Semi-quantitative rapid detection device and detection method for food allergen protein

Abstract

The invention provides a food allergen protein semi-quantitative rapid detection device and method, the detection device comprises a pretreatment tube, a reaction tube and a color development tube, the pretreatment tube is internally provided with a stirrer for grinding and stirring a sample to be detected and a filter membrane containing a free labeled antibody, and the bottom of the pretreatment tube is provided with a sample liquid outlet; the sample solution slowly passes through the filter membrane under the action of gravity, the target protein and the labeled antibody are combined to form a compound, and the compound and the sample solution flow into a reaction tube below together; the reaction tube is used for capturing an antigen-antibody complex formed in the pretreatment tube, converting the TMB base solution into a golden yellow reaction product and introducing the reaction product into the color development tube. The device is simple to operate, does not need to depend on professionals and equipment, can quickly obtain a detection result, and provides a new method which is quick, economical, convenient and user-friendly for detection of food allergens.

Description

Technical Field

[0001] This invention relates to a protein detection device and method, specifically to a semi-quantitative rapid detection device and method for food allergen proteins. Background Technology

[0002] Reducing exposure to foods containing allergens is an effective way to lower the risk of food allergies. As a preventative measure, on-site testing for allergens in food can effectively prevent allergic reactions.

[0003] In analytical biochemistry, enzyme-linked immunosorbent assay (ELISA) is a commonly used technique for measuring and quantifying specific proteins. Due to its high specificity and sensitivity, it is considered the gold standard for diagnostic purposes, including allergen detection. However, this method requires complex instruments to interpret the results, limiting its potential for on-site testing. Colloidal gold assays can also rapidly detect allergens on-site; however, the results are not precise enough, only indicating the presence or absence of allergens without providing specific concentration information, resulting in lower reliability.

[0004] Enzyme-linked immunosorbent assay (ELISA): As a widely used protein detection method, it boasts advantages such as high accuracy, high sensitivity, and high specificity. However, this method requires specialized laboratory equipment and professional operation, and the testing time is long, making it unsuitable for rapid detection in food.

[0005] Colloidal gold immunochromatography: This method is ideal for point-of-care testing due to its simplicity, rapid detection, portability, and good stability. However, its limited sensitivity and inability to perform quantitative detection result in lower reliability. Furthermore, considering that human tolerance to allergens is dynamic, simply detecting whether protein levels exceed a certain threshold cannot meet the testing needs of the general public.

[0006] Existing food allergy testing equipment includes the NIMA portable food allergen detector from the United States, which can quickly detect the presence of gluten in food and displays the results using indicator lights. It boasts a sensitivity of up to 20 ppm and an accuracy of 99.5%. However, it is limited to gluten detection and cannot meet the testing needs of individuals allergic to other substances. Similarly, this device only provides quantitative results and is expensive.

[0007] In ELISA testing, TMB 2+The optical density of proteins in ELISA samples typically ranges from 0.1 to 3, and this pattern applies to various samples. However, the general public often lacks the skills and resources for laboratory procedures, particularly in on-site testing environments where ideal protein extraction conditions are often unavailable, such as appropriate extraction temperature, sufficient extraction time, and adequate sample grinding, leading to low extraction efficiency. Furthermore, on-site testing usually requires rapid completion, necessitating significantly shorter incubation times and simplified washing steps, typically within 15 minutes, which further weakens the intensity of the optical density signal. Therefore, even if the food sample contains a high level of allergen proteins, their actual concentration in the test sample may be too low due to insufficient extraction efficiency and shortened reaction time. This results in a lower TMB concentration measured by ELISA. 2+ The concentration is extremely low, making it difficult to distinguish from background noise, which may lead to false negatives or other detection errors.

[0008] In ELISA assays, the recognition accuracy of microplate readers is limited when processing extremely low signals, especially when these signals are close to background noise (optical density 0.03), making it difficult for the instrument to distinguish between background noise and the true signal. Furthermore, minute differences between low-concentration samples often fail to be accurately identified. Summary of the Invention

[0009] To address the aforementioned technical problems, the present invention aims to provide a semi-quantitative rapid detection device and method for food allergen proteins. This detection device is simple to operate, requires no specialized personnel or equipment, and can rapidly obtain test results, providing an economical, convenient, and user-friendly new method for detecting food allergens.

[0010] This invention provides a semi-quantitative food allergen protein detection device, the detection device comprising: a pretreatment tube, a reaction tube, and a colorimetric tube, wherein:

[0011] The pretreatment tube is equipped with a stirrer for grinding and stirring the sample to be tested and a filter membrane containing free labeling antibody. The sample solution outlet is located at the bottom of the pretreatment tube.

[0012] The sample solution slowly passes through the filter membrane under the influence of gravity. The target protein in the sample solution binds to the labeled antibody to form a complex, which flows into the reaction tube below along with the sample solution.

[0013] The reaction tube is used to capture the antigen-antibody complex formed in the pretreatment tube, convert the TMB substrate into a golden-yellow reaction product, and introduce the reaction product into the development tube.

[0014] According to a specific embodiment of the present invention, preferably, the pretreatment tube is further provided with an extraction solution for extracting the target protein from the sample to be tested.

[0015] According to a specific embodiment of the present invention, preferably, the colorimetric tube contains a nanoparticle reagent.

[0016] According to a specific embodiment of the present invention, preferably, the extract can be added to the pretreatment tube in advance, or it can be stored in advance and added to the pretreatment tube before use; the nanoparticles can be added to the colorimetric tube in advance, or they can be stored in advance and added to the colorimetric tube before use; wherein, the containers such as the colorimetric tubes containing the nanoparticles need to be stored away from light.

[0017] According to a specific embodiment of the present invention, preferably, the reaction tube has openings at the top and bottom. The top opening is used for the input of sample solution and is connected to the pretreatment tube, while the bottom opening slowly discharges liquid through a filter membrane with fixed capture antibodies and is connected to the colorimetric tube.

[0018] According to a specific embodiment of the present invention, preferably, the filter membrane containing the free labeling antibody and the filter membrane with the fixed antibody are protected and fixed by grooves on the inner walls of the pretreatment tube and the reaction tube, respectively.

[0019] According to a specific embodiment of the present invention, preferably, the connection between the reaction tube and the pretreatment tube is detachable.

[0020] According to a specific embodiment of the present invention, preferably, the reaction tube is designed to be light-proof.

[0021] According to a specific embodiment of the present invention, preferably, the bottom opening of the pretreatment tube is provided with a switch for controlling the inflow and outflow of liquid.

[0022] According to a specific embodiment of the present invention, preferably, the detection device includes a reagent kit containing nanoparticle reagents for reacting with the reaction products in the colorimetric tube to produce a color change.

[0023] According to a specific embodiment of the present invention, preferably, the nanoparticle reagent is a gold nanorod reagent with a concentration of 30 μM to 480 μM, preferably 60 μM.

[0024] This invention utilizes the TMB corresponding to the wavelength change of the optimized gold nanorods. 2+ The characteristic of a narrowing concentration range is related to the generation of TMB after shortening the reaction time. 2+ This principle of "less is more" allows for the successful detection of signals that were previously undetectable after accelerating the reaction time. It is a technique that speeds up the testing process and is not limited by the original ELISA protein concentration. This technique can be applied to reduce the reaction time of ELISA, enabling rapid protein detection without sacrificing detection accuracy.

[0025] According to a specific embodiment of the present invention, preferably, the aspect ratio of the gold nanorod is about 3.3.

[0026] According to a specific embodiment of the present invention, preferably, the detection device includes a standard colorimetric card for comparing the detection results in the colorimetric tube to obtain semi-quantitative analysis results of protein concentration.

[0027] According to a specific embodiment of the present invention, preferably, the free-labeled antibody is capable of specifically binding to the target protein in the sample solution to generate an antigen-antibody complex, and the free-labeled antibody carries horseradish peroxidase.

[0028] According to a specific embodiment of the present invention, preferably, the immobilized capture antibody is capable of capturing antigen-antibody complexes and immobilizing them on a filter membrane inside the reaction tube.

[0029] The working process of the detection tube of the present invention is as follows: The target of the present invention is allergen proteins in food samples. During the test, the food is ground and stirred in the pretreatment tube, while the target protein is extracted with protein extraction solution; the extracted target protein slowly passes through the first membrane under the action of gravity, first binding with free antibodies carrying peroxidase to form a protein-antibody complex, and then flowing into the reaction tube; the complex binds to the fixed antibody through the second membrane in the reaction tube and is fixed in the reaction tube; colorless base solution (TMB) is added to the reaction tube for color development, and the TMB is catalyzed by the peroxidase of the complex on the second membrane to turn blue, producing TMB. 2+ TMB 2+ The concentration of TMB was positively correlated with the target protein in the food sample; 2+ The base solution flows into the colorimetric tube containing gold nanorods, and the TMB... 2+ The nanorods are corrosive and preferentially etch their long sides, causing a blue shift in the corresponding absorption wavelength, which is reflected in a visible color change. The color change or blue shift wavelength is positively correlated with the concentration of the target protein. By comparing with a preset colorimetric card, the specific concentration value can be obtained semi-quantitatively.

[0030] The present invention also provides a protein detection method, which specifically includes the following steps:

[0031] The test sample solution containing the target protein is reacted with a specific antibody that captures the target protein.

[0032] Add a labeled antibody reagent, wherein the labeled antibody carries horseradish peroxidase;

[0033] Add the substrate reagent to obtain the reaction solution;

[0034] The reaction solution is mixed with nanoparticles to obtain a colorimetric solution;

[0035] The colorimetric solution after the reaction was analyzed to obtain the detection results.

[0036] The present invention also provides a method for detecting food allergen proteins using the above-mentioned semi-quantitative rapid detection device, specifically including the following steps:

[0037] The sample to be tested is placed in a pretreatment tube, and the target protein is ground and extracted by a stirrer in the pretreatment tube to obtain a sample solution. The sample solution slowly passes through a filter membrane containing a free labeling antibody under the action of gravity. The target protein binds with the labeling antibody to form an antigen-antibody complex, which flows into the reaction tube below along with the sample solution.

[0038] In the reaction tube, the antigen-antibody complex in the sample solution is captured and fixed by the capture antibody on the filter membrane, and the remaining liquid continues to flow to the outlet by gravity and is discharged into the waste liquid tube.

[0039] After the sample solution is drained, replace the colorimetric tube with the waste liquid tube connected to the bottom outlet;

[0040] The TMB substrate is added to the reaction tube, where it reacts with the peroxidase-containing antigen-antibody complex immobilized on the filter membrane to generate TMB. 2+ And it enters the colorimetric tube along with the solution;

[0041] TMB 2+ The protein was reacted with nanoparticles to obtain detection results, which were then compared with a standard colorimetric card to obtain semi-quantitative analysis results of protein concentration.

[0042] According to a specific embodiment of the present invention, preferably, the pretreatment tube is further provided with an extraction solution for extracting the target protein from the sample to be tested.

[0043] According to a specific embodiment of the present invention, preferably, the colorimetric tube contains a nanoparticle reagent.

[0044] According to a specific embodiment of the present invention, preferably, the nanoparticles are gold nanorods with a concentration of 30 μM to 480 μM, preferably 60 μM.

[0045] According to a specific embodiment of the present invention, preferably, the time taken for the semi-quantitative rapid detection device for food allergen proteins to perform the detection is 15 to 30 minutes. This time refers to the total detection time from sample addition to final comparison results, wherein the reaction time for incubating the sample solution in the pretreatment tube is generally 15 minutes.

[0046] The detection device and detection method of the present invention can adjust TMB 2+The ratio of nanoparticles to total nanoparticles, such as reducing (i.e., "optimizing") the amount of nanoparticles, results in stronger etching of each nanoparticle, exhibiting a more pronounced color change. This allows samples where the wavelength change was previously undetectable due to low concentration to have its wavelength change increased, making the change detectable. Introducing more TMB substrate into the peroxidase can also generate more TMB. 2+ This further enhances the blue shift or color change, achieving signal amplification. Combining the signal amplification effect of nanoparticles with the enrichment effect of the thin film on the chromogenic tube and other signal enhancement methods can achieve the detection of low protein concentrations.

[0047] The beneficial effects of this invention are:

[0048] Food allergies, as an increasingly serious health problem, urgently require a method for rapid food analysis. The semi-quantitative rapid detection device for food allergen proteins provided by this invention is a device for rapid on-site detection of allergen proteins in food. In particular, it is a semi-quantitative rapid detection tube for food allergen proteins that can be operated by non-professionals without the need for complex laboratory equipment. It can overcome the shortcomings of existing technologies and equipment, such as complex operation, reliance on professional personnel and equipment, and inability to obtain semi-quantitative results quickly. It provides an economical, convenient, and user-friendly new solution for the detection of food allergens.

[0049] This invention utilizes the TMB corresponding to the optimized gold nanorods. 2+ The narrowing range is related to the increased TMB generated after shortening the reaction time. 2+ This technique, combining the principle of "less is more," successfully detects signals that were previously undetectable after accelerating the reaction time. It is a method to speed up the testing process and is not limited by the original ELISA protein concentration. This approach allows for rapid protein detection with a shorter ELISA reaction time without sacrificing accuracy.

[0050] Regarding the detection conditions of this invention: The detection device of this invention features naked-eye analysis and semi-automatic testing, allowing even ordinary people to conduct tests anytime and anywhere, such as at campsites, restaurants, or homes. Generally, protein extraction efficiency is low for untrained individuals. However, the membrane inside the reaction tube provided by this invention not only filters out impurities but also enriches the protein. Combined with the signal amplification effect of the gold nanorods, this effectively compensates for the low protein concentration resulting from low extraction efficiency. Furthermore, the detection device of this invention is a disposable device, avoiding potential cross-contamination from reuse.

[0051] The detection device of this invention can enable on-site detection of potential allergens in food without the need for laboratory facilities. The main features of this detection device include:

[0052] 1. The test results are presented in different colors, which realizes the visualization of protein test results.

[0053] 2. The operation is simple and suitable for non-professional users. Users only need to directly participate in two experimental steps: sample addition, grinding, and liquid addition, and simple assembly. This invention provides an assembly design through separately designed pretreatment tubes, reaction tubes, waste liquid tubes, and colorimetric tubes, requiring only simple operation and component combination to complete the test. The reaction tube, as the main component for the immune reaction, can be combined with other components to complete the detection, simplifying the equipment structure, improving reliability, and lowering the barrier to entry.

[0054] 3. The testing conditions are relaxed, requiring no professional laboratory personnel or analytical instruments, enabling semi-automatic testing. Combined with its simple operation and portability, its application scenarios can be extended to the dining table, allowing testing to be conducted without time or space limitations.

[0055] 4. Similar to immunochromatographic assay kits, neither the reaction tubes nor the colorimetric tubes have moving parts, resulting in low production costs.

[0056] 5. The detection device employs a special design, concentrating and storing the labeled and capture antibodies within two circular membranes. These membranes are protected and secured by grooves in the inner wall, ensuring that external pressure or vibration will not cause deformation or malfunction during transport or storage. Furthermore, the inner wall not only provides additional protection but also allows some components (such as the pretreatment tube and colorimetric tube) to simultaneously function as the detection device and store reagents, directly participating in the detection process, thus reducing device size and operational steps. During detection, the inner wall effectively isolates the detection process from the external environment, preventing external contamination and enhancing the protective capabilities of the detection device, thereby ensuring the accuracy of the test results.

[0057] Existing colloidal gold test strips with low sensitivity and NIMA with high sensitivity can only detect the presence of allergens. If the food contains protein concentrations below the threshold, it cannot be detected. Even for the same allergen, the sensitizing concentration varies greatly among different individuals. The detection device of this invention is a portable analytical tool that can operate independently of a laboratory. Its key difference from existing detection devices is that it is not limited by testing conditions such as time, space, and personnel, and it provides semi-quantitative analysis results at a relatively low cost. The technical solution of this invention is based on ELISA, which has higher sensitivity than colloidal gold test strips and can provide semi-quantitative results, meeting the testing needs of different populations and enabling personalized testing. Moreover, thanks to semi-automatic detection and signal amplification, users can independently complete the operation and result analysis without being familiar with laboratory operations, making it highly user-friendly. At the same time, the detection device is small and portable, providing convenient and quick sample analysis for allergy sufferers who need to perform food testing. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of the detection tube process.

[0059] Figure 2 This is a schematic diagram of the dimensions of the testing tube.

[0060] Figure 3 Performance test diagram of the improved high aspect ratio gold nanorod.

[0061] Figure 4 For conventional TMB before and after nanoparticle optimization 2+ Comparison of signal before and after concentration range optimization.

[0062] Figure 5 For low TMB before and after nanoparticle optimization 2+ Comparison of signal before and after concentration range optimization.

[0063] Figure 6 This represents the variation in optical density range produced by standard ELISA at different incubation times.

[0064] Figure 7 The results show the wavelength variation range of the standard ELISA under different incubation times after optimization with gold nanorods.

[0065] Figure 8 The results are from a routine 30-minute protein assay (using micro-gold nanorods).

[0066] Figure 9 This is the result of a routine ELISA protein assay over 2 hours.

[0067] Figure 10 The results are from a routine protein assay (using a standard gold nanorod) taken 30 minutes ago.

[0068] Figure 11 The results show the concentration of cheese bread samples measured under different protein detection conditions.

[0069] Figure 12 The results show the concentration and detection limit of sweet bun samples measured under different protein detection conditions.

[0070] Explanation of reference numerals in the attached figures:

[0071] 201: Stirrer; 202: Pretreatment tube; 203: Reaction tube; 204: Waste liquid tube; 205: Colorimetric tube; 206: Filter membrane containing free-labeled antibody; 207: Sample solution; 208: Filter membrane containing immobilized capture antibody; 209: Waste liquid; 210: TMB base solution; 211: TMB 2+ Solution, 212: Nanoparticles, 213: Switch. Detailed Implementation

[0072] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0073] In a specific embodiment of the present invention, the device structure and reaction process of the semi-quantitative rapid detection device for food allergen proteins of the present invention are as follows: This detection device mainly consists of a pretreatment tube 202, a reaction tube 203, and a colorimetric tube 205 (see details). Figure 1 ).

[0074] (1) The pretreatment tube 202 is mainly used for grinding various forms of food and extracting proteins. It contains a stirrer 201 for grinding and stirring the food, an extractant for extracting the target protein, and a filter membrane 206 containing a free-labeled antibody (the free-labeled antibody contains peroxidase). A small amount of food is placed in the pretreatment tube 202 and ground and stirred simultaneously with the stirrer 201. The extractant will extract the protein from the food. After extraction, the switch 213 at the bottom of the pretreatment tube 202 is turned on. The sample solution 207 slowly passes through the filter membrane 206 containing the free-labeled antibody under the action of gravity. The target protein binds with the labeled antibody to form a complex and flows into the reaction tube 203 connected below along with the sample solution 207.

[0075] (2) Reaction tube 203 is mainly used to capture the antigen-antibody complex formed in pretreatment tube 202, and then convert the TMB base solution 210 into TMB. 2+Solution 211 is provided, and the liquid can be introduced into different tubes. Reaction tube 203 has upper and lower openings. The upper opening is for the input of sample solution 207 and can be connected to the outlet of pretreatment tube 202. The lower opening slowly discharges liquid through a filter membrane with immobilized capture antibodies and can be connected to waste tube 204 or colorimetric tube 205. Reaction tube 203 is designed to be light-proof to prevent photocatalysis of TMB base solution 210 during the reaction, which could affect detection accuracy. When sample solution 207 slowly flows to the filter membrane inside reaction tube 203, the complexes in sample solution 207 are secondary captured and immobilized by the filter membrane 208 containing immobilized capture antibodies. The remaining waste liquid 209 continues to flow to the outlet by gravity and is discharged into waste tube 204. After sample solution 207 is discharged, replace waste liquid tube 204 connected to the bottom outlet with colorimetric tube 205 containing nanoparticles 212. Then, add TMB base solution 210 to reaction tube 203. TMB base solution 210 will react with the peroxidase-containing complex immobilized on the filter membrane to generate TMB. 2+ Solution 211, and along with the solution, enters the colorimetric tube 205.

[0076] (3) The chromogenic tube 205 can transmit protein signals from TMB. 2+ Concentration is converted into a color change, transforming the signal that needs to be distinguished by a spectrometer into a signal that can be analyzed with the naked eye, thus completing semi-quantitative analysis. The upper opening of the colorimetric tube 205 can be connected to the reaction tube 203, which contains nanoparticles 212, i.e., a solution of gold nanorods. TMB 2+ Solution 211 enters the chromogenic tube 205 to etch the gold nanorods. Different protein concentrations will produce different concentrations of TMB. 2+ This results in different etching degrees and color changes. After all the substrate has flowed into the colorimetric tube 205, wait for 5 minutes to complete the test.

[0077] (4) In addition to the main pretreatment tube 202, reaction tube 203, and colorimetric tube 205, the detection device may also include other accessories, such as a standard colorimetric card and a waste liquid tube 204. The colorimetric card contains various colors after the nanogold rods are hand-etched, each corresponding to a different concentration range. These colors are measured by laboratory analytical instruments and laboratory personnel, and the results are reliable and accurate. Users can compare and match the detection results (i.e., colors) with the colors on the colorimetric card to obtain the corresponding protein concentration range.

[0078] In one specific embodiment of the present invention, the top view, side view, bottom view, and dimensions of each component of the protein detection device of the present invention are as follows: Figure 2 As shown, the stirring section of the stirrer 201 has a length of L1, which is equal to 19.65 mm, and the diameter of the top circular component of the stirrer 201 is... The length of the pretreatment tube 202 is L4, which is 65 mm; the length of the upper part of the pretreatment tube 202 is L2, which is 50 mm; the thickness of the free antibody-labeled filter membrane in the lower part of the pretreatment tube 202 is L3, which is 0.5 mm; the diameter of the opening at the top of the pretreatment tube 202 is... Equal to 10mm; the diameter of the lower opening of the pretreatment tube 202 is The length of reaction tube 203 is equal to 3mm; the length of pretreatment tube 202 is the same, L4, and L4 equals 65mm; the diameter of the opening on reaction tube 203 is... Equal to 9.8mm; the diameter of the lower opening of reaction tube 203 is The diameter of the upper opening of the waste liquid pipe 204 is 3.4 mm; the length of the waste liquid pipe 204 is L5, and L5 equals 50.5 mm; the diameter of the upper opening of the waste liquid pipe 204 is... The length of the colorimetric tube 205 is L6, which is equal to 25mm; the diameter of the upper opening of the colorimetric tube 205 is... =7mm; the wall thickness of each of the above tubes is L7, which equals 0.5mm.

[0079] In one specific embodiment of the present invention, the gold nanorods in the protein detection device provided by the present invention are prepared by a seed crystal growth method, specifically including the following steps:

[0080] 1. Preparation of seed solution

[0081] 0.5 mL of 0.5 mM chloroauric acid HAuCl4 was mixed with 0.5 mL of 0.2 mM hexadecyltrimethylammonium bromide CTAB solution to obtain Au(III)CTAB solution; 0.06 mL of fresh 0.01 mM sodium borohydride NaBH4 was diluted to 0.1 mL with water, and then injected into the Au(III)CTAB solution with vigorous stirring (1200 rpm); the color of the solution changed from yellow to brownish-yellow, and stirring was stopped after 2 minutes to obtain seed solution; the seed solution was placed at room temperature for growth for 30 minutes for later use.

[0082] 2. Preparation of gold salt growth solution

[0083] Dissolve 0.09 g of CTAB and 0.006 g of 2,6-dihydroxybenzoic acid in 2.5 mL of warm water (50-70 °C). When the solution cools to 30 °C, add 0.12 mL of 4 mM AgNO3 solution to obtain a mixed solution. Let the mixed solution stand at 30 °C for 15 minutes, then add 2.5 mL of 1 mM HAuCl4 solution and stir slowly (400 rpm) for 15 minutes. Then add 0.02 mL of 0.064 mM ascorbic acid and stir vigorously for 30 seconds until the solution becomes colorless to obtain the gold salt growth solution. At this time, the concentration of CTAB in the growth solution is about 0.05 M.

[0084] 3. Seed growth

[0085] 0.008 mL of seed solution was injected into the gold salt growth solution and stirred for 30 seconds. Finally, the solution was left to stand at 30 °C for 12 hours to grow gold nanorods.

[0086] 4. Nanoparticle purification

[0087] Before use, the gold nanorod solution can be processed as needed, specifically as follows: Take 2 mL of the gold nanorod solution, centrifuge at 12000 rpm for 10 minutes, and then remove the supernatant; add an equal volume of distilled water to the precipitate, then disperse it uniformly by ultrasonication, and centrifuge again under the same conditions; CTAB is removed by this method. Then, the resulting concentrated gold nanorod solution is mixed with a known concentration of CTAB to make the final concentration of CTAB 0.16 M. The gold nanorods prepared by this method have longitudinal and transverse absorption peak wavelengths of 710 nm and 520 nm, respectively, with a variable wavelength range of 190 nm and an aspect ratio of approximately 3.3.

[0088] In one specific embodiment of the present invention, the synthesis of gold nanorods was improved. To enhance the detection range, the present invention synthesized gold nanoparticles with a higher aspect ratio and a variable wavelength range of 350 nm using a dual surfactant method. Specifically, in the gold salt growth solution preparation stage, 0.09 g of CTAB was replaced with 0.045 g of CTAB and 0.045 g of benzylhexadecyl dimethyl ammonium chloride (BDAC), forming a total of 0.09 g of dual surfactant system; the remaining steps remained the same. During seed crystal growth in the gold salt growth solution, specific crystal faces were covered by CTAB. With the help of silver ions and aromatic additives, the reduced gold atoms accumulated on the un-CTAB-covered sides of the seed crystal, and the spherical seed crystals gradually grew into rod-shaped nanoparticles, such as... Figure 3 As shown in the figure, each curve corresponds to the absorbance of the gold nanorod for different wavelengths of light under a specific shape. In testing the performance of this gold nanorod, an antibody containing HRP was used to generate different concentrations of TMB. 2+The solution etched the gold nanorods to varying degrees, thereby altering their shape, absorption peak positions, and absorbance. The improved gold nanorods exhibited longitudinal and transverse absorption peak wavelengths of 870 nm and 520 nm, respectively, while maintaining similarities to TMB. 2+ The etching reaction.

[0089] After adding a certain amount of BDAC, the aspect ratio of the gold nanorods increased significantly. A possible explanation is that after BDAC replaced one of the methyl groups of CTAB with benzyl groups, the steric hindrance was greater, and the binding with the gold nanorods was weaker. Therefore, it preferentially grew at both ends of the gold nanorods. Because of the weak binding of benzyl groups, the two ends grew faster, thereby increasing the aspect ratio and improving the upper limit of the detection concentration range.

[0090] In one specific embodiment of the present invention, the method for optimizing the concentration of gold nanorods is as follows: during the purification step of the gold nanorods, the obtained concentrated gold nanorod solution is mixed with an additional amount of CTAB of known concentration, ensuring that the final concentration of CTAB is maintained at 0.16M. In this embodiment, specifically, 2 mL of the concentrated gold nanorod solution is mixed with a CTAB solution of known concentration to obtain 16 mL of gold nanorod solution. Since the yield of gold nanorods synthesized by different synthesis processes or batches varies, the dilution ratio will also vary, and experimental verification of the optimal ratio is required.

[0091] In one specific embodiment of the present invention, the protein detection device of the present invention can shorten the reaction time of conventional ELISA. ELISA typically requires a reaction time of 0.5 to 3 hours. The concentration of the gold nanorods depends on the synthesis method; when using the gold seed growth method, the obtained gold nanorod solution can be in the micromolar (μM) range. The gold nanorods synthesized in this invention have a concentration of approximately 480 μM and an aspect ratio of approximately 3.3. When applied directly without treatment (1x)... The FAST β-lactoglobulin ELISA kit requires a detection time of 30 minutes. After processing (0.125x), the concentration is approximately 60 μM, which further reduces the detection time to 8 minutes. Specifically, the correspondence between the concentration of the gold nanorods and the protein concentration is shown in Table 1 below:

[0092] Table 1

[0093]

[0094] This invention mainly utilizes the TMB corresponding to the optimized gold nanorods. 2+ The narrowing range is related to the increased TMB generated after shortening the reaction time. 2+The principle of "less" combined with this allows for the successful detection of signals that were previously undetectable after accelerating the reaction time. This technique accelerates the testing process and is not limited by the original ELISA protein concentration. It can be applied to reduce the reaction time of ELISA, achieving rapid protein detection without sacrificing accuracy. Since this technique directly reduces the reaction time required to achieve the same signal concentration, the term "accelerated reaction" will be used to refer to this process in the following text. Furthermore, due to TMB... 2+ The concentration of TMB is positively correlated with the reaction time; therefore, in the following text, "conventional TMB" will be used. 2+ "Concentration" is used to refer to the signal response under routine testing; "low TMB" 2+ "Concentration" is used to refer to the signal response under rapid testing.

[0095] TMB generated during the standard ELISA reaction time 2+ The concentration and signal output per unit time are already optimized. Using optimized gold nanorods will not accelerate the reaction; instead, it will result in a proportional or compressed color conversion. Based on the common sense of technicians, this corresponds to a TMB (transient molecular weight) of [a specific value, likely related to reaction time]. 2+ Within the concentration range, reducing the amount of gold nanorods should enhance etching while maintaining linear correlation.

[0096] Experiment 1

[0097] When TMB 2+ When the corresponding optical density is greater than 1.4, such as Figure 4 As shown, regardless of TMB 2+ Regardless of the concentration change, the signal response to the wavelength change after treatment was limited to a maximum wavelength change of 179 nm (depending on the aspect ratio of the gold nanorods; the aspect ratio of the gold nanorods used in the experiment was approximately 3.3), and could not be increased further, thus losing its linear relationship with the concentration.

[0098] Before optimization, the concentration of conventional gold nanorods was 480 μM. After optimization, the concentration of trace gold nanorods was 60 μM. This resulted in a TMB increase during the conventional reaction time. 2+ Within the specified range, trace amounts of gold nanorods will be affected by excessive TMB. 2+ Etching to the point of losing aspect ratio makes it impossible to observe effectively. Only with trace amounts of TMB... 2+ Only through proper reaction can over-etching be avoided. Diluted nanoparticles cannot generate corresponding signal changes within this range, instead leading to signal loss and limiting wavelength changes to below 179 nm. Therefore, whether increasing TMB... 2+ Neither increasing the amount of gold nanorods nor optimizing the amount used can substantially improve the etching effect within this range. Therefore, based on the common sense of technicians, the above conclusion can be inferred that "the TMB corresponding to the conventional reaction time..." 2+The conclusion that "reducing the amount of gold nanorods within the concentration range should enhance etching while maintaining linear correlation" is not valid.

[0099] Experiment 2

[0100] Detection was performed under conditions with an optical density range of 0–0.9 to represent the small amount of TMB generated under rapid protein detection. 2 + The test results are as follows: Figure 5 The results show that within this range, the wavelength change after optimization with gold nanorods is more significant than before optimization. The same intensity of wavelength change can be achieved with lower optical density, meaning the time to obtain the same signal level is shortened. Before optimization, the detection response with gold nanorods was low, occupying only 18 nm of wavelength change at an optical density of 0.9, resulting in significant signal loss. After optimization, the signal response with gold nanorods, at the same optical density of 0.9, already occupies approximately 90 nm of change, half of the maximum wavelength change, more effectively utilizing the TMB generated after shortening the response time. 2+ By compressing the TMB corresponding to the gold nanorods. 2+ A wide concentration range can effectively shorten the reaction time to reach a detectable signal level, enabling rapid protein detection. Therefore, the technical solution of this invention exists in specific TMB (tumor monoxide) requirements. 2+ Within this range, it is suitable for rapid protein detection.

[0101] Experiment 3

[0102] The commercially available β-lactoglobulin concentration ELISA kit (trade name: [product name missing]) with a calibrated average detection limit of 0.042 μg / mL was used. FASTβ-Lactoglobulin (catalog number: R4912, specification: 48T, from: R-Biopharm AG) was used in the experiment. As a control, a series of standard solutions with different concentrations (0–4.5 μg / mL) and a micro-protein solution with a concentration of 0.1 μg / mL were tested, with total incubation times of 40, 30, 19, 13.5, 8, and 5.5 minutes, respectively.

[0103] The detection results for the standard solution with a concentration of 4.5 μg / mL are as follows: Figure 6As shown, when the incubation time exceeds the calibrated incubation time by 30 minutes, the signal response of the standard solution loses its linearity and enters a plateau phase. As the incubation time gradually decreases, the corresponding signal range also gradually shrinks. Taking an 8-minute reaction time as an example, the corresponding optical density has decreased to 0–1.08, making it impossible to identify trace protein solutions with a concentration of 0.1 μg / mL. This is because the sensor's optical density resolution is limited and fixed at ±0.001. As the corresponding optical density value range decreases, the relative resolution also decreases, resulting in the inability to distinguish trace concentrations of the target protein.

[0104] Experiment 4

[0105] The optimized gold nanorods were added to the test conditions of Experiment 3 above, and the experiment was conducted in the same manner as Experiment 3. The optimized gold nanorods can convert different optical density signal ranges into fixed wavelength variation ranges, which can significantly shorten the incubation time and successfully identify the concentration of trace proteins that were originally indistinguishable.

[0106] Test results as follows Figure 7 As shown, taking incubation times of 30, 19, 13.5, and 8 minutes as examples, the original signal ranges of 0–3, 0–2.5, 0–1.8, and 0–1.1 nm were all converted to signal levels (0–170 nm) equivalent to the 30-minute reaction time after using gold nanorods optimized to varying degrees. Even with a total incubation time of only 8 minutes, the wavelength variation range remained comparable to the 30-minute reaction time, allowing the detection of trace protein concentrations as low as 0.12 μg / mL (wavelength variation = 3 nm), close to the actual concentration of 0.1 μg / mL, effectively accelerating the protein detection reaction. The corresponding TMB of the gold nanorods after dilution... 2+ The scope is also narrowed accordingly; the optimized nano-gold rods can handle different TMB generated at different times. 2+ Concentration ranges were all converted into wavelength variation signals from 0 to 170 nm. Optimized gold nanorods were added according to different optical density ranges; these optimized gold nanorods required less TMB. 2+ It reaches the signal plateau period, i.e., 170nm, more quickly, thereby significantly reducing the required reaction time.

[0107] Therefore, through a similar optimization process, the technical solution of this invention can effectively accelerate the reaction time of any ELISA-based detection kit. The technical solution of this invention is applicable to detection needs within the conventional ELISA concentration range, and for specific detection needs (such as toxic substances, allergenic proteins, etc.), it has the effect of accelerating reaction time.

[0108] Example 1

[0109] This embodiment provides a method such as Figure 1The detection method shown in the diagram for detecting the target protein involves the following specific steps:

[0110] (1) Sample selection: Two food items were selected as test samples: cheese bread made from dairy products and sweet buns containing no milk.

[0111] (2) Pretreatment: The above samples were placed into pretreatment tubes 202 and ground and stirred using a stirrer 201. Protein was extracted from the food using the extraction buffer. After extraction, the switch 213 at the bottom of the pretreatment tube 202 was turned on. The sample solution 207 slowly passed through the filter membrane 206 containing the free labeling antibody under gravity. The target protein and the labeling antibody bound to form a complex, which flowed into the reaction tube 203 connected below along with the sample solution 207. To demonstrate the accuracy of the detection method, 10 μg / mL of β-lactoglobulin standard was added to an additional cheese bread sample in this embodiment. This is referred to as: spiked cheese bread. All cheese bread samples were additionally diluted 10-fold with the extraction buffer to adapt to the ELISA detection range.

[0112] (3) Reaction: When the sample solution 207 slowly flows to the filter membrane 208 containing the immobilized capture antibody in the reaction tube 203, the complex in the sample solution 207 will be captured and immobilized again by the immobilized capture antibody on the filter membrane. The remaining liquid continues to flow to the outlet by gravity and is discharged into the waste liquid tube 204. After the sample solution 207 is discharged, the waste liquid tube 204 connected to the bottom outlet is replaced with a colorimetric tube 205 with a gold nanorod. Then, the TMB base solution 210 is added to the reaction tube 203. TMB reacts with the peroxidase-containing complex immobilized on the filter membrane to generate TMB. 2+ Solution 211, and along with the solution, enters the colorimetric tube 205.

[0113] (4) Color development: The upper opening of the color development tube 205 is connected to the reaction tube 203. The tube contains a trace amount of gold nanorod solution with a concentration of 60 μM and a volume of 100 μL; the aspect ratio of the gold nanorods is approximately 3.3; TMB 2+ Solution 211 enters the chromogenic tube 205 to etch the gold nanorods; different concentrations of protein will produce different concentrations of TMB. 2+ The solution is then processed to produce different etching degrees and color changes; after all the base solution has flowed into the colorimetric tube 205 and 5 minutes have elapsed, the test is complete; by comparing the test results with the colorimetric card, the protein concentration in the food to be tested can be obtained as 160 μg / g.

[0114] (5) To accurately verify the accuracy of the detection results in this embodiment, the color change of the colorimetric tube 205 obtained after the reaction in step (4) was quantitatively detected. The overall detection time was 30 minutes. The detection results are as follows: Figure 8As shown. In this embodiment, using micro-gold nanorods, the β-lactoglobulin content in cheese bread, spiked cheese bread, and sweet buns was measured to be 165.54 μg / g, 173.33 μg / g, and 0.11 μg / g, respectively. The standard recovery rate and detection limit were 102.2% and 0.90 μg / g, respectively. Micro-gold nanorods can more fully utilize the limited TMB after shortening the time. 2+ The content was increased to a higher etching level, successfully distinguishing between spiked and unspiked samples, with wavelength changes of 17 nm and 18 nm, respectively. The target protein in the sweet bun was also successfully identified, with a wavelength change of 1 nm, which can be considered a positive reaction, but it could not be accurately quantified because the signal was below the detection limit.

[0115] Comparative Example 1

[0116] This comparative example used a β-lactoglobulin ELISA kit (trade name: Beta-lactoglobulin ELISA KitII, catalog number: M2112, specification: 96T, from: Morinaga Institute of Biological Science, Inc.) with a detection limit of 0.31 μg / g and a detection range of 0.31 μg / g to 20 μg / g, which detects bovine β-lactoglobulin.

[0117] Using the same test samples as in Example 1, the tests were performed according to the above kit operation method, with a total incubation time of 2 hours. Quantitative analysis was performed on the obtained test results, as shown below. Figure 9 As shown.

[0118] like Figure 9 As shown, under a standard ELISA reaction time of 2 hours, the β-lactoglobulin content in cheese bread, spiked cheese bread, and sweet bread was measured to be 161.64 μg / g, 170.19 μg / g, and 0.38 μg / g, respectively. The standard recovery rate was 85.5%, and the limit of detection was 0.58 μg / g. The sweet bread showed a positive signal at the limit of detection, indicating the presence of the protein, but the quantification result may be inaccurate.

[0119] Comparative Example 2

[0120] The detection method used in this comparative example was the same as that in Comparative Example 1, using the same sample and the same kit. The difference was that the total incubation time was 30 min, and conventional gold nanorods were used for protein detection. The concentration of the conventional gold nanorods was 480 μM, the volume was 100 μL, and the aspect ratio of the gold nanorods was approximately 3.3.

[0121] Test results as follows Figure 10As shown. The β-lactoglobulin content in cheese bread, spiked cheese bread, and sweet dinner rolls was measured to be 181.07 μg / g, 181.07 μg / g, and 0 μg / g, respectively. The detection limit was measured to be 2.32 μg / g. The above detection limits were all determined as follows: When establishing the standard curve, the fitted curve formula y = mx + b was obtained. Data information was obtained using the LINEST function in Excel and substituted into the following formula:

[0122] Detection limit = 3.3 × (σ / m)

[0123] σ refers to the standard deviation of the blank value, and m is the slope. Due to the shortened detection time, the resulting TMB... 2+ The amount was significantly reduced, resulting in a maximum wavelength shift of only 5 to 6 nm for the gold nanorods. Even when the β-lactoglobulin content in cheese bread was successfully detected, the spiked and unspecified samples could not be effectively distinguished, both corresponding to a wavelength change of 5 nm. The difference in wavelength etching change was less than 1 nm, which is below the detection resolution of 1 nm. Similarly, the wavelength change caused by β-lactoglobulin in dessert buns was too small (<1 nm) to be effectively observed.

[0124] Figure 11 and Figure 12 This paper presents a comparison of the detection results of three different protein assays: Example 1 (where 2 in the figure represents a micro-gold nanorod ELISA), Comparative Example 1 (where 1 in the figure represents a conventional ELISA), and Comparative Example 2 (where 3 in the figure represents a conventional gold nanorod ELISA). The results show that Comparative Example 2 reduced the reaction time to 30 minutes and used a conventional gold nanorod ELISA. The results indicate that not only were the protein detection results for cheese bread inflated, but it also failed to distinguish between spiked and unspecified samples. Furthermore, for sweet bread with even lower protein content, no signal response was detected, resulting in a significantly increased detection limit and a significant decrease in overall detection performance.

[0125] Similarly, by reducing the reaction time from 2 hours to 30 minutes, the results of the two cheese bread samples detected using micro-gold nanorods in Example 1 were comparable to or close to the results of the conventional ELISA in Comparative Example 1, with higher standard recoveries and a low detection limit. For the sweet bread with very low target protein content, the detection results of the micro-gold nanorods were similar to those of the conventional ELISA, achieving a positive signal below the detection limit and detecting the presence of the protein. Therefore, micro-gold nanorods can significantly shorten the detection time while maintaining detection performance. Compared to the 2-hour conventional ELISA in Comparative Example 1, the micro-gold nanorod method can shorten the detection time by 90 minutes.

Claims

1. A semi-quantitative rapid detection device for food allergen proteins, the detection device comprising: Pretreatment tubes, reaction tubes, and colorimetric tubes, including: The pretreatment tube is equipped with a stirrer for grinding and stirring the sample to be tested and a filter membrane containing free labeling antibody. The sample solution outlet is located at the bottom of the pretreatment tube. The sample solution slowly passes through the filter membrane under the influence of gravity. The target protein in the sample solution binds to the labeled antibody to form a complex, which flows into the reaction tube below along with the sample solution. The reaction tube is used to capture the antigen-antibody complex formed in the pretreatment tube, convert the TMB substrate into a golden-yellow reaction product, and introduce the reaction product into the development tube.

2. The detection device according to claim 1, wherein, The pretreatment tube is also equipped with an extraction solution for extracting the target protein from the sample to be tested; the colorimetric tube is pre-filled with nanoparticle reagent.

3. The detection device according to claim 1, wherein, The reaction tube has openings at the top and bottom. The top opening is used for the input of sample solution and is connected to the pretreatment tube. The bottom opening slowly discharges liquid through a filter membrane with fixed capture antibodies and is connected to the colorimetric tube.

4. The detection device according to claim 3, wherein, The connection between the reaction tube and the pretreatment tube is detachable.

5. The detection device according to claim 1, wherein, The reaction tube is designed to be completely light-proof.

6. The detection device according to claim 1, wherein, The detection device includes a reagent kit containing nanoparticle reagents, which react with the reaction products in the colorimetric tube to produce a color change.

7. The detection device according to claim 6, wherein, The nanoparticle reagent is a gold nanorod reagent with a concentration of 30 μM to 480 μM, preferably 60 μM.

8. The detection device according to claim 1, wherein, The detection device includes a standard colorimetric card, which is used to compare the detection results in the colorimetric tube to obtain semi-quantitative analysis results of protein concentration.

9. The detection device according to claim 1, wherein, The free-labeled antibody can specifically bind to the target protein in the sample solution to generate an antigen-antibody complex, and the free-labeled antibody carries horseradish peroxidase.

10. The detection device according to claim 3, wherein, The immobilized capture antibody can capture antigen-antibody complexes and immobilize them on the filter membrane inside the reaction tube.

11. A protein detection method, specifically comprising the following steps: The test sample solution containing the target protein is reacted with an antibody that specifically captures the target protein. Add labeled antibody reagent, wherein, The labeled antibody carries horseradish peroxidase; Add the substrate reagent to obtain the reaction solution; The reaction solution is mixed with nanoparticles to obtain a colorimetric solution; The colorimetric solution after the reaction was analyzed to obtain the detection results.

12. The method according to claim 11, wherein, This method uses the detection device according to any one of claims 1-10 for detection, and specifically includes the following steps: The sample to be tested is placed in a pretreatment tube, and the target protein is ground and extracted by a stirrer in the pretreatment tube to obtain a sample solution. The sample solution slowly passes through a filter membrane containing a free labeling antibody under the action of gravity. The target protein binds with the labeling antibody to form an antigen-antibody complex, which flows into the reaction tube below along with the sample solution. In the reaction tube, the antigen-antibody complex in the sample solution is captured and fixed by the capture antibody on the filter membrane, and the remaining liquid continues to flow to the outlet by gravity and is discharged into the waste liquid tube. After the sample solution is discharged, replace the waste liquid tube connected to the bottom outlet with a colorimetric tube; The TMB substrate is added to the reaction tube, where it reacts with the peroxidase-containing antigen-antibody complex immobilized on the filter membrane to generate TMB. 2+ And it enters the colorimetric tube along with the solution; TMB 2+ The reaction with nanoparticles yields detection results, which are then compared with a standard colorimetric card to obtain semi-quantitative analysis results of protein concentration. Preferably, the pretreatment tube is further provided with an extraction solution for extracting the target protein from the sample to be tested; Preferably, the colorimetric tube contains a nanoparticle reagent.

13. The method according to claim 11 or 12, wherein, The nanoparticles are gold nanorods with a concentration of 30 μM to 480 μM, preferably 60 μM.

14. The method according to any one of claims 11-13, wherein, The detection time of the method is 15 to 30 minutes.

Images