Electrochemical aptamer sensor based on microelectrode array as well as preparation method and application of electrochemical aptamer sensor

Abstract

The invention relates to an electrochemical aptamer sensor based on a microelectrode array and a preparation method and application thereof, and belongs to the technical field of sensors. The electrochemical aptamer sensor comprises a microelectrode array which is formed by orderly arranging a plurality of micron-sized gold electrode units, and each electrode unit independently realizes signal acquisition and analysis; the modification layer comprises a GO-PPy / dsDNASA modification layer and an AuNPs / CHI-Au / dsDNADC modification layer, and the GO-PPy / dsDNASA modification layer and the AuNPs / CHI-Au / dsDNADC modification layer are respectively and electrically deposited on the surfaces of different electrode units of the microelectrode array; the redox molecule is an electroactive molecule with a nitrogen-containing heteroaromatic ring structure and is embedded into the dsDNASA and the dsDNAEC, so that high-sensitivity and high-specificity synchronous detection on the staphylococcus aureus and the escherichia coli is realized.

Description

Technical Field

[0001] This invention belongs to the field of sensor technology, and particularly relates to an electrochemical aptamer sensor based on a microelectrode array, its preparation method, and its application. Background Technology

[0002] Foodborne and environmental pathogens such as Staphylococcus aureus and Escherichia coli are widely present in water, food, and soil. They can cause food poisoning, intestinal infections, and other diseases through ingestion or contact, seriously threatening human health and public health safety. While traditional pathogen detection techniques such as microbial isolation and culture, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) offer high detection accuracy, they are limited by time consumption, complex operation, and high cost, and also make it difficult to achieve simultaneous detection of multiple targets.

[0003] Against this backdrop, electrochemical biosensors have become a research hotspot due to their advantages such as rapid response, ease of operation, and low cost. However, traditional single-electrode sensors still have significant drawbacks: they cannot distinguish signal changes caused by different targets, making it difficult to achieve multi-target analysis; the macroscopic electrode structure leads to large sample consumption, making them unsuitable for rare samples; at the same time, the single-channel detection mode is easily affected by individual differences and operational fluctuations, resulting in high detection errors. These limitations severely restrict their practical application in the high-sensitivity detection of multiple targets of pathogens.

[0004] Microelectrode arrays (MEAs) consist of multiple micrometer-sized electrode units arranged in an orderly manner. Different electrode units can be equipped with different biometric elements, enabling simultaneous detection of multiple targets while reducing sample consumption and detection errors. However, achieving multi-target detection with a single MEA still faces challenges: on the one hand, functional modification of the MEA surface by fixing biometric elements can easily cause crosstalk between microelectrode units, leading to irreversible dissociation and detachment of the modified layer; on the other hand, the small effective sensing area severely limits the number of identification probes that can be fixed, thus fundamentally limiting the detection sensitivity of low-abundance microbial analytes.

[0005] Therefore, there is an urgent need to develop a multi-target pathogen electrochemical aptamer sensor based on a microelectrode array to achieve rapid, highly specific, and highly sensitive simultaneous detection of various pathogens. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an electrochemical aptamer sensor based on a microelectrode array (MEA), its fabrication method, and its application. Using site-selective electrodeposition (SSE) technology, electroactive materials can be directionally deposited onto the surface of a MEA to form a uniform, stable, strongly adherent, and controllable modification layer. Combining this technology with a directional molecular distribution strategy, an MEA-based electrochemical aptamer sensor is constructed, enabling highly sensitive and specific simultaneous detection of Staphylococcus aureus and Escherichia coli. Specifically, site-selective electrodeposition technology can be used to construct graphene oxide-polypyrrole (GO-PPy) layers and gold nanoparticles (AuNPs) / chitosan-gold (CHI-Au) composite material layers on different electrode units of a single MEA. The GO-PPy layer, with its abundant oxygen-containing functional groups, especially carboxyl groups, readily binds to the Staphylococcus aureus aptamer Apt. SA The amino groups on the probe undergo covalent amidation, thus providing a stable substrate for Apt synthesis. SA The CHI-Au layer is fixed, while its amino-rich molecular structure interacts with the E. coli aptamer Apt. EC The carboxyl group on the probe undergoes a covalent amidation reaction to achieve Apt EC The site-selective electrodeposition of this composite layer not only fixes the two aptamers in different MEA units, effectively avoiding signal crosstalk, but also significantly increases the active surface area of ​​the electrode, thereby greatly improving the probe loading capacity.

[0007] The first objective of this invention is to provide an electrochemical aptamer sensor based on a microelectrode array, comprising: A microelectrode array (MEA) is composed of several micrometer-sized gold electrode units arranged in an orderly manner, with each electrode unit independently realizing signal acquisition and analysis. Modification layer, including GO-PPy / dsDNA SA Modified layer and AuNPs / CHI-Au / dsDNA EC The modification layer is electrodeposited on the surface of different electrode units of the microelectrode array; the GO-PPy / dsDNA SA The modification layer consists of graphene oxide-polypyrrole composite material and dsDNA. SA Composition; the AuNPs / CHI-Au / dsDNA EC The modification layer consists of gold nanoparticles, chitosan-gold composite material, and dsDNA. EC composition; Redox molecules, which are electroactive molecules with nitrogen-containing heteroaromatic ring structures, are embedded in the dsDNA. SA and the dsDNA EC internal.

[0008] In one embodiment of the present invention, the dsDNA SA It is composed of aptamers of equal volume, Apt. SA With cDNA SA The mixture was prepared by hybridization with PBS buffer and then annealing. And / or, the dsDNA EC It is composed of aptamers of equal volume, Apt. EC With cDNA EC The mixture was prepared by hybridization with PBS buffer and then annealing. And / or, the electroactive molecule with the nitrogen-containing heteroaromatic ring structure is selected from one or more of methylene blue (MB), Nile blue (NB), and anthraquinone (AQ).

[0009] Furthermore, the electroactive molecule with a nitrogen-containing heteroaromatic ring structure is methylene blue (MB). Methylene blue (MB) is a typical electroactive molecule whose molecular structure contains a nitrogen-containing heteroaromatic ring capable of electron gain and loss. When a specific potential is applied to the electrode, MB can undergo a reversible redox reaction on the electrode surface, and electrons are subsequently transferred between MB and the electrode to form a detectable current signal. The signal intensity is related to the MB concentration. When MB is used as an electroactive mediator to intercalate into dsDNA, the change in current is directly related to the amount of dsDNA unwinding. The change in current generated by this intercalation-release process shows a good linear relationship with the target concentration (R0). 2 Based on this characteristic (≥0.991 / 0.996), MB can be used as an electroactive probe to monitor the changes in electrical signals after the aptamer binds to the pathogen using square wave voltammetry (SWV), thereby enabling the quantitative detection of two target pathogens.

[0010] In one embodiment of the present invention, the aptamer Apt SA The sequence is: 5'-NH2-GCAATGGTACGGTACTTCCTCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCAGCTACGTCAAAAGTGCACGCTACTTTGCTAA-3'; aptamer Apt SA The 5' end of the sequence is modified with an amino group and then modified on the GO-PPy electrodeposition electrode via a -CO-NH- bond; And / or, the cDNA SA The sequence is: 5'-TTAGCAAAGTAGCCACGTGAGC-3'; And / or, the aptamer Apt EC The sequence is: 5'-COOH-CCATGAGTGTTGTGAAATGTTGGGACACTAGGTGGCATAGAGCCG-3'; aptamer Apt ECThe 5' end of the sequence is modified with a carboxyl group and modified on the electrode of Example 1 via a -CO-NH- bond; And / or, the cDNA EC The sequence is: 5'-GCTCTATGCCACCTAGTGTC-3'.

[0011] A second objective of this invention is to provide a method for fabricating the aforementioned electrochemical aptamer sensor based on a microelectrode array, comprising the following steps: S1. Immerse the microelectrode array in GO-PPy solution, apply a potential to one side of the electrode unit of the microelectrode array to perform electrodeposition, activate the resulting electrode after electrodeposition, and then add dsDNA. SA The solution was incubated to form GO-PPy / dsDNA SA Modification layer; S2. Electrodeposition is performed by sequentially applying potentials to the other electrode units of the microelectrode array obtained in S1 in HAuCl4 solution and CHI-Au solution; after electrodeposition, activated dsDNA is added. EC Incubation in solution forms AuNPs / CHI-Au / dsDNA EC Modification layer; S3. The microelectrode array obtained in S2 is immersed in a redox molecular solution for incubation to obtain the electrochemical aptamer sensor based on the microelectrode array.

[0012] In one embodiment of the present invention, in S1, the concentration of graphene oxide in the GO-PPy solution is 0.08 mg / mL-0.12 mg / mL, the concentration of lithium perchlorate is 18 mmol / L-22 mmol / L, and the concentration of pyrrole is 2.8 wt‰-3.2 wt‰. And / or, the electrodeposition process is as follows: potential of 0.45V-0.6V, time of 580s-620s.

[0013] In one embodiment of the present invention, in S2, the concentration of chloroauric acid in the HAuCl4 solution is 1.8 mg / mL-2.2 mg / mL, and the concentration of sodium acetate is 9.5 mmol / L-10.5 mmol / L; And / or, the electrodeposition process corresponding to the HAuCl4 solution is: a potential of 0.55V-0.65V and a time of 350s-370s; And / or, the CHI-Au solution is obtained by mixing chitosan solution and chloroauric acid solution at a volume ratio of (6.5-7.5):1; the concentration of the chitosan solution is 0.18wt%-0.22wt%; and the concentration of the chloroauric acid solution is 1.8mg / mL-2.2mg / mL. And / or, the electrodeposition process corresponding to the CHI-Au solution is: a potential of -1.1V to -1.4V and a time of 85s to 95s.

[0014] In one embodiment of the present invention, in S3, the concentration of redox molecules in the redox molecule solution is 1.3 mmol / L-1.5 mmol / L.

[0015] In one embodiment of the present invention, the activation solution used in S1 is an EDC / NHS mixture, which can activate the carboxyl terminus of the target compound, thereby facilitating the formation of -CO-NH- bonds; when activating the GO-PPy electrodeposition electrode, the concentration of EDC in the EDC / NHS mixture is 0.35 mol / L-0.45 mol / L, and the concentration of NHS is 0.08 mol / L-0.12 mol / L; for dsDNA EC During activation treatment, the concentration of EDC in the EDC / NHS mixture is 45 mmol / L-55 mmol / L, and the concentration of NHS is 20 mmol / L-30 mmol / L.

[0016] In one embodiment of the present invention, the dsDNA SA Solution and the dsDNA EC The concentrations of the solutions independently ranged from 2.8 µmol / L to 3.2 µmol / L.

[0017] A third objective of this invention is to provide an application of the aforementioned microelectrode array-based electrochemical aptamer sensor in the detection of Staphylococcus aureus and Escherichia coli.

[0018] The technical solution of the present invention has the following advantages compared with the prior art: (1) The electrochemical aptamer sensor described in this invention relies on the multi-channel characteristics of the microelectrode array. By constructing specific recognition layers for Staphylococcus aureus and Escherichia coli on the surface of different electrode units, it can realize the simultaneous detection of the two pathogens. This breaks through the limitation of traditional single-electrode sensors that can only target one target at a time, and solves the problem of detecting rare samples or trace amounts of contamination. Its core lies in the fact that the independent electrode units of the microelectrode array have physical spacing and insulating substrate design, which can isolate the diffusion of different recognition probes in space. Combined with site-selective electrodeposition and directional molecular distribution strategies, it can completely avoid crosstalk between units in multi-target detection, ensure the stability and uniformity of the modified layer, and thus obtain high detection efficiency and specificity.

[0019] (2) The GO-PPy / dsDNA in the electrochemical aptamer sensor of the present invention SA Modified layer and AuNPs / CHI-Au / dsDNA ECThe modified layers all possess the advantages of high conductivity and high specific surface area: In the GO-PPy composite material, the layered structure of GO provides a high specific surface area, which can increase dsDNA. SA With a fixed amount, the conjugated π-π structure of PPy constructs an efficient electron transport channel, reducing electron transfer impedance; in the AuNPs / CHI-Au composite material, the high conductivity of AuNPs and the hydrophilicity of CHI work synergistically to accelerate electron transport and improve biocompatibility, thereby enhancing dsDNA. EC The fixed efficiency of the two, together with the synergistic effect, significantly enhances the sensor's response signal to low-abundance targets, enabling accurate detection of low-abundance pathogens, with the detection limit of Staphylococcus aureus as low as 25.4 CFU / mL and the detection limit of Escherichia coli as low as 8.8 CFU / mL.

[0020] (3) The preparation method of the present invention constructs the modification layer through site-selective electrodeposition technology, wherein GO-PPy is directionally polymerized on a designated electrode unit under electric field induction; AuNPs / CHI-Au forms a dense structure through bilayer electrodeposition, and the amino groups of CHI can form coordination bonds with gold atoms on the electrode surface, thereby further enhancing the adhesion of the modification layer. From a mechanistic perspective, the electric field force during the electrodeposition process can drive GO, Py, and Au. 3+ Active substances such as CHI migrate in a directional manner and undergo in-situ reactions, resulting in interfacial chemical bonds and strong intermolecular interactions between the modified layer and the electrode substrate, as well as between the components within the modified layer. This significantly improves the stability of the sensor and effectively solves the technical problems of traditional modified layers being prone to detachment and having a short lifespan. Attached Figure Description

[0021] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein: Figure 1 This is a schematic diagram of the electrochemical aptamer sensor based on a microelectrode array according to the present invention; wherein, AB is a borosilicate / gold microelectrode array chip with a glass ring as the electrochemical reservoir, C is a schematic diagram of an 8×8 microelectrode array layout, and DE is GO-PPy / dsDNA. SA Modified layer and AuNPs / CHI-Au / dsDNA EC Scanning electron microscope image of the modified layer; schematic diagram of the preparation and detection mechanism of the F-electrochemical aptamer sensor. Figure 2 The effect of different deposition potentials on GO-PPy / dsDNA in Test Example 1 of this invention SA The influence of the modified layer on the electrochemical signal; Figure 3 This invention provides a test example 2 for the effect of different deposition potentials on AuNPs / CHI-Au / dsDNA.EC The influence of the modified layer on the electrochemical signal; Figure 4 The GO-PPy / dsDNA used in Example 1 and Comparative Example 1 of Test Example 3 of this invention SA Electrochemical behavior of the modified layer in the selective electrodeposition step at each point; where A is the CV curve of the electrode of Example 1, B is the CV curve of the electrode of Comparative Example 1, C is the SWV curve of the electrode of Example 1, and D is the SWV curve of Comparative Example 1. Figure 5 The AuNPs / CHI-Au / dsDNA in Example 1 and Comparative Examples 2-3 of this invention are used in Test Example 3 of this invention. EC Electrochemical behavior of the modified layer in the selective electrodeposition step at each point; where A is the CV curve of the electrode of Example 1, B is the CV curve of the electrode of Comparative Example 2, C is the CV curve of the electrode of Comparative Example 3, D is the SWV curve of the electrode of Example 1, E is the SWV curve of the electrode of Comparative Example 2, and F is the SWV curve of the electrode of Comparative Example 3. Figure 6 The above are the signal response and detection standard curves of the electrochemical aptamer sensor based on microelectrode array in Example 1 of Test Example 3 of the present invention for different concentrations of Staphylococcus aureus and Escherichia coli. The left figure shows the relationship between the change of SWV peak current and the concentration of Staphylococcus aureus, and the inset shows the relationship between the SWV decrease intensity and the logarithmic concentration of bacteria. The right figure shows the relationship between the change of SWV peak current and the concentration of Escherichia coli, and the inset shows the relationship between the SWV decrease intensity and the logarithmic concentration of bacteria. Figure 7 The results show the specific detection results of Staphylococcus aureus or Escherichia coli by the electrochemical aptamer sensor based on microelectrode array in Example 1 of Test Example 4 of the present invention; wherein, A is Staphylococcus aureus and B is Escherichia coli. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0023] In this invention, unless otherwise stated, the experimental methods used in the embodiments are all conventional methods, and the materials and reagents used are all commercially available.

[0024] In this invention, unless otherwise stated, the microelectrode array (MEA) used in the embodiments is an 8×8 microelectrode array, and the central part of the chip contains gold feed lines and 64 gold microelectrodes.

[0025] In this invention, unless otherwise stated, the chitosan solution used in the examples is made by dissolving chitosan in deionized water containing acetic acid (the volume ratio of acetic acid to deionized water is 1:500), and then adjusting the pH of the system to about 5.

[0026] In this invention, unless otherwise stated, the sequences used in the embodiments are as follows: aptamer Apt SA The sequence is: 5'-NH2-GCAATGGTACGGTACTTCCTCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACAGCTACGTCAAAAGTGCACGCTACTTTGCTAA-3'; cDNA SA The sequence is: 5'-TTAGCAAAGTAGCCACGTGAGC-3'; aptamer Apt EC The sequence is: 5'-COOH-CCATGAGTGTTGTGAAATGTTGGGACACTAGGTGGCATAGAGCCG-3'; cDNA EC The sequence is: 5'-GCTCTATGCCACCTAGTGTC-3'. Example 1

[0027] Reference Figure 1 As shown, the electrochemical aptamer sensor based on a microelectrode array and its fabrication method in this embodiment specifically include the following steps: S1. Microelectrode array (MEA) regeneration: The MEA was sequentially placed in n-propanol, anhydrous ethanol, and deionized water and sonicated for 5 min each to complete physical cleaning. Then, electrochemical cleaning was carried out using a platinum electrode as the auxiliary electrode and an Ag / AgCl electrode as the reference electrode. First, it was cyclically scanned 10 times in a 0.1 mol / L sodium hydroxide solution at a potential range of -1.0 V to -0.3 V and a scan rate of 1 V / s. Then, it was transferred to a 0.05 M sulfuric acid solution and cyclically scanned 20 times at a potential range of -0.3 V to 1.5 V and a scan rate of 1 V / s. After regeneration was completed, it was rinsed with deionized water and dried with nitrogen gas for later use.

[0028] S2, GO-PPy / dsDNA SAModification layer: First, the regenerated MEA was immersed in GO-PPy electrodeposition solution (0.1 mg / mL GO, 20 mmol / L LiClO4 and 3 wt‰ pyrrole), and a constant potential of 0.55 V (vs. Ag / AgCl) was applied to some electrode units. After deposition at room temperature for 600 s, it was rinsed with deionized water. Then, the electrode was immersed in EDC / NHS mixed solution (0.4 mol / L EDC and 0.1 mol / L NHS) and activated at 37 °C for 2 h. After washing, 3 µM dsDNA was added. SA Solution (aptamer Apt) SA With cDNA SA Mix the contents into 1×PBS in equal volume, maintain at 95°C for 5 min, then slowly cool to room temperature, incubate at 37°C for 12 h, and then rinse with PBS.

[0029] S3, AuNPs / CHI-Au / dsDNA EC Modification layer: First, the MEA electrode prepared by S2 was immersed in a 10 mmol / L sodium acetate solution containing 2 mg / mL HAuCl4. A 0.6 V potential was applied to the electrode unit, which was different from that of S2, for 360 s to deposit AuNPs layer. Then, the electrode was immersed in a CHI-Au mixture (0.2 wt% chitosan solution and 2 mg / mL HAuCl4 solution mixed at a volume ratio of 7:1) at pH approximately 5, and a -1.2 V potential was applied for deposition for 90 s. The CHI-Au layer was then stabilized by soaking in PBS for 20 min. Finally, the electrode was immersed in 3 µM dsDNA. EC Solution (aptamer Apt) EC With cDNA EC Mix the contents in equal volumes and place them in 1×PBS. Incubate at 95°C for 5 min, then slowly cool to room temperature. Add the mixture to an EDC / NHS mixture (50 mmol / L EDC and 25 mmol / L NHS, activated at 37°C for 2 h, then rinse). Incubate at 37°C for 12 h and then rinse with PBS.

[0030] S4, MB embedding: The MEA prepared in S2 was immersed in HEPES buffer (20 mM HEPES, 10 mM KCl and pH about 7) containing 1.4 mmol / L MB and incubated at 25°C for 1 h. Comparative Example 1

[0031] Basically the same as Example 1, except that: GO-PPy / dsDNA SA The preparation of the modification layer specifically includes the following steps: First, the regenerated MEA was immersed in GO-PPy electrodeposition solution (0.1 mg / mL GO, 20 mmol / L LiClO4, and 3 wt‰ pyrrole) and kept at room temperature for 600 s, then rinsed with deionized water. Next, the electrode was immersed in an EDC / NHS mixture and activated at 37°C for 2 h. After washing, 3 µM dsDNA was added. SA Solution (aptamer Apt) SA With cDNA SA Mix the contents into 1×PBS in equal volume, maintain at 95°C for 5 min, then slowly cool to room temperature, incubate at 37°C for 12 h, and then rinse with PBS. Comparative Example 2

[0032] Basically the same as Example 1, except that: AuNPs / CHI-Au / dsDNA EC The preparation of the modification layer specifically includes the following steps: First, the MEA electrode prepared by S2 was immersed in a 10 mmol / L sodium acetate solution containing 2 mg / mL HAuCl4, and kept at room temperature for 360 s for different electrode units than S2 to form an AuNPs layer. Then, the electrode was immersed in a CHI-Au mixture (0.2 wt% chitosan solution and 2 mg / mL HAuCl4 solution mixed at a volume ratio of 7:1) at pH approximately 5, and a -1.2 V potential was applied for deposition for 90 s. It was then soaked in PBS for 20 min to stabilize the CHI-Au layer. Finally, the electrode was immersed in 3 µM dsDNA. EC Solution (aptamer Apt) EC With cDNA EC Mix the ingredients in equal volumes and place them in 1×PBS. Incubate at 95°C for 5 min, then slowly cool to room temperature. Add EDC / NHS mixture and activate at 37°C for 2 h before rinsing. Incubate at 37°C for 12 h before rinsing with PBS. Comparative Example 3

[0033] Basically the same as Example 1, except that: AuNPs / CHI-Au / dsDNA EC The preparation of the modification layer specifically includes the following steps: First, the MEA electrode prepared by S2 was immersed in a 10 mmol / L sodium acetate solution containing 2 mg / mL HAuCl4. A 0.6 V potential was applied to the electrode unit (different from S2) for 360 s to deposit AuNPs. Then, the electrode was immersed in a CHI-Au mixture (0.2 wt% chitosan solution and 2 mg / mL HAuCl4 solution mixed at a volume ratio of 7:1) at approximately pH 5, kept at room temperature for 90 s, and then soaked in PBS for 20 min to stabilize the CHI-Au layer. Finally, the electrode was immersed in 3 µM dsDNA. ECSolution (aptamer Apt) EC With cDNA EC Mix the ingredients in equal volumes and place them in 1×PBS. Incubate at 95°C for 5 min, then slowly cool to room temperature. Add EDC / NHS mixture and activate at 37°C for 2 h before rinsing. Incubate at 37°C for 12 h before rinsing with PBS. Test Example 1

[0034] Based on Example 1, the preparation of GO-PPy / dsDNA at different potentials (0.4V-0.6V) (vs. Ag / AgCl) was investigated. SA The effect of the modification layer on the electrochemical signal; preparation of GO-PPy / dsDNA SA After modification, the initial peak current was recorded using square wave voltammetry (SWV), with detection parameters set to a potential range of -0.5V to 0V, a frequency of 15Hz, and an amplitude of 25mV. Subsequently, a concentration of 10... 5 Staphylococcus aureus at CFU / mL was incubated at 37℃ for 1 h, washed with PBS, and the peak current was detected again by SWV. The change in peak current ΔI between the two tests was calculated, and the results are as follows: Figure 2 As shown. From Figure 2 It can be seen that when the deposition potential increases from 0.4V to 0.55V, ΔI shows a continuous upward trend, indicating that increasing the potential within this range is beneficial to enhancing the electrochemical response signal of the electrode. However, when the potential exceeds 0.55V, ΔI decreases significantly, and the signal performance weakens. This is because when the potential is too low, the deposition amount of GO / PPy is insufficient, resulting in fewer active sites on the electrode; while when the potential is too high, the deposition of GO-PPy structures may lead to increased aggregation / defects, which in turn reduces the electrochemical response capability. Test Example 2

[0035] Based on Example 1, the effects of CHI-Au layers prepared at different potentials (-1.0V to -1.4V) on AuNPs / CHI-Au / dsDNA were investigated. EC The influence of the modified layer on the electrochemical signal; preparation of AuNPs / CHI-Au / dsDNA EC After modification, the initial peak current was recorded using square wave voltammetry (SWV); subsequently, a concentration of 10% was added to the system. 5 CFU / mL of E. coli was incubated at 37°C for 1 hour, washed with PBS, and the peak current was detected again by SWV. The change in peak current ΔI between the two measurements was calculated, and the results are as follows: Figure 3 As shown. From Figure 3It can be seen that when the deposition potential increases from -1.0V to -1.2V, ΔI shows a continuous upward trend, indicating that increasing the potential within this range is beneficial to enhancing the electrochemical response signal of the electrode. However, when the potential exceeds -1.2V, ΔI decreases significantly, and the signal performance weakens. This is because when the potential is too low, the deposition amount of CHI-Au is insufficient, resulting in fewer active sites on the electrode; while when the potential is too high, the deposition of CHI-Au structures may lead to increased aggregation / defects, which in turn reduces the electrochemical response capability. Test Example 3

[0036] Based on Example 1, cyclic voltammetry (CV) was used in a solution of 5 mmol / L potassium ferricyanide / potassium ferrocyanide ([Fe(CN)6]). 3 / 4- In an electrolyte solution composed of potassium chloride and 100 mmol / L, the changes in current signal of the electrode after each modification step were measured, with the scanning range set from -0.2 V to 0.6 V. Under optimal electrochemical analysis conditions, the electrochemical biosensors constructed in Example 1 and Comparative Examples 1-3 were used to detect Staphylococcus aureus and Escherichia coli, with the bacterial concentration gradient set to 10. 1 -10 8 The concentration was CFU / mL. The initial peak current was recorded using square wave voltammetry (SWV). Subsequently, different concentrations of Staphylococcus aureus and Escherichia coli were added to the detection system and incubated at 37°C for 1 hour. After rinsing with phosphate-buffered saline (PBS), the peak current was detected again using SWV. The change in peak current ΔI between the two measurements was calculated. A linear curve was obtained by fitting the data points with the logarithm of the Staphylococcus aureus concentration as the x-axis and ΔI as the y-axis. The relevant results are shown below. Figures 4-6 As shown.

[0037] from Figure 4 It can be seen that, compared with the unmodified electrode, the redox peak signal of the modified electrode after GO-PPy electrodeposition is weakened. This phenomenon, combined with the extreme semiconductor properties of GO, confirms that the GO-PPy interface has been successfully deposited on the gold electrode surface; dsDNA with a negatively charged phosphate backbone was introduced. SA Subsequently, electrostatic repulsion occurs between it and the similarly negatively charged iron / ferricyanide redox probe, causing a further decrease in peak current, thus confirming the presence of dsDNA. SA Successfully immobilized on the GO-PPy electrodeposition electrode; when MB molecules insert into dsDNA SA Following the structural design, a distinct oxidation peak appeared at approximately -0.30V, indicating that MB successfully inserted into dsDNA. SA It also generates a stable electrochemical signal; after incubating Staphylococcus aureus on the surface of the GO-PPy electrodeposition electrode, the SWV signal peak is significantly reduced, which proves that... S. aureus With aptamer Apt SASpecific binding occurs, leading to the dissociation of complementary DNA and MB from the electrode surface. In contrast, the GO-PPy electrode modified by the impregnation method did not show significant changes in its cyclic voltammetric redox peak after dsDNA immobilization, and no obvious MB signal was detected near -0.30V during SWV measurement, indicating the site-selective electrodeposition characteristics of GO-PPy.

[0038] from Figure 5 It can be seen that, due to the high active surface area formed by the aggregation of AuNPs, the electrodeposition significantly enhances the redox peak current, confirming that Au has been successfully deposited on the gold electrode surface; after the deposition of the CHI-Au layer, the electrode exhibits improved performance against [Fe(CN)6]. 3 / 4- The electrochemical performance of the redox pair was enhanced, specifically by increased current intensity and a narrowing of the redox peak potential difference. The reduced peak spacing further corroborated the successful deposition of the coating. Subsequently, dsDNA with a negatively charged phosphate backbone was immobilized. EC This caused the peak current to decrease further, confirming dsDNA EC The MB was successfully immobilized on the surface of the modified layer; after MB insertion, an oxidation peak of approximately 11 nA appeared at approximately -0.30 V, indicating that the MB had been successfully inserted into the double-stranded DNA. EC It generates a stable electrochemical signal; after incubating E. coli on the electrode surface, the SWV signal peak is significantly reduced, indicating that E. coli interacts with the aptamer Apt. EC Specific binding occurs, leading to the dissociation of complementary DNA and MB from the electrode surface. The CV and SWV curves for the control group, corresponding to two modification methods—immersion in AuNPs / electrodeposition of CHI-Au and electrodeposition of AuNPs / immersion in CHI-Au—show that the redox peaks of the CV curves did not change significantly before and after immersion modification, and even when MB molecules are embedded in dsDNA... EC The SWV curve did not show a significant signal peak near -0.3V. The above series of experimental results successfully confirmed that selective modification of the electrode with AuNPs and CHI-Au can be achieved by site-selective electrodeposition.

[0039] from Figure 6 It can be seen that the logarithmic value of the Staphylococcus aureus concentration has a significant linear relationship with the change in its current response (ΔI), and the corresponding log-linear regression equation is y=0.331 lgC. S. aureus +0.204 (R) 2 =0.991, y represents the change in current ΔI*, C S. aureusThe concentration of Staphylococcus aureus (σ represents the standard deviation) was used to calculate the limit of detection (LOD) for Staphylococcus aureus using the 3σ / slope rule, which yielded a result of 25.4 CFU / mL. Similarly, the logarithm of the Escherichia coli concentration showed a significant linear relationship with the change in its current response (ΔI*), with the corresponding log-linear regression equation being y = 1.551lgC. E. coli +1.022 (R) 2 =0.996, y represents the change in current ΔI*, C E. coli (representing the concentration of E. coli), and calculated using the same method, the limit of detection (LOD) for E. coli by the sensor is 8.8 CFU / mL. Test Example 4

[0040] Staphylococcus aureus / Escherichia coli, Pseudomonas aeruginosa, Salmonella paratyphi, Enterococcus faecalis, and a blank control were used as detection targets. Square wave voltammetry (SWV) was used to determine the current change (ΔI) corresponding to different samples: First, the initial peak current of the electrochemical aptamer sensor based on the microelectrode array in Example 1 was recorded using square wave voltammetry (SWV). Then, a concentration of 10... 3 Each bacterial sample at CFU / mL was incubated at 37℃ for 1 h, washed with PBS, and then the peak current was detected again by SWV. The change in peak current ΔI between the two measurements was calculated, and the data points were fitted with the logarithm of the Staphylococcus aureus concentration on the x-axis and ΔI on the y-axis to obtain the corresponding linear curve. The results are as follows: Figure 7 As shown. From Figure 7 As can be seen from the detection graph for Staphylococcus aureus on the left, its corresponding ΔI (approximately 1.0 nA) is significantly higher than that of Escherichia coli (ΔI≈0.1 nA), Pseudomonas aeruginosa (ΔI≈0.3 nA), Salmonella paratyphi (ΔI≈0.2 nA), Enterococcus faecalis (ΔI≈0.05 nA), and the blank group (ΔI≈0.1 nA). On the right, in the detection graph for Escherichia coli, its ΔI (approximately 4.5 nA) is also much higher than that of Staphylococcus aureus (ΔI≈0.8 nA), Pseudomonas aeruginosa (ΔI≈0.2 nA), Salmonella paratyphi (ΔI≈0.1 nA), Enterococcus faecalis (ΔI≈0.3 nA), and the blank group (ΔI≈0.1 nA). The ΔI values ​​for the other bacteria are not significantly different from those of the blank group. This indicates that the microelectrode array electrochemical aptamer sensor specifically binds to Staphylococcus aureus and Escherichia coli, triggering a significant electrochemical signal response, while having no specific recognition effect on other interfering bacteria, thus demonstrating the high selectivity of the sensor for target bacteria. Test Example 5

[0041] Lake water was used as the actual sample for analysis. The standard additive method was used to evaluate the sensor's detection performance on the actual sample, and three parallel tests were performed for each sample. The results are shown in Table 1. Table 1

[0042] As shown in Table 1, in lake water supplemented with standard strains, the recovery rate of Staphylococcus aureus fluctuated between 93.53% and 99.33%, with a relative standard deviation (RSD) of 2.91% to 6.31%; the recovery rate of Escherichia coli ranged from 96.1% to 100.47%, with a corresponding RSD of 3.4% to 9.51%. This indicates that the standard strains possess both high accuracy and precision in actual water sample testing, effectively addressing the drawbacks of traditional testing methods, such as complex operation, long processing time, and weak anti-interference capabilities. This demonstrates its promising application prospects in rapid on-site screening scenarios with complex matrices.

[0043] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An electrochemical aptamer sensor based on a microelectrode array, characterized in that, include: The microelectrode array is composed of several micron-sized gold electrode units arranged in an orderly manner, with each electrode unit independently realizing signal acquisition and analysis. Modification layer, including GO-PPy / dsDNA SA Modified layer and AuNPs / CHI-Au / dsDNA EC The modification layer is electrodeposited on the surface of different electrode units of the microelectrode array; the GO-PPy / dsDNA SA The modification layer consists of graphene oxide-polypyrrole composite material and dsDNA. SA Composition; the AuNPs / CHI-Au / dsDNA EC The modification layer consists of gold nanoparticles, chitosan-gold composite material, and dsDNA. EC composition; Redox molecules, which are electroactive molecules with nitrogen-containing heteroaromatic ring structures, are embedded in the dsDNA. SA and the dsDNA EC internal.

2. The electrochemical aptamer sensor based on a microelectrode array according to claim 1, characterized in that, The dsDNA SA It is composed of aptamers of equal volume, Apt. SA With cDNA SA The mixture was prepared by hybridization with PBS buffer and then annealing. And / or, the dsDNA EC It is composed of aptamers of equal volume, Apt. EC With cDNA EC The mixture was prepared by hybridization with PBS buffer and then annealing. And / or, the electroactive molecule with the nitrogen-containing heteroaromatic ring structure is selected from one or more of methylene blue, Nile blue, and anthraquinone.

3. The electrochemical aptamer sensor based on a microelectrode array according to claim 2, characterized in that, The aptor Apt SA The sequence is: 5'-NH2-GCAATGGTACGGTACTTCCTCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACAGCTACGTCAAAAGTGCACGCTACTTTGCTAA-3'; And / or, the cDNA SA The sequence is: 5'-TTAGCAAAGTAGCCACGTGAGC-3'; And / or, the aptamer Apt EC The sequence is: 5'-COOH-CCATGAGTGTTGTGAAATGTTGGGACACTAGGTGGCATAGAGCCG-3'; And / or, the cDNA EC The sequence is: 5'-GCTCTATGCCACCTAGTGTC-3'.

4. The method for fabricating an electrochemical aptamer sensor based on a microelectrode array as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Immerse the microelectrode array in GO-PPy solution, apply a potential to one side of the electrode unit of the microelectrode array to perform electrodeposition, activate the resulting electrode after electrodeposition, and then add dsDNA. SA The solution was incubated to form GO-PPy / dsDNA SA Modification layer; S2. Electrodeposition is performed by sequentially applying potentials to the other electrode units of the microelectrode array obtained in S1 in HAuCl4 solution and CHI-Au solution; after electrodeposition, activated dsDNA is added. EC Incubation in solution forms AuNPs / CHI-Au / dsDNA EC Modification layer; S3. The microelectrode array obtained in S2 is immersed in a redox molecular solution for incubation to obtain the electrochemical aptamer sensor based on the microelectrode array.

5. The method for fabricating an electrochemical aptamer sensor based on a microelectrode array according to claim 4, characterized in that, In S1, the concentration of graphene oxide in the GO-PPy solution is 0.08 mg / mL-0.12 mg / mL, the concentration of lithium perchlorate is 18 mmol / L-22 mmol / L, and the concentration of pyrrole is 2.8 wt‰-3.2 wt‰. And / or, the electrodeposition process is as follows: potential of 0.45V-0.6V, time of 580s-620s.

6. The method for fabricating an electrochemical aptamer sensor based on a microelectrode array according to claim 4, characterized in that, In S2, the concentration of chloroauric acid in the HAuCl4 solution is 1.8 mg / mL-2.2 mg / mL, and the concentration of sodium acetate is 9.5 mmol / L-10.5 mmol / L; And / or, the electrodeposition process corresponding to the HAuCl4 solution is: a potential of 0.55V-0.65V and a time of 350s-370s; And / or, the CHI-Au solution is obtained by mixing chitosan solution and chloroauric acid solution at a volume ratio of (6.5-7.5):1; the concentration of the chitosan solution is 0.18wt%-0.22wt%; and the concentration of the chloroauric acid solution is 1.8mg / mL-2.2mg / mL. And / or, the electrodeposition process corresponding to the CHI-Au solution is: a potential of -1.1V to -1.4V and a time of 85s to 95s.

7. The method for fabricating an electrochemical aptamer sensor based on a microelectrode array according to claim 4, characterized in that, In S3, the concentration of redox molecules in the redox molecule solution is 1.3 mmol / L-1.5 mmol / L.

8. The method for fabricating an electrochemical aptamer sensor based on a microelectrode array according to claim 4, characterized in that, The activation solution used is an EDC / NHS mixture.

9. The method for fabricating an electrochemical aptamer sensor based on a microelectrode array according to claim 4, characterized in that, The dsDNA SA Solution and the dsDNA EC The concentrations of the solutions independently ranged from 2.8 µmol / L to 3.2 µmol / L.

10. The application of the electrochemical aptamer sensor based on microelectrode array as described in any one of claims 1-3 in the detection of Staphylococcus aureus and Escherichia coli.

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