Electrochemical sensing platform for detecting lead ions and malathion as well as preparation method and detection method of electrochemical sensing platform

Abstract

The invention belongs to the field of detection, and particularly relates to an electrochemical sensing platform for detecting lead ions and malathion as well as a preparation method and a detection method of the electrochemical sensing platform. The preparation method of the electrochemical sensing platform comprises the steps of designing a three-chain DNA compound, preparing a gold nanoparticle electrode, preparing the electrochemical sensing platform and the like. The electrochemical sensing platform realizes dual detection of lead ions (in a signal enhancement mode) and malathion (in a signal weakening mode) through specific recognition of DNAzyme and an aptamer on the basis of a three-chain DNA compound and a gold nanoparticle electrode. The method has the advantages of no label, high sensitivity (the detection limits respectively reach 5.42 pM and 0.34 pM), strong anti-interference capability and good stability, and is suitable for rapid detection of trace pollutants in environment and food samples.

Description

Technical Field

[0001] This invention belongs to the field of detection, specifically relating to an electrochemical sensing platform for detecting lead ions and malathion, as well as its preparation and detection methods. Background Technology

[0002] In the fields of environmental monitoring and food safety, lead ions and organophosphorus pesticide residues are two important pollutants. Lead ions exhibit significant bioaccumulation and toxicity, while organophosphorus pesticides such as malathion can damage the human nervous system. Establishing effective pollutant monitoring technologies is a key measure to ensure food safety and environmental protection. Traditional detection techniques, such as gas chromatography (GC), high-performance liquid chromatography (HPLC), and atomic absorption spectrometry (AAS), have the advantages of sensitivity and accuracy. However, electrochemical analysis methods demonstrate unique value in the field of trace pollutant detection due to their lower detection limits, faster response, better portability, simpler operation, and lower cost. However, the significant differences in chemical properties between pesticide residues and heavy metal ions lead to serious interference in electrochemical reactions, making single-platform multi-target detection a technical challenge.

[0003] While existing studies have achieved electrochemical detection of multiple targets, they generally rely on the use of signal-labeled molecules, leading to significant drawbacks in the sensor platform construction process: First, the labeling steps are cumbersome, typically requiring complex probe modification procedures, which prolongs the sensor assembly cycle; second, the cost remains high, with labeling reagents and purification consumables being expensive; third, stability is limited, as labeled molecules are prone to degradation during storage. More importantly, the labeling process alters the conformation of biorecognition elements, reducing detection sensitivity. Given that both types of pollutants often coexist in real-world environmental samples, developing a label-free electrochemical sensing platform capable of detecting pesticide residues and heavy metal ions has significant scientific and practical value.

[0004] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides a method for preparing an electrochemical sensing platform for detecting lead ions and malathion, the preparation method comprising the following steps:

[0006] (I) Design of triple-stranded DNA complex (Apt-Dz-P1)

[0007] The triple-stranded DNA complex comprises a fixed strand (P1), a recognition strand (Apt, malathion aptamer), and a functional strand (Dz, a lead-dependent DNA zyme); wherein:

[0008] The sequence of the fixed chain is as follows:

[0009] 5'-HS-(CH2)6-TTTTTTTTTTGCAGTCAAGAAGTTAAGAGA-3', with a thiol group modified at the 5' end; (SEQ ID NO.1)

[0010] The sequence of the identification chain is:

[0011] 5'-ATCCGTCACAACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTCTTGACTGCTGGTGTTGGCTCCCGTAT-3'; (SEQ ID NO. 2)

[0012] The sequence of the functional chain is as follows:

[0013] 5'-TTGTGTATAAGAGCAGGTGTTTTTTTTTTTCATCTCTGAAGTAGCGCCGCCGTATAGTGAG-3'; (SEQ ID NO. 3)

[0014] (II) Fabrication of gold nanoparticle electrodes

[0015] (II-1) The glassy carbon electrode is polished, cleaned and dried in sequence to obtain a dry electrode;

[0016] (II-2) The dried electrode was immersed in HAuCl4 solution and a voltage was applied to obtain the gold nanoparticle electrode (depAu / GCE);

[0017] (III) Preparation of electrochemical sensing platform

[0018] (III-1) Anneal the fixed strand, recognition strand and functional strand to obtain a triple-stranded DNA complex;

[0019] (III-2) The triple-stranded DNA complex and hairpin deoxyribonucleic acid were co-incubated on the surface of the gold nanoparticle electrode to obtain the electrochemical sensing platform.

[0020] Preferably, in step (II-1):

[0021] The polishing method is as follows: polishing with 0.3μm and 0.05μm alumina in sequence;

[0022] And / or, the cleaning method is: ultrasonic cleaning in deionized water and ethanol solution in sequence;

[0023] And / or, the drying method is: drying with nitrogen gas.

[0024] Preferably, in step (II-2):

[0025] The concentration of the HAuCl4 solution is 1 wt%.

[0026] And / or, the voltage is applied by applying a constant potential of -0.2V for 30 seconds.

[0027] Preferably, in step (III-1):

[0028] The molar ratio of the fixed chain, the identification chain, and the functional chain is 1:1:1;

[0029] And / or, the annealing method is as follows: hold at 94°C for 5 min, and then cool to 4°C at a rate of 6°C / min.

[0030] Preferably, in step (III-2):

[0031] The sequence of the hairpin deoxyribonucleic acid (H1, stem-loop structure) is as follows:

[0032] 5'-HS-(CH2)6-TTTGGGTTGGGCGGGATGGGTCTCACTAT / rA / GGAAGAGATTGTTTTTCCCGCCC-3', with a thiol group modified at the 5' end, and rA being the cleavage site; (SEQ ID NO.4)

[0033] And / or, the concentration ratio of the triple-stranded DNA complex to hairpin deoxyribonucleic acid is 1:50;

[0034] And / or, the incubation method is: incubation at 4°C for 8 hours.

[0035] Based on the same technical concept, the present invention further provides an electrochemical sensing platform for detecting lead ions and malathion obtained by the above preparation method.

[0036] Based on the same technical concept, the present invention provides a detection method, which includes the following steps:

[0037] (I) Lead ion detection

[0038] (I-1) Samples of different lead ion concentrations are dropped onto the electrochemical sensing platform for reaction;

[0039] (I-2) Then the electrochemical sensing platform was washed with phosphate buffer and then hemin was added dropwise to the electrochemical sensing platform to carry out the reaction;

[0040] (I-3) Continue to clean the electrochemical sensing platform with phosphate buffer solution, then immerse the electrochemical sensing platform in phosphate buffer solution, and use square wave voltammetry to test the current signal to obtain the lead ion concentration;

[0041] (II) Detection of Malathion

[0042] (II-1) Lead ions of a certain concentration and malathion of different concentrations of the test sample are added dropwise to the electrochemical sensing platform for reaction;

[0043] (II-2) Then the electrochemical sensing platform was washed with phosphate buffer and then hemin was added dropwise to the electrochemical sensing platform to carry out the reaction;

[0044] (II-3) Continue to clean the electrochemical sensing platform with phosphate buffer solution, then immerse the electrochemical sensing platform in phosphate buffer solution, and use square wave voltammetry to test the current signal to obtain the malathion concentration.

[0045] Preferably, in steps (I-1) and (II-1), the reaction temperature is 25°C and the reaction time is 90 min.

[0046] Preferably, in steps (I-2) and (II-2), the reaction temperature is 25°C and the reaction time is 45 min.

[0047] Preferably, in steps (I-3) and (II-3), the test conditions are: potential range 0 to -0.5V, frequency 15Hz, and amplitude 25mV.

[0048] To facilitate understanding of this invention, the principles of this invention will be explained as follows:

[0049] like Figure 1 As shown, the triple-stranded DNA complex (Apt-Dz-P1) acts as both a target recognition element and the walking strand of the DNA walker. Specifically:

[0050] (a) Pb 2+ Detection mechanism (signal on mode):

[0051] 1. Molecular recognition: Pb 2+ The DNAzyme is specifically activated, cleaving H1 and releasing a guanine (G)-rich sequence (S1). Simultaneously, a triple-stranded complex is released, triggering the DNAwalker cycle to cleave H1, ultimately yielding a large amount of S1.

[0052] 2. Signal output: S1 folds in a potassium-containing buffer solution to form a G4 quadruplex, and then G4 forms a G4 / hemin complex with heme, generating a strong current signal.

[0053] (II) Malathion detection mechanism (signal off mode):

[0054] 1. Competitive combination: Combining malathion and Pb2+ Simultaneously, malathion binds to the aptamer, disrupting the triple-stranded structure of Apt-Dz-P1, causing the DNAwalker to be unable to move and thus preventing the large-scale cleavage of H1.

[0055] 2. Signal suppression: A large amount of G4 / hemin complex cannot be obtained on the electrode surface, resulting in a decrease in current signal.

[0056] The beneficial effects of this invention are as follows:

[0057] 1. Highly efficient dual-contaminant detection capability: Through the unique design of the triple-stranded DNA complex, it achieves single-platform detection of lead ions (Pb). 2+ The parallel detection of phosmet and malathion solves the technical challenge of multi-target detection in traditional methods.

[0058] 2. Innovative signal transduction mechanism: When using the same signal molecule (G4 / hemin), lead ion detection adopts a signal enhancement mode, while malathion detection adopts a signal attenuation mode, which avoids the complexity of introducing multiple signal molecules in traditional methods and provides a new approach for multi-target detection.

[0059] 3. Label-free detection: It eliminates the need for complex labeling steps or expensive labeling reagents, simplifying the construction process of the sensing platform, reducing costs, and avoiding the influence of labeling molecules on the conformation of biometric elements, thereby further improving detection efficiency.

[0060] 4. High sensitivity and low detection limit: By combining DNA walker with DNAzyme for cyclic cleavage, signal amplification is achieved, with the detection limit of lead ions as low as 5.42 pM and malathion as low as 0.34 pM, which can meet the needs of trace pollutant detection.

[0061] 5. Excellent stability and reproducibility: The sensing platform can still maintain 89.91% of the initial signal strength after being stored at 4℃ for 11 days. The relative standard deviation (RSD) of inter-batch and intra-batch tests is only 2.51% and 3.03%, respectively, demonstrating good stability and reproducibility.

[0062] 6. Strong anti-interference capability: Through the introduction of specific DNAzymes and aptamers, the sensing platform exhibits high selectivity for common metal ions and pesticide interfering substances, ensuring the reliability of detection results.

[0063] 7. Broad potential for practical application: In the detection of environmental water and soil samples, the spiked recoveries were between 92.82% and 108.82%, which were highly consistent with the results of existing traditional methods (such as ICP-MS and HPLC), verifying its practical value in environmental monitoring and food safety. Attached Figure Description

[0064] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0065] Figure 1 This is a schematic diagram of the detection principle of the electrochemical sensing platform.

[0066] Figure 2 This is a gel electrophoresis characterization of the cleavage of hairpin probe H1 by a lead-ion-dependent DNAzyme and the disruption of the triple-stranded complex by malathion.

[0067] Figure 3 This is a graph showing the electrochemical response results of the electrochemical sensing platform under different assembly states. Among them:

[0068] Curve a is: (Apt-Dz-P1, H1) / depAu / GCE;

[0069] Curve b is: Pb 2+ / (Apt-Dz-P1,H1) / depAu / GCE,

[0070] Curve c is: hemin / Pb 2+ / (Apt-Dz-P1,H1) / depAu / GCE,

[0071] Curve d is: hemin / (Pb) 2+ +malathion) / (Apt-Dz-P1,H1) / depAu / GCE,

[0072] Pb 2+ The concentration was 500 nM, and the concentration of malathion was 50 nM.

[0073] Figure 4 The constructed electrochemical sensing platform is in the presence or absence of Pb 2+ Electrochemical response results when present.

[0074] Figure 5 It is a fixed Pb 2+ The electrochemical response of the electrochemical sensing platform to different concentrations of malathion at a concentration of 0.01 mM is shown in the figure.

[0075] Figure 6 The graph shows the EIS test results of the electrochemical sensing platform under different assembly states (the inset shows the equivalent fitting circuit of the electrochemical sensing platform). Among them:

[0076] Curve a is: depAu / GCE;

[0077] Curve b is: (Apt-Dz-P1, H1) / depAu / GCE;

[0078] Curve c is: Pb 2+ / (Apt-Dz-P1,H1) / depAu / GCE;

[0079] Curve d is: hemin / Pb 2+ / (Apt-Dz-P1,H1) / depAu / GCE;

[0080] Curve e is: hemin / (Pb) 2+ +malathion) / (Apt-Dz-P1,H1) / depAu / GCE;

[0081] The test solution was 5 mM [Fe(CN)6] containing 0.10 M KCl. 3- / 4- The solution, in which curves c and d show Pb 2+ At a concentration of 0.5 μM, curve e shows the Pb content. 2+ The concentration was 0.01 mM, and the concentration of malathion was 50 nM.

[0082] Figure 7 These are CV test results of the electrochemical sensing platform under different assembly states. The meanings of the curves and the test solutions are the same. Figure 6 .

[0083] Figure 8 The electrochemical sensing platform can detect different concentrations of Pb. 2+ The electrochemical response results are shown in the figure.

[0084] Figure 9 It is ΔI and Pb 2+ Linear fitting curve of concentration logarithmic value.

[0085] Figure 10 This is a graph showing the electrochemical response results of the electrochemical sensing platform to different concentrations of malathion.

[0086] Figure 11 It is a linear fit curve of ΔI versus the logarithm of malathion (Mal) concentration.

[0087] Figure 12 This is a graph showing the stability evaluation results of the constructed electrochemical sensing platform.

[0088] Figure 13 This is a graph showing the batch-to-batch reproducibility evaluation results of the constructed electrochemical sensing platform.

[0089] Figure 14 This is a graph showing the results of the batch reproducibility evaluation of the constructed electrochemical sensing platform.

[0090] Figure 15 It is the Pb of the constructed electrochemical sensing platform 2+ Graph showing the results of the selective evaluation of the test.

[0091] Figure 16 This is a graph showing the selectivity evaluation results of malathion detection using the constructed electrochemical sensing platform.

[0092] Figure 17 Pb in water samples from different environments 2+ The results of the spiked recovery experiment are shown in the figure.

[0093] Figure 18 Pb in different soil samples 2+ The results of the spiked recovery experiment are shown in the figure.

[0094] Figure 19 The constructed sensing platform and ICP-MS method were used to directly detect Pb in water samples from different environments. 2+ Content comparison chart.

[0095] Figure 20 The constructed sensing platform and ICP-MS method were used to directly detect Pb in different soil samples. 2+ Content comparison chart.

[0096] Figure 21 This is a graph showing the results of spiked recovery experiments of malathion in water samples from different environments.

[0097] Figure 22 This is a graph showing the results of spiked recovery experiments of malathion in different soil samples.

[0098] Figure 23 This is a comparison chart showing the direct detection results of malathion in different environmental water samples using the constructed sensing platform and HPLC method.

[0099] Figure 24 This is a comparison chart showing the direct detection results of malathion in different soil samples using the constructed sensing platform and HPLC method. Detailed Implementation

[0100] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0101] Example 1

[0102] This embodiment provides a method for preparing an electrochemical sensing platform for detecting lead ions and malathion, the preparation method comprising the following steps:

[0103] (I) Design of triple-stranded DNA complex (Apt-Dz-P1)

[0104] The triple-stranded DNA complex comprises a fixed strand (P1), a recognition strand (Apt, malathion aptamer), and a functional strand (Dz, a lead-dependent DNA zyme); wherein:

[0105] The sequence of the fixed chain is as follows:

[0106] 5'-HS-(CH2)6-TTTTTTTTTTGCAGTCAAGAAGTTAAGAGA-3', with a thiol group modified at the 5' end; (SEQ ID NO.1)

[0107] The sequence of the identification chain is:

[0108] 5'-ATCCGTCACAACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTCTTGACTGCTGGTGTTGGCTCCCGTAT-3'; (SEQ ID NO. 2)

[0109] The sequence of the functional chain is as follows:

[0110] 5'-TTGTGTATAAGAGCAGGTGTTTTTTTTTTTCATCTCTGAAGTAGCGCCGCCGTATAGTGAG-3'; (SEQ ID NO. 3)

[0111] (II) Fabrication of gold nanoparticle electrodes

[0112] (II-1) The glassy carbon electrode (GCE, Φ=3mm) was polished with 0.3μm and 0.05μm aluminum oxide in sequence, then ultrasonically cleaned in deionized water and ethanol solution respectively, and then dried with nitrogen gas to obtain a dry electrode;

[0113] (II-2) The dried electrode was immersed in a 1% HAuCl4 solution and a constant potential of -0.2V was applied for 30s to obtain the gold nanoparticle electrode (depAu / GCE);

[0114] (III) Preparation of electrochemical sensing platform

[0115] (III-1) The Apt:Dz:P1 complex was annealed at a molar ratio of 1:1:1 to form a triple-stranded DNA complex. The annealing procedure was as follows: hold at 94°C for 5 minutes, then cool to 4°C at a rate of 6°C / min, and store at this temperature for later use to obtain the triple-stranded DNA complex.

[0116] (III-2) The triple-stranded DNA complex was co-incubated with H1 (at a concentration ratio of 1:50) on the electrode surface (4°C, 8 h) to obtain the electrochemical sensing platform.

[0117] Example 2

[0118] This embodiment provides a method for detecting lead ions and malathion, the method comprising the following steps:

[0119] (I) Lead ion detection

[0120] (I-1) Add 10 μL of samples containing different lead ion concentrations to the surface of the electrochemical sensing platform and react for 90 min (reaction temperature 25℃);

[0121] (I-2) After the reaction was completed, the sample was washed three times with 0.1M phosphate buffer (prepared from Na2HPO4 and NaH2PO4, containing 0.10M KCl, pH 7.0), and then 10μL of 5mM hemin was added dropwise and the reaction was carried out at 25℃ for 45min.

[0122] (I-3) Continue to clean the electrode three times with 0.1M phosphate buffer solution, and then immerse the electrode in the 0.1M phosphate buffer solution. Use square wave voltammetry to test the current signal. The test conditions are: potential range 0 to -0.5V, frequency 15Hz, amplitude 25mV, to obtain the lead ion concentration.

[0123] (II) Detection of Malathion

[0124] (II-1) Add 10 μL of a sample containing 0.01 mM lead ions and different concentrations of malathion to the electrode surface and react for 90 min (25℃);

[0125] (I-2) After the reaction was completed, the sample was washed three times with 0.1M phosphate buffer (prepared from Na2HPO4 and NaH2PO4, containing 0.10M KCl, pH 7.0), and then 10μL of 5mM hemin was added dropwise and the reaction was carried out at 25℃ for 45min.

[0126] (I-3) Continue to clean the electrode three times with 0.1M phosphate buffer solution, and then immerse the electrode in the 0.1M phosphate buffer solution. Use square wave voltammetry to test the current signal. The test conditions are: potential range 0 to -0.5V, frequency 15Hz, amplitude 25mV, to obtain the malathion concentration.

[0127] Verification Example 1: Feasibility Analysis

[0128] like Figure 2 As shown, non-denaturing polyacrylamide gel electrophoresis confirmed the successful assembly of the triple-stranded complex (Apt-Dz-P1) (band 5). When Pb is present... 2+ At this time, the DNAzyme is activated and cleaves substrate H1 (band 7), releasing G-rich sequences that form a G-quadruplex / hemin complex, generating a significant electrochemical signal. Figure 3 (Signal enhancement). Malathion inhibits signal generation by competitively binding to the aptamer and disrupting the triple-chain structure (band 8). Figure 3 (Signal weakening). Furthermore, with Pb... 2+ As the concentration increased from 0 to 0.20 μM, the current signal showed Pb 2+ Increased concentration dependence Figure 4 When Pb is fixed 2+ At a concentration of 0.01 mM, the current signal showed a concentration-dependent decay as the malathion concentration increased. Figure 5 These experimental results fully demonstrate that the sensing strategy developed in this invention can be used to detect Pb. 2+ Malathion is feasible.

[0129] Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) tests further validated the feasibility of assembling the sensing platform. Figures 6-7 As shown, the gold nanoparticle-modified electrode (depAu / GCE) exhibits a low charge transfer resistance (Rct) due to its excellent conductivity. Figure 6 Curve a) and a stronger peak current signal ( Figure 7 Curve a). When Apt-Dz-P1 and H1 are assembled on the electrode surface, Rct increases significantly due to the negative charge of the nucleic acid backbone. Figure 6 Curve b), the peak current signal is weakened ( Figure 7 Curve b). Add Pb 2+ After that, the impedance continued to increase ( Figure 6 Curve c), the current further decreases ( Figure 7 Curve c), which is mainly due to Pb 2+ Activation of the DNAzyme cleavage reaction and triggering the continuous operation of the DNA walker result in a large amount of single-stranded DNA (S1) being randomly distributed on the electrode surface, thereby increasing the steric hindrance for mass transfer and electron transfer. The addition of hemin further increases the impedance. Figure 6 Curve d), the current signal continues to weaken ( Figure 7Curve d) indicates that a G4 / hemin supramolecular structure formed on the electrode surface, further enhancing the steric hindrance effect on mass transfer and electron transfer. In contrast, when 0.01 mM Pb was simultaneously added to the sensing platform... 2+ After incubation with malathion and heme, the resistance was significantly reduced. Figure 6 Curve e), the current increases sharply ( Figure 7 Curve e) shows that the binding of malathion to the aptamer hinders the formation of the DNA walker, thereby inhibiting the generation of the G4 / hemin complex and maintaining the ordered assembly of H1 on the electrode surface. Furthermore, the ineffective formation of the triple-stranded DNA structure also leads to a decrease in the negative charge density of nucleic acid molecules on the electrode surface. These results fully demonstrate that the sensing platform can be successfully assembled.

[0130] Verification Example 2: Performance Analysis

[0131] For Pb 2+ The detection showed that the electrochemical signal intensity increased with Pb. 2+ The concentration increases gradually (and the concentration gradually increases). Figure 8 Within the concentration range of 10 pM to 1 μM, the signal difference (ΔI), i.e., the target signal (I0), is... t The difference between the signal (I0) and the blank signal (ΔI=I) t -I0), with Pb 2+ The logarithm of the concentration shows a good linear relationship. Figure 9 The fitted curve equation is ΔI. Pb2+ =1216.42*lgC+3459.23(R) 2 =0.9989), the detection limit reaches 5.42pM (signal-to-noise ratio S / N = 3). When Pb is fixed 2+ At a concentration of 0.01 mM, the electrochemical signal of the sensing platform decreased as the malathion concentration increased from 1 pM to 1 μM. Figure 10 Within the range of 1 pM to 100 nM, ΔI showed a linear correlation with the logarithm of malathion concentration. Figure 11 The linear equation is ΔI. Mal = -1054.94*lgC - 4529.03(R) 2 =0.9965), the detection limit is 0.34 pM (S / N = 3).

[0132] Verification Example 3: Evaluation of the stability, reproducibility, and selectivity of the sensing platform

[0133] With Pb 2+ To analyze the model, the stability of the sensing platform was first evaluated. The prepared sensor was stored at 4°C and tested every two days, with results as follows: Figure 12As shown, the ΔI value remained essentially unchanged for the first three days, and by day 11 it still maintained 89.91% of the initial signal strength, indicating that the platform has excellent stability. Reproducibility was evaluated through inter-batch and intra-batch tests; the relative standard deviation (RSD) of the test results from six different batches was 2.51%. Figure 13 The RSD of the signals from the same batch of 8 sensors was 3.03%. Figure 14 This confirms that the platform has good reproducibility.

[0134] Regarding selective evaluation, for Pb 2+ The detection process involved selecting multiple common metal ions as interfering substances (concentration of Pb). 2+ 10 times (e.g.) Figure 15 As shown, Pb 2+ The ΔI value of Pb was significantly higher than that of the interfering ions, and there was no significant difference between it and the signal of the mixed ion group, indicating that Pb 2+ The introduction of DNA-dependent DNAzymes makes the sensing platform highly resistant to ion interference. This is especially relevant considering the potential presence of Pb in real-world samples. 2+ When malathion coexists with the target analyte, its interference can be effectively avoided by adjusting the sample pH to 10 to hydrolyze the analyte, and then adjusting it back to pH 7.0 for detection. For malathion detection, a common pesticide at a concentration 10 times higher than the target analyte is selected as the interfering agent. Figure 16 The results showed that malathion caused significantly stronger signal attenuation than other pesticides, proving that the sensing platform also has excellent selectivity for malathion detection.

[0135] Verification Example 4: Practicality Assessment

[0136] To verify the practicality of the sensing platform, water samples from urban rivers such as the Jinjiang, Shahe, and Jiang'an Rivers, agricultural water samples including agricultural drainage and greenhouse runoff, and soil samples from vegetable gardens, orchards, and roadside green belts were selected for testing. The entire research process was divided into two parts: First, a blank spiked recovery experiment was conducted, using PBS buffer as a blank control, and 1, 10, and 100 nM Pb were added to the water samples, respectively. 2+ Adding 1, 10, and 100 μmol / kg of Pb to soil samples 2+ To eliminate the effect of malathion on Pb 2+ To prevent interference with the detection, the sample pH should be adjusted to 10 beforehand to decompose it before detection. Figures 17-18 As shown, the recoveries of spiked samples for different water samples ranged from 94.09% to 107.67%, while those for soil samples ranged from 92.82% to 107.51%. Further analysis was conducted using inductively coupled plasma mass spectrometry (ICP-MS) as a reference method to directly detect spiked samples (…). Figures 19-20 The results obtained by the two methods showed good consistency, confirming that the sensing platform can detect Pb in environmental water and soil. 2+The test has excellent accuracy.

[0137] Similarly, with fixed Pb 2+ Malathion was tested in urban river water samples, agricultural water samples, and various soil samples at a concentration of 0.01 mM. Figures 21-22 As shown, the recoveries of spiked water samples ranged from 93.22% to 107.79%, and those of soil samples ranged from 93.59% to 108.82%, confirming the reliability of this sensing strategy for the detection of malathion in real samples. When validated using high-performance liquid chromatography (HPLC) as a reference method, it was found that the malathion content (<0.5 nM) in urban river water samples was below the HPLC detection limit, but the electrochemical sensor could still effectively detect it, highlighting its advantages in ultra-trace detection. Figures 23-24 The detection results for the remaining environmental water and soil samples showed no significant difference from HPLC, further demonstrating the reliability of this sensing platform in the detection of real-world environmental samples. These results indicate that the sensing platform constructed in this study can effectively detect Pb in real-world environmental samples. 2+ Both malathion and malathion detection offer both accuracy and reliability, demonstrating significant application potential in the field of heavy metal and pesticide pollution monitoring.

[0138] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing an electrochemical sensing platform for detecting lead ions and malathion, characterized in that, The preparation method includes the following steps: (I) Design of triple-stranded DNA complexes The triple-stranded DNA complex comprises a stationary strand, a recognition strand, and a functional strand; wherein: The sequence of the fixed chain is as follows: 5'-HS-(CH2)6-TTTTTTTTTTGCAGTCAAGAAGTTAAGAGA-3', with a thiol group modified at the 5' end; The sequence of the identification chain is: 5'-ATCCGTCACAACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTCTTGACTGCTGTGTTGGCTCCCGTAT-3'; The sequence of the functional chain is as follows: 5'-TTGTGTATAAGAGCAGGGTTTTTTTTTTTCATCTCTGAAGTAGCGCCGCCGTATAGTGAG-3'; (II) Fabrication of gold nanoparticle electrodes (II-1) The glassy carbon electrode is polished, cleaned and dried in sequence to obtain a dry electrode; (II-2) The dried electrode was immersed in HAuCl4 solution and a voltage was applied to obtain the gold nanoparticle electrode; (III) Preparation of electrochemical sensing platform (III-1) Anneal the fixed strand, recognition strand and functional strand to obtain a triple-stranded DNA complex; (III-2) The triple-stranded DNA complex and hairpin deoxyribonucleic acid were co-incubated on the surface of the gold nanoparticle electrode to obtain the electrochemical sensing platform.

2. The method for preparing the electrochemical sensing platform for detecting lead ions and malathion according to claim 1, characterized in that, In step (II-1): The polishing method is as follows: polishing with 0.3μm and 0.05μm alumina in sequence; And / or, the cleaning method is: ultrasonic cleaning in deionized water and ethanol solution in sequence; And / or, the drying method is: drying with nitrogen gas.

3. The method for preparing the electrochemical sensing platform for detecting lead ions and malathion according to claim 1, characterized in that, In step (II-2): The concentration of the HAuCl4 solution is 1 wt%. And / or, the voltage is applied by applying a constant potential of -0.2V for 30 seconds.

4. The method for preparing the electrochemical sensing platform for detecting lead ions and malathion according to claim 1, characterized in that, In step (III-1): The molar ratio of the fixed chain, the identification chain, and the functional chain is 1:1:1; And / or, the annealing method is as follows: hold at 94°C for 5 min, and then cool to 4°C at a rate of 6°C / min.

5. The method for preparing the electrochemical sensing platform for detecting lead ions and malathion according to claim 1, characterized in that, In step (III-2): The sequence of the hairpin deoxyribonucleic acid is as follows: 5'-HS-(CH2)6-TTTGGGTTGGGCGGGATGGGTCTCACTAT / rA / GGAAGAG ATGTTTTTCCCGCCC-3', with a thiol group modified at the 5' end, and rA being the cleavage site; And / or, the concentration ratio of the triple-stranded DNA complex to hairpin deoxyribonucleic acid is 1:50; And / or, the incubation method is: incubation at 4°C for 8 hours.

6. An electrochemical sensing platform for detecting lead ions and malathion obtained by the preparation method according to any one of claims 1 to 5.

7. A detection method, characterized in that, The detection method for lead ions and malathion using the electrochemical sensing platform described in claim 6 includes the following steps: (I) Lead ion detection (I-1) Samples of different lead ion concentrations are dropped onto the electrochemical sensing platform for reaction; (I-2) Then the electrochemical sensing platform was washed with phosphate buffer and then hemin was added dropwise to the electrochemical sensing platform to carry out the reaction; (I-3) Continue to clean the electrochemical sensing platform with phosphate buffer solution, then immerse the electrochemical sensing platform in phosphate buffer solution, and use square wave voltammetry to test the current signal to obtain the lead ion concentration; (II) Detection of Malathion (II-1) Lead ions of a certain concentration and malathion of different concentrations of the test sample are added dropwise to the electrochemical sensing platform for reaction; (II-2) Then the electrochemical sensing platform was washed with phosphate buffer and then hemin was added dropwise to the electrochemical sensing platform to carry out the reaction; (II-3) Continue to clean the electrochemical sensing platform with phosphate buffer solution, then immerse the electrochemical sensing platform in phosphate buffer solution, and use square wave voltammetry to test the current signal to obtain the malathion concentration.

8. The detection method according to claim 7, characterized in that, In steps (I-1) and (II-1), the reaction temperature is 25°C and the reaction time is 90 min.

9. The detection method according to claim 7, characterized in that, In steps (I-2) and (II-2), the reaction temperature is 25°C and the reaction time is 45 min.

10. The detection method according to claim 7, characterized in that, In steps (I-3) and (II-3), the test conditions are: potential range 0 to -0.5V, frequency 15Hz, and amplitude 25mV.

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