation

[0044] The technical solutions in the embodiments of this application will now be described with reference to the accompanying drawings.

[0045] It should be understood that the scope of protection of this application is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of this application is for describing specific embodiments and not for limiting the scope of protection of this application; in the specification and claims of this application, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0046] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this application, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description in this application, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this application may be used to implement this application.

[0047] It should be specifically noted that, unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this application pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are all conventional biochemical reagents; the raw materials, instruments, and equipment used in the following embodiments can all be obtained through commercial purchase or by existing methods; unless otherwise specified, the amounts of experimental reagents used are the amounts used in conventional experimental operations; unless otherwise specified, the experimental methods are all conventional methods.

[0048] Unless otherwise stated, the terms used in the specification and claims shall have the following meanings.

[0049] The term "room temperature" as used in this application has a meaning known in the art and generally refers to 24-28°C.

[0050] Multichannel electrochemical immunoassay is a high-throughput, high-sensitivity, and high-specificity rapid analytical technique. It combines highly sensitive electrochemical sensing with highly specific immunoreaction. Different antigen or antibody probes modified on the surface of multichannel electrodes specifically recognize and react with the target molecules in the sample, capturing them. The multichannel electrochemical sensor then enables highly sensitive simultaneous quantitative detection of multiple biomarkers. This technology requires only a small sample size and can achieve simultaneous on-site detection and analysis of multiple biomarkers within 0.5 hours. It offers advantages such as low sample requirement, speed, simplicity, high sensitivity, high throughput, low cost, miniaturization, and portability, perfectly meeting the development needs of multi-biomarker detection technology in clinical biological samples.

[0051] Next, the solution provided in this application will be described in detail with reference to specific embodiments.

[0052] The method for preparing an integrated multichannel electrochemical aptamer sensor for simultaneous detection of transformation biomarkers in post-infarction hemorrhage provided in this application includes the following steps:

[0053] I. Fabrication of Electrochemical Aptamer Sensors

[0054] Step 1: Prepare a mixed solution of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) PEDOT:PSS and polyvinyl alcohol (PVA). Take 0.05 g of a 3% PVA aqueous solution melted at 95°C and add it to 1 g of PEDOT:PSS solution. Stir at 200 rpm / s for at least 6 hours to obtain a PVA crosslinked PEDOT:PSS mixed solution, and store at room temperature.

[0055] Step 2, prepare polypyrrole (PPy) electrolyte. Mix 0.1M sodium carbonate and 0.1M p-toluenesulfonic acid sequentially in water until homogeneous. Then add a 1% (v / v) pyrrole monomer solution and mix thoroughly. Set aside at 4°C.

[0056] Step 3: Drop the solution prepared in Step 1 onto the surface of the working electrode, and allow it to dry and swell at room temperature to obtain a PEDOT:PSS / PVA hydrogel (DPH) substrate with a three-dimensional network structure.

[0057] Step 4: Using Ag / AgCl as the reference electrode, a platinum wire electrode as the counter electrode, the DPH-modified electrode prepared in Step 3 as the working electrode, and the mixed solution prepared in Step 2 as the electrolyte, the final PPy / DPH-modified electrode is obtained by deposition at a potential of 1.2V for 700s using a chronoamperometric method.

[0058] Step 5, the electrode prepared in step 4 is then treated with 0.1 mM Au. + The solution is an electrolyte, and an Au / PPy / DPH modified electrode loaded with gold nanoparticles can be obtained by deposition for 40 s using a chronoamperometric method at a potential of -0.2 V.

[0059] Step 6: Incubate the electrode prepared in Step 5 with the corresponding aptamer at 4°C for a certain period of time to obtain a specifically detectable electrode. For example, the aptamer incubation time is 2 hours, and the aptamer concentration is 1 μg / mL: this duration allows the specific aptamer to be fully immobilized on the surface of the gold nanoparticles through Au-S bonds, ensuring sufficient specific binding sites are formed on the electrode surface and guaranteeing the sensitivity of subsequent detection.

[0060] Step 7: Incubate the electrode prepared in Step 6 with a 1% bovine serum albumin (BSA) solution at 4°C for a certain period of time to block non-specific binding sites, thus obtaining the final detection electrode. In this step, the incubation time with the BSA solution is 1 hour: this duration sufficiently blocks non-specific binding sites on the electrode surface while avoiding interference with the specific binding activity of the aptamers due to excessively long incubation.

[0061] II. Electrochemical Detection Methods

[0062] When performing tests on the Chenhua CHI660e electrochemical workstation, differential pulse voltammetry (DPV) was used with a three-electrode system. Ag / AgCl was used as the reference electrode, a platinum wire electrode as the counter electrode, and the prepared electrode was used as the working electrode. A 5mM [Fe(CN)6] electrolyte containing 0.1M KCl was employed. 4- / 3- The solution was an electrolyte, and three markers of hemorrhage transformation in acute ischemic stroke—MMP-9, FN, and PAI—were detected sequentially. The detection voltage was 0–0.5 V, and the sample incubation time was 20 min.

[0063] When performing detection on a portable electrochemical device, DPV is also used for detection. The detection voltage is 0-0.5V, the sample incubation time is 20min, and the detection channels are 1-3. The final results are displayed on a mobile terminal or in the cloud, and the DPV curve data is stored in the cloud.

[0064] Figure 1 The morphology of different substrate materials was shown using SEM, among which Figure 1 a is a SEM image of DPH, showing the typical honeycomb structure of the hydrogel. This structure fills the interior of the material with pores, providing it with good permeability and ion transport properties. Figure 1 b is a pure PPy material. This interface material exhibits a dense, scaly distribution, providing more active sites for the subsequent deposition of gold nanoparticles. Figure 1 c represents the PPy / DPH composite interface substrate material. As shown in the figure, its surface morphology is mainly similar to that of pure PPy material, exhibiting a dense, scaly distribution. However, compared to the PPy interface, its scaly structure is more uniformly distributed and smaller in size. Figure 1 In Figure d, Au nanoparticles attached to the PPy / DPH composite material are clearly visible. They are tightly adhered to the substrate material, providing connection sites for subsequent aptamer fixation. These results indicate that the formation of the hydrogel network facilitates the growth of the PPy flake structure, and DPH, as the framework of the three-dimensional network structure, is composited with PPy to obtain a dense, flexible polymer substrate material with more active sites. Ultimately, gold nanoparticles are successfully loaded, providing conditions for aptamer fixation.

[0065] To further demonstrate that Au was successfully loaded onto the PPy / DPH composite material, such as Figure 2 As shown, EDS was used to investigate the elemental composition, content, and corresponding elemental distribution maps of the Au / PPy / DPH composite material. The results show that the Au / PPy / DPH composite material contains C, N, O, S, and Au, and their elemental contents are indicated. Furthermore, the elemental distribution maps show that C, N, O, S, and Au are all uniformly distributed, indicating successful composite formation. These results further demonstrate the successful deposition of Au nanoparticles in the PPy / DPH composite material.

[0066] Figure 3 XPS full spectra of single PPy, DPH, and PPy / DPH composites were presented. The corresponding fine-spectral analysis provided molecular-level evidence confirming the successful composite formation.

[0067] In the C1s spectrum ( Figure 3 (b) The characteristic peaks of DPH correspond to CC / CH (284.8 eV), CO / CS (286.2 eV), OC=O (287.7 eV), and the π-π* satellite peak (289.2 eV), respectively. In contrast, the spectrum of PPy shows a distinct CN peak at 285.9 eV, and the characteristic peak of its doped state (attributed to the CN peak adjacent to the cation nitrogen) is observed at 288.6 eV. + This demonstrates the conductivity state of PPy. In the PPy / DPH composite, the C 1s spectrum combines the characteristics of both: in addition to the base CC / CH peaks, the CN peak of PPy shifts to 285.5 eV, and its doped CN state... + The peak shifted to 286.5 eV, while the CO and OC=O peaks of the DPH component remained visible. These systematic shifts indicate a significant electronic interaction between PPy and the DPH matrix.

[0068] N 1s spectrum ( Figure 3 c) This provides direct evidence for the successful introduction of PPy. The binding energies of 399.9 eV and 401.1 eV in pure PPy correspond to the neutral nitrogen (-NH-, N-pyrrole) and the positively charged doped nitrogen (-N-) in its chain, respectively. + =, N-pyridine+). After recombination, both shifted towards lower binding energies (to 399.8 eV and 400.7 eV, respectively), indicating an increase in the electron cloud density around the nitrogen atom, further confirming that PPy interacted with DPH as an electron donor.

[0069] S2p spectrum ( Figure 3 d) reveals the electronic reconstruction of the interface from the perspective of DPH. In pure DPH, the doublets at 163.7 / 164.7 eV and 167.8 / 169.0 eV are attributed to the thiophene ring sulfur of PEDOT and the sulfur of the sulfonate group of PSS, respectively. After recombination, the S2p doublet of PEDOT shifts significantly to 162.2 / 163.5 eV, and the significant decrease in binding energy directly confirms that its sulfur atom has gained electrons from the π-conjugated system of PPy or the nitrogen atom; while the sulfur peak shift of PSS is small, indicating that its interaction with PPy is weak.

[0070] In summary, the systematic binding energy shifts observed in the C 1s, N 1s, and S2p core energy level spectra all point to a strong charge interaction between PPy and DPH, rather than a simple physical mixing, which strongly proves the successful preparation of the PPy / DPH composite material.

[0071] To comprehensively evaluate the electrochemical performance of bare GCE, DPH / GCE, PPy / GCE, and PPy / DPH / GCE electrodes with different modifications, a series of tests and comparative analyses were conducted. In this experiment, 0.1 mM KCl and 5 mM [Fe(CN)6] were used. 4- / 3- (1:1) solution is used as electrolyte.

[0072] Measurements were performed on different electrodes using CV. For example... Figure 4 As shown, different modified electrodes exhibited different current responses within the potential range of -0.2 to 0.7 V. While the bare GCE, as the substrate electrode, showed some electrochemical activity in its CV curve, its overall performance was relatively average, with poor conductivity. However, the DPH / GCE and PPy / GCE, due to the addition of DPH and PPy conductive materials, showed significantly improved CV curves compared to bare GCE, displaying higher current densities and richer redox peaks. Most notably, the CV performance of PPy / DPH / GCE was the most remarkable. Compared to pure GCE, the current density was approximately 73.9 times higher; compared to DPH / GCE and PPy / GCE, it was also approximately 4.3 times and 3.8 times higher, respectively. This result fully demonstrates the significant advantages of the PPy / DPH composite material in improving the electrochemical performance of the electrode.

[0073] Furthermore, to further investigate the charge transport performance of different modified electrodes, EIS was used to detect the different modified electrodes, and the charge transfer resistance (Rct) of each electrode was measured. For example... Figure 5 As shown, the Rct values ​​of bare GCE and DPH / GCE are relatively large, indicating their relatively weak charge transport capabilities. In contrast, the Rct value of PPy / DPH / GCE is significantly smaller, further demonstrating the superior electronic conductivity of this electrode. This result is consistent with the CV test results, further validating the effectiveness of the PPy / DPH composite material in improving the electrochemical performance of the electrode.

[0074] In addition to conductivity, the cycling stability of the electrode was also tested. Through 100 CV scans, it was found that the oxidation peak current intensity of the PPy / DPH / GCE electrode remained almost unchanged after multiple scans. Figure 6 This indicates that the electrode has excellent cycling stability and can maintain stable electrochemical performance during long-term use.

[0075] The performance of a sensor critically depends on its fabrication and detection conditions. To obtain optimal performance, this invention systematically optimized the deposition time of gold nanoparticles, aptamer incubation time, BSA blocking time, aptamer concentration, and biomarker incubation time. The results are shown in... Figure 7 .

[0076] First, the electrochemical deposition time of gold nanoparticles was optimized. Experimental results showed that deposition time significantly affected the number and morphology of active sites on the electrode surface. When the deposition time was too short (e.g., 10 s, 20 s, 30 s), insufficient gold nanoparticle deposition resulted in limited active sites on the electrode surface, making it difficult to adequately immobilize aptamer molecules, thus leading to a weaker observed current response signal. When the deposition time was too long (e.g., 50 s, 60 s), excessive deposition caused gold nanoparticle aggregation, which reduced the effective specific surface area and active sites, also hindering aptamer immobilization and resulting in a decreased current response. Overall, the modified electrode exhibited the highest current response at a deposition time of 40 s. Therefore, 40 s was determined to be the optimal deposition time for gold nanoparticles.

[0077] Based on the same principle, we optimized other key conditions and determined their optimal values: the aptamer incubation time is preferably 2 hours, at which point the fixation amount reaches saturation and the current response tends to stabilize; the BSA blocking time is preferably 1 hour, which can fully block non-specific sites, minimize background interference, and maximize the signal-to-noise ratio; the aptamer concentration is preferably 1 μg / mL, which effectively avoids intermolecular steric hindrance while achieving high sensitivity, resulting in the largest current difference; the biomarker incubation time is preferably 20 minutes, at which point the binding reaction reaches equilibrium and the current response stabilizes. The combination of these optimized conditions ensures that the sensor achieves the best overall performance.

[0078] Figure 8 A comparison of detection results from different electrochemical sensors was presented. It was found that only the final Au / PPy / DPH / GCE-apt sensor showed a significant current change, which also confirms the sensor's detection principle: signal amplification via the substrate material, followed by Au-S bond fixation of the corresponding aptamer to the material surface, thereby achieving specific binding of the aptamer to the corresponding biomarker and generating an electrochemical signal for detection.

[0079] like Figure 9 The figure shows the DPV detection curves and corresponding linear fitting results for three biomarkers: MMP-9, FN, and PAI-1. As the concentration of each biomarker increases, the DPV peak current signal exhibits a regular decreasing trend. Further analysis shows a good linear relationship between the peak current response value and the logarithm (Log C) of the biomarker concentration, RC. 2All achieved a linearity of 0.999. Based on this linear relationship, and using the signal-to-noise ratio (S / N = 3), the limits of detection (LOD) for MMP-9, FN, and PAI-1 were calculated to be as low as 4.043 fg / mL, 0.296 fg / mL, and 2.790 fg / mL, respectively. The sensor's excellent sensitivity and wide linear range lay the foundation for its accurate and stable detection of low-concentration target analytes in complex real-world samples.

[0080] Figure 10 The selectivity (anti-interference) test results of the electrochemical sensor of this invention are presented. The tests were conducted under conditions where the target markers (MMP-9: 1 pg / mL; FN: 1 ng / mL; PAI-1: 1 pg / mL) coexisted with high concentrations of potential interfering substances (CEA: 10 ng / mL; PSA: 10 ng / mL; BSA: 0.1 mg / mL). The results show that for any target analyte, the specific current response signal is significantly stronger than the non-specific signal caused by the high concentration of interfering substances. This result fully demonstrates that even in the presence of high background interference, the sensor of this invention can still achieve highly selective and highly anti-interference detection of the target analyte.

[0081] Figure 11 This paper demonstrates the performance of the present invention in simultaneously detecting MMP-9, FN, and PAI-1 using a multi-channel electrochemical device. Detection was performed simultaneously on three independent signal channels, and the DPV curves are shown in the figure. The results show that the peak current maintains a good linear relationship with concentration (R² = 0.999) across the concentration range of each target analyte. Compared with individual detection, the sensitivity and linear range of simultaneous detection did not change significantly. These results confirm that the present invention, based on a multi-channel design, successfully achieves rapid, highly specific, and highly sensitive simultaneous analysis of multiple biomarkers.

[0082] Figure 12 The reproducibility and stability of the sensor were evaluated. We tested the responses of three groups of sensors modified with different aptamers, with six electrodes in each group, for a total of 18 electrodes, to the same concentration of sample. The relative standard deviation (RSD) of the current values ​​was less than 5% for all of them. In addition, the RSD of six consecutive detections by the same electrode was also less than 5%. These data fully demonstrate that the sensor of this invention has excellent reproducibility and stability, providing a solid theoretical basis for its practical application.

[0083] Figure 13 This study analyzed the correlation between the combined detection results of three biomarkers (MMP-9, FN, and PAI-1) using a multi-channel electrochemical sensor and the ELISA results. The detection results of the three biomarkers showed a high degree of agreement with the ELISA results, with correlation coefficients as follows: MMP-9 R0... 2 =0.9912, R of FN 2=0.9794 and R of PAI-1 2 =0.9786, which indicates that the two methods have equal value in medical diagnosis.

[0084] Figure 14 This is a schematic diagram showing the detection results of a multichannel electrochemical aptamer sensor and three biomarkers (MMP-9, FN, and PAI-1) for hemorrhagic transformation after acute ischemic stroke. Figure 14 As shown above, the multi-channel electrochemical aptamer sensor integrates electrochemical sensor hardware with three detection channels, such as screen-printed electrodes and portable device integration. Each channel corresponds to one target biomarker (MMP-9, FN, PAI-1), enabling the simultaneous detection of three biomarkers by a single device.

[0085] Furthermore, the fabrication process of this multichannel electrochemical aptamer sensor focuses on material modification and electrode functionalization as its core steps: first, a PVA-crosslinked PEDOT:PSS mixture is prepared and drop-coated to form a DPH substrate electrode; then, polypyrrole (PPy) and gold nanoparticles are introduced via electrochemical deposition to construct a signal amplification substrate; finally, a specific aptamer is incubated and blocked with BSA to complete the sensor functionalization modification. The detection method employs differential pulse voltammetry (DPV) with a 5mM [Fe(CN)6] solution containing 0.1M KCl. 4- / 3- The solution is an electrolyte, and multi-channel synchronous detection is achieved through a three-electrode system (the modified electrode is the working electrode).

[0086] Thus, the multi-channel electrochemical aptamer sensor of this application, through its multi-channel integrated design, can simultaneously detect three biomarkers: MMP-9, FN, and PAI-1. The detection time and labor costs are comparable to those for single-biomarker detection, overcoming the limitations of traditional single-channel detection methods that require individual testing and are time-consuming and labor-intensive. Furthermore, the detection range covers 10 fg / mL to 1 ng / mL (down to the femtogram level), adapting to the low concentration characteristics of biomarkers in clinical samples. The linear correlation coefficient between concentration and electrochemical signal is R²≈0.999 (as shown in the image calibration curve), indicating extremely strong linear correlation. The detection results are highly consistent with commonly used clinical ELISA methods, ensuring the reliability of clinical applications. The modification with composite materials (PPy / DPH) and gold nanoparticles amplifies the electrochemical signal, improving the detection capability of low-concentration biomarkers. The treatment of non-specific sites by BSA blocking effectively reduces interference from extraneous proteins, ensuring the specificity and stability of the detection signal.

[0087] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A multichannel electrochemical aptamer sensor, characterized in that, The device includes a screen-printed electrode; the surface of the screen-printed electrode is modified with a polymer substrate material that amplifies electrochemical signals, the polymer substrate material is loaded with gold nanoparticles and a specific aptamer is fixed by chemical bonds; wherein the specific aptamer specifically recognizes three biomarkers: human matrix metalloproteinase, fibrinogen, and plasminogen activator inhibitor, and the multi-channel electrochemical aptamer sensor is integrated with a multi-channel electrochemical detection device.

2. The multichannel electrochemical aptamer sensor according to claim 1, characterized in that, The polymer substrate material is a composite material of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid), polyvinyl alcohol and polypyrrole, PPy / DPH.

3. The multichannel electrochemical aptamer sensor according to claim 1, characterized in that, The gold nanoparticles are loaded onto the surface of the polymer substrate material by electrochemical deposition, and the specific aptamer is fixed to the surface of the gold nanoparticles by Au-S bonds; the non-specific binding sites on the surface of the screen-printed electrode are sealed with 1% BSA solution.

4. A method for preparing the multichannel electrochemical aptamer sensor according to any one of claims 1-3, characterized in that, Includes the following steps: Step (1): Design and prepare polymer substrate materials to amplify electrochemical signals; Step (2): Based on the polymer substrate material modified electrode, a specific aptamer is modified to prepare a multi-channel electrochemical aptamer sensor.

5. The method according to claim 4, characterized in that, The method includes: (1) Preparation of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) crosslinked polyvinyl alcohol: Take 0.05 g of 3% polyvinyl alcohol aqueous solution melted at 95℃, add 1 g of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) solution, stir at 200 rpm / s for more than 6 h, and keep at room temperature (24℃) for later use; (2) Preparation of polypyrrole electrolyte: Mix 0.1 M sodium carbonate and 0.1 M p-toluenesulfonic acid in water until homogeneous, add 1% volume fraction of pyrrole monomer solution and mix until homogeneous, and store at 4°C for later use; (3) Preparation of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol hydrogel modified electrode: The mixed solution in step (1) was drop-coated onto the surface of the working electrode, dried at room temperature (24°C) and swollen to obtain poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol hydrogel substrate modified electrode. (4) Preparation of polypyrrole / poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol modified electrode: using Ag / AgCl as reference electrode, platinum wire as counter electrode, and the poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol hydrogel modified electrode of step (3) as working electrode, and using the polypyrrole electrolyte of step (2) as electrolyte, the electrode was deposited by time-amperometric method at a potential of 1.2V for 700 s to obtain the polypyrrole / poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol modified electrode; (5) Preparation of gold / polypyrrole / poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol modified electrode: with 0.1 mM gold ions (Au + The solution is used as the electrolyte. The electrode in step (4) is deposited by chronoamperometry at a potential of -0.2 V to obtain a gold / polypyrrole / poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) / polyvinyl alcohol modified electrode loaded with gold nanoparticles. (6) Preparation of specific detection electrode: Incubate the electrode from step (5) with the corresponding specific aptamer at 4°C; (7) Sealing treatment: The electrode from step (6) is incubated with 1% bovine serum albumin (BSA) solution at 4°C to seal the non-specific binding sites and obtain the sensor.

6. The method according to claim 5, characterized in that, The deposition time of the chronoamperometry method in step (5) is 40 s; the aptamer incubation time in step (6) is 2 h and the aptamer concentration is 1 μg / mL; the BSA blocking time in step (7) is 1 h.

7. The use of the multichannel electrochemical aptamer sensor as described in any one of claims 1-3 in the combined and simultaneous detection of hemorrhage transformation biomarkers after acute ischemic stroke, characterized in that, The purpose is to simultaneously detect the biomarkers human matrix metalloproteinase MMP, fibrinogen FN, and plasminogen activator inhibitor PAI-1, with detection concentration ranges of 1 fg / mL to 1 ng / mL, 1 fg / mL to 10 ng / mL, and 10 fg / mL to 10 ng / mL, respectively.

8. The use according to claim 7, characterized in that, The detection method employed differential pulse voltammetry (DPV) using a three-electrode system. The electrolyte was 5 mM [Fe(CN)6] containing 0.1 M KCl. 4- / 3- The solution, wherein the detection voltage of the differential pulse voltammetry (DPV) is 0~0.5 V, and the sample incubation time is 20 min.

9. The use according to claim 8, characterized in that, The three-electrode system uses Ag / AgCl as the reference electrode, platinum wire as the counter electrode, and the modified electrode of the multichannel electrochemical aptamer sensor according to any one of claims 1-3 as the working electrode.

10. A method for combined and simultaneous detection of hemorrhage biomarkers after acute ischemic stroke, characterized in that, The multi-channel electrochemical aptamer sensor described in any one of claims 1-3 is used for detection via a portable multi-channel electrochemical detection device or a Chenhua CHI660e electrochemical workstation. The detection channels are 1 to 3, and the detection data is displayed and stored by a mobile terminal or the cloud.

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