A preparation method of an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposites

Abstract

The application relates to a preparation method and application of an electrochemical sensor for sensitive detection of lead ions based on a metal nanocomposite. 2+ The introduction of the target Pb 2+ triggers the S-DNA cleavage of the DNAzyme to cut off a single-strand DNA, and simultaneously initiates the DNA Walker effect to obtain a large amount of single-strand DNA. The supernatant after magnetic separation is added dropwise on an electrode modified with HP1, after incubation, the single-strand DNA serves as an initiation chain to initiate the CHA signal amplification reaction, so that more and more signal probes are connected to the electrode surface, and the electrical signal in the system is enhanced. The quantitative analysis of lead ions is realized by using the change of the electrical signal corresponding to the change of the methylene blue concentration on the electrode surface before and after the addition of Pb 2+ The prepared electrochemical sensor has high sensitivity, a wide detection range, good selectivity and excellent practical application capability.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposite materials. Background Technology

[0002] With the rapid development of industry and agriculture, heavy metal pollution, due to its widespread distribution, accumulation, and high toxicity, has become a serious threat to human health and ecosystems. Lead typically enters the human body through the respiratory tract and mouth, and cannot be metabolized or biodegraded; it is one of the most toxic and persistent heavy metal ions. Even exposure to extremely low levels of lead can lead to many health problems, including nerve damage, cardiovascular damage, kidney dysfunction, and decreased fertility. In particular, lead can impair normal brain development in children, causing symptoms such as memory loss, irritability, anemia, and intellectual disability. The U.S. Environmental Protection Agency (EPA) and the World Health Organization (WHO) have set limits on lead levels in drinking water. 2+ The concentration limits were 72 nM and 48 nM, respectively. Some researchers have found Pb in fruit and vegetable samples. 2+ Cd 2+ and Hg 2+ Heavy metal ions coexist, with concentrations mostly ranging from 100 to 200 nM. Therefore, there is an urgent need to develop sensitive and effective methods for detecting Pb in contaminated samples. 2+ Analysis is performed. Currently, atomic absorption spectrometry, inductively coupled plasma mass spectrometry, inductively coupled plasma atomic emission spectrometry, and high-performance liquid chromatography are commonly used to analyze Pb. 2+ The aforementioned traditional detection methods can provide good sensitivity, but they rely on large, precision instruments and suffer from drawbacks such as time-consuming and labor-intensive operation, expensive and complex instruments, and the inability to perform real-time detection. Therefore, from a practical application perspective, establishing a sensitive, reliable, and convenient detection method is of great significance.

[0003] Electrochemical sensors offer advantages such as high sensitivity, simple operation, and low cost in quantitative analysis, but they often suffer from instability. To further improve detection performance, functional nucleic acids are frequently introduced as target recognition elements, offering good selectivity, high biostability, and flexible design. Currently, electrochemical methods based on functional nucleic acids for the detection of heavy metal ions are becoming a research hotspot. Furthermore, various signal amplification strategies have been developed, such as CHA (catalytic hairpin self-assembly), HCR (hybridization chain reaction), RCA (rolling circle amplification), and enzyme-assisted signal amplification methods. Among these, CHA, as an enzyme-free isothermal signal amplification method, is simple to design, cost-effective, and has attracted widespread attention. Metal nanocomposites, due to their tunable nanoscale size, diverse structures, high porosity, and good conductivity, are applied in electrochemical detection research. They can not only carry electrochemical signal tags but also serve as immobilization matrices for biomolecules, thus also amplifying signals.

[0004] This invention addresses the problems of poor stability in existing detection technologies. First, it introduces magnetic separation technology to reduce interference from false positive signals. Second, it utilizes metal nanocomposite materials to carry more signal tags, effectively amplifying the electrochemical signal. Finally, it cleverly employs a catalytic hairpin assembly strategy to further amplify the signal. Based on this invention, the electrochemical sensing strategy overcomes the problems of the existing technologies while possessing the characteristics of high sensitivity, good selectivity, excellent stability, and convenience and economy, which is conducive to the widespread application of this invention. Summary of the Invention

[0005] A method for preparing an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposite materials, comprising the following steps:

[0006] (1) Preparation of metal nanocomposites: A one-pot solvothermal method was used for preparation. First, transition metal chlorides and lauric acid were dissolved in dimethylformamide and ultrasonically mixed. Then, aminophthalic acid was added and ultrasonically treated. The above mixed solution was transferred to a high-pressure reactor and incubated overnight. The product was collected by high-speed centrifugation and washed successively with N,N-dimethylformamide and ethanol to remove unreacted solvent. The product was then vacuum dried to constant weight to obtain transition metal-organic framework nanomaterials. Noble metal nanoparticles were generated in situ on the above nanomaterials using a redox method. First, the above nanomaterials were ultrasonically dispersed in water and a noble metal solution was added and mixed evenly. Then, a reducing agent was added and stirred thoroughly. The mixture was centrifuged at high speed and dried to constant weight to obtain metal nanocomposites.

[0007] (2) Surface modification of metal nanocomposite material with HP2 and signal tag: The metal nanocomposite material prepared above was dispersed in a signal tag solution and mixed overnight with a magnetic stirrer. The resulting product was centrifuged at high speed and washed with buffer to remove the physically adsorbed signal tag. Then HP2 was added, stirred overnight at 4 °C, centrifuged at high speed and washed with buffer to remove unbound HP2, thus obtaining the metal nanocomposite material with surface modification of HP2 and signal tag.

[0008] (3) Preparation of MBs@S-DNA / E-DNA system: S-DNA is loaded onto MBs by the strong binding between biotin on S-DNA and streptavidin coated on the surface of magnetic beads (MBs). Then, E-DNA forms a double-stranded structure by complementary base pairing with S-DNA in the manner of DNA walking. After magnetic separation and washing with buffer, the supernatant is removed and the MBs@S-DNA / E-DNA system is successfully constructed.

[0009] (4) Construction of electrochemical sensors: Pb-containing molecules were added to the MBs@S-DNA / E-DNA system. 2+ standard solution, Pb 2 + -DNAzyme is activated, the rA site of S-DNA is cleaved, and single-stranded DNA is released. Simultaneously, the released E-DNA acts as a walking strand, binding to the next S-DNA, ultimately cleaving off more single-stranded DNA. After magnetic separation, the supernatant is collected. Thiol-modified HP1 binds to the gold electrode surface via gold-sulfur bonds. When the supernatant is dropped onto the electrode, the single-stranded DNA acts as the initiating strand, opening HP1 to form a double-stranded structure and protruding a sequence. Then, a metal nanocomposite material modified with HP2 and a signal tag is dropped on. This protruding sequence further opens HP2, releasing the single-stranded DNA into the next CHA cycle. Ultimately, a large number of signal tags are attached to the electrode surface. Finally, the electrode is placed in a buffer solution, and the electrochemical signal of the signal tag is detected using differential pulse voltammetry. The addition of Pb... 2+ The change in the response signal before and after can affect Pb 2+ Perform quantitative analysis.

[0010] Further specifying, in step (1), the transition metal chloride is one or more of zirconium tetrachloride, copper chloride, and nickel chloride; the noble metal solution is one or more of tetrachloroauric acid, palladium acetate, and rhodium nitrate; and the reducing agent is one or more of sodium borohydride, ascorbic acid, citrate, and hydrazine hydrate.

[0011] Further specifying, in step (2), the signal tag is one or more of thionine, methylene blue, and ferrocene.

[0012] Further specifying, in step (3), the volume of MBs is 1~10 μL.

[0013] Further specifying, in steps (2), (3), and (4), the sequence of HP2 is: 5'-GAG ATG CAG TCA CTCGAA CAA AAA AAA AAG TGA CTG CAT CTC TTC C-3'; the sequence of S-DNA is: 5'-TTT TTACAC TAT C / rA / G GAA GAG ATG CAG TCA CTC GAA CAC G-3', wherein the 5' end is modified with biotin and contains an rA site in the middle; the sequence of E-DNA is: 5'-CAT CTC TTC TCC GAG CCG GTC GAA ATA GTG TTTTTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CAT CTC TTC TCC GAG CCG GTC GAAATA GTG T-3'; and the sequence of HP1 is: 5'- TTT TTG AGA TGC AGT CAC TTT TTT TTT TGTTCG AGT GAC TGC ATC TCT TCC-3'.

[0014] Further specifying, in steps (2), (3), and (4), the concentration of the DNA strand is 1-2 μM, the volume used is 5-10 μL, and the incubation time is 0.5 h-2.5 h.

[0015] Further specifying, in steps (2), (3), and (4), the buffer solution is one or more of Buffer I, Tris-HCl, PBS, HEPE, and phosphate buffer solution.

[0016] Compared with the prior art, the present invention has the following significant advantages:

[0017] 1. This invention utilizes the specific recognition of lead ions by lead ion DNAzyme, thereby improving the selectivity and accuracy of electrochemical sensors.

[0018] 2. This invention utilizes magnetic separation technology to reduce system background signal and improve the reliability of electrochemical sensors.

[0019] 3. This invention successfully prepared a metal nanocomposite material that can carry more signal tags and achieve signal amplification.

[0020] 4. This invention ingeniously utilizes catalytic hairpin assembly technology to further amplify signals and enhance the sensitivity of electrochemical biosensors.

[0021] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the fabrication of an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposite materials.

[0023] Figure 2 The sensor constructed in Example 1 of this invention, when supplemented with different concentrations of Pb... 2+ The differential pulse results before (solid line) and after (dashed line).

[0024] Figure 3 This is the standard curve for the detection of lead ions by the sensor constructed in Embodiment 1 of the present invention.

[0025] Figure 4 The sensor constructed in Embodiment 1 of this invention exhibits selectivity for lead ions in the presence of other interfering ions. Implementation

[0026] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Example

[0027] A method for preparing and applying an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposite materials is described below. Figure 1 As shown.

[0028] A method for preparing an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposite materials includes the following steps:

[0029] (1) Zirconium-based metal-organic framework nanomaterials (Zr-MOF) were prepared by a one-pot solvothermal method. 0.32 g of zirconium tetrachloride and 9.6 g of lauric acid were weighed, dissolved in 80 mL of dimethylformamide, and ultrasonically mixed for 30 min. Then, 0.496 g of aminophthalic acid was added and ultrasonically treated for 5 min. The above mixed solution was transferred to a high-pressure reactor and kept at 120 °C for 48 h. The product was collected by centrifugation at 8000 rpm for 5 min. The product was washed with 10 mL of N,N-dimethylformamide and ethanol to remove unreacted solvent. The product was dried in a vacuum drying oven at 70 °C to constant weight to obtain Zr-MOF. Noble metal nanoparticles were generated in situ on the above nanomaterials by redox method. 2 mg of Zr-MOF was weighed and ultrasonically dispersed in 2 mL of water. Then, 20 μL of 1% tetrachloroauric acid solution was added to the mixture and stirred thoroughly. Add 2 mL of 2 mM sodium borohydride to the above solution and stir at 25 °C for 1 h. Centrifuge at high speed and vacuum dry to constant weight to obtain AuNPs / Zr-MOF nanocomposite material.

[0030] (2) 24 mg of the AuNPs / Zr-MOF prepared above was dispersed in 8 mL of 1 mM methylene blue (MB) solution and mixed with a magnetic stirrer for 24 h to obtain AuNPs / Zr-MOF with MB surface modification. The obtained product was centrifuged at high speed and washed with PBS buffer to remove physically adsorbed MB. 10 μL of 2 μM HP2 was added to 10 μL of the above mixed solution, stirred at 4 °C for 12 h, then centrifuged at high speed and washed with PBS buffer to remove unbound HP2, thus obtaining AuNPs / Zr-MOF nanocomposite material with HP2 and MB surface modification.

[0031] (3) Add 5 μL of 10 mg / mL MBs to a 200 μL centrifuge tube, vortex to mix, and wash three times with 50 μL of Buffer I buffer. Add 10 μL of 2 μM S-DNA to the centrifuge tube, incubate at 37 ℃ with shaking for 1 h, magnetically separate the mixture, and wash with Buffer I buffer. Then, add 10 μL of 1 μM E-DNA to the precipitate, incubate at 37 ℃ for 1 h, magnetically separate the reactants, wash with Buffer I buffer, and remove the supernatant to successfully construct the MBs@S-DNA / E-DNA system.

[0032] (4) Add 5 μL of 2 μM thiol-modified HP1 to the surface of the gold electrode and incubate at 37 °C for 1 h in a constant temperature and humidity chamber. Then wash the electrode with PBS buffer to remove HP1 that has not bound to the electrode surface. Add 5 μL of 0.1 mM MCH blocking agent and incubate at 37 °C for 1 h. Add 10 μL of lead ion standard solutions of different concentrations to the MBs@S-DNA / E-DNA system and incubate at 37 °C for 30 min. 2+ Single-stranded DNA was cleaved by activating the rA site on S-DNA. After magnetic separation, the supernatant was collected and dropped onto the modified electrode. The single-stranded DNA, acting as the initiator, opened HP1, forming a double-stranded structure and protruding a sequence. Then, 5 μL of AuNPs / Zr-MOF nanocomposite material modified with HP2 and MB was added and incubated for 1 h. This protruding sequence further opened HP2, releasing the single-stranded DNA into the next CHA cycle. Ultimately, a large number of signal tags were attached to the electrode surface. Finally, the electrode was placed in PBS buffer, and the electrochemical signal of the signal tags was detected using differential pulse voltammetry. The addition of Pb... 2+ The change in the response signal before and after can affect Pb 2+ Perform quantitative analysis.

[0033] (5) Establishment of standard curve: Add 10 μL of lead ion standard solutions of different concentrations to step (4) to obtain sample detection solutions of different gradients. After incubation, an electrical signal is obtained. Linear fitting is performed with the logarithm of lead ion concentration as the abscissa and the difference of current signal as the ordinate to establish the standard curve of the sensor for lead ions.

[0034] The parameters for detecting the sample current signal were set as follows: scan range of -0.6 V to 0.15 V, scan rate of 50 mV / s, amplitude of 0.05 V, and pulse width of 0.05 s.

[0035] like Figure 2 As shown, this is the sensor constructed in Example 1 of the present invention when different concentrations of Pb are added. 2+ The differential pulse results before (solid line) and after (dashed line).

[0036] like Figure 3 As shown, this is the standard curve for the detection of lead ions by the sensor constructed in Embodiment 1 of the present invention. Example

[0037] A method for preparing an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposite materials and its application, the practical application of which includes the following steps:

[0038] (1) To verify the specific recognition of lead ions by the prepared electrochemical sensor based on metal nanocomposite materials, lead ion standards were added to the phosphate buffer solution to make the concentration of lead ions in the sample 10 nM; other interfering ion standard solutions were prepared using the phosphate buffer solution, with a concentration of 1 μM for each. The detection system constructed in Example 1 was used to detect the above-mentioned different interfering ion standards, and the detection results are as follows: Figure 4 As shown, this illustrates the method of the present invention for Pb 2+ It has good selectivity. Example

[0039] A method for preparing an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposite materials and its application, the practical application of which includes the following steps:

[0040] (1) Food sample processing: The actual samples were pretreated by microwave digestion. 0.5 g of spiked food samples (rice flour, tea, honey) were weighed and added to the microwave digestion furnace along with 8 mL of HNO3 and 2 mL of H2O2. The reaction temperature, pressure, heating time, and holding time were set to 180 ℃, 400 psi, 12 min, and 15 min, respectively. After digestion, the container containing the digestion solution was heated on a heating plate to remove the acid from the digestion solution. Finally, it was cooled to room temperature, and the food extract was obtained by standard addition method.

[0041] (2) Sample detection: Take 10 μL of food extract and measure the electrical signal according to steps (1)-(4) of Example 1. Substitute the signal into the standard curve to obtain the concentration of lead ions in the sample.

[0042] (3) When using rice flour as a food sample for determination, the lead ion standard was added to the rice flour at a dosage of 10 nmol / L, with 0.5 times and 5 times the dosage of the standard amount respectively. 10 μL of sample solution was taken, and the electrical signal was measured according to steps (1)-(4) of Example 1. The lead ion concentration in the sample was obtained by substituting the signal into the standard curve detected in Example 1. Each sample was measured three times, and the average value was taken. The RSD and recovery rate were calculated as shown in the table below:

[0043]

[0044] The prepared electrochemical sensor has been verified to exhibit advantages such as fast response, high sensitivity, good selectivity, wide detection range, and good reproducibility and stability in the detection of lead ions. Detection of actual samples demonstrates that the prepared sensor has excellent practical application value.

[0045] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0046] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposite materials, characterized in that, Includes the following steps: (1) A transition metal-organic framework was prepared by a one-pot solvothermal method using transition metal chlorides and organic ligands. A noble metal solution and a reducing agent were added to obtain a metal nanocomposite material with good biocompatibility and high porosity. An electrochemical signal probe was obtained by modifying the surface of the above composite material with a signal tag and HP2. The sequence of HP2 is 5'-GAG ATG CAGTCA CTC GAA CAA AAA AAA AAG TGA CTG CAT CTC TTC C-3'; (2) Recognition process of target lead ions: Through the strong binding force between streptavidin and biotin, DNAzyme structures in the form of DNA Walker are immobilized on the surface of MBs, and Pb 2+ The presence of this activates the DNAzyme, causing the rA site of the S-DNA to be cleaved and single-stranded DNA to be excised. Simultaneously, the released E-DNA acts as a walk strand, binding to the next S-DNA, ultimately yielding a large amount of single-stranded DNA to construct Pb. 2+ -DNAzyme-mediated bipedal DNA Walker, the S-DNA sequence is 5'-TTT TTA CAC TATC / rA / G GAA GAG ATG CAG TCA CTC GAA CAC G-3', where the 5' end is modified with biotin and the middle contains an rA site. The E-DNA sequence is 5'-CAT CTC TTC TCC GAG CCG GTC GAA ATA GTG TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CAT CTC TTC TCC GAG CCG GTC GAA ATA GTG T-3'; (3) Construction of electrochemical sensor: The supernatant after magnetic separation was dropped onto the surface of a gold electrode modified with HP1. Single-stranded DNA was used as the initiator to start the CHA cycle reaction, which resulted in a large number of signal probes being attached to the electrode surface. The electrochemical signal was detected by differential pulse voltammetry to realize Pb 2+ For quantitative detection, the HP1 sequence is 5'- TTT TTG AGA TGC AGT CACTTT TTT TTT TGT TCG AGT GAC TGC ATC TCT TCC-3'.

2. The method for preparing an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposite materials according to claim 1, characterized in that, In step (1), the transition metal chloride is one or more of zirconium tetrachloride, copper chloride, and nickel chloride; the organic ligand is one or more of aminophthalic acid, lauric acid, terephthalic acid, and dimethylimidazole; the noble metal solution is one or more of tetrachloroauric acid, palladium acetate, and rhodium nitrate; the reducing agent is one or more of sodium borohydride, ascorbic acid, citrate, and hydrazine hydrate; and the signal tag is one or more of thionine, methylene blue, and ferrocene.

3. The method for preparing an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposite materials according to claim 1, characterized in that, In step (2), the volume of MBs is 1~10 μL.

4. The method for preparing an electrochemical sensor for sensitive detection of lead ions based on metal nanocomposite materials according to claim 1, characterized in that, In steps (1), (2), and (3), the concentrations of DNA strand HP2, S-DNA, E-DNA, and HP1 are 1–2 μM; the volume used is 5–10 μL; and the incubation time is 0.5–2.5 h.

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