A paper-based electrochemical chip, its preparation method and application

Abstract

The present invention provides a preparation method of a paper-based electrochemical chip and its simultaneous detection for lead, cadmium, mercury, and arsenic ions. The paper-based electrochemical chip prepared according to the preparation method provided by the present invention has the advantages of rich raw materials, light weight, small volume, low cost, easy folding, convenient to carry, degradable, and simple to manufacture. The simultaneous detection method for lead, cadmium, mercury, and arsenic ions provided by the present invention has low detection operation cost, short detection time, and high detection accuracy, and can be used for on-site simultaneous detection of lead, cadmium, mercury, and arsenic ions, and the interference of the paper itself is excluded in this detection method.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to a paper-based electrochemical chip, a preparation method thereof, and an application thereof in the simultaneous detection of lead, cadmium, mercury, and arsenic ions. Background Art

[0002] At present, heavy metal ion pollution has gradually developed into an important global problem. Heavy metal ions have high toxicity and non-degradability. Cadmium is a non-essential element for the human body and often exists in the form of compounds in nature. Under normal environmental conditions, it will not directly affect human health. However, once cadmium enters the human body, it will gradually accumulate, causing chronic cadmium poisoning and damaging various organs such as the kidneys, spleen, pancreas, thyroid gland, and the nervous system. Lead, mercury, and arsenic ions are currently well-documented toxic substances and have potential toxicity to many cell tissues.

[0003] Traditional heavy metal ion detection technologies mainly include atomic fluorescence spectrometry, stripping voltammetry, atomic absorption spectrometry, inductively coupled plasma method, etc. However, the detection equipment used in these methods is expensive, not easy to carry, and the pretreatment process of the detected samples is complex, and the detection cost is high, which is not suitable for detection in large areas.

[0004] In the past few decades, with the development of miniaturization and integration, microfluidic chip point-of-care testing systems have attracted worldwide research interest. The application of microfluidic technology in chemical and biological analysis reduces the amount of samples and reagents required, reduces the consumption of expensive reagents, and greatly reduces the costs of sample preparation and detection. Since the first application of microfluidic technology in analytical detection, various materials have been used as substrates to fabricate microfluidic chips, such as silicon, glass, polymers, and paper. Among them, paper, as an ideal material, can be used to fabricate low-cost, high-efficiency, and disposable analytical devices. By using wax printing or other technologies to create hydrophobic barriers and hydrophilic channels, microfluidic channels on the paper surface can be generated, and paper-based microfluidic analytical devices (μPADs) can be simply fabricated. Then, the sample is dropped onto the defined detection area for detection. μPADs allow complex biological or chemical assay operations to be performed on one device, including various complex sample preparation, reaction, and detection steps, thus eliminating the need for time-consuming and cumbersome traditional bench-top assay operations. μPADs are widely used in point-of-care testing platforms due to their relatively simple fabrication technology and compatibility with various detection methods.

[0005] More and more detection technologies and methods have been applied to the paper-based microfluidic chip system. Electrochemical detection based on aptamers has developed into a detection technology with great application potential for paper-based microfluidic chip systems due to its advantages such as small size, convenient carrying, high sensitivity, and good selectivity. Summary of the Invention

[0006] To solve the above problems, the present invention applies the electrochemical detection technology based on nucleic acid aptamers to the paper-based microfluidic chip system, and provides a preparation method of a paper-based electrochemical chip, which is characterized by including the following steps: Step S1-1, draw a chip pattern, which includes a working electrode, a reference electrode, a counter electrode and a detection area; Step S1-2, print the chip pattern onto chromatography paper with low-tack tape; Step S1-3, for the chromatography paper, apply carbon paste ink to the working electrode and the counter electrode, apply paraffin to the hydrophobic area, and apply Ag / AgCl ink to the reference electrode to obtain a pretreated paper-based electrochemical chip; Step S1-4, place the pretreated paper-based electrochemical chip on a hot press for hot pressing to obtain a hot-pressed paper-based electrochemical chip; Step S1-5, drop a gold nanoparticle solution onto the detection area of the hot-pressed paper-based electrochemical chip, and then increase the size of the gold nanoparticles in the detection area of the hot-pressed paper-based electrochemical chip by the seed solution growth method to obtain a gold nanoparticle paper-based electrochemical chip; Step S1-6, perform a functionalization dropping operation on the detection area of the gold nanoparticle paper-based electrochemical chip to obtain a paper-based electrochemical chip, wherein the detection area is a hydrophilic area, and the peripheral area of the detection area is a hydrophobic area.

[0007] The preparation method of the paper-based electrochemical chip provided by the present invention may also have the following technical feature, wherein Step S1-6 includes the following steps: Step S1-6-1, drop CdCP, PbCP, HgCP and AsCP solutions onto the four corner areas of the detection area of the gold nanoparticle paper-based electrochemical chip respectively, and react at room temperature to obtain a functionalized gold nanoparticle paper-based electrochemical chip; Step S1-6-2, drop 6-mercaptohexanol onto the four corner areas of the detection area of the functionalized gold nanoparticle paper-based electrochemical chip, and incubate at room temperature to obtain a 6-mercaptohexanol gold nanoparticle paper-based electrochemical chip; Step S1-6-3, drop CdApt, G4, AsApt and HgApt solutions onto the four corner areas of the detection area of the 6-mercaptohexanol gold nanoparticle paper-based electrochemical chip respectively, and react at a constant temperature.

[0008] The preparation method of the paper-based electrochemical chip provided by the present invention may further have the following technical features. In step S1-6-1, among the CdCP, PbCP, HgCP, and AsCP solutions, the nucleic acid sequence of CdCP is: 5’-SH-ACTAATGACAACGGACTGTGAACCCGTCGTCCTGAG-3’, the nucleic acid sequence of PbCP is: 5’-SH-TAGTTTAGCCCAACCACACCAACC-3’, the nucleic acid sequence of HgCP is: 5’-SH-TTCTTTCTTCCCCTTGTTTGTT-3’, and the nucleic acid sequence of AsCP is: 5′-SH-ACAGAACAACCAACGTCGCTCCGGGTACTTCTTC-3’. In step S1-6-3, among the CdApt, G4, AsApt, and HgApt solutions, the nucleic acid sequence of CdApt is: 5’-CTCAGGACGACGGGTTCACAGTAGGTTGTC-MB-3’, the nucleic acid sequence of G4 is: 5’-GGTTGGTGTGGTTGG-MB-3’, the nucleic acid sequence of AsApt is: 5’-GTTGCAGCGAG-MB-3’, and the nucleic acid sequence of HgApt is: 5’-SH-AAGAAAGAAGGGGAACAAACAA-MB-3’.

[0009] The present invention also provides a paper-based electrochemical chip prepared by the above preparation method.

[0010] The present invention also provides a method for simultaneously detecting lead, cadmium, mercury, and arsenic ions by using the above paper-based electrochemical chip for detection.

[0011] The simultaneous detection method for lead, cadmium, mercury, and arsenic ions provided by the present invention is characterized by the following steps: Step S2-1, drop PBS buffer solution at the central area of the detection region of the paper-based electrochemical chip to obtain a PBS-treated paper-based electrochemical chip; Step S2-2, drop a mixed solution of lead, cadmium, mercury, and arsenic ions with different concentrations at the central area of the detection region of the PBS-treated paper-based electrochemical chip. After reacting for 1 to 30 minutes, connect the PBS-treated paper-based electrochemical chip to an electrochemical workstation, and measure the change in the electrochemical signal intensity of the lead, cadmium, mercury, and arsenic ion regions at the four corners of the detection region of the PBS-treated paper-based electrochemical chip; Step S2-3, respectively draw standard curves of the change in electrochemical signal intensity and the concentrations of lead, cadmium, mercury, and arsenic ions in the mixed solution of lead, cadmium, mercury, and arsenic ions according to the measurement results of Step S2-2; Step S2-4, drop the sample to be tested at the central area of the detection region of the PBS-treated paper-based electrochemical chip. After reacting for 1 to 30 minutes, connect the PBS-treated paper-based electrochemical chip to an electrochemical workstation, measure the change in the electrochemical signal intensity of the sample to be tested, and calculate the concentrations of lead, cadmium, mercury, and arsenic ions in the sample to be tested according to the standard curve.

[0012] Functions and effects of the invention

[0013] According to the preparation method of the paper-based electrochemical chip provided by the present invention, since the chip pattern is printed on the chromatographic paper with low-tack tape, the chip pattern includes a working electrode, a reference electrode, a counter electrode, and a detection region. The working electrode and the counter electrode are filled with carbon paste ink, the hydrophobic region is filled with paraffin, the reference electrode is filled with Ag / AgCl ink, and the detection region is functionalized after growing gold nanoparticles. Nucleic acids for detecting lead ions and cadmium ions are used in the functionalization process. Therefore, this preparation method actually combines the electrochemical detection technology based on nucleic acid aptamers and the paper-based microfluidic chip technology, so it also combines the respective advantages of these two technologies, making the paper-based electrochemical chip prepared according to the preparation method provided by the present invention have the advantages of rich raw materials, light weight, small volume, low cost, easy folding, convenient to carry, degradable, and simple to manufacture.

[0014] The simultaneous detection method for lead, cadmium, mercury, and arsenic ions using the paper-based electrochemical chip provided by the present invention has the following advantages:

[0015] 1. The operation cost of detection is low and the detection time is short;

[0016] 2. It can be used for on-site simultaneous detection of lead, cadmium, mercury, and arsenic ions with high detection accuracy;

[0017] 3. Since the concentrations of lead, cadmium, mercury, and arsenic ions are calculated by measuring the change in the electrochemical signal intensity of the sample to be tested, the interference of the paper itself, such as the color of the paper, is excluded. Brief Description of the Drawings

[0018] Figure 1 is a schematic diagram of the chip pattern structure of an embodiment of the present invention;

[0019] Figure 2 is a flowchart for preparing a paper-based electrochemical chip according to an embodiment of the present invention;

[0020] Figure 3 is a schematic diagram of the detection principle of a method for simultaneously detecting lead, cadmium, mercury, and arsenic ions according to an embodiment of the present invention;

[0021] Figure 4 is a cyclic voltammogram of a hot-pressed paper-based electrochemical chip before and after adding gold nanoparticle solution and seed solution according to an embodiment of the present invention;

[0022] Figure 5 is an electrochemical impedance spectrum of a hot-pressed paper-based electrochemical chip before and after adding gold nanoparticle solution and seed solution according to an embodiment of the present invention;

[0023] Figure 6 is a standard curve of lead, cadmium, mercury, and arsenic ions according to an embodiment of the present invention. Detailed Description of the Invention

[0024] The following illustrates the specific steps of the present invention through examples, but is not limited by the examples.

[0025] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.

[0026] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0027] The reagents used in the following examples were purchased through ordinary commercial channels, and the experimental operations and experimental conditions not specified refer to the conventional operations and conventional conditions in the art.

[0028] The following describes the specific embodiments of the present invention with reference to the accompanying drawings.

[0029] <Example>

[0030] This example provides a method for preparing a paper-based electrochemical chip and its application in the simultaneous detection of lead, cadmium, mercury, and arsenic ions.

[0031] The preparation method of this example includes the following steps:

[0032] Step S1-1, draw the chip pattern on a computer using Adobe Illustrator software.

[0033] Figure 1It is a schematic diagram of the chip pattern structure of an embodiment of the present invention. As Figure 1 shown, the chip pattern of the present invention includes four working electrodes 1-4, four reference electrodes 5-8, four counter electrodes 9-12, and a detection area 13. Among them, the detection area 13 includes four corner areas 14-17, a central area 18, and connection areas 19-22. At each of the four corner areas 14, there is a working electrode 1, a reference electrode 5, and a counter electrode 9. The working electrode is located in the middle of the corner area, the reference electrode is located on the left side of the working electrode, and the counter electrode is located on the right side of the working electrode. In addition, the detection area 13 is a hydrophilic area, and the peripheral area of the detection area is a hydrophobic area.

[0034] Step S1-2: Use a process cutting printer to print the chip pattern onto a whatman No.1 chromatographic paper with a low-tack tape.

[0035] Step S1-3: For the chromatographic paper, apply carbon paste ink to the working electrodes and counter electrodes, apply paraffin to the hydrophobic areas, and apply Ag / AgCl ink to the reference electrodes to obtain a pretreated paper-based electrochemical chip.

[0036] Step S1-4: Place the pretreated paper-based electrochemical chip on a hot press plate and hot press it at 90 °C for 5 s so that the paraffin penetrates the paper to form a hydrophobic barrier, obtaining a hot-pressed paper-based electrochemical chip.

[0037] The preparation process of the paper-based electrochemical chip in this embodiment is as Figure 2 shown.

[0038] Step S1-5: Drop a gold nanoparticle solution on the detection area of the hot-pressed paper-based electrochemical chip, and then increase the size of the gold nanoparticles in the detection area of the hot-pressed paper-based electrochemical chip by the seed solution growth method to obtain a gold nanoparticle paper-based electrochemical chip.

[0039] Among them, the specific preparation process of the gold nanoparticle solution in Step S1-5 is as follows: Place 50 mL of 1 mmol / L HAuCl4 aqueous solution at 140 °C, mix evenly, stir and heat repeatedly for 15 min, add 5 mL of 38.8 mmol / L trisodium citrate, and heat to boiling for 20 min to obtain the gold nanoparticle solution. The specific operation process of the seed solution growth method is as follows: Take 30 μL of 5 nM gold nanoparticle solution and drop it on the detection area of the hot-pressed paper-based electrochemical chip, react at room temperature for 2 h, drop 30 μL of a mixed solution (seed solution) of 2.0 mM HAuCl4 and 20 mM NH2OH·HCl, incubate at room temperature for 1 h, wash 3 times with deionized water, and dry for 30 min.

[0040] The detection area of the hot-pressed paper-based electrochemical chip in steps S1-5 was subjected to electrochemical impedance spectroscopy and cyclic voltammetry measurements before and after dropping the gold nanoparticle solution and before and after treatment by the seed solution growth method, and both measurements were carried out in a mixed solution of 5 mmol / L [Fe(CN)6] 3- / 4- and 0.1 mol / L KCl, and the measurement results are as Figure 4 and Figure 5 shown: Among them, Figure 4 is the cyclic voltammogram of the hot-pressed paper-based electrochemical chip of this embodiment before and after dropping the gold nanoparticle solution and dropping the seed solution, as Figure 4 shown. After the growth of the dropped seed solution, the current of the hot-pressed paper-based electrochemical chip increased significantly. Figure 5 is the electrochemical impedance spectroscopy of the hot-pressed paper-based electrochemical chip of this embodiment before and after dropping the gold nanoparticle solution and dropping the seed solution, as Figure 5 shown. After the growth of the dropped seed solution, the resistance of the hot-pressed paper-based electrochemical chip decreased significantly, indicating that after dropping the gold nanoparticle solution and dropping the seed solution on the hot-pressed paper-based electrochemical chip of this embodiment, the size of the gold nanoparticles increased, and the conductivity increased significantly, enabling the originally weakly conductive paper to conduct current better.

[0041] Step S1-6: Prepare another batch of gold nanoparticle paper-based electrochemical chips according to steps S1-1 to S1-5, perform a functionalized dropping operation on the detection area of the gold nanoparticle paper-based electrochemical chip, and then obtain the paper-based electrochemical chip. Store this batch of paper-based electrochemical chips in a refrigerator at 4°C.

[0042] Among them, the specific operation of the functionalized dropping operation in step S1-6 is as follows:

[0043] Step S1-6-1: Drop 60 μL of 14 mmol / L CdCP, PbCP, HgCP, and AsCP solutions at the four corner areas of the detection area of the gold nanoparticle paper-based electrochemical chip, and react at room temperature for 16 h to obtain a functionalized gold nanoparticle paper-based electrochemical chip;

[0044] Step S1-6-2: Drop 60 μL of 2 mmol / L 6-mercaptohexanol (MCH) at the four corner areas of the detection area of the functionalized gold nanoparticle paper-based electrochemical chip, and incubate at room temperature for 1 h to reduce non-specific adsorption, obtaining a mercaptohexanol gold nanoparticle paper-based electrochemical chip;

[0045] Step S1-6-3: Drop 60 μL of 7 mmol / L CdApt, G4, AsApt, and HgApt solutions at the four corner areas of the detection area of the mercaptohexanol gold nanoparticle paper-based electrochemical chip, and react at a constant temperature of 25°C for 15 min.

[0046] Among them, the nucleic acid sequence of CdCP is: 5'-SH-ACTAATGACAACGGACTGTGAACCCGTCGTCCTGAG-3', the nucleic acid sequence of PbCP is: 5'-SH-TAGTTTAGCCCAACCACACCAACC-3', the nucleic acid sequence of HgCP is: 5'-SH-TTCTTTCTTCCCCTTGTTTGTT-3', the nucleic acid sequence of AsCP is: 5′-SH-ACAGAACAACCAACGTCGCTCCGGGTACTTCTTC-3', the nucleic acid sequence of CdApt is: 5'-CTCAGGACGACGGGTTCACAGTAGGTTGTC-MB-3', the nucleic acid sequence of G4 is: 5'-GGTTGGTGTGGTTGG-MB-3', the nucleic acid sequence of AsApt is: 5'-GTTGCAGCG AG-MB-3', and the nucleic acid sequence of HgApt is: 5'-SH-AAGAAAGAAGGGGAACAAACAA-MB-3'.

[0047] The detection principle of the simultaneous detection method for lead, cadmium, mercury, and arsenic ions in this embodiment is as Figure 3 shown.

[0048] The prepared paper-based electrochemical chip is used for the simultaneous detection of lead, cadmium, mercury, and arsenic ions in fruit and vegetable samples, including the following steps:

[0049] Step S2-1: 60 μL of 10 mmol / L PBS buffer (containing 10 mmol / L disodium hydrogen phosphate, 10 mmol / L sodium dihydrogen phosphate, 0.1 mol / L NaCl, 25 mmol / L MgCl2, pH 7.5) is respectively dropped onto the central area of the detection region of 7 paper-based electrochemical chips to obtain PBS-treated paper-based electrochemical chips;

[0050] Step S2-2: 1, 20, 40, 60, 80, 100, and 300 nmol / L of lead, cadmium, mercury, and arsenic ion mixed solutions are respectively dropped onto the central area of the detection region of 7 paper-based electrochemical chips. After reacting for 15 min, the PBS-treated paper-based electrochemical chips are connected to an electrochemical workstation, and the electrochemical signal intensity changes in the lead, cadmium, mercury, and arsenic ion regions at the four corners of the detection region of the PBS-treated paper-based electrochemical chips are measured. The electrochemical signal intensity changes of lead, cadmium, mercury, and arsenic ions are obtained from the square wave voltammetry -0.3 V peak position (methylene blue is modified on the aptamer, and the signal peak position of methylene blue is around -0.3 V);

[0051] Step S2-3: According to the measurement results of Step S2-2, standard curves of the electrochemical signal intensity changes and the concentrations of lead, cadmium, mercury, and arsenic ions in the lead, cadmium, mercury, and arsenic ion mixed solutions are respectively plotted;

[0052] Step S2-4, measure the concentrations of lead, cadmium, mercury, and arsenic ions in the fruit and vegetable samples.

[0053] Step S2-4-1, pretreat the fruit and vegetable samples by wet digestion. The specific pretreatment steps are as follows:

[0054] Wash the apples and lettuce, drain them, take the edible parts of the apples and lettuce, and break them into homogenates with a blender respectively. Take 2 portions of 20 g of homogenized apples and 2 portions of 20 g of homogenized lettuce and place them in 4 glass dishes. Add 1 mL of mixed solutions of lead, cadmium, mercury, and arsenic ions with different concentrations to the 4 glass dishes, let them stand for 30 min, then add 20 mL of concentrated HNO3 (mass fraction 65%) to the 4 glass dishes, cover the glass dishes and digest for 24 h. Then heat the 4 portions of digested liquid to boiling, and then add 10 mL of concentrated HNO3 (mass fraction 65%) and 10 mL of HClO4 (mass fraction 72%) to the 4 glass dishes respectively, heat up to about 180 °C. When the 4 portions of solutions turn brownish, add 10 mL of HNO3 (mass fraction 65%) respectively, continue heating until the solutions become transparent, then turn off the power, and cool down until the white smoke has dissipated. Next, add 1 mL of HNO3 (mass fraction 1%) to the 4 glass dishes respectively, and then adjust the pH of the 4 portions of solutions to 7 with 10 mmol / L PBS buffer solution (containing 0.1 mol / L NaCl, 25 mmol / L MgCl2, pH 7.5). Finally, dilute the 4 portions of neutral solutions to 10 mL with 10 mmol / L PBS buffer solution respectively as the test samples.

[0055] Step S2-4-2, drop the 4 test samples onto the central areas of the detection regions of 4 PBS-treated paper-based electrochemical chips respectively. After reacting for 15 min, connect the PBS-treated paper-based electrochemical chips to an electrochemical workstation, measure the change in the electrochemical signal intensity of the test samples, and calculate the concentrations of lead, cadmium, mercury, and arsenic ions in the 4 test samples according to the standard curve.

[0056] Figure 6 is the standard curve of lead, cadmium, mercury, and arsenic ions in the embodiment of the present invention. As Figure 6 can be seen, there is a good linear relationship between the change in the electrochemical signal intensity in the lead, cadmium, mercury, and arsenic ion regions around the detection region of the PBS-treated paper-based electrochemical chip and the concentrations of lead, cadmium, mercury, and arsenic ions. The 2+ linear regression equation of Cd is: ΔI = 0.61C Cd 2+ + 15.37, and the correlation coefficient R 2 = 0.9965. The 2+ linear regression equation of Pb is: ΔI = 1.11C Pb 2++23.97, correlation coefficient R 2 = 0.9980, Hg 2+ 's linear regression equation is: ΔI = 1.44C Hg 2+ +6.45, correlation coefficient R 2 = 0.9847, AsSO 3- 's linear regression equation is: ΔI = 1.03C AsSO 3- +12.51, correlation coefficient R 2 = 0.9941, △I is the change in current intensity, unit is nA, C Pb 2+ 、C Cd 2+ 、C Hg 2+ and C AsSO 3- are the concentrations of lead, cadmium, mercury, and arsenic ions respectively, unit is nM.

[0057] The measured and theoretical values of the concentrations of lead, cadmium, mercury, and arsenic ions in 4 test samples are listed in the following table respectively,

[0058]

[0059]

[0060] As can be seen from the table, the paper-based electrochemical chip can simultaneously detect lead, cadmium, mercury, and arsenic ions in fruit and vegetable samples, with the error between the measured value and the theoretical value within 10%, high detection accuracy, and fast detection speed.

Claims

1. A preparation method of a paper-based electrochemical chip, characterized in that It includes the following steps: Step S1-1: Draw a chip pattern, where the chip pattern includes a working electrode, a reference electrode, a counter electrode, and a detection area; Step S1-2: Print the chip pattern onto a chromatographic paper with a low-tack tape; Step S1-3: For the chromatographic paper, apply carbon paste ink to the working electrode and the counter electrode, apply paraffin to the hydrophobic area, and apply Ag / AgCl ink to the reference electrode to obtain a pretreated paper-based electrochemical chip; Step S1-4: Place the pretreated paper-based electrochemical chip on a hot press for hot pressing to obtain a hot-pressed paper-based electrochemical chip; Step S1-5: Drop a gold nanoparticle solution onto the detection area of the hot-pressed paper-based electrochemical chip, and then increase the size of the gold nanoparticles in the detection area of the hot-pressed paper-based electrochemical chip by the seed solution growth method to obtain a gold nanoparticle paper-based electrochemical chip; Step S1-6: Perform a functionalization dropping operation on the detection area of the gold nanoparticle paper-based electrochemical chip to obtain the paper-based electrochemical chip, wherein the detection area is a hydrophilic area, and the peripheral area of the detection area is a hydrophobic area, The specific operation of the functionalization dropping operation in Step S1-6 is as follows: Step S1-6-1: Drop CdCP, PbCP, HgCP, and AsCP solutions onto the four corner areas of the detection area of the gold nanoparticle paper-based electrochemical chip respectively, and react at room temperature to obtain a functionalized gold nanoparticle paper-based electrochemical chip. Among the CdCP, PbCP, HgCP, and AsCP solutions, The nucleic acid sequence of CdCP is: 5’-SH-ACTAATGACAACGGACTGTGAACCCGTCGTCCTGAG-3’; The nucleic acid sequence of PbCP is: 5’-SH-TAGTTTAGCCCAACCACACCAACC-3’; The nucleic acid sequence of HgCP is: 5’-SH-TTCTTTCTTCCCCTTGTTTGTT-3’; The nucleic acid sequence of AsCP is: 5′-SH-ACAGAACAACCAACGTCGCTCCGGGTACTTCTTC-3’; Step S1-6-2: Drop 6-mercaptohexanol onto the four corner areas of the detection area of the functionalized gold nanoparticle paper-based electrochemical chip, and incubate at room temperature to obtain a 6-mercaptohexanol gold nanoparticle paper-based electrochemical chip; Step S1-6-3: Drop CdApt, G4, AsApt, and HgApt solutions onto the four corner areas of the detection area of the 6-mercaptohexanol gold nanoparticle paper-based electrochemical chip respectively, and react at a constant temperature. Among the CdApt, G4, AsApt, and HgApt solutions, The nucleic acid sequence of CdApt is: 5’-CTCAGGACGACGGGTTCACAGTAGGTTGTC-MB-3’; The nucleic acid sequence of G4 is: 5’-GGTTGGTGTGGTTGG-MB-3’, and the nucleic acid sequence of AsApt is: 5’-GTTGCAGCGAG-MB-3’ The nucleic acid sequence of the HgApt is: 5'-SH-AAGAAAGAAGGGGAACAAACAA-MB-3'.

2. A paper-based electrochemical chip, characterized in that, It is prepared by the preparation method as claimed in claim 1.

3. A method for simultaneously detecting lead, cadmium, mercury, and arsenic ions, characterized in that, Detection is carried out using the paper-based electrochemical chip as described in claim 2.

4. The simultaneous detection method for lead, cadmium, mercury, and arsenic ions according to claim 3, wherein It includes the following steps: Step S2-1, PBS buffer solution is dropped at the central area of the detection area of the paper-based electrochemical chip to obtain a PBS-treated paper-based electrochemical chip; Step S2-2, a mixed solution of lead, cadmium, mercury, and arsenic ions with different concentrations is dropped at the central area of the detection area of the PBS-treated paper-based electrochemical chip. After reacting for 1 to 30 minutes, the PBS-treated paper-based electrochemical chip is connected to an electrochemical workstation, and the change in the electrochemical signal intensity of the lead, cadmium, mercury, and arsenic ion areas at the four corners of the detection area of the PBS-treated paper-based electrochemical chip is measured; Step S2-3, according to the measurement results of step S2-2, standard curves of the change in the electrochemical signal intensity and the concentrations of lead, cadmium, mercury, and arsenic ions in the mixed solution of lead, cadmium, mercury, and arsenic ions are respectively drawn; Step S2-4, the sample to be tested is dropped at the central area of the detection area of the PBS-treated paper-based electrochemical chip. After reacting for 1 to 30 minutes, the PBS-treated paper-based electrochemical chip is connected to an electrochemical workstation, and the change in the electrochemical signal intensity of the sample to be tested is measured. According to the standard curve, the concentrations of lead, cadmium, mercury, and arsenic ions in the sample to be tested are calculated.

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