Preparation method of electrochemical sensor for detecting acrylamide based on Ce-MOF and AuPd nanoflower composite material

Abstract

The invention designs a preparation method of an electrochemical sensor for detecting acrylamide based on a Ce-MOF and AuPd nanoflower composite material. The advantages of large surface area and good stability of Ce-MOF are utilized to load a large amount of AuPd NFs, and the composite material Ce-MOF (at) AuPd NFs with high conductivity is obtained. The preparation method comprises the following steps: modifying an electrode by virtue of a physical adsorption effect, fixing a triple-helix molecular switch THMS on the electrode by virtue of a gold-sulfur bond and a palladium-sulfur bond, when acrylamide exists, combining acrylamide with an aptamer, forming DNAzyme by virtue of single-stranded CDNA1 and single-stranded CDNA2, and driving a DNA walker amplification strategy in the presence of Mg < 2 + >, so that a hairpin HP1 is sheared, and a large amount of single-stranded S1 is obtained. According to the method, S1, signal tags combined on an electrode are reduced and signals are reduced by destroying a triple helix structure, so that acrylamide detection is realized. The electrochemical biosensor constructed by the method disclosed by the invention has relatively high specificity and relatively high sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a method for preparing an electrochemical sensor for detecting acrylamide based on a Ce-MOF and AuPd nanoflower composite material. Background Art

[0002] Acrylamide (AA) is an environmental and food contaminant that can be exposed to humans through the skin, mucous membranes, respiratory tract, and digestive tract. Diet is a significant route of human exposure. Acrylamide is commonly found in fried and baked foods (coffee, potato chips, biscuits, etc.) that undergo high-temperature processing. It is primarily formed by the Maillard reaction between reducing sugars and asparagine at high temperatures (greater than 120°C). Due to its neurotoxicity in humans, acrylamide is considered a potential neurotoxin, as well as due to its mutagenicity, genotoxicity, and carcinogenicity in animals. Acrylamide is closely related to food safety issues and poses potential risks to human health. Therefore, the development of a simple and sensitive method for the detection of acrylamide in food is crucial.

[0003] Currently, there are numerous methods for detecting acrylamide, including liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-tandem mass spectrometry (GC-MS), ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), and supercritical fluid chromatography-tandem mass spectrometry. These methods offer high sensitivity and specificity, but they are costly, require complex sample pretreatment procedures, and rely on trained personnel, making them difficult to meet the requirements for accurate on-site detection. Fluorescence analysis offers excellent stability and rapid response, but suffers from limited sensitivity and expensive equipment. Electrochemical sensors, however, offer advantages such as low cost, simple operation, and rapid analysis, potentially overcoming these challenges. However, to further enhance the specificity and sensitivity of electrochemical sensors, electrochemical aptamer sensors have been employed. Aptamers are single-stranded DNA or RNA molecules derived in vitro through the systematic evolution of ligands by exponential enrichment (SELEX) process. Aptamers offer advantages such as good stability, strong specificity, and ease of storage, thereby enhancing the specificity of electrochemical sensors. Secondly, a composite material of Ce-MOF and AuPd nanoflowers was designed. The advantages of Ce-MOF's large surface area and good stability were used to load a large amount of gold-palladium nanoflowers, thereby improving the conductivity of the electrode. 2+ A DNA walker cyclic amplification strategy driven by a DNAzyme-dependent mechanism has been proposed, which has excellent signal amplification performance and higher reaction rate, thereby improving the sensitivity of the electrochemical sensor. Summary of the Invention

[0004] The present invention provides a method for preparing an electrochemical sensor for detecting acrylamide based on a Ce-MOF and AuPd nanoflower composite material.

[0005] A method for preparing an electrochemical sensor for detecting acrylamide based on a Ce-MOF and AuPd nanoflower composite material is carried out according to the following steps: (1) Preparation of AuPd nanoflowers: First, hexadecylpyridinium chloride monohydrate and ascorbic acid were dissolved in water. After magnetic stirring at a constant temperature for a certain period of time, gold salt was added to the solution and the reaction continued for 20 minutes. Subsequently, palladium salt was added to the mixture and the reaction was continued under magnetic stirring for 3 hours. The product was collected by centrifugation and washed several times with ultrapure water. The resulting precipitate was dispersed in water, namely AuPd NFs.

[0006] (2) Preparation of Ce-MOF and AuPd nanoflower composites: First, cerium nitrate hexahydrate was dissolved in water, and 1,3,5-benzenetricarboxylic acid was dissolved in an equal volume solution of ultrapure water and anhydrous ethanol. The two solutions were mixed and further sonicated to obtain a mixture. After the mixture was heated in a water bath for a certain period of time, it was centrifuged and washed with an equal volume solution of ultrapure water and anhydrous ethanol, and dried to a constant weight to obtain a white product, namely Ce-MOF. The synthesized Ce-MOF and AuPd NFs were magnetically stirred for a certain period of time to obtain Ce-MOF@AuPd NFs.

[0007] (3) Determination of acrylamide: Ce-MOF@AuPd NFs were modified on the surface of the gold electrode by physical adsorption, and the triple helical molecular switch THMS (composed of A1 and A2 labeled with thiol) was fixed on the material through gold-sulfur bonds and palladium-sulfur bonds, and the active site was blocked with 6-mercapto-1-ethanol. When the target is present, the target binds to the aptamer Apt, and the CDNA1 sequence labeled with biotin is exposed. At this time, it is combined with HP1 also labeled with biotin to the magnetic beads through streptavidin-biotin, and CDNA2 is introduced. CDNA2 can form a DNAzyme with CDNA1 and is activated on the Mg 2+ In the presence of , the DNAzyme is activated, driving the DNA walker and cleaving HP1. The supernatant obtained by magnetic separation contains a large amount of S1. When added to the electrode, S1 binds to A1 in the triple helix on the electrode, disrupting the triple helix. When the electrode is immersed in a methylene blue solution, the intact triple helix can bind a large amount of methylene blue. However, in the presence of acrylamide, S1 disrupts the triple helix, reducing the amount of bound methylene blue and the signal.

[0008] It is further defined that in step (1), the constant temperature is 30-40°C; the time is 5-10 min; the gold salt is one or more of tetrachloroauric acid and potassium aurous cyanide, and the palladium salt is one or more of palladium chloride, palladium nitrate, and sodium tetrachloropalladate.

[0009] It is further defined that in step (2), the water bath heating temperature is 50-80°C; the time is 0.5-2 h; and the stirring time is 20-30 h.

[0010] It is further defined that in step (3), the concentration of the DNA chain is 1~5 μM; the volume of the DNA chain is 2~10 μL; the sequence of the CDNA1 is 5'-ATC ACG ACA GCG ATC GAG GAT TTG CCG TTT CCG GTT TTT TTTTTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-3', wherein the 3' end is labeled with biotin; the sequence of the CDNA2 is 5'-CGG CAA ATC CTC GCA CCC ATG TAT GCG CAA T-3'; the sequence of the HP1 is 5'-TTT TTT TTT TTT TTT TTT AAC TTC ACT ACA AA T TAT TGC GCA TT / rA / GGT CGT GATGAA GGG ATG TGA AGA AGT GAG GAG GGA AG-3', wherein the 5' end is modified with biotin and contains an rA site in the middle. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a preparation method of an electrochemical sensor for detecting acrylamide based on a Ce-MOF and AuPd nanoflower composite material and a schematic diagram of detecting acrylamide.

[0012] Figure 2 Square wave voltammetry curves of the sensor constructed in the present invention before (solid line) and after (dashed line) the addition of 250 nM acrylamide.

[0013] Figure 3 This is the standard curve for detecting acrylamide by the sensor constructed in the present invention.

[0014] Figure 4 This is the specificity of the sensor constructed by the present invention to acrylamide. DETAILED DESCRIPTION

[0015] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Example

[0016] A method for preparing an electrochemical sensor for detecting acrylamide based on a Ce-MOF and AuPd nanoflower composite material, wherein the method is as follows Figure 1 shown.

[0017] The specific steps are as follows: (1) Preparation of AuPd nanoflowers: First, 144 mg of chlorohexadecylpyridinium monohydrate and 4.0 mL of ascorbic acid (100 mM) were dissolved in 10 mL of ultrapure water. After stirring at 35°C for 6 min, 256 μL of tetrachloroauric acid (10 mM) was added to the solution and stirred at this temperature for 20 min. Subsequently, 3.2 mL of sodium tetrachloropalladate (10 mM) solution was added to the mixture and reacted under magnetic stirring for 3 h. The product was collected by centrifugation at 10,000 rpm for 20 min and washed several times with ultrapure water. The obtained product was dispersed in ultrapure water and stored at 4°C for later use.

[0018] (2) Preparation of Ce-MOF and AuPd nanoflower composites: First, 0.434 g of cerium nitrate hexahydrate was dissolved in 15 mL of ultrapure water, and 0.23 g of 1,3,5-benzenetricarboxylic acid was dissolved in 10 mL of a solution of equal volumes of distilled water and ethanol. The two solutions were mixed and dissolved by ultrasonication. After the mixture was kept in a water bath at 60°C for 1 hour, the product was collected by centrifugation at 8000 rpm for 10 minutes and washed several times with equal volumes of distilled water and ethanol to remove the unreacted solvent. The precipitate obtained by centrifugation was dried at 60°C for 12 hours to obtain a white product, namely Ce-MOF. 500 μL of Ce-MOF (2 mg / mL) and 500 μL of AuPd NFs were mixed, and the mixture was magnetically stirred for 24 hours. The product was centrifuged at 8000 rpm for 10 minutes and washed several times with ultrapure water to obtain Ce-MOF@AuPd NFs.

[0019] (3) Identification of the target: Add 5 μL of 4.8 μM Apt and 5 μL of acrylamide solutions of different concentrations to a centrifuge tube, shake at a constant temperature of 37°C for 1 hour. After the reaction is completed, add 10 μL of 2 μM CDNA1 to the centrifuge tube and shake at a constant temperature of 37°C for 1 hour to obtain mixture A. Add 15 μL of 10 mg / mL magnetic beads MBs to a 200 μL centrifuge tube, vortex to mix, and wash three times with 50 μL of Buffer I buffer. Add mixture A and 10 μL of 3 μM HP1 to the washed magnetic beads, vortex to mix, shake at a constant temperature of 37°C for 1 hour, magnetically separate and wash with Buffer I buffer to obtain a precipitate. Add 5 μL of 4 μM CDNA2 and 20 μL of 10 mM Mg to the precipitate. 2+ The solution was shaken at a constant temperature of 37 °C for 1.5 h, and the supernatant was transferred to another centrifuge tube for subsequent use after magnetic separation.

[0020] (4) Construction of electrochemical sensor: First, 5 μL of Ce-MOF@AuPd NFs was added to the surface of the gold electrode and incubated at 37°C for 1 h in a constant temperature and humidity chamber. The electrode was then washed with Tris-HCl buffer to remove the unadsorbed Ce-MOF@AuPd NFs. Then, 5 μL of 2 μM THMS (triple helix) was added, incubated at 37°C for 2 h, and the electrode was washed. 5 μL of 0.1 mM MCH blocking agent was added and incubated at 37°C for 1 h to block the unbound sites. The electrode was then washed with Tris-HCl buffer to remove the MCH that was not bound to the electrode surface. 5 μL of the supernatant obtained in (3) was added to the electrode surface and incubated at 37°C for 2 h. The above electrode was immersed in a 16 mM methylene blue solution to allow the methylene blue to be embedded in the DNA chain. Finally, the electrode was placed in a PBS buffer solution, and the electrochemical signal of the signal tag was detected by square wave voltammetry. Acrylamide can be quantitatively analyzed by the change in response signal before and after the addition of acrylamide.

[0021] (5) Establishment of standard curve: Add 5 μL of acrylamide standard solution of different concentrations to (3) and perform step (4) to obtain the corresponding electrical signal. Use the logarithmic value of acrylamide concentration as the horizontal axis and the current signal as the vertical axis to perform linear fitting to establish the standard curve of the sensor for acrylamide.

[0022] like Figure 2 Shown are the square wave voltammetry curves of the sensor constructed in Example 1 of the present invention before (solid line) and after (dashed line) the addition of 250 nM acrylamide.

[0023] like Figure 3 As shown, this is the standard curve for detecting acrylamide using the sensor constructed in Example 1 of the present invention. Example

[0024] A method for preparing an electrochemical sensor for detecting acrylamide based on a Ce-MOF and AuPd nanoflower composite material, and its practical application, comprising the following steps: (1) In order to verify that the prepared Ce-MOF and AuPd nanoflower composite electrochemical sensor for sensitive detection of acrylamide has specific recognition of acrylamide, acrylamide and other interfering substance standards were added to Tris-HCl buffer solution to prepare standard solutions, with the concentration of each being 250 nM. The above-mentioned several different interfering substance standard solutions were tested according to the detection system constructed in Example 1, and the test results are as follows: Figure 4 As shown, it is shown that the method of the present invention has high selectivity for acrylamide. Example

[0025] A method for preparing an electrochemical sensor for detecting acrylamide based on a Ce-MOF and AuPd nanoflower composite material, and its practical application, comprising the following steps: (1) Actual sample processing: Coffee samples and ground potato chip samples were dispersed in water (200 mg / mL) and ultrasonically treated for 30 min. Then, 2.5 mL of n-hexane was added, and the mixture was placed in a constant temperature oscillator for 15 min. After centrifugation at 4000 rpm for 15 min, the operation was repeated to complete the defatting process. The resulting precipitate was treated with Carrez I and II (1 mL each) to break the emulsion and precipitate proteins and carbohydrates. After centrifugation at 4000 rpm for 15 min, the supernatant was obtained, filtered through a 0.45 μm microporous membrane, and diluted 500 times with Tris-HCl. The food extract was obtained by the standard addition method.

[0026] (2) Sample detection: The electrical signal was measured according to steps (1) to (4) of Example 1, and the concentration of acrylamide in the sample was obtained by substituting the standard curve into the electrical signal.

[0027] (3) When coffee was used as the actual sample for measurement, 0.01 times and 20 times the standard amount of acrylamide were added to the coffee, respectively, with a 5 nM addition amount as the reference. 5 μL of the sample solution was taken, and the electrical signal was measured according to steps (1) to (4) of Example 1. The standard curve detected in Example 1 was used to obtain the acrylamide concentration in the sample. Each sample was measured three times and the average value was taken. The average recovery rate of the prepared electrochemical sensor was calculated to be 101.2% to 110.1%.

[0028] The prepared electrochemical sensor has been verified to have the advantages of fast response, high sensitivity, good selectivity, wide detection range, good reproducibility and stability for the detection of acrylamide. The detection of actual samples shows that the prepared sensor has very good practical application value.

[0029] 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 embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0030] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for preparing an electrochemical sensor for detecting acrylamide based on Ce-MOF and AuPd nanoflower composite materials, characterized in that: The following steps are involved: (1) Preparation of AuPd nanoflowers: First, hexadecylpyridinium chloride monohydrate and ascorbic acid are dissolved in water, and gold salt and palladium salt are added to the solution in sequence. The product is collected by centrifugation and washed with ultrapure water for multiple times. Finally, it is dispersed in water to obtain AuPd nanoflowers, i.e., AuPd NFs. As an electrode modification material, compared with single precious metal particles, the bimetallic composite material has better conductivity due to the synergistic effect. (2) Preparation of Ce-MOF and AuPd nanoflower composite materials: Ce nitrate hexahydrate was dissolved in water, and 1,3,5-benzenetricarboxylic acid was dissolved in an equal volume solution of ultrapure water and anhydrous ethanol. The two solutions were mixed and heated in a water bath to obtain Ce-MOF. Taking advantage of the large surface area and good stability of Ce-MOF, it was stirred and mixed with the prepared AuPd NFs to obtain Ce-MOF@AuPd NFs with good conductivity, which was used as an electrode modification material. (3) Electrochemical sensor for detecting acrylamide: Ce-MOF@AuPd NFs were added dropwise onto a gold electrode to be modified on the gold electrode surface by physical adsorption. The triple-helix molecular switch THMS, which was composed of a single-stranded A1 and a single-stranded A2 labeled with a thiol group, was fixed on the material through a gold-sulfur bond and a palladium-sulfur bond. When the target was present, the target bound to the aptamer chain Apt, and the single-stranded CDNA1 sequence was exposed. At this time, it was fixed to the surface of the magnetic beads together with the hairpin sequence HP1, and the single-stranded CDNA2 was introduced, so that the single-stranded CDNA1 and the single-stranded CDNA2 were combined to form a DNAzyme. 2+ Under the activation of , the DNA walker amplification strategy is driven, so that the hairpin sequence HP1 is sheared. The supernatant obtained by magnetic separation contains a large amount of single-stranded S1. When it is added to the electrode surface, the triple helix structure can be destroyed, the methylene blue bound to the electrode surface is reduced, and the signal is reduced.

2. The method for preparing an electrochemical sensor for detecting acrylamide based on Ce-MOF and AuPd nanoflower composite materials according to claim 1, characterized in that: In step (1), the gold salt is one or more of tetrachloroauric acid and potassium aurous cyanide, and the palladium salt is one or more of palladium chloride, palladium nitrate, and sodium tetrachloropalladate.

3. The method for preparing an electrochemical sensor for detecting acrylamide based on a Ce-MOF and AuPd nanoflower composite material according to claim 1, characterized in that: In step (2), the water bath heating temperature is 50-80°C; the time is 0.5-2 h; and the stirring time is 24 h.

4. The method for preparing an electrochemical sensor for detecting acrylamide based on a Ce-MOF and AuPd nanoflower composite material according to claim 1, characterized in that: In step (3), the volume of the DNA chain is 2-10 μL; the volume of the magnetic beads is 10-20 μL; the volume of the Mg 2+ The concentration is 5~15 mM.

5. The method for preparing an electrochemical sensor for detecting acrylamide based on a Ce-MOF and AuPd nanoflower composite material according to claim 1, characterized in that: In step (3), the sequence of the single-stranded CDNA1 is 5'-ATC ACG ACAGCG ATC GAG GAT TTG CCG TTT CCG GTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTTTTT TTT TT-3', wherein the 3' end is labeled with biotin; the sequence of the single-stranded CDNA2 is 5'-CGG CAA ATCCTC GCA CCC ATG TAT GCG CAA T-3'; the sequence of the hairpin sequence HP1 is 5'-TTT TTT TTT TTTTTT TTT AAC TTC ACT ACA AA T TAT TGC GCA TT / rA / GGT CGT GAT GAA GGG ATG TGAAGA AGT GAG GAG GGA AG-3', wherein the 5' end is modified with biotin and contains an rA site in the middle.

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