CN117761300A

CN117761300A - Detection kit, rapid detection method and application thereof in aflatoxin detection

Info

Publication number: CN117761300A
Application number: CN202311760034.6A
Authority: CN
Inventors: 闫肃; 孙璞; 杨树青
Original assignee: Suzhou Yichuang Biotechnology Co ltd
Current assignee: Suzhou Yichuang Biotechnology Co ltd
Priority date: 2023-12-20
Filing date: 2023-12-20
Publication date: 2024-03-26

Abstract

The invention provides a detection kit, a rapid detection method and application thereof in detecting aflatoxin, wherein the detection kit comprises a microfluidic chip with a sample adding port, a first reagent, a second reagent, a magnetic particle cleaning solution and a luminescent substrate solution which are mutually and independently arranged in the microfluidic chip; the first reagent comprises magnetic particles coated with an antigen, the second reagent comprises an enzyme-labeled antibody, the luminescent substrate solution comprises a luminescent substrate and a substrate buffer, and the substrate buffer comprises bis (N-methylacridine) nitrate, 2-amino-2-methyl-1, 3-propanediol, 3-indoxyl disodium phosphate, 2-methyl-4-isothiazolin-3-one hydrochloride and chloroacetamide. The substrate of the invention is more stable, so that the shelf life of the kit can be prolonged. In addition, the kit has the advantages of simplicity and convenience in operation, high sensitivity, high accuracy, good repeatability, no reagent pollution, no waste and the like.

Description

Detection kit, rapid detection method and application thereof in aflatoxin detection

Technical Field

The invention belongs to the field of biotechnology and food safety rapid detection, and particularly relates to a detection kit, a rapid detection method and application thereof in detecting aflatoxin.

Background

Aflatoxins (AFs) are a class of highly toxic mycotoxins that are widely found in feed and food storage processes, and are secondary metabolites of aspergillus flavus and aspergillus parasiticus. These mycotoxins are a serious hazard to animal and human health due to their carcinogenicity, hepatotoxicity, and mutagenicity. The cancer research institute of World Health Organization (WHO) in 1993 is classified as class 1 carcinogen, which is 75 times as much as standard carcinogen dimethyl nitrosamine, 68 times as toxic as arsenic, 10 times as much as potassium cyanide, and has extremely strong destructiveness to liver tissue. Among the several aflatoxins that have been found, aflatoxin B1 has the strongest toxicity, which is widely present in mildewed feeds, and cows metabolize to aflatoxin M1 in vivo after intake of aflatoxin B1, thus causing contamination of the milk source.

At present, the detection method of aflatoxin M1 mainly comprises three methods of liquid chromatography-tandem mass spectrometry, liquid chromatography and enzyme-linked immunosorbent assay, wherein the sample pretreatment of the liquid chromatography and the liquid chromatography-tandem mass spectrometry is complex and requires an organic solvent, pollutes the environment, has high requirements on operators and has high detection cost; the ELISA method has low cost, is a mainstream detection method at present, but has long detection time and complicated steps, requires professional personnel to operate in a laboratory, is suitable for occasions with a large number of single detection samples, is not suitable for spot check and batch detection with a small number of samples, is easy to cause reagent pollution and waste, and cannot meet the requirement of on-site rapid detection.

Therefore, in order to solve the problem of milk source pollution caused by environmental pollution, feed mildew and the like at present, an aflatoxin M1 detection method which is rapid in detection, convenient to operate and accurate in result is urgently needed, detection of residual aflatoxin M1 in a raw milk source is facilitated, and the flow of 'toxic milk' to the market is forbidden from the source.

Disclosure of Invention

The invention aims to provide a detection kit and a rapid detection method which are rapid in detection speed, convenient to operate and free of reagent pollution and waste, and application of the detection kit and the rapid detection method in aflatoxin detection.

In order to solve the technical problems, the invention adopts the following technical scheme:

the first aspect of the invention provides a detection kit, which comprises a microfluidic chip with a sample adding port, a first reagent, a second reagent, a magnetic particle cleaning solution and a luminescent substrate solution which are mutually and independently arranged in the microfluidic chip; wherein the first reagent comprises magnetic particles coated with an antigen, the second reagent comprises an enzyme-labeled antibody, the luminescent substrate solution comprises a luminescent substrate and a substrate buffer, and the substrate buffer comprises bis (N-methylacridine) nitrate (CAS: 2315-97-1), 2-amino-2-methyl-1, 3-propanediol (CAS: 115-69-5), 3-indoxyl disodium phosphate (CAS: 3318-43-2), 2-methyl-4-isothiazolin-3-one hydrochloride (CAS: 26172-54-3), and chloroacetamide (CAS: 79-07-2).

According to the invention, bis (N-methylacridine) nitrate and 2-amino-2-methyl-1, 3-propanediol are added into a substrate buffer solution to improve the stability of a luminous substrate solution, the luminous efficiency is enhanced by using 3-indoxyl disodium phosphate, and 2-methyl-4-isothiazolin-3-one hydrochloride and chloroacetamide are added as a preservative combination, so that the substrate solution is more stable.

According to some embodiments, the mass of bis (N-methylacridine) nitrate is 0.01% to 0.5% of the total mass of the substrate buffer, e.g., 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49% or 0.5%.

According to some embodiments, the concentration of the 2-amino-2-methyl-1, 3-propanediol in the substrate buffer is 10 to 20g/L, e.g., 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L, or 20g/L.

According to some embodiments, the disodium 3-indoxyl phosphate is present in an amount of 0.01% -0.2% by mass, e.g., 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19% or 0.2% by mass of the total mass of the substrate buffer.

According to some embodiments, the mass of the 2-methyl-4-isothiazolin-3-one hydrochloride salt is 0.01% -0.2%, e.g., 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19% or 0.2% of the total mass of the substrate buffer.

According to some embodiments, the chloroacetamide comprises 0.01% -0.5% by mass of the total mass of the substrate buffer, e.g., 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49% or 0.5%.

Further, the substrate buffer may further comprise 65 to 85g/L Tris (hydroxymethyl) aminomethane (Tris), for example 65g/L, 66g/L, 67g/L, 68g/L, 69g/L, 70g/L, 71g/L, 72g/L, 73g/L, 74g/L, 75g/L, 76g/L, 77g/L, 78g/L, 79g/L, 80g/L, 81g/L, 82g/L, 83g/L, 84g/L or 85g/L.

Further, the substrate buffer may further comprise 20 to 30g/L Tris (hydroxymethyl) aminomethane hydrochloride (Tris HCl), for example 20g/L, 21g/L, 22g/L, 23g/L, 24g/L, 25g/L, 26g/L, 27g/L, 28g/L, 29g/L or 30g/L.

Further, the substrate buffer may further comprise 5 to 10g/L of 1, 3-bis ((trimethylol) methylamino) propane (CAS: 64431-96-5), for example 5g/L, 6g/L, 7g/L, 8g/L, 9g/L or 10g/L.

Further, the substrate buffer also includes dimethyl sulfoxide (DMSO) at 0.1% -5% of the total mass of the substrate buffer, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9% or 5%.

According to some embodiments, the pH of the substrate buffer is 8 to 9, e.g., 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0.

According to some embodiments, the concentration of the luminescent substrate in the luminescent substrate liquid is 0.2-2 g/L.

According to some embodiments, the second reagent further comprises an enzyme conjugate diluent comprising 0.01% to 0.5% of phenylmethylsulfonyl fluoride (PMSF) by total mass of the enzyme conjugate diluent, 0.1% to 3% of maltose by total mass of the enzyme conjugate diluent, 0.01% to 5% of propyl p-hydroxybenzoate by total mass of the enzyme conjugate diluent, and 0.1% to 5% of ProClin300 or ProClin150 by total mass of the enzyme conjugate diluent.

Wherein the phenylmethylsulfonyl fluoride comprises 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49% or 0.5% of the total mass of the enzyme conjugate diluent.

The maltose may comprise 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9% or 3% of the total mass of the enzyme conjugate diluent.

The propyl p-hydroxybenzoate may comprise 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.2% by weight of the total enzyme conjugate diluent 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9% or 5%.

The ProClin300 or ProClin150 may comprise 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46% of the total mass of the enzyme conjugate diluent 0.47%, 0.48%, 0.49%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9% or 5%.

According to the invention, the combination of phenylmethylsulfonyl fluoride and maltose is added into the enzyme conjugate diluent to serve as a protease stabilizer, and the combination of propyl p-hydroxybenzoate and ProClin300 or ProClin150 serving as a preservative is added, so that the stability of the enzyme-labeled antibody in a microfluidic chip is improved.

Further, the enzyme conjugate diluent may further comprise 5 to 15g/L sodium caseinate, e.g. 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L or 15g/L.

Further, the enzyme conjugate diluent may also include 5 to 10g/L sodium chloride, such as 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, or 10g/L.

Further, the enzyme conjugate diluent may also include 0.1 to 0.5g/L sodium dihydrogen phosphate dihydrate, such as 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, or 0.5g/L.

Further, the enzyme conjugate diluent may also include 1 to 5g/L disodium phosphate dodecahydrate, such as 1g/L, 2g/L, 3g/L, 4g/L, or 5g/L.

Further, the enzyme conjugate diluent has a pH of 7.5 to 8.5, such as 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5.

According to some embodiments, the magnetic particle cleaning solution includes 0.01% to 0.1% nonionic detergent NP-40 by weight of the total magnetic particle cleaning solution, e.g., the nonionic detergent NP-40 is present at a concentration of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%.

According to some embodiments, the magnetic particle cleaning solution comprises 0.1% -5% by total mass of the magnetic particle cleaning solution of non-ionic detergent Brij-35, e.g., the concentration of non-ionic detergent Brij-35 is 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.6%, 0.7%, 0.8%, 1.9%, 1.2%, 1.3%, 4.2%, 3%, 4.2%, 3.2%, 4%, 3.2%, 4.3%, 4.2%, 3.3.3%, 4.3.2%, 4.3%, 4.3.3%, 4.3.2%, 4.3.3.3%, 4.3.3.3.3.3.3%, 4% and/or the concentration of the non-ionic detergent Brij-35.

According to some embodiments, the magnetic particle cleaning fluid comprises 0.1% -3.0% of the total mass of the magnetic particle cleaning fluid, such as a concentration of the non-ionic detergent mepol SH of 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.2%, 1.3%, 1.2%, 2%, 2.2%, 1% of the non-ionic detergent mepol SH.

According to the invention, the combination of the nonionic detergent NP-40, brij-35 and MERPOL SH is further added into the magnetic particle cleaning liquid, so that the cleaning efficiency of the magnetic particle cleaning liquid can be well improved, and the problems of low magnetic particle cleaning efficiency, nonuniform cleaning efficiency and poor result repeatability caused by incapability of oscillating during cleaning due to tiny internal space of a film microfluidic chip are avoided.

Wherein the non-ionic detergent NP-40 is known as ethylphenyl polyethylene glycol, also known as Nonidet P40 or Nonidet P-40, and is commercially available, for example, from the following Syngnathus, shanghai Ji-Qi, sigma-Aldrich, etc.

The nonionic detergent Brij-35 is polyoxyethylene lauryl ether, which is commercially available, for example, from Jiangsu sea Ann petrochemical plant, shanghai Ke Raman reagent Co., ltd., sigma-Aldrich, etc.

Nonionic detergent MERPOL SH is available from Sigma-Aldrich, stepan, aledine.

Further, the magnetic particle cleaning liquid further comprises 1 to 10g/L of tris (hydroxymethyl) aminomethane, for example, the concentration of tris (hydroxymethyl) aminomethane may be 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L or 10g/L.

Further, the magnetic particle cleaning solution further comprises 10-15 g/L of tris (hydroxymethyl) aminomethane hydrochloride, for example, the concentration of tris (hydroxymethyl) aminomethane hydrochloride can be 10g/L, 11g/L, 12g/L, 13g/L, 14g/L or 15g/L.

Further, the magnetic particle cleaning fluid further includes procalin 300 in an amount of 0.1% -5% by total mass of the magnetic particle cleaning fluid, e.g., the concentration of procalin 300 may be 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.1%, 1.2%, 1.3%, 4%, 3.3%, 4.3%, 3.2%, 3.3%, 4.3.2%, 3.3%, 4%, 3.2%, 3.3.2%, 4%, 4.3.3.3%, 4%, 4.3.2%, 4.3.3.2%, 4%, 3.2%, 4.3.3.2%, 4%, 4.3.3.2%, 4.3.3%, 4.2.3%, 4.3.3.3.3.3.3%.

Further, the pH of the magnetic particle cleaning liquid is 8 to 9, for example 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

According to some embodiments, the difference between the pH of the magnetic particle cleaning solution and the pH of the substrate buffer is less than 0.4.

When the magnetic particle cleaning liquid needs to enter the capsule cavity of the first reagent by means of the instrument to clean the magnetic particles for multiple times, a small amount of residual magnetic particle cleaning liquid is difficult to avoid in the capsule cavity of the first reagent, and the residual magnetic particle cleaning liquid can be mixed with a luminescent substrate entering the capsule cavity in the next step, so that if the magnetic particle cleaning liquid is improperly selected, the luminescent substrate is possibly decomposed, the detection result is affected, the residual amount is different, the influence degree is different, and the detection repeatability is poor.

Therefore, the invention further adopts a buffer solution component similar to the substrate buffer solution, and controls the pH of the magnetic particle cleaning solution to be close to the pH of the substrate buffer solution, so that the residual magnetic particle cleaning solution can not influence the acid-base property of the luminous substrate solution, can not cause the decomposition of the substrate, and is beneficial to further ensuring the accuracy and the repeatability of the detection result.

According to some embodiments, the volume of the first reagent is 20 to 50 μl, and the concentration of the magnetic particles in the first reagent is 0.1mg/mL to 1mg/mL; the volume of the second reagent is 30-300 mu L, and the concentration of the enzyme-labeled antibody in the second reagent is 0.2-10 mu g/mL; the volume of the magnetic particle cleaning liquid is 1-3 mL; the volume of the luminescent substrate liquid is 100-300 mu L.

According to some embodiments, the mass ratio of the magnetic particles coated with antigen to the antigen is 100-300:1, such as 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190:1, 200:1, 210:1, 220:1, 230:1, 240:1, 250:1, 260:1, 270:1, 280:1, 290:1 or 300:1; in the enzyme-labeled antibody, the mass ratio of the antibody to the enzyme is 1:1-10, e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

According to some specific embodiments, the magnetic particles can be selected from carboxyl magnetic particles and tosyl magnetic particles, and the particle size is 1-3 μm.

According to some embodiments, the enzyme is alkaline phosphatase; the luminescent substrate is APS-5 powder ((4-chlorobenzenesulfonyl) (10-methyl-9, 10-dihydroacridine methylene) disodium phosphate).

According to some embodiments, the microfluidic chip comprises a plurality of independent capsules formed by hot pressing a first film and a second film, and a disposable valve is arranged between each of the capsules. Each independent capsule is used for pre-loading the first reagent, the second reagent, the magnetic particle cleaning solution and the luminous substrate solution, and the microfluidic chip is also provided with a capsule for storing waste liquid.

According to some embodiments, the antigen and the antibody are an antigen and an antibody capable of specific binding. The antigen and antibody in the kit of the invention can be adjusted according to the detection requirements.

According to some embodiments, the antigen is an aflatoxin M1 antigen and the antibody is an aflatoxin M1 antibody. Wherein, the aflatoxin M1 antigen can be one of M1-BSA or M1-HSA. The aflatoxin M1 antibody can be one of a mouse monoclonal antibody, a sheep monoclonal antibody and a rabbit polyclonal antibody.

The second aspect of the invention provides a rapid detection method, wherein a sample to be detected is added to a sample adding port of the detection kit, and then the detection kit is put into a microfluidic chemiluminescent immunoassay analyzer for detection, and a detection result is output.

According to some embodiments, the time from adding the sample to outputting the detection result is less than or equal to 10 minutes.

The third aspect of the invention provides an application of the detection kit in detecting the content of aflatoxin M1 in a dairy product.

Wherein the milk product comprises milk, milk powder, etc.

The detection principle of the invention is a competition method in a microfluidic magnetic particle chemiluminescence immunoassay method, and specifically comprises the following steps: the antigen coated on the magnetic particles competes with the antigen in the sample to be detected for binding the enzyme-labeled antibody, the more the antigen in the sample is, the more the alkaline phosphatase-labeled antibody is bound, the less the alkaline phosphatase-labeled antibody is immobilized on the magnetic particles, after the free alkaline phosphatase-coated antibody is washed off, the luminescent substrate and the alkaline phosphatase bound on the magnetic particles act to emit light, and the luminescence value is inversely proportional to the concentration of the antigen in the sample.

Compared with the prior art, the invention has the following advantages:

according to the invention, the substrate is more stable by improving the formula of the luminescent substrate liquid, so that the shelf life of the kit can be prolonged. In addition, the kit has the advantages of simplicity and convenience in operation, high sensitivity, high accuracy, good repeatability, no reagent pollution, no waste and the like.

Drawings

Fig. 1 is a schematic structural diagram of a microfluidic chip, wherein 1 is a sample inlet, 2 is a capsule for storing a second reagent, 3 is a capsule for storing a first reagent, 4 is a capsule for storing a luminescent substrate liquid, 5 is a capsule for storing a magnetic particle cleaning liquid, and 6 is a capsule for storing waste liquid;

FIG. 2 is a graph of the fit of application example 3;

fig. 3 is an IC50 graph of application example 3.

Detailed Description

The invention is further described below with reference to examples. The present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not collide with each other.

In the following examples and comparative examples, room temperature means 25.+ -. 5 ℃. In the following examples and comparative examples, "%" is mass percent, as specified.

In the following examples and comparative examples, unless otherwise specified, the raw materials, reagents and the like used were either conventional commercially available products or products prepared according to methods conventional in the art. The aflatoxin M1 antigen used in the examples and comparative examples described below is an antigen from ZD6018, a company of bloolon technologies, su zhou; the aflatoxin M1 antibody is an antibody purchased from mybiosource, product number MBS603134; alkaline phosphatase was alkaline phosphatase commercially available from rogowski under accession number 0313703110; APS-5, namely "(4-chlorobenzenesulfide) (10-methyl-9, 10-dihydroacridine methylene) disodium phosphate", manufacturer: a xibao organism; melphalan organisms; CAS number 193884-53-6.

Example 1

1. Preparing a first reagent:

(1) carboxyl magnetic particles (5 mg) were collected and placed on a magnetic rack, and the supernatant was discarded. Washing the carboxyl magnetic particles 3 times by using a buffer solution, and then uniformly suspending and mixing the magnetic particles by using 325 mu L of MES buffer solution; (2) preparing 10mg/mL NHS and 10mg/mL EDC with MES solution respectively; (3) adding 115 mu L of the EDC and NHS solution into the system in the step (1), uniformly mixing for 30min at room temperature, after the reaction is finished, performing magnetic separation, removing the supernatant, repeatedly cleaning for 3 times by using an MES solution, and adding 500 mu L of the MES solution for suspension and uniform mixing; (4) adding 0.025mg aflatoxin M1 antigen, mixing uniformly by vortex, rotating at room temperature, mixing uniformly for 2 hours, performing magnetic separation, removing supernatant after the reaction is finished, performing magnetic separation after repeated washing for 3 times by using MES solution, and removing supernatant; (5) adding 500 mu L of sealing liquid into the magnetic particles, uniformly mixing by vortex, rotating at room temperature, uniformly mixing for 1h, sealing, performing magnetic separation after the reaction is finished, cleaning the magnetic particles for 3 times by using Tris-HCl solution, re-suspending the magnetic particles by using 500 mu LTris-HCl solution, and diluting the magnetic particles to the concentration of 0.2mg/mL by using magnetic particle diluent for standby.

The formula of the magnetic particle diluent is as follows: 1000ml of solution comprises 6g Tris, 15g sodium chloride, 5g sodium caseinate and 2ml Proclin-300, the solvent being water.

2. Preparing a second reagent:

(1) taking 0.5mg of aflatoxin M1 antibody, and diluting to 2mg/mL with 0.1M PBS solution; (2) adding 2-iminothiolane hydrochloride (2-IT) into the antibody solution, and performing oscillation reaction at room temperature for 30min; (3) dialyzing the activated antibody solution; (4) taking 0.5mg of alkaline phosphatase, and diluting to 10mg/mL by using a triethanolamine solution with pH of 7.6; (5) adding 2.5 mu L of SM (PEG) 8 solution into the alkaline phosphatase solution, performing oscillation reaction at room temperature for 15min, and performing dialysis treatment; (6) mixing the sulfhydrylation antibody with the alkaline phosphatase, adding 0.6 mu L of 1M magnesium chloride solution into the mixed solution, and carrying out shaking reaction at 2-8 ℃ for overnight; (7) adding 3 mu L of NEM solution and 3 mu L of glycol solution into the solution, and performing oscillation reaction at room temperature for 10min; (8) dialyzing the solution; (9) adding equal volume of glycerol into the obtained enzyme conjugate, mixing, preparing enzyme conjugate, and diluting with enzyme conjugate diluent to enzyme conjugate concentration of 0.6 μg/mL for use.

The preparation method of the enzyme conjugate diluent comprises the following steps:

about 700mL of purified water was added to a beaker, the beaker was placed on a magnetic stirrer, 10g of sodium caseinate, 8g of sodium chloride, 0.31g of sodium dihydrogen phosphate dihydrate and 3.49g of disodium hydrogen phosphate dodecahydrate were added while stirring, after stirring until fully dissolved, 0.5g of propyl p-hydroxybenzoate and 1mL of ProClin300, 0.2mL of PMSF and 3g of maltose were added to the solution, respectively, and stirring was fully carried out until fully dissolved and mixed, the pH of the solution was measured at 25.+ -. 1 ℃ to 8.0.+ -. 0.2, otherwise, the solution was adjusted with hydrochloric acid or sodium hydroxide and fixed to 1000mL with purified water, and after filtration with a filter membrane of 0.22 μm, the solution was preserved for use.

3. Preparing a luminescent substrate solution:

a brown capped Erlenmeyer flask was prepared, about 700mL of purified water was added, and placed on a magnetic stirrer, 71g of Tris and 26g of Tris HCl, 15.8g of 2-amino-2-methyl-1, 3-propanediol, 7g of 1, 3-bis ((trimethylol) methylamino) propane, 0.5g of 2-methyl-4-isothiazolin-3-one hydrochloride, 0.5g of chloroacetamide were sequentially added while stirring, stirred for 60min until complete dissolution was achieved, the pH of the solution was determined to be 8.5.+ -. 0.2 at 22.+ -. 1 ℃ or adjusted with 50% hydrochloric acid or sodium hydroxide, and the solution was placed at 2-8 ℃ for 12 hours after filtration with a 0.22 μm filter membrane. And adding 0.8g of 3-indolyloxy disodium phosphate into the mixed solution taken out at the temperature of 2-8 ℃ and stirring until the disodium phosphate is fully dissolved. Dissolving 0.4g of bis (N-methylacridine) nitrate in 1mL of DMSO, adding the solution into the DMSO, stirring for 60min, fixing the volume to 1000mL by using purified water, fully and uniformly mixing, and keeping away from light for later use to prepare the substrate buffer solution.

1g of APS-5 powder was uniformly mixed with 1000mL of the above substrate buffer to obtain a luminescent substrate solution.

4. Preparing magnetic particle cleaning liquid

Preparing non-ionic detergent NP-40/Brij-35/MERPOL SH, setting a constant-temperature water bath at 45+/-2 ℃, weighing 1g Brij35, adding into a beaker, adding 0.25mL NP-40 and 1mL MERPOL SH into the beaker, placing the beaker into the constant-temperature water bath, and stirring until the mixture is dissolved; preparation of Tris buffer: about 500mL of purified water was added to another beaker, placed on a magnetic stirrer, 5g of Tris was added while stirring, after dissolution 12g of Tris-HCl was added and stirring was continued for 30min, 1mL of Proclin300 and the prepared nonionic detergent NP-40/Brij-35/MERPOL SH were added to the solution, the pH of the solution was determined to be 8.5.+ -. 0.2 at 25.+ -. 1 ℃, otherwise, it was adjusted with hydrochloric acid or sodium hydroxide and fixed to 1000mL with purified water, filtered with a 0.22 μm filter membrane and stored for use.

Comparative example 1

Comparative example 1 is substantially the same as example 1 except that the substrate buffer, the formulation of which is shown in table 1 below, is used to adjust the pH of the substrate buffer to 8.5±0.2 with 50% hydrochloric acid or sodium hydroxide.

TABLE 1

Application example 1

The same sample test was performed by taking 200 μl of each of the luminescent substrate solution prepared in example 1 and the luminescent substrate solution prepared in comparative example 1, respectively, pre-filling the respective cavities of the thin film microfluidic chip, storing the sample in the respective cavities of the thin film microfluidic chip at 2-8deg.C for 3 months, and then filling the respective cavities of the thin film microfluidic chip with the freshly prepared other reagents of example 1, wherein the volume of the first reagent was 30 μl, the volume of the second reagent was 50 μl, and the volume of the magnetic particle cleaning solution was 2000 μl, and the sample loading amount was 100 μl, and comparing the luminescent values with the initially pre-filled substrates, with the results shown in tables 2 and 3 below. From the results of tables 2 and 3, it can be seen that the substrates of the present invention are more stable and the luminescence value is higher when samples of the same concentration are tested.

The detection result of the initial pre-loaded substrate is the detection result of pre-loading the freshly prepared luminescent substrate liquid, the first reagent and the second reagent kit magnetic particle reagent into the thin film microfluidic chip according to the same dosage.

TABLE 2

TABLE 3 Table 3

Comparative example 2

Comparative example 2 is essentially the same as example 1 except that the enzyme conjugate diluent, the formulation of which is shown in Table 4 below, is 50% hydrochloric acid or sodium hydroxide to adjust the pH of the diluent to 8.0.+ -. 0.2.

TABLE 4 Table 4

Sequence number	Name of the name	Formula amount (1000 ml solution)
			1	Casein acid sodium salt	10.00g
2	Sodium chloride	8.00g
			3	Sodium dihydrogen phosphate dihydrate	0.31g
4	Disodium hydrogen phosphate dodecahydrate	3.49g
			5	Propyl p-hydroxybenzoate	0.5g
6	ProClin300	1ml
			7	Maltose	3g
8	50％NaOH	/
			9	50％HCl	/

Application example 2

The second reagent prepared according to the protocol of example 1 and the second reagent prepared according to the protocol of comparative example 2 were each 50 μl and pre-loaded into the corresponding pocket of the thin film microfluidic chip, respectively, and subjected to a thermal acceleration test at 37 ℃ for 7 days and a preservation test at 2-8 ℃ for 7 days, and then the other freshly prepared reagents of example 1 were added into the corresponding pocket of the thin film microfluidic chip, wherein the volume of the first reagent was 30 μl, the volume of the luminescent substrate solution was 200 μl, and the volume of the magnetic particle cleaning solution was 2000 μl, and the same sample test was performed, wherein the sample loading was 100 μl, and the luminescent value comparison data results are shown in tables 5 and 6 below, and it was seen from tables 5 and 6 that the enzyme conjugates to which PMSF was added were better in stability.

TABLE 5

TABLE 6

Application example 3

Each reagent prepared according to the protocol of example 1 was loaded into the corresponding pocket of the thin film microfluidic chip, wherein the volume of the first reagent was 30. Mu.L, the volume of the second reagent was 50. Mu.L, the volume of the luminescent substrate solution was 200. Mu.L, and the volume of the magnetic particle cleaning solution was 2000. Mu.L.

1. Sensitivity test

The calibration materials with different concentrations are used as samples to be tested, wherein the sample loading amount is 100 mu L, the test results are shown in the following table 7, the fitting curve is shown in FIG. 2, and the IC50 curve is shown in FIG. 3. As can be seen from FIG. 3, the IC50 thereof was 0.0156ppb. And the IC50 obtained by detection with an ELISA kit of the inlet r-bipharm is 0.03ppb. It can be seen that the sensitivity of the kit of example 1 is higher.

TABLE 7

Calibration material	Concentration (ppb)	RLU-1	RLU-2	RLU-3	RLU average	B/B0
							S0	0	3279466	3553084	3434171	3422240.333	1.000
S1	0.002	2939569	2949584	2910222	2933125	0.857
							S2	0.0039	2493601	2562686	2478208	2511498.333	0.734
S3	0.0156	1810341	1810391	1810365	1810365.667	0.529
							S4	0.0625	741083	743629	734943	739885	0.216
S5	0.25	110706	103779	110121	108202	0.032

2. Repeatability test

Two milk samples were used as samples to be tested, wherein the sample loading was 100 μl, the reproducibility data is shown in table 6 below, and the reproducibility of the kit of example 1 is good as seen in table 8.

TABLE 8

3. Detection limit test

A blank sample was used as a sample to be tested, wherein the sample loading amount was 100. Mu.L, and the test results are shown in Table 9 below, and the detection limit of the kit of example 1 was 0.00236ppb as seen in Table 9 below.

TABLE 9

4. Recovery test

(1) Marked 0.5%: taking 400 mu L of milk sample 1 and 400 mu L of milk sample 2 respectively, adding 2 mu L of aflatoxin M1 calibrator of 1ppb respectively, and fully and uniformly mixing;

(2) Sample adding: sucking 100 mu L of sample by using a pipette, adding the sample into a sample adding port of a reagent chip, placing the sample into an instrument for detection, and repeatedly testing the sample for 3 times after the sample is subjected to original milk and the sample is subjected to marking;

(3) Reading a luminescence value, substituting the luminescence value into a calibration curve, calculating the original milk and the concentration of the labeled aflatoxin M1, and calculating the labeled recovery rate according to a formula: wherein, denominator "scalar" is the theoretical concentration of the calibrator after the sample is added.

The test results are shown in tables 10 and 11 below. As can be seen from tables 10 and 11, the recovery rate of the kit of example 1 was high.

Table 10

TABLE 11

The present invention has been described in detail with the purpose of enabling those skilled in the art to understand the contents of the present invention and to implement the same, but not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.

Claims

1. A test kit, characterized in that: the device comprises a microfluidic chip with a sample adding port, a first reagent, a second reagent, a magnetic particle cleaning solution and a luminescent substrate solution which are mutually and independently arranged in the microfluidic chip; wherein the first reagent comprises magnetic particles coated with an antigen, the second reagent comprises an enzyme-labeled antibody, the luminescent substrate solution comprises a luminescent substrate and a substrate buffer solution, and the substrate buffer solution comprises bis (N-methylacridine) nitrate, 2-amino-2-methyl-1, 3-propanediol, 3-indoxyl disodium phosphate, 2-methyl-4-isothiazolin-3-one hydrochloride and chloroacetamide.

2. The test kit of claim 1, wherein: the mass of the bis (N-methylacridine) nitrate accounts for 0.01-0.5% of the total mass of the substrate buffer, the concentration of the 2-amino-2-methyl-1, 3-propanediol in the substrate buffer is 10-20 g/L, the mass of the 3-indoxyl disodium phosphate salt accounts for 0.01-0.2% of the total mass of the substrate buffer, the mass of the 2-methyl-4-isothiazolin-3-one hydrochloride salt accounts for 0.01-0.2% of the total mass of the substrate buffer, and the mass of the chloroacetamide accounts for 0.01-0.5% of the total mass of the substrate buffer.

3. The test kit of claim 2, wherein: the substrate buffer solution also comprises 65-85 g/L of tris (hydroxymethyl) aminomethane, 20-30 g/L of tris (hydroxymethyl) aminomethane hydrochloride, 5-10 g/L of 1, 3-bis ((trimethylol) methylamino) propane and dimethyl sulfoxide accounting for 0.1-5% of the total mass of the substrate buffer solution; and/or the number of the groups of groups,

the pH of the substrate buffer is 8-9.

4. The test kit of claim 1, wherein: the second reagent further comprises an enzyme conjugate diluent comprising 0.01-0.5% of phenylmethylsulfonyl fluoride, 0.1-3% of maltose, 0.01-5% of propyl p-hydroxybenzoate and 0.1-5% of ProClin300 or ProClin150 by weight of the enzyme conjugate diluent.

5. The test kit of claim 4, wherein: the enzyme conjugate diluent also comprises 5-15 g/L sodium caseinate, 5-10 g/L sodium chloride, 0.1-0.5 g/L sodium dihydrogen phosphate dihydrate and 1-5 g/L disodium hydrogen phosphate dodecahydrate; and/or the pH of the enzyme conjugate diluent is 7.5-8.5.

6. The test kit of claim 1, wherein: the magnetic particle cleaning liquid comprises nonionic detergent NP-40 accounting for 0.01-0.1% of the total mass of the magnetic particle cleaning liquid, nonionic detergent Brij-35 accounting for 0.1-5% of the total mass of the magnetic particle cleaning liquid and nonionic detergent MERPOL SH accounting for 0.1-3.0% of the total mass of the magnetic particle cleaning liquid.

7. The test kit of claim 6, wherein: the magnetic particle cleaning liquid also comprises 1-10 g/L of tris (hydroxymethyl) aminomethane, 10-15 g/L of tris (hydroxymethyl) aminomethane hydrochloride and Proclin300 accounting for 0.1-5% of the total mass of the magnetic particle cleaning liquid; and/or the pH value of the magnetic particle cleaning liquid is 8-9.

8. The test kit of claim 1, wherein: the difference between the pH of the magnetic particle cleaning solution and the pH of the substrate buffer solution is less than 0.4;

and/or the volume of the first reagent is 20-50 mu L, and the concentration of the magnetic particles in the first reagent is 0.1-1 mg/mL; the volume of the second reagent is 30-300 mu L, and the concentration of the enzyme-labeled antibody in the second reagent is 0.2-10 mu g/mL; the volume of the magnetic particle cleaning liquid is 1-3 mL; the volume of the luminescent substrate liquid is 100-300 mu L, and the concentration of the luminescent substrate in the luminescent substrate liquid is 0.2-2 g/L;

and/or, in the magnetic particles coated with the antigen, the mass ratio of the magnetic particles to the antigen is 100-300:1; in the enzyme-labeled antibody, the mass ratio of the antibody to the enzyme is 1:1-10;

and/or, the enzyme is alkaline phosphatase; the luminescent substrate is APS-5 powder;

and/or the microfluidic chip comprises a plurality of independent capsule cavities formed by hot pressing of a first film and a second film, and a disposable valve is arranged between each two capsule cavities;

and/or the antigen is aflatoxin M1 antigen, and the antibody is aflatoxin M1 antibody.

9. A rapid detection method is characterized in that: adding a sample to be detected to a sample adding port of the detection kit according to any one of claims 1 to 8, then placing the detection kit into a microfluidic chemiluminescent immunoassay analyzer for detection, and outputting a detection result.

10. Use of a detection kit according to any one of claims 1 to 8 for detecting the content of aflatoxin M1 in a dairy product.