Aptamer sensor, preparation method thereof and method for detecting metal ions by using aptamer sensor

Abstract

The invention relates to the technical field of electrochemical sensors, in particular to an aptamer sensor, a preparation method of the aptamer sensor and a detection method of the aptamer sensor on metal ions, and the method comprises the following steps: S1, spin-coating photoresist on the surface of a gold electrode, performing soft baking, covering the surface of the photoresist with a mask plate, and performing exposure, then post-baking the photoresist, and constructing a detection site through developing solution treatment; s2, immersing the gold electrode into a zinc salt-containing benzyl alcohol solution, and circularly scanning potential under an oil bath condition to form a nano-porous gold structure; s3, depositing a reference electrode and a counter electrode at each detection site; s4, dispensing a specific aptamer solution of target metal ions at each detection site, self-assembling and fixing an aptamer through an Au-S bond, and performing room-temperature incubation; then flushing the aptamer by using a PBS (Phosphate Buffer Solution), and sealing a non-specific site of the aptamer by using 1mM of mercaptoethanol; according to the invention, high-precision synchronous fast detection of four kinds of target metal ions can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of electrochemical sensors, in particular to an aptamer sensor, a preparation method thereof and a detection method of metal ions. BACKGROUND

[0002] In the fields of food safety monitoring, drinking water quality control and industrial wastewater treatment, there is an increasing demand for high-sensitivity and multi-target simultaneous detection of heavy metal ions. For example, it is necessary to monitor the heavy metal residues in raw materials in real time during food processing, to accurately quantify trace metal ions in drinking water sources, and to simultaneously analyze the concentrations of various ions in wastewater to meet environmental protection standards in the electroplating industry. However, traditional detection methods such as atomic absorption spectroscopy and inductively coupled plasma mass spectrometry rely on large equipment and complex sample pretreatment, making it difficult to meet the demand for on-site rapid screening.

[0003] Through retrieval, Chinese Patent Publication No. CN115825175A discloses an electrochemical POCT aptamer sensor and its construction method and application. The electrochemical detection sensor of the invention uses PET as the substrate, PDMS as the electrolytic cell, and methylene blue doped metal organic framework modified aptamer probes. The modified electrode uses gold nanoparticles as the substrate, one end of which is modified with an electroactive molecule, nucleic acid aptamer, connected to the gold nanoparticles through a chemical bond. The four working electrodes share the same pair of electrodes and reference electrodes. The electrochemical sensor is placed in a solution containing the heavy metal ions to be detected. After the heavy metal ions are specifically recognized by the electrode, the aptamer configuration changes, causing the electroactive molecule to approach the surface of the working electrode, resulting in an increase in the current signal. The invention can efficiently and sensitively detect heavy metal ions in samples on site.

[0004] The related technology in the above has the following defects: although the invention can simultaneously detect Cd²⁺, Hg²⁺, Pb²⁺ and As³⁺, the competitive binding of different ion aptamers on the same electrode surface will cause cross interference and potential crosstalk problems. The current path between adjacent electrodes overlaps, and when Pb²⁺ / Cd²⁺ is simultaneously detected, the current increase caused by high concentration of Cd²⁺ will be coupled to the Pb²⁺ detection area through the shared electrolytic cell, causing false positive signals. At the same time, the aptamer competition failure phenomenon is significant. When As³⁺ and Hg²⁺ coexist, the Hg²⁺ aptamer preferentially occupies the active sites on the surface of the gold nanoparticles, reducing the binding efficiency of the As³⁺ aptamer. In addition, the invention does not consider the interference of sulfides and organic acids in industrial wastewater. Sulfides form a passivation layer with gold nanoparticles, increasing the electron transfer resistance and causing the Pb²⁺ detection signal to decay. Organic acids form complexes with Hg²⁺, blocking the binding of aptamers, resulting in low Hg²⁺ recovery rate in wastewater samples and affecting the accuracy of metal ion detection. Therefore, improvement is needed. SUMMARY

[0005] In order to realize high-precision synchronous rapid detection of four target metal ions, the application provides an aptamer sensor, a preparation method thereof and a detection method of metal ions.

[0006] In a first aspect, the application provides a preparation method of an aptamer sensor, which adopts the following technical scheme: the preparation method of the aptamer sensor comprises the following steps: S1, first spin-coat photoresist on the surface of a gold electrode and perform soft baking, then cover a mask plate with a 4-region pattern on the surface of the photoresist and perform exposure, then post-bake the photoresist and treat it with a developing solution to expose the region pattern, thereby constructing four physically isolated detection sites; S2, immerse the exposed gold electrode in a zinc salt-containing benzyl alcohol solution and perform cyclic potential scanning under oil bath conditions, so that the gold electrode forms a nanoporous gold structure through the embedding and dissolution of zinc on the gold electrode; S3, deposit a reference electrode and a counter electrode at each detection site, and the reference electrode, the counter electrode and the gold electrode together form a locally independent three-electrode system; S4, drop-coat a specific aptamer solution of a target metal ion at each detection site, fix the aptamer through Au-S self-assembly and perform room temperature incubation, then rinse the aptamer with a PBS buffer and block the non-specific sites of the aptamer with 1 mM mercaptoethanol.

[0007] Optionally, in the step S1, after the detection sites are constructed, etch a separation groove between adjacent detection sites to form a physical separation strip, and fill perfluorosilane in the separation groove to form a hydrophobic barrier.

[0008] In a second aspect, the application provides an aptamer sensor, which adopts the following technical scheme: the aptamer sensor is prepared by the above-mentioned preparation method of the aptamer sensor.

[0009] In a third aspect, the application provides a detection method of metal ions by the aptamer sensor, which adopts the following technical scheme: the detection method of metal ions by the aptamer sensor, which adopts the above-mentioned aptamer sensor to detect metal ions, comprises the following steps: S1, divide the sample solution to be detected into four independent sample inlets through a microfluidic channel, and combine the separation grooves to limit the diffusion of droplets, so that the sample solution enters the corresponding detection sites; S2, use a multi-channel electrochemical workstation to synchronously collect the differential pulse voltammetry signals of each detection site; S3, analyzing the current change value based on the differential signal response mechanism: at the detection site of Pb²⁺ and As³⁺, the conformation of the aptamer sensor changes after binding to the target ion, the electron transfer resistance increases, the current decreases, and the current change value is negatively correlated with the concentration; at the detection site of Cd²⁺ and Hg²⁺, the electroactive group is exposed after the aptamer sensor binds to the target ion, promoting electron transfer, increasing the current, and the current change value is positively correlated with the concentration; S4, quantifying the concentration of each metal ion by a pre-calibration curve to realize the synchronous detection of Pb²⁺, Cd²⁺, Hg²⁺ and As³⁺.

[0010] Optionally, the step S1 comprises the following steps: S11, bonding the polydimethylsiloxane substrate and the polymethyl methacrylate cover sheet by oxygen plasma bonding technology, and then designing four independent sample inlets on the cover sheet, each inlet being connected to a corresponding detection site through a split microchannel; S2, injecting the sample solution to be tested into the sample inlet by gravity driving or a micro piezoelectric pump, and realizing the synchronous delivery of the sample solution and the buffer by combining the double-channel design, i.e. the upper channel delivers the sample and the lower channel delivers the buffer.

[0011] Optionally, in the step S2, 0.2 mM EDTA is added to the detection buffer of the multi-channel electrochemical workstation to selectively chelate Cd²⁺ and Hg²⁺ interference ions and eliminate their cross-reaction on the detection area of Pb²⁺ and As³⁺.

[0012] Optionally, a cross-validation mechanism is added between the step S3 and the step S4, and the cross-validation mechanism comprises the following steps: S31, at the detection site of Pb²⁺ and As³⁺, the hairpin loop part of the aptamer sensor is designed as a G-quadruplex sequence, then a thiol group is modified at the 5' end to fix it on the gold electrode surface, and then a T7 promoter sequence is designed at the 3' end; At the detection site of Cd²⁺ and Hg²⁺, the hairpin stem part of the aptamer sensor is embedded into a rolling circle amplification primer binding domain, then a biotin is modified at the 5' end, and then the bonding with the magnetic beads is realized through the specific binding of biotin and streptavidin; S32, when Pb²⁺ or As³⁺ binds to the aptamer sensor, the hairpin structure is first triggered to open to expose the T7 promoter, then T7 RNA polymerase and circular DNA template are added, and a rolling circle amplification reaction is started to generate long single-stranded DNA rich in G-quadruplex structure; When Cd²⁺ or Hg²⁺ binds to the aptamer sensor, the hairpin structure is first triggered to unwind to release the rolling circle amplification primer, then the primer binds to the circular DNA template, and then the Phi29 DNA polymerase catalyzes the amplification to generate repetitive sequence DNA; S33. First, add thioflavone T to the rolling circle amplification product to allow thioflavone T to intercalate into the G-quadruplex or double-stranded DNA; simultaneously, use differential pulse voltammetry to detect the oxidation peak current of thioflavone T, and then construct a self-validation model by the ratio of fluorescence intensity change value to current change value to eliminate background noise interference. S34. First, a magnetic separation unit is integrated into the bonding structure between the substrate and the cover plate in step S11. Cd²⁺ and Hg²⁺ are enriched by magnetic beads during sample injection. Then, lyophilized microspheres preloaded with circular DNA templates are used to compress the rolling circle amplification reaction time. Finally, the ion concentration signal is output through dual-mode cross-validation.

[0013] Optionally, it is suitable for the simultaneous detection of Pb²⁺, Cd²⁺, Hg²⁺, and As³⁺ in water, food, and industrial samples. When the sample is solid, the sample is ultrasonically extracted with 0.1 M HNO₃ solution for 10 minutes, and the supernatant is taken for detection after centrifugation.

[0014] In summary, this application includes the following beneficial technical effects: This application integrates physical isolation, nanoporous gold structure, microfluidic splitting, chelation anti-interference and cross-validation machine to achieve simultaneous detection of four target ions. It only takes 15 minutes from sample processing to output results, which greatly improves detection efficiency. The detection result deviation is less than 5% even with 200 times the interference ions, thus meeting the high-precision on-site rapid detection needs of food, water quality and industrial scenarios. The photolithography process enables precise microscale patterning of detection sites. The synergistic effect of isolation trenches and hydrophobic barriers significantly reduces the solution diffusion rate of adjacent detection sites, completely eliminating cross-contamination in multi-target detection. At the same time, it enhances the mechanical stability of the electrode surface, thereby improving the uniformity of subsequent electrochemical deposition of nanoporous gold structures. Oil bath temperature control reduced the viscosity of the benzyl alcohol solution, accelerated the zinc ion mass transfer rate, and improved the uniformity of zinc ion insertion and dissolution reactions. At the same time, precise control of pore depth through 100 cycles of potential scanning expanded the effective specific surface area of ​​the gold electrode to more than 100 times that of the original gold electrode. It also formed a vertically oriented pore structure, providing high-density anchoring points for subsequent aptamer fixation, and significantly enhanced electron transfer efficiency and improved the detection current signal. By physically isolating and designing independent micro-area circuits, potential crosstalk during multi-channel detection is completely eliminated, reducing impedance fluctuations between electrodes. At the same time, the microscale integrated local independent three-electrode system reduces electrolyte consumption and significantly improves detection efficiency. The sample solution is precisely diverted to the corresponding detection site through capillary action and gravity, while the hydrophobic barrier of the isolation groove limits the diffusion area of ​​the droplets, completely avoiding crosstalk in multi-target detection. The cross-validation mechanism triggers a specific rolling circle amplification path after the target metal ions bind, and a self-validation model is constructed by the ratio of fluorescence intensity change to current change, eliminating most of the background noise interference. At the same time, this mechanism significantly reduces the false positive rate and improves the anti-interference ability in complex samples such as industrial wastewater. Attached Figure Description

[0015] Figure 1 This is a flowchart of the preparation method of the aptamer sensor in Embodiment 1 of this application; Figure 2 This is a flowchart of the method for detecting metal ions using an aptamer sensor according to Embodiment 5 of this application; Figure 3 This is a flowchart of step S3 in embodiment 5 of this application. Detailed Implementation

[0016] The following is in conjunction with the appendix Figures 1-3 This application will be described in further detail. Example 1

[0017] This application discloses a method for preparing an aptamer sensor. For example... Figure 1 As shown, the method for fabricating an aptamer sensor includes the following steps: S1. First, spin-coat photoresist onto the surface of the gold electrode and perform soft baking. Then, cover the photoresist surface with a mask having 4 area patterns and expose it. Then, bake the photoresist and treat it with a developer to expose the area patterns, thus constructing 4 physically isolated detection sites. S2. Immerse the gold electrode in the exposed area in a benzyl alcohol solution containing zinc salt, and scan the potential cyclically under oil bath conditions. Through the embedding and dissolution of zinc in the gold electrode, the gold electrode forms a nanoporous gold structure. S3. Deposit a reference electrode and a counter electrode at each detection site. The reference electrode, counter electrode, and gold electrode together form a locally independent three-electrode system. S4. Drop a solution of the target metal ion specific aptamer onto each detection site, fix the aptamer by self-assembly via Au-S bonds, and incubate at room temperature; then rinse the aptamer with PBS buffer and block the non-specific sites of the aptamer with 1 mM mercaptoethanol.

[0018] Specifically, the implementation method of step S1 is as follows: First, a 1.5 μm thick positive photoresist is spin-coated onto the gold electrode surface at 1500 rpm, followed by soft baking on an 80℃ hot plate for 90 seconds to remove solvent; then, a chromium mask with four circular area patterns is tightly covered on the photoresist surface, the four circular area patterns having a diameter of 500 μm and a spacing of 200 μm, and then exposed to a 365 nm ultraviolet light source at an intensity of 10 mW / cm² for 8 seconds to induce a photochemical reaction in the photoresist of the mask pattern area; immediately after exposure, the photoresist is post-baked on a 110℃ hot plate for 60 seconds to strengthen cross-linking, and then immersed in a 2.38% tetramethylammonium hydroxide developer solution with vibration treatment for 40 seconds to completely dissolve the photoresist in the unexposed areas, thereby precisely constructing four physically isolated circular detection sites on the gold electrode surface; subsequently, reactive ion etching technology is used to etch between adjacent detection sites with a depth of 10 μm and a width of 50 μm. A μm isolation trench is formed, and perfluorooctyltriethoxysilane is filled in the isolation trench to form a hydrophobic barrier.

[0019] Step S1 achieves precise microscale patterning of detection sites through photolithography. The synergistic effect of isolation trenches and hydrophobic barriers significantly reduces the solution diffusion rate of adjacent detection sites, completely eliminating cross-contamination in multi-target detection. At the same time, it enhances the mechanical stability of the electrode surface to improve the uniformity of subsequent electrochemical deposition of nanoporous gold structures.

[0020] Specifically, the implementation method of step S2 is as follows: First, the exposed area of ​​the gold electrode after photolithography treatment in step S1 is immersed in a benzyl alcohol solution containing 0.8 M zinc chloride. Then, the solution is placed in a constant temperature oil bath at 80°C to maintain the stability of the reaction temperature. Subsequently, an electrochemical workstation is used to perform 100 cyclic potential scans in the range of -1.0 V to +0.5 V at a scan rate of 20 mV / s. Zinc ions are embedded in the gold electrode lattice at a negative potential to form an Au-Zn alloy phase, and zinc ions are selectively dissolved at a positive potential. Finally, a pore size of 50-80 nm and a pore density of 5 × 10¹ are formed in the exposed area. 0 Three-dimensional interconnected nanoporous gold structures with pores / cm².

[0021] Step S2 reduces the viscosity of the benzyl alcohol solution by controlling the temperature in an oil bath, accelerating the zinc ion mass transfer rate and improving the uniformity of zinc ion insertion and dissolution reactions. At the same time, step S2 precisely controls the pore depth through 100 cycles of potential scanning, increasing the effective specific surface area of ​​the gold electrode to more than 100 times that of the original gold electrode. It also forms a vertically oriented pore structure, providing high-density anchoring points for subsequent aptamer fixation and significantly enhancing electron transfer efficiency and improving the detection current signal.

[0022] Specifically, the implementation method of step S3 is as follows: First, on the detection site of the nanoporous gold structure formed in step S2, an Ag / AgCl reference electrode with a diameter of 200 μm is deposited at a distance of 30 μm from the edge of the gold electrode using magnetron sputtering technology. At the same time, platinum paste is coated on the opposite side at a distance of 40 μm from the gold electrode using microdispensing technology. The platinum paste is sintered at 350℃ for 30 minutes to form a platinum counter electrode array with a diameter of 300 μm, so that each detection site forms a locally independent three-electrode system centered on the nanoporous gold electrode.

[0023] Step S3 completely eliminates potential crosstalk during multi-channel detection through physical isolation and independent micro-area circuit design, reduces impedance fluctuations between electrodes, and at the same time, the microscale integrated local independent three-electrode system reduces the amount of electrolyte used and significantly improves detection efficiency.

[0024] Specifically, the implementation method of step S4 is as follows: First, 0.5 μL of a specific aptamer solution of the target metal ion is precisely drop-coated onto each detection site of the nanoporous gold structure. Then, utilizing the high specific surface area and vertical pore structure of the nanoporous gold structure, the thiol groups at the ends of the aptamers are self-assembled and fixed to the inner wall of the pores via Au-S bonds. The assembled parts are then incubated at room temperature in the dark for 12 hours to achieve directional anchoring of the aptamers in three-dimensional space. After incubation, the assembled parts are rinsed three times with 0.1 M PBS buffer at a flow rate of 50 μL / s to completely remove unbound aptamer molecules. Subsequently, the gold electrode is immersed in a 1 mM mercaptoethanol solution for 1 hour to block it, allowing short-chain thiol molecules to competitively occupy the remaining vacancy sites on the surface of the gold electrode, forming an anti-biocontamination monolayer.

[0025] Compared to planar electrodes, step S4 significantly improves aptamer binding efficiency through three-dimensional construction, and the encapsulation of mercaptoethanol solution reduces non-specific adsorption of gold electrodes in complex samples, ensuring the specificity of multi-target simultaneous detection.

[0026] In summary, this method reduces the solution diffusion rate of adjacent detection sites by constructing four physically isolated detection sites, isolation trenches, and a hydrophobic barrier, thus completely eliminating cross-contamination in multi-target detection. The effective specific surface area is expanded through a three-dimensional nanoporous gold structure, and high-density anchoring points are provided for aptamer fixation through vertical channels, improving electron transfer rate. A locally independent three-electrode system reduces multi-channel potential crosstalk and reduces electrolyte consumption. The directional self-assembly of aptamers and Au-S bonds within the nanopores, along with the encapsulation with mercaptoethanol, reduces non-specific adsorption and improves anti-interference performance in complex samples. Furthermore, the aptamer binding stability and mechanical strength ensure the long-term reliability of the aptamer sensor in food, water, and industrial applications. Example 2

[0027] Before step S1 in Example 1, the gold electrode needs to be pretreated, that is, the gold electrode is first polished by sandpaper, and then soaked in Piranha solution to remove impurities on the surface of the gold electrode, thereby obtaining a clean and flat surface. Example 3

[0028] The aptamer preparation method in step S4 of Example 1 is as follows: aptamer sequences with high specific binding ability to target metal ions are screened by exponential enrichment ligand system evolution technology, and then a professional biotechnology company is commissioned to synthesize the aptamers. The synthesized aptamers are purified by high performance liquid chromatography. Example 4

[0029] This embodiment discloses an aptamer sensor. The aptamer sensor is prepared by the method of Example 1. Example 5

[0030] This embodiment discloses a method for detecting metal ions using an aptamer sensor. For example... Figure 2 As shown, the method for detecting metal ions using an aptamer sensor, employing the aptamer sensor of Example 4, includes the following steps: S1. The sample solution to be tested is diverted to 4 independent inlets through the microfluidic channel, and the droplet diffusion is restricted by the isolation groove, so that the sample solution enters the corresponding detection site respectively; S2. A multi-channel electrochemical workstation is used to simultaneously acquire differential pulse voltammetric signals at each detection site; S3. Analysis of current change values ​​based on differential signal response mechanism: At the detection sites of Pb²⁺ and As³⁺, the aptamer sensor will undergo a conformational change after binding to the target ion, increasing the electron transfer resistance and decreasing the current, and the current change value is negatively correlated with the concentration; at the detection sites of Cd²⁺ and Hg²⁺, the aptamer sensor will expose electroactive groups after binding to the target ion, promoting electron transfer, increasing the current, and the current change value is positively correlated with the concentration. S4. The concentration of each metal ion is quantified by pre-calibration curve, realizing the simultaneous detection of four ions: Pb²⁺, Cd²⁺, Hg²⁺, and As³⁺.

[0031] Specifically, step S1 includes the following steps: S11. The polydimethylsiloxane substrate is bonded to the polymethyl methacrylate cover using oxygen plasma bonding technology. Then, four independent injection ports are designed on the cover, and each injection port is connected to the corresponding detection site through a split microchannel. S2. The sample solution to be tested is injected into the inlet by gravity or a micro piezoelectric pump. Combined with the dual-channel design, the sample solution and buffer solution are delivered synchronously, that is, the upper channel delivers the sample and the lower channel delivers the buffer solution.

[0032] Step S1 uses capillary action and gravity to precisely divert the sample solution to the corresponding detection site. At the same time, the hydrophobic barrier of the isolation groove limits the diffusion area of ​​the droplets, thus completely avoiding crosstalk in multi-target detection.

[0033] Specifically, step S2 includes the following steps: S21. Add 0.2 mM EDTA to the detection buffer of the multi-channel electrochemical workstation and mix thoroughly by shaking to form a chelation buffer system. Based on the ultra-high binding constant of EDTA with Cd²⁺ and Hg²⁺, EDTA will selectively chelate free Cd²⁺ and Hg²⁺ interfering ions in the solution and generate stable water-soluble complexes, reducing the free concentration of interfering ions to below the detection limit, thereby completely blocking the non-specific adsorption and cross-reaction of interfering ions to the detection sites of Pb²⁺ and As³⁺. S22. The detection buffer containing EDTA is delivered to each detection site through the microfluidic lower layer channel, while the sample solution to be tested is injected synchronously through the upper layer channel. Then, the multi-channel electrochemical workstation is started, the differential pulse voltammetry parameters are set, and the current response signals of the four detection sites are acquired synchronously. Then, the stability of the current change values ​​of Pb²⁺ and As³⁺ detection sites before and after the addition of EDTA is compared to verify the elimination effect of cross-interference.

[0034] Step S2 involves adding 0.2 mM EDTA to form a chelation system, completely blocking the non-specific adsorption of interfering ions on the detection sites of Pb²⁺ and As³⁺. The dual-channel microfluidic design enables simultaneous delivery of the sample solution and buffer solution, improving the signal-to-noise ratio of the Pb²⁺ and As³⁺ detection signals. In industrial wastewater containing 200 times the concentration of coexisting ions, the cross-interference intensity of Cd²⁺ and Hg²⁺ on Pb²⁺ and As³⁺ will be significantly reduced, the spiked recovery rate of Pb²⁺ will be significantly improved, and the repeatability RSD of the current change value will be optimized to below 3%, thereby achieving accurate detection of the sample and reducing the overall detection time to 15 minutes, meeting the needs of rapid on-site screening.

[0035] like Figure 3 As shown, a cross-validation mechanism is added between step S3 and step S4. The cross-validation mechanism includes the following steps: S31. At the detection sites of Pb²⁺ and As³⁺, the hairpin loop of the aptamer sensor is first designed as a G-quadruplex sequence, then a thiol group is modified at the 5' end to fix it to the surface of the gold electrode, and finally a T7 promoter sequence is designed at the 3' end. At the detection sites of Cd²⁺ and Hg²⁺, the hairpin stem of the aptamer sensor is first embedded with the rolling loop amplification primer binding domain, and then biotin is modified at the 5' end. Finally, the binding with the magnetic beads is achieved through the specific binding of biotin and streptavidin. S32. When Pb²⁺ or As³⁺ binds to the aptamer sensor, the hairpin structure is first triggered to open and expose the T7 promoter. Then, T7 RNA polymerase and circular DNA template are added to initiate the rolling circle amplification reaction to generate long single-stranded DNA rich in G-quadruplex structures. When Cd²⁺ or Hg²⁺ binds to the aptamer sensor, the hairpin structure is first triggered to unwind and release the rolling circle amplification primers. The primers then bind to the circular DNA template and are amplified by Phi29 DNA polymerase to generate repetitive sequence DNA. S33. First, add thioflavone T to the rolling circle amplification product to allow thioflavone T to intercalate into the G-quadruplex or double-stranded DNA; simultaneously, use differential pulse voltammetry to detect the oxidation peak current of thioflavone T, and then construct a self-validation model by the ratio of fluorescence intensity change value to current change value to eliminate background noise interference. S34. First, a magnetic separation unit is integrated into the bonding structure between the substrate and the cover plate in step S11. Cd²⁺ and Hg²⁺ are enriched by magnetic beads during sample injection. Then, lyophilized microspheres preloaded with circular DNA templates are used to compress the rolling circle amplification reaction time. Finally, the ion concentration signal is output through dual-mode cross-validation.

[0036] The cross-validation mechanism enables the target metal ion to bind and trigger a specific rolling circle amplification pathway. It also constructs a self-validation model by comparing the ratio of fluorescence intensity change to current change, thus eliminating most background noise interference. At the same time, this mechanism significantly reduces the false positive rate and improves the anti-interference performance in complex samples such as industrial wastewater.

[0037] In summary, this detection method achieves precise sample solution splitting, and the hydrophobic barrier of the isolation trench suppresses the cross-contamination rate of adjacent detection sites, completely eliminating multi-target crosstalk. Furthermore, the chelation system formed by EDTA significantly reduces the signal interference in the Pb²⁺ and As³⁺ detection regions. A cross-validation mechanism simultaneously collects fluorescence intensity and current changes, thereby constructing a self-validating model to eliminate background noise and reduce the false positive rate. Ultimately, it achieves simultaneous detection of Pb²⁺, Cd²⁺, Hg²⁺, and As³⁺ within 15 minutes, with a detection result deviation of less than 5% even with 200 times the interference ions, thus meeting the high-precision on-site rapid detection needs in food, water quality, and industrial scenarios. Example 6

[0038] The detection method in Example 5 is applicable to the simultaneous detection of Pb²⁺, Cd²⁺, Hg²⁺, and As³⁺ in water, food, and industrial samples. When the sample is solid, it is ultrasonically extracted with 0.1 M HNO₃ solution for 10 minutes. This process efficiently releases bound heavy metal ions through acid hydrolysis, while the ultrasonic cavitation effect significantly improves ion dissolution efficiency. The sample is then centrifuged at 10,000 rpm for 5 minutes, and the supernatant is directly used for detection. The entire pretreatment process takes only 15 minutes. Compared to the national standard microwave digestion method, this method significantly improves efficiency and avoids reliance on high-temperature and high-pressure equipment, meeting the needs for rapid on-site screening of solid samples.

[0039] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for preparing an aptamer sensor, characterized in that: Includes the following steps: S1. First, spin-coat photoresist onto the surface of the gold electrode and perform soft baking. Then, cover the photoresist surface with a mask having 4 area patterns and expose it. Then, bake the photoresist and treat it with a developer to expose the area patterns, thus constructing 4 physically isolated detection sites. S2. Immerse the gold electrode in the exposed area in a benzyl alcohol solution containing zinc salt, and scan the potential cyclically under oil bath conditions. Through the embedding and dissolution of zinc in the gold electrode, the gold electrode forms a nanoporous gold structure. S3. Deposit a reference electrode and a counter electrode at each detection site. The reference electrode, counter electrode, and gold electrode together form a locally independent three-electrode system. S4. Drop a solution of the target metal ion specific aptamer onto each detection site, fix the aptamer by self-assembly via Au-S bonds, and incubate at room temperature; then rinse the aptamer with PBS buffer and block the non-specific sites of the aptamer with 1 mM mercaptoethanol.

2. The method for preparing the aptamer sensor according to claim 1, characterized in that: In step S1, after the detection sites are constructed, isolation trenches are etched between adjacent detection sites to form a physical separation zone, and perfluorosilane is filled in the isolation trenches to form a hydrophobic barrier.

3. An aptamer sensor, characterized in that: It is prepared by the method described in claim 2.

4. A method for detecting metal ions using an aptamer sensor, characterized in that: The detection of metal ions using the aptamer sensor as described in claim 3 includes the following steps: S1. The sample solution to be tested is diverted to 4 independent inlets through the microfluidic channel, and the droplet diffusion is restricted by the isolation groove, so that the sample solution enters the corresponding detection site respectively; S2. A multi-channel electrochemical workstation is used to simultaneously acquire differential pulse voltammetric signals at each detection site; S3. Analysis of current change values ​​based on differential signal response mechanism: At the detection sites of Pb²⁺ and As³⁺, the aptamer sensor will undergo a conformational change after binding to the target ion, increasing the electron transfer resistance and decreasing the current, and the current change value is negatively correlated with the concentration; at the detection sites of Cd²⁺ and Hg²⁺, the aptamer sensor will expose electroactive groups after binding to the target ion, promoting electron transfer, increasing the current, and the current change value is positively correlated with the concentration. S4. The concentration of each metal ion is quantified by pre-calibration curve, realizing the simultaneous detection of four ions: Pb²⁺, Cd²⁺, Hg²⁺, and As³⁺.

5. The detection method according to claim 4, characterized in that: Step S1 includes the following steps: S11. The polydimethylsiloxane substrate is bonded to the polymethyl methacrylate cover using oxygen plasma bonding technology. Then, four independent injection ports are designed on the cover, and each injection port is connected to the corresponding detection site through a split microchannel. S2. The sample solution to be tested is injected into the inlet by gravity or a micro piezoelectric pump. Combined with the dual-channel design, the sample solution and buffer solution are delivered synchronously, that is, the upper channel delivers the sample and the lower channel delivers the buffer solution.

6. The detection method according to claim 4, characterized in that: In step S2, 0.2 mM EDTA is added to the detection buffer of the multichannel electrochemical workstation to selectively chelate Cd²⁺ and Hg²⁺ interfering ions and eliminate their cross-reaction with the detection regions of Pb²⁺ and As³⁺.

7. The detection method according to claim 5, characterized in that: A cross-validation mechanism is added between steps S3 and S4. The cross-validation mechanism includes the following steps: S31. At the detection sites of Pb²⁺ and As³⁺, the hairpin loop of the aptamer sensor is first designed as a G-quadruplex sequence, then a thiol group is modified at the 5' end to fix it to the surface of the gold electrode, and finally a T7 promoter sequence is designed at the 3' end. At the detection sites of Cd²⁺ and Hg²⁺, the hairpin stem of the aptamer sensor is first embedded with the rolling loop amplification primer binding domain, and then biotin is modified at the 5' end. Finally, the binding with the magnetic beads is achieved through the specific binding of biotin and streptavidin. S32. When Pb²⁺ or As³⁺ binds to the aptamer sensor, the hairpin structure is first triggered to open and expose the T7 promoter. Then, T7 RNA polymerase and circular DNA template are added to initiate the rolling circle amplification reaction to generate long single-stranded DNA rich in G-quadruplex structures. When Cd²⁺ or Hg²⁺ binds to the aptamer sensor, the hairpin structure is first triggered to unwind and release the rolling circle amplification primers. The primers then bind to the circular DNA template and are amplified by Phi29 DNA polymerase to generate repetitive sequence DNA. S33. First, add thioflavone T to the rolling circle amplification product to allow thioflavone T to intercalate into the G-quadruplex or double-stranded DNA; simultaneously, use differential pulse voltammetry to detect the oxidation peak current of thioflavone T, and then construct a self-validation model by the ratio of fluorescence intensity change value to current change value to eliminate background noise interference. S34. First, a magnetic separation unit is integrated into the bonding structure between the substrate and the cover plate in step S11. Cd²⁺ and Hg²⁺ are enriched by magnetic beads during sample injection. Then, lyophilized microspheres preloaded with circular DNA templates are used to compress the rolling circle amplification reaction time. Finally, the ion concentration signal is output through dual-mode cross-validation.

8. The detection method according to any one of claims 4-7, characterized in that: It is suitable for the simultaneous detection of Pb²⁺, Cd²⁺, Hg²⁺ and As³⁺ in water, food and industrial samples. When the sample is solid, the sample is ultrasonically extracted with 0.1 M HNO3 solution for 10 minutes, and the supernatant is taken for detection after centrifugation.

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