A composite material based on platinum nanoparticles loaded with nickel-cobalt double metal hydroxide, an electrochemical sensor and a detection method
By designing an electrochemical sensor using Pt@NiCo-LDH signal amplification material, the complexity and high cost of existing OTA detection technologies have been solved, achieving sensitive and rapid detection results suitable for environmental monitoring and food safety analysis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- DALIAN INSTITUTE OF CHEMICAL PHYSICS CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2021-12-15
- Publication Date
- 2026-05-15
AI Technical Summary
Existing OTA detection technologies require large instruments, are complex to operate, have poor portability, and are costly, which limits their application in rapid real-time detection and on-site analysis.
Pt@NiCo-LDH was designed as a signal amplification material, combined with a paper-based electrochemical sensing platform, and an electrochemical sensor was constructed by labeling with nucleic acid aptamers to detect ochratoxin A.
It achieves high sensitivity, fast analysis speed, and simple operation in OTA detection, and has good selectivity and specificity, making it suitable for environmental monitoring, disease diagnosis, and food safety analysis.
Smart Images
Figure HDA0003413722440000011 
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Abstract
Description
Technical Field
[0001] This invention relates to the field of novel signal amplification material design and electrochemical analysis and detection technology, and more specifically to a method for preparing an electrochemical sensor based on platinum nanoparticle-loaded nickel-cobalt bimetallic hydroxide as a signal amplification material and a method for detecting biotoxins. Background Technology
[0002] Ochratoxin A (OTA) is widely found in nature and is a secondary metabolite produced by arsenic and penicillin. It has strong teratogenic and carcinogenic properties. Because OTA is difficult to destroy during processing and storage, it poses a serious threat to food safety and, more importantly, to human health and life. Therefore, it is necessary to design a sensitive and rapid OTA detection technology.
[0003] Currently, traditional techniques for detecting OTA (over-the-counter) mainly include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and atomic absorption spectrometry (AAS). However, these methods generally require large instruments and have drawbacks such as complex operation, poor portability, and high detection costs, which severely limit their application in rapid real-time detection and on-site analysis.
[0004] Electrochemical sensors have advantages such as high sensitivity, fast analysis speed, and simple operation. With the help of paper-based sensing platforms and aptamer recognition technology, they have played an important role in fields such as environmental monitoring, disease diagnosis, and food safety analysis. Summary of the Invention
[0005] The purpose of this invention is to design Pt@NiCo-LDH as a signal amplification material to label aptamers, and to combine it with a paper-based electrochemical sensing platform to construct an electrochemical sensor for detecting ochratoxin A.
[0006] According to one aspect of the present invention, a composite material is provided, the composite material comprising an electrical signal amplification material, the surface of which is modified with a nucleic acid aptamer;
[0007] The electrical signal amplification material includes a carrier and an active component; the carrier is a bimetallic hydroxide, and the bimetallic hydroxide is a nickel-cobalt hydroxide;
[0008] The active metal element in the active component is selected from at least one of Pt and Au.
[0009] Optionally, the active metal element accounts for 1% to 5% of the signal amplification material.
[0010] According to one aspect of the present invention, a method for preparing the above-mentioned composite material is provided, comprising preparing an aqueous solution a containing a bimetallic hydroxide, adding an aqueous solution b containing an active component precursor, sonicating, adding a mixed solution c containing sodium borohydride and sodium hydroxide, reacting, centrifuging and washing, and drying to obtain the electrical signal amplification material;
[0011] A phosphate buffer solution containing an electrical signal amplification material was prepared, and a nucleic acid aptamer solution was added. The mixture was then shaken, centrifuged, and washed to obtain the composite material.
[0012] Optionally, the mass ratio of the bimetallic hydroxide to the active component precursor is 20:1 to 40:1;
[0013] The mass ratio of sodium borohydride, sodium hydroxide and bimetallic hydroxide is 100:2:4 to 100:4:8;
[0014] The ultrasound duration is 20–40 minutes.
[0015] The reaction time is 1 to 2 hours;
[0016] The drying temperature is 55-70℃, and the drying time is 20-30h;
[0017] The mass ratio of the electrical signal amplification material to the nucleic acid aptamer is 50:1 to 100:1;
[0018] The oscillation temperature is 3-6℃, and the oscillation time is 10-15h.
[0019] According to one aspect of the present invention, a method for preparing an electrochemical sensor for detecting biotoxins is provided, comprising the following steps:
[0020] (1) Design the printing pattern of the paper chip, print the printing pattern on the colorimetric paper by wax printing, heat it to form a hydrophilic working area with hydrophobic surroundings.
[0021] A carbon working electrode, an Ag / AgCl reference electrode, and a carbon counter electrode were printed in the hydrophilic working area using screen printing to obtain a paper chip.
[0022] (2) The solution containing Mxene-Au, the complementary chain solution containing nucleic acid aptamers, and the solution containing aptamer complexes are sequentially transferred to the surface of the carbon working electrode to obtain the electrochemical sensor;
[0023] The aptamer complex is selected from the above-mentioned composite material and the composite material prepared by the above-mentioned preparation method.
[0024] Optionally, in the Mxene-Au solution, the concentration of Mxene-Au is 1-2 mg / mL; in the complementary strand solution containing the nucleic acid aptamer, the concentration of the complementary strand of the nucleic acid aptamer is 5-10 μM; and in the aptamer complex solution, the concentration of the aptamer complex is 1-2 mg / mL.
[0025] The volume ratio of the Mxene-Au solution, the complementary strand solution containing the nucleic acid aptamer, and the solution containing the aptamer complex is 1:1:1 to 1:2:2.
[0026] Optionally, the preparation method of the Mxene-Au includes the following steps:
[0027] (i) Prepare a hydrofluoric acid solution containing Ti3AlC2, stir, centrifuge, wash and dry to obtain Mxene;
[0028] (ii) Prepare an aqueous solution containing Mxene, add chloroauric acid solution while stirring, and after the reaction, centrifuge, wash and dry to obtain the Mxene-Au.
[0029] Optionally, in steps (i) and (ii), the drying temperature is independently 50–70°C and the drying time is independently 10–15 h.
[0030] In step (i), the concentration of the aqueous solution containing Mxene is 0.2–0.5 mg / mL;
[0031] The stirring time is 20-30 hours, and the stirring temperature is 20-30°C.
[0032] The centrifugal washing was performed using ultrapure water until the pH of the supernatant was 6.
[0033] In step (ii), the mass ratio of Mxene to chloroauric acid is 1:1 to 5:1;
[0034] The reaction temperature is 3–6 min.
[0035] According to one aspect of the present invention, a method for detecting biotoxins is provided, using an electrochemical sensor prepared by the above-described method;
[0036] A standard solution containing biotoxins is transferred to the surface of the carbon working electrode. A phosphate buffer solution containing hydrogen peroxide and o-phenylenediamine is added dropwise to the hydrophilic working area. Electrochemical signals are detected by differential pulse voltammetry. A standard curve of current intensity versus biotoxin concentration is plotted. The current intensity of the sample to be tested is compared with the standard curve to calculate the biotoxin concentration.
[0037] Optionally, the phosphate buffer solution has a pH of 6.4 to 7.0, a hydrogen peroxide concentration of 2 to 5 mM, and an o-phenylenediamine concentration of 5 to 10 mM.
[0038] Optionally, the biotoxin is ochratoxin A.
[0039] As one embodiment of the present invention, a method for preparing an electrochemical sensor based on platinum nanoparticle-supported nickel-cobalt bimetallic hydroxide as a signal amplification material includes the following steps:
[0040] (1) Design the printing pattern of the paper chip using Adobe Illustrator CS4 software on a computer;
[0041] (2) Print the pattern designed in step (1) onto A4 chromatographic paper using a wax printing machine. Then place the printed chromatographic paper in an oven and heat it at 200°C for 60-180 seconds to melt the wax and allow it to penetrate the entire thickness of the paper, forming a hydrophilic working area in the hydrophobic region.
[0042] (3) The carbon working electrode, Ag / AgCl reference electrode and carbon counter electrode are sequentially printed onto the paper chip obtained in step (2) using screen printing.
[0043] (4) Transfer the Mxene-Au solution to the surface of the carbon working electrode;
[0044] (5) Continue to transfer the complementary strand DNA solution containing the aptamer to the surface of the carbon working electrode;
[0045] (6) Continue to transfer the solution containing the aptamer complex apta-Pt@NiCo-LDH to the surface of the carbon working electrode;
[0046] (7) Continue to transfer the standard solution containing ochratoxin A to the surface of the carbon working electrode, add a phosphate buffer solution with pH 6.4-7.0 containing hydrogen peroxide and o-phenylenediamine to the electrochemical working area, detect the electrochemical signal by differential pulse voltammetry, plot the standard curve of current intensity versus ochratoxin A concentration, compare the current intensity of the sample to be tested with the standard curve, and complete the detection of ochratoxin A.
[0047] The preparation steps of Mxene-Au in step (4) are as follows:
[0048] (i) Preparation of Mxene
[0049] 3–5 mg of Ti3AlC2 powder was slowly added to 20 mL of hydrofluoric acid solution with a mass fraction of 30%–50%. The solution was then stirred at room temperature for 24 h to selectively etch the Al atomic layer. The resulting product was collected by centrifugation and washed with ultrapure water until the pH of the supernatant became 6. Mxene was obtained by drying in a vacuum drying oven at 60 °C for 15 h.
[0050] (ii) Preparation of Mxene-Au
[0051] Dissolve 1-3 mg of Mxene obtained in step (1) in 10 mL of ultrapure water, and then slowly add 1 mL of chloroauric acid solution with a mass fraction of 0.8%-1% under stirring, and continue the reaction for 5 min; collect the product by centrifugation and wash it three times with ultrapure water; finally dry it in a vacuum drying oven at 60 °C for 12 h to obtain the Mxene-Au composite material.
[0052] The preparation steps of apta-Pt@NiCo-LDH in step (6) are as follows:
[0053] (a) Preparation of ZIF-67
[0054] 0.58–1.16 g of cobalt nitrate and 0.65–1.31 g of 2-methylimidazole were dissolved in 50 mL of methanol solution and sonicated for 10 min. The two solutions were then mixed and stirred at room temperature for 30 min. The mixture was then aged at room temperature for 24 h. The resulting purple product was collected by centrifugation and washed three times with methanol. The product was then dried in a vacuum drying oven at 70 °C for 24 h to obtain ZIF-67 powder.
[0055] (b) Preparation of NiCo-LDH
[0056] Dissolve 0.1–0.3 g of ZIF-67 powder obtained in step (a) in 50 mL of ethanol solution and heat to 80 °C. Slowly add 50 mL of ethanol solution containing 0.365–0.73 g of nickel nitrate under stirring, and reflux for 1–3 h. Then collect the resulting pale green product by centrifugation and wash three times with anhydrous ethanol. Finally, dry in a vacuum drying oven at 60 °C for 24 h to obtain NiCo-LDH powder.
[0057] (c) Preparation of Pt@NiCo-LDH
[0058] Dissolve 0.1–0.3 g of NiCo-LDH powder obtained in step (b) in 30 mL of ultrapure water; add 0.5 mL of 0.8%–1% chloroplatinic acid solution and sonicate for 30 min; then add 3 mL of a mixed solution containing sodium borohydride (0.1–0.2 M) and sodium hydroxide (0.2–0.4 M) and continue the reaction for 1 h; collect the product by centrifugation and wash three times with ultrapure water and anhydrous ethanol; finally, dry in a vacuum drying oven at 60 °C for 24 h to obtain Pt@NiCo-LDH;
[0059] (d) Preparation of apta-Pt@NiCo-LDH
[0060] Dissolve 4 mg of Pt@NiCo-LDH powder obtained in step (c) in 2 mL of phosphate buffer solution with pH 7.0; add 1.0 mL of aptamer solution with a concentration of 10–20 μg / mL; incubate in a constant temperature shaking incubator at 4 °C for 12 h; after centrifugation and washing, redisperse the solution in 2.0 mL of phosphate buffer solution with pH 7.0 to obtain apta-Pt@NiCo-LDH solution, and store at 4 °C for later use.
[0061] In this application, the nucleotide sequence of the OTA aptamer is as follows:
[0062] '5′-SH-(CH2)6-GATCGGGTGTGGTGGCGTAAAGGGAGCATCGGACA-3';
[0063] The nucleotide sequence of the complementary strand of the OTA aptamer is as follows:
[0064] 5′-SH-(CH2)6-CCCCCCTGTCCGATGCT-3.
[0065] The beneficial effects of this invention include:
[0066] (1) The synthesized Pt@NiCo-LDH composite material is used as a signal amplification material. It has good peroxidase activity and can accelerate the oxidation reaction of o-phenylenediamine by catalyzing the reduction of hydrogen peroxide, thereby realizing the amplification of electrochemical signals.
[0067] (3) Electrochemical sensors have the advantages of good selectivity, fast analysis speed, simple operation and high sensitivity in the detection of OTA. Attached Figure Description
[0068] Figure 1 This is a schematic diagram illustrating the experimental principle of the present invention.
[0069] Figure 2 Differential pulse voltammetry curves of the sensors corresponding to different concentrations of ochratoxin A;
[0070] Figure 3 Linear fitting curve of the sensor for detecting ochratoxin A. Detailed Implementation
[0071] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.
[0072] like Figure 1 As shown, this invention uses platinum nanoparticles loaded with nickel-cobalt bimetallic hydroxide (Pt@NiCo-LDH) as a signal amplification material to label nucleic acid aptamers. The nucleic acid aptamer-modified Pt@NiCo-LDH is immobilized on the electrode surface using complementary strand DNA of the nucleic acid aptamer, and o-phenylenediamine is used as a signal molecule to obtain a distinct electrochemical signal. In the presence of ochratoxin A, the binding of the target analyte to the nucleic acid aptamer leads to the detachment of Pt@NiCo-LDH from the electrode interface, thereby causing a change in the current signal and achieving quantitative detection of ochratoxin A. The electrochemical sensor constructed by this method exhibits advantages such as simple operation, high sensitivity, and strong specificity in OTA detection, and also provides a pathway for the rapid detection of other biotoxins.
[0073] Example 1
[0074] A method for preparing an electrochemical sensor based on platinum nanoparticles loaded with nickel-cobalt bimetallic hydroxide as a signal amplification material, comprising the following steps:
[0075] (1) Design the printing pattern of the paper chip using Adobe Illustrator CS4 software on a computer;
[0076] (2) Print the pattern designed in step (1) onto A4 chromatographic paper using a wax printing machine. Then place the printed chromatographic paper in an oven and heat it at 200°C for 180 seconds to melt the wax and penetrate the entire thickness of the paper, forming a hydrophilic working area in the hydrophobic region.
[0077] (3) The carbon working electrode, Ag / AgCl reference electrode and carbon counter electrode are sequentially printed onto the paper chip obtained in step (2) using screen printing.
[0078] (4) Transfer the Mxene-Au solution to the surface of the carbon working electrode;
[0079] (5) Continue to transfer the complementary strand DNA solution containing the aptamer to the surface of the carbon working electrode;
[0080] (6) Continue to transfer the solution containing the aptamer complex apta-Pt@NiCo-LDH to the surface of the carbon working electrode;
[0081] (7) Continue to transfer the standard solution containing ochratoxin A to the surface of the carbon working electrode, add a phosphate buffer solution with pH 7.0 containing hydrogen peroxide and o-phenylenediamine to the hydrophilic working area, detect the electrochemical signal by differential pulse voltammetry, and plot the standard curve of current intensity versus ochratoxin A concentration.
[0082] The differential pulse voltammetric curves of the sensor corresponding to different concentrations of ochratoxin A (a: 0; b: 20 fg / mL; c: 100 fg / mL; d: 1 pg / mL; e: 10 pg / mL; f: 100 pg / mL; g: 1 ng / mL; h: 10 ng / mL; i: 100 ng / mL) are shown below. Figure 2 As shown, Figure 3 The results indicate that the logarithm of the measured ochratoxin A concentration is positively correlated with the current value, and the linear equation is: I = 3.84lgc OTA +15.99, R 2 =0.996, the linear range is 20 fg / mL to 100 ng / mL, and the detection limit is 8.7 fg / mL.
[0083] The preparation of Mxene-Au in step (4) is as follows:
[0084] (i) Preparation of Mxene
[0085] 5 mg of Ti3AlC2 powder was slowly added to 20 mL of 40% hydrofluoric acid solution, and the solution was stirred at room temperature for 24 h to selectively etch the Al atomic layer. The product was collected by centrifugation and washed with ultrapure water until the pH of the supernatant became 6. Mxene was obtained by drying in a vacuum drying oven at 60 °C for 15 h.
[0086] (ii) Preparation of Mxene-Au
[0087] Dissolve 2 mg of Mxene obtained in step (1) in 10 mL of ultrapure water, and then slowly add 1 mL of 1% chloroauric acid solution under stirring. Continue the reaction for 5 min. Collect the product by centrifugation and wash it three times with ultrapure water. Finally, dry it in a vacuum drying oven at 60 °C for 12 h to obtain the Mxene-Au composite material.
[0088] The preparation of apta-Pt@NiCo-LDH in step (6) is as follows:
[0089] (1) Preparation of ZIF-67
[0090] 1.16 g of cobalt nitrate and 1.31 g of 2-methylimidazole were dissolved in 50 mL of methanol solution and sonicated for 10 min. The two solutions were then mixed and stirred at room temperature for 30 min. The mixture was then aged at room temperature for 24 h. The resulting purple product was collected by centrifugation and washed three times with methanol. The product was dried in a vacuum drying oven at 70 °C for 24 h to obtain ZIF-67 powder.
[0091] (2) Preparation of NiCo-LDH
[0092] Dissolve 0.1g ZIF-67 powder obtained in step (1) in 50mL of ethanol solution and heat to 80℃. Slowly add 50mL of ethanol solution containing 0.365g nickel nitrate under stirring and reflux for 1h. Then collect the light green product by centrifugation and wash it three times with anhydrous ethanol. Finally, dry it in a vacuum drying oven at 60℃ for 24h to obtain NiCo-LDH powder.
[0093] (3) Preparation of Pt@NiCo-LDH
[0094] Dissolve 0.1 g of NiCo-LDH powder obtained in step (2) in 30 mL of ultrapure water; add 0.5 mL of 1% chloroplatinic acid solution and sonicate for 30 min; then add 3 mL of a mixed solution containing sodium borohydride (0.1 M) and sodium hydroxide (0.2 M) and continue the reaction for 1 h; collect the product by centrifugation and wash it three times with ultrapure water and anhydrous ethanol; finally dry it in a vacuum drying oven at 60 °C for 24 h to obtain Pt@NiCo-LDH;
[0095] (4) Preparation of apta-Pt@NiCo-LDH
[0096] Dissolve 4 mg of Pt@NiCo-LDH powder obtained in step (3) in 2 mL of phosphate buffer solution with pH 7.0; add 1.0 mL of aptamer solution with a concentration of 20 μg / mL; incubate in a constant temperature shaking incubator at 4 °C for 12 h; after centrifugation and washing, redisperse the solution in 2.0 mL of phosphate buffer solution with pH 7.0 to obtain apta-Pt@NiCo-LDH solution, and store at 4 °C for later use.
[0097] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.
Claims
1. An electrochemical sensor based on a paper chip, characterized in that: A paper chip having a hydrophilic working area formed by wax printing, the hydrophilic working area being surrounded by a hydrophobic area; a carbon working electrode, an Ag / AgCl reference electrode, and a carbon counter electrode printed on the hydrophilic working area; wherein the surface of the carbon working electrode is modified with Mxene-Au; The surface of the Mxene-Au is immobilized with a complementary strand of a nucleic acid aptamer; the complementary strand of the nucleic acid aptamer is modified with an aptamer complex through base complementary pairing. The aptamer complex includes an electrical signal amplification material, the surface of which is modified with a nucleic acid aptamer. The electrical signal amplification material includes a carrier and an active component; the carrier is a bimetallic hydroxide, and the bimetallic hydroxide is a nickel-cobalt hydroxide; The active metal element in the active component is selected from at least one of Pt and Au.
2. The electrochemical sensor according to claim 1, characterized in that, The active metal element accounts for 1% to 5% of the electrical signal amplification material.
3. A method for preparing the aptamer complex in the electrochemical sensor according to claim 1 or 2, characterized in that, Prepare an aqueous solution a containing a bimetallic hydroxide, add an aqueous solution b containing an active component precursor, sonicate, add a mixed solution c containing sodium borohydride and sodium hydroxide, react, centrifuge, wash, and dry to obtain the electrical signal amplification material; A phosphate buffer solution containing an electrical signal amplification material was prepared, and a nucleic acid aptamer solution was added. The mixture was shaken, centrifuged, and washed to obtain the aptamer complex.
4. The preparation method according to claim 3, characterized in that, The mass ratio of the bimetallic hydroxide to the active component precursor is 20:1 to 40:1; The mass ratio of sodium borohydride, sodium hydroxide and bimetallic hydroxide is 100:2:4 to 100:4:8; The ultrasound duration is 20-40 minutes. The reaction time is 1-2 hours; The drying temperature is 55~70°C, and the drying time is 20~30h; The mass ratio of the electrical signal amplification material to the nucleic acid aptamer is 50:1 to 100:1; The oscillation temperature is 3~6°C, and the oscillation time is 10~15h.
5. A method for preparing an electrochemical sensor for detecting biotoxins, characterized in that, Includes the following steps: (1) Design the printing pattern of the paper chip, print the printing pattern on the colorimetric paper by wax printing, heat it to form a hydrophilic working area with hydrophobic surroundings; A carbon working electrode, an Ag / AgCl reference electrode, and a carbon counter electrode were printed in the hydrophilic working area using screen printing to obtain a paper chip. (2) The solution containing Mxene-Au, the complementary chain solution containing nucleic acid aptamers, and the solution containing aptamer complexes are sequentially transferred to the surface of the carbon working electrode to obtain the electrochemical sensor; The aptamer complex is selected from the aptamer complex of claim 1 or 2, or the aptamer complex prepared by the preparation method of any one of claims 3 to 4.
6. The preparation method according to claim 5, characterized in that, The concentration of Mxene-Au in the Mxene-Au solution is 1-2 mg / mL; the concentration of the complementary strand of the nucleic acid aptamer in the nucleic acid aptamer solution is 5-10 μM; and the concentration of the aptamer complex in the aptamer complex solution is 1-2 mg / mL. The volume ratio of the Mxene-Au solution, the complementary strand solution containing the nucleic acid aptamer, and the solution containing the aptamer complex is 1:1:1 to 1:2:
2.
7. The preparation method according to claim 5, characterized in that, The preparation method of the Mxene-Au includes the following steps: (i) Prepare a hydrofluoric acid solution containing Ti3AlC2, stir, centrifuge, wash and dry to obtain Mxene; (ii) Prepare an aqueous solution containing Mxene, add chloroauric acid solution while stirring, after reaction, centrifuge, wash, and dry to obtain the Mxene-Au; In steps (i) and (ii), the drying temperature is independently 50~70°C and the drying time is independently 10~15h. In step (i), the concentration of the aqueous solution containing Mxene is 0.2~0.5 mg / mL; The stirring time is 20-30 hours, and the stirring temperature is 20-30°C. The centrifugal washing was performed using ultrapure water until the pH of the supernatant was 6. In step (ii), the mass ratio of Mxene to chloroauric acid is 1:1 to 5:1; The reaction time is 3 to 6 minutes.
8. A method for detecting biotoxins, characterized in that, An electrochemical sensor prepared by the preparation method according to any one of claims 5 to 7; A standard solution containing biotoxins is transferred to the surface of the carbon working electrode. A phosphate buffer solution containing hydrogen peroxide and o-phenylenediamine is added dropwise to the hydrophilic working region. Electrochemical signals are detected by differential pulse voltammetry, and a standard curve of current intensity versus biotoxin concentration is plotted. The current intensity of the sample to be tested is compared with the standard curve to calculate the biotoxin concentration.
9. The method according to claim 8, characterized in that, The phosphate buffer solution has a pH of 6.4-7.0, a hydrogen peroxide concentration of 2-5 mM, and an o-phenylenediamine concentration of 5-10 mM.
10. The method according to claim 8, characterized in that, The biotoxin in question is ochratoxin A.