Detection method and kit for gluten content in food

Abstract

The present invention relates to a method and a kit for detecting the content of gluten in food. The kit comprises a Raman enhanced substrate solution and a quartz sheet, wherein the Raman enhanced substrate solution comprises a gold nanorod dimer and a Raman signal molecule, wherein the gold nanorod dimer is self-assembled by aptamer-functionalized gold nanorods and aptamer complementary chain-functionalized gold nanorods. The kit is easy to operate, convenient in instrumentation, and can detect the content of gluten in food quickly, stably, and sensitively.

Description

Technical Field

[0001] The invention relates to the technical field of bioanalysis, and in particular to a method and a kit for detecting the content of wheat gluten in food. Background Art

[0002] Wheat is the most commonly used food raw material and is widely used in food and food industry. However, wheat, as one of the eight common allergic foods, can cause severe allergic reactions. Wheat gluten has been clearly identified as the protein in wheat that causes celiac disease, mainly including gliadin and glutenin, which account for about 80% to 85% of the total wheat protein. However, there is currently no specific treatment for wheat allergy, and strictly avoiding the consumption of foods containing allergens is the most effective treatment. Therefore, the detection of gluten is closely related to the production and labeling of food. For the detection of food allergen components in food, there are mainly protein-based methods (such as ELISA detection) and gene-based methods (such as PCR detection). These methods have the advantages of high detection sensitivity and high accuracy, but there are limitations such as long time consumption, large and expensive equipment, and the need for professional operators. Therefore, it is necessary to establish a rapid, portable, accurate and sensitive method for detecting wheat gluten. Summary of the invention

[0003] The present invention aims to solve one of the technical problems in the related art to at least a certain extent. To this end, the purpose of the present invention is to provide a method and a kit for detecting the content of gluten in food. The kit is easy to operate, convenient in instrumentation, and can detect the content of gluten in food quickly, stably and sensitively.

[0004] To this end, in a first aspect of the present invention, a kit for detecting the gluten content in food is proposed, comprising a Raman enhanced substrate solution and a quartz sheet, wherein the Raman enhanced substrate solution comprises a gold nanorod dimer and a Raman signal molecule, wherein the gold nanorod dimer is self-assembled by aptamer-functionalized gold nanorods and aptamer complementary chain-functionalized gold nanorods.

[0005] According to a kit of the present invention, a gold nanorod dimer is prepared as a Raman probe by using self-assembly technology, and the gold nanorod dimer is disaggregated to form a single dispersed gold nanorod based on the specific recognition of wheat alcohol-soluble protein and aptamer. In the sensing system, the Raman intensity is proportional to the yield of the gold nanorod dimer. Therefore, the wheat gluten concentration is inversely proportional to the Raman signal intensity. The kit is easy to operate, the instrument is convenient, and can quickly, stably and sensitively detect the wheat gluten content in food.

[0006] Optionally, the nucleic acid sequence of the aptamer is shown as SEQ ID NO: 1, and the nucleic acid sequence of the complementary strand of the aptamer is shown as SEQ ID NO: 2.

[0007] Optionally, the molar ratio of the gold nanorods to the aptamer or the aptamer complementary chain is 1:60.

[0008] Optionally, the gold nanorods have an aspect ratio of 3.7.

[0009] In a second aspect of the present invention, the present invention provides a method for detecting the content of gluten in food using the above-mentioned kit, which comprises:

[0010] Take 20 μL of Raman enhancement substrate solution, add 1 μL of the sample solution to be tested, incubate at room temperature to form a mixed solution, take 10 μL of the mixed solution and drop it on a quartz plate and dry it;

[0011] The dried quartz plate is placed in a Raman spectrometer to measure the Raman signal intensity and calculate the total content of wheat gluten in the sample to be tested.

[0012] The detection method according to the embodiment of the present invention can quickly and sensitively detect the gluten content in food.

[0013] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a standard curve for detection of wheat gluten according to an embodiment of the present invention;

[0015] Figure 2 It is the specificity of the kit according to the embodiment of the present invention to detect the standard sample. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0017] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0018] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0019] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0020] Example 1 Preparation of gold nanorod dimers

[0021] Gold nanorod dimers were prepared by self-assembly method, with the following specific steps:

[0022] (1) Add 12 μL ultrapure water, 8 μL 0.2% sodium dodecyl sulfate (SDS), 4 μL 10×TBE buffer, 2.4 μL 1 μM aptamer solution (Apt) (add 19.9 μL TE buffer to each OD of aptamer to make a 100 μM stock solution and dilute to 1 μM) or 2.4 μL 1 μM aptamer complementary solution (RC) (add 22.6 μL TE buffer to each OD of aptamer complementary chain to make a 100 μM stock solution and dilute to 1 μM), mix well, and incubate at room temperature with shaking for 12 h.

[0023] (2) The reaction solution in step (1) was centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. The supernatant was washed once with a 0.5×TBE solution containing 0.02% SDS to remove excess Apt or RC, and the precipitate was dispersed in 10 μL of a 0.5×TBE solution containing 0.02% SDS to obtain AuNRs-Apt or AuNRs-RC. The solution was stored at 4°C for later use.

[0024] (3) The AuNRs-Apt and AuNRs-RC prepared in step (2) were mixed in a ratio of 1:1, and incubated with shaking at room temperature for 12 h to prepare a gold nanorod dimer.

[0025] (4) Adding a Raman signal molecule 4-MBA with a final concentration of 10 μM to the gold nanorod dimer solution prepared in step (3), incubating the mixture under shaking for 4 h at room temperature, thereby obtaining a Raman enhanced substrate solution. The Raman enhanced substrate solution was stored at 4° C. for subsequent gluten detection.

[0026] Example 2 Standard curve for wheat gluten detection

[0027] The detection performance of the kit was investigated using wheat alcohol-soluble protein as a representative. The detection steps are as follows: take 20 μL of reagent 1 (the Raman enhanced substrate solution obtained in Example 1), add 1 μL of sample solution, incubate at room temperature for 45 minutes, take 10 μL of solution and drop it on a quartz plate and dry it, and use a Raman spectrometer to measure the Raman signal intensity. The laser wavelength for Raman intensity measurement is 785 nm. According to the Raman signal molecule at 1078 cm -1 The Raman signal intensity at the location is plotted as a standard curve (e.g. Figure 1 Calculate the total content of wheat prolamin in the sample.

[0028] The test kit is stable and time-saving, with a detection limit as low as 35.06 ng / mL and a quantification limit of 116.87 ng / mL. The test results can be quantitatively detected by a portable Raman spectrometer. All experiments were repeated three times, and the results had good reproducibility.

[0029] Example 3 Stability and accuracy of the kit for testing standard samples

[0030] Three groups of wheat alcohol-soluble protein standard samples of different concentrations were prepared respectively, and each group of samples was tested with this kit. Three parallels were taken for each group of samples, and the experiment was repeated 3 times to calculate the inter-batch and intra-batch differences. The results showed that the inter-batch difference was 2.27% to 8.50%, and the intra-batch difference was 3.01% to 6.58%. The detection method has good stability and precision. The specific results are shown in Table 1 below:

[0031] Table 1

[0032]

[0033] Example 4 Specificity of the kit for detecting standard samples

[0034] The KGI Plant Protein Extraction Kit was used to extract soybean, nut, and peanut protein extracts, and the KGI Protein Extraction Kit was used to extract fish and shrimp protein extracts. Milk powder and whole egg powder were prepared into a 10 mg / mL solution to obtain a protein extract. The specificity of the kit was verified according to the steps in Example 2. The results are as follows: Figure 2 As shown, the SERS intensity of blank samples (peanuts, soybeans, nuts, fish, shrimp, milk and eggs) is close to that of the negative control, indicating that the kit has excellent specificity for detecting wheat gluten.

[0035] Example 5 Accuracy of the kit for testing standard samples

[0036] Take 0.5mL of the whole protein extract of soybean, peanut and nut extracted in Example 4, add different amounts of gliadin, so that the final concentration of gliadin is 0.5, 1.0, 5.0μg / mL, and 3 parallel samples are prepared for each concentration, and the blank spike recovery rate is determined. The test results are shown in Table 2. In the range of 0.5-5.0μg / mL concentration, the recovery rate is 89.64%-108.49%, and the detection method has good accuracy. The specific results are shown in the following table:

[0037] Table 2

[0038]

[0039] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0040] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A kit for detecting the content of gluten in food, characterized in that: The invention comprises a Raman enhanced substrate solution and a quartz sheet. The Raman enhanced substrate solution comprises a gold nanorod dimer and a Raman signal molecule. The gold nanorod dimer is self-assembled by gold nanorods functionalized with an aptamer and gold nanorods functionalized with an aptamer complementary chain. The nucleic acid sequence of the aptamer is shown in SEQ ID NO: 1, and the nucleic acid sequence of the aptamer complementary chain is shown in SEQ ID NO:

2.

2. The kit according to claim 1, characterized in that The molar ratio of the gold nanorods to the aptamer or the aptamer complementary chain is 1:

60.

3. The kit according to claim 1, characterized in that The aspect ratio of the gold nanorods is 3.

7.

4. A method for detecting the gluten content in food using the kit according to any one of claims 1 to 3, characterized in that: include: Take 20 μL of Raman enhancement substrate solution, add 1 μL of the sample solution to be tested, incubate at room temperature to form a mixed solution, take 10 μL of the mixed solution and drop it on a quartz plate and dry it; The dried quartz plate is placed in a Raman spectrometer to measure the Raman signal intensity and calculate the total content of wheat gluten in the sample to be tested.

5. The method according to claim 4, characterized in that The incubation time was 45 min.

6. The method according to claim 4, characterized in that The laser wavelength for Raman intensity measurement was 785 nm.

7. The method according to claim 4, characterized in that Raman signal intensity is the Raman signal molecule at 1078cm -1 The signal strength at the

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