CN211577172U - Acrylamide sensor - Google Patents

Abstract

The utility model discloses an acrylamide sensor, including the modified electrode, chitosan and stannic oxide/carbon nano chain are added to the modified electrode surface, then add the acrylamide antigen who uses Bovine Serum Albumin (BSA) as the carrier to chicken Ovalbumin (OVA) seals the active site that does not combine. The gold nanoparticle labeled primary antibody is fixed on the surface of an electrode through immunoreaction, acrylamide to be detected is dripped into the modified electrode to form a three-electrode system with a platinum electrode and a saturated calomel electrode, the acrylamide causes system current change, an electrochemical workstation captures signals, and a detection method is established according to the relation between the concentration of the acrylamide to be detected and the electrochemical signals. The sensor can be used for quantitative analysis of acrylamide in food. The utility model discloses an acrylamide electrochemical sensor detects fast, sensitivity is high, the specificity is strong, has fine popularization application prospect.

Description

Acrylamide sensor

Technical Field

The utility model relates to an analysis and detection technical field especially relates to an acrylamide sensor.

Background

Acrylamide is a compound with neurotoxicity, genotoxicity and potential carcinogenicity. In 4 months 2002, the swedish National Food Administration (NFA) and scientists at stockholm university found that acrylamide is detected in some fried foods such as french fries, potato chips, bread, etc., and has attracted widespread attention from international organization and society. In 2002, 6 months, the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) of the united nations have made an urgent conference to investigate the safety of acrylamide consumption. In 2 months 2005, the joint food additives Joint Experts Committee (JECFA) under the food and agriculture organization and the world health organization of the united nations performed a systematic risk assessment of acrylamide. A large number of animal experimental studies show that acrylamide can cause tumors in various organs of rats, such as oral cavity, thyroid gland, mammary gland, testis, uterus, pituitary gland, and the like. The World Health Organization (WHO) stipulates that the maximum allowable residual amount (MRL) of acrylamide in drinking water is 0.5. mu.g/kg. Therefore, the method for detecting acrylamide in food with high sensitivity, high speed and high efficiency is of great significance.

At present, the detection of acrylamide residue in food mainly comprises an instrumental analysis method, an immunoassay method, an electrochemical sensor method and the like. The instrumental analysis method is a traditional analysis method of acrylamide and mainly comprises gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS) and other methods. The instrumental analysis method has the advantages of high sensitivity, good selectivity, easy realization of automation and the like, but the detection methods need expensive and complicated instruments and equipment and professional operators, and are complicated in sample treatment, complex in detection process and high in detection cost, so that the requirements of high-throughput and on-site rapid detection are difficult to meet. Compared with an instrument analysis method, the immunosensor has the advantages of rapidness, specificity, low detection cost, low requirement on operators and the like.

SUMMERY OF THE UTILITY MODEL

An object of the utility model is to provide a quick, accurate, high sensitive acrylamide sensor provides the foundation for realizing the on-the-spot short-term test of acrylamide.

In order to achieve the above purpose, the utility model adopts the following technical scheme.

An acrylamide sensor comprises a modified electrode and is characterized in that a chitosan layer is arranged on the surface of the modified electrode, a tin dioxide/carbon nano-chain layer is arranged in the chitosan layer, and a coating original layer is arranged on the outer surface of the chitosan layer; the unbound active sites in the coating layer are blocked by chicken ovalbumin.

More preferably, the modified electrode is a glassy carbon electrode.

More preferably, the coating antigen layer is an acrylamide antigen layer, and the acrylamide antigen layer takes bovine serum albumin as a carrier.

More preferably, the acrylamide sensor, the gold nanoparticle labeled primary antibody compound, the object to be detected and the colloidal gold labeled horseradish peroxidase-gold nanoparticle-secondary antibody compound jointly form a working electrode, and the working electrode, the counter electrode and the reference electrode form a three-electrode system; acrylamide causes electrochemical signals of the three-electrode system, the electrochemical workstation captures the electrochemical signals, and a detection method is established according to the relation between the concentration of the acrylamide to be detected and the electrochemical signals.

More preferably, the counter electrode is a platinum electrode and the reference electrode is a saturated calomel electrode or an Ag/AgCl electrode.

The utility model has the advantages that:

the utility model deposits a chitosan layer, a stannic oxide/carbon nano-chain layer and a coating original layer on the surface of the modified electrode, and the stannic oxide/carbon nano-chain layer is used as a signal amplifying element; in actual detection, the acrylamide immunosensor with high sensitivity is constructed by combining high-specificity immunoreaction, so that a basis is provided for realizing on-site rapid detection of the acrylamide immunosensor. Through practical test, the utility model provides an acrylamide sensor is to the half inhibitory concentration (IC50) of detection of acrylamide is 0.04ng/kg, and linear range is 0.007ng/kg-0.24ng/kg, and the detection limit is 0.0028 ng/kg.

Drawings

Fig. 1 shows a flow chart for preparing an acrylamide sensor according to the present invention.

Description of reference numerals:

1: modified electrode, 2: chitosan layer, 3: tin dioxide/carbon nanochain, 4: coating original layer, 5: casein, 6: gold nanoparticle-labeled primary antibody complex, 7: analyte, 8: and (3) marking the horseradish peroxidase-gold nanoparticle-secondary antibody compound by using colloidal gold.

Detailed Description

The following description will be further made in conjunction with the accompanying drawings of the specification, so that the technical solution and the advantages of the present invention are clearer and clearer. The embodiments described below are exemplary and are intended to be illustrative of the present invention, but should not be construed as limiting the invention.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, or may be learned by practice of the invention.

An acrylamide sensor made by:

1) synthesis of tin dioxide/carbon nanochain

Tin dioxide/carbon nanochains (SnO)₂Preparation of @ C NCs): 0.153g of sodium stannate and 2.68g D-glucose are dissolved in 18mL of deionized water, then the solution is transferred into a polytetrafluoroethylene reaction kettle and heated for 5 hours at the constant temperature of 180 ℃, and the product is washed by the deionized water and ethanol after centrifugation and dried. The tube furnace was placed at 700 ℃ for 3h under low flow argon as shielding gas.

2) Synthesis of gold nanoparticles

The synthesis of gold nanoparticles (AuNP) employs trisodium citrate reduction: 100mL of a 0.01 wt% aqueous chloroauric acid solution was placed in a round bottom flask, and heated to boiling on an electric mantle. 1.9mL of a 1 wt% aqueous solution of trisodium citrate was added rapidly and accurately with continued stirring, and heating was continued for 2.5min until the solution was a clear red color. After cooling at room temperature, the solution was returned to the original volume with deionized water and stored at 4 ℃ for further use.

3) Synthesis of gold nanoparticle-labeled primary antibody

Gold nanoparticle labeled primary antibody complex (AuNP-Ab)₁) The preparation of (1): 0.1mol/L of K₂CO₃The pH of the gold nanorod solution was adjusted to 9.2. Adding 1mL and 9 mu g/mL of Acylamide-4-MPA antibody into the gold nanorod solution, fully mixing for 20min, blocking for 1h by 0.45mL of 5% BSA solution, centrifuging at 4 ℃ for 20min, and repeatedly washing the precipitate with 1mL of 0.01mol/L PBS (phosphate buffer solution) with pH 7.0. The final product was dispersed in 1mL of 0.01mol/L PBST pH 7.4 and stored at 4 ℃ until use.

4) Synthesis of gold nanoparticle-labeled secondary antibody and enzyme

Horse radish peroxidase-gold nanoparticle-secondary antibody (HRP-AuNP-Ab) marked by colloidal gold₂) The preparation process of the compound is as follows: taking 250 mu L of the gold nanoparticle solution AuNP prepared above, and adding 0.1mol/L of K₂CO₃The pH was adjusted to 9.2. Then, 1mL of 10. mu.g/mL secondary antibody and 1mL of 0.4mg/mL horseradish peroxidase HRP were added to the AuNP solution, the mixture was thoroughly mixed for 20min, then blocked with 0.5mL of 5 wt% BSA solution for 1h, centrifuged at 12,000r/min at 4 ℃ for 30min, and the precipitate was washed repeatedly with 1mL of 0.01mol/L, pH value-7.4 PBS. The final product was dispersed in 1mL of PBS with a value of 0.01mol/L, pH of 7.4 and stored at 4 deg.CThe application is as follows.

5) Preparation of acrylamide sensor

As shown in a combined figure 1, chitosan and the tin dioxide/carbon nanochain are added to the surface of a modified electrode 1, the chitosan is deposited on the surface of the modified electrode 1, the tin dioxide/carbon nanochain 3 is clamped in a chitosan layer 2, then a coating original layer 4 is fixed on the outer surface of the chitosan layer 2, and the unbound active sites on the coating original layer 4 are sealed by casein 5, so that the acrylamide sensor is prepared.

Wherein the modified electrode is a glassy carbon electrode. The steps of depositing the chitosan and the tin dioxide/carbon nano chain on the glassy carbon electrode are as follows: 1) and polishing the surface of the glassy carbon electrode, and airing at room temperature. 2)0.5mg, SnO₂And ultrasonically dispersing the @ C NCs in 1mL of 0.2 wt% chitosan aqueous solution, dropwise adding 5 mu L of chitosan aqueous solution onto the surface of the pretreated glassy carbon electrode, and airing at room temperature. 3) Washing the electrode surface with deionized water, dripping 5 μ L of coating antigen with certain concentration, and incubating at 37 deg.C for 1 h. Unadsorbed coating antigen was washed with 0.01mol/L, pH value 7.4 PBST, dried with nitrogen and incubated with 6. mu.L of 10 wt% chicken ovalbumin solution for 1h to block the unbound sites and prevent non-specific adsorption. 4) The electrode was rinsed with 0.01mol/L, pH PBST with a value of 7.4 and placed in a refrigerator at 4 ℃ until use.

In actual detection, gold nanoparticle labeled primary antibody complex 6 (AuNP-Ab) is added into the acrylamide sensor₁) And a substance to be detected 7, adopting a competitive reaction mode, wherein the concentration of the gold nanoparticle marked primary antibody compound 6 fixed on the electrode is inversely proportional to the concentration of the substance to be detected 7, and the colloidal gold marked horse radish peroxidase-gold nanoparticle-secondary antibody compound 8 (HRP-AuNP-Ab)₂) And primary antibody is fixed on the surface of the electrode through a specific reaction. The acrylamide sensor serves as a working electrode, the acrylamide sensor, a platinum electrode and a saturated calomel electrode form a three-electrode system, and an HRP enzyme catalysis substrate on a gold nanorod-marked secondary antibody probe generates an electrochemical signal which is recorded by an electrochemical workstation. And establishing a standard curve of detection according to the relation between the electrochemical signal and the concentration of the drug.

From the above description, it should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and modifications and substitutions based on the known art are all within the scope of the present invention, which should be defined by the terms of the appended claims and their equivalents. The details not described in the detailed description are prior art or common general knowledge.

Claims (4)

1. An acrylamide sensor comprises a modified electrode and is characterized in that a chitosan layer is arranged on the surface of the modified electrode, a tin dioxide/carbon nano-chain layer is arranged in the chitosan layer, and a coating original layer is arranged on the outer surface of the chitosan layer; the unbound active sites in the coating layer are blocked by chicken ovalbumin.

2. The acrylamide sensor according to claim 1 wherein the modified electrode is a glassy carbon electrode.

3. The acrylamide sensor according to claim 1 wherein the coating layer is an acrylamide antigen layer.

4. The acrylamide sensor according to claim 3 wherein the acrylamide antigen layer is supported by bovine serum albumin.

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