CN111077319A - Micro-fluidic chip immunodetection kit and detection method thereof - Google Patents
Micro-fluidic chip immunodetection kit and detection method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
- G01N33/5304—Reaction vessels, e.g. agglutination plates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
Abstract
The invention discloses a micro-fluidic chip immunodetection kit and a detection method thereof. The kit comprises a microfluidic chip, a wafer cover plate and a wafer base plate, wherein the wafer cover plate and the wafer base plate are formed by bonding up and down; one or more detection units are arranged in the wafer bottom plate, and each detection unit comprises a liquid storage tank, a mixed reaction tank, a detection tank, a waste liquid tank and a first vent hole which are connected in sequence; wherein the mixed reaction pool is fixed with a fluorescent probe corresponding to the detection drug; and a goat anti-mouse immunoglobulin G and a corresponding drug antigen are fixed in the detection pool. The detection method utilizing the kit has the characteristics of high flux, less sample consumption, high detection speed, simple and convenient operation and the like, does not need to use electrodes, voltage or an external magnetic field, does not need to be driven by electroosmosis drive, hot gas pump drive or optical capture micropump and the like, can simultaneously detect the residues of various veterinary drugs in various matrixes, is suitable for rapid screening analysis on site, and can greatly relieve the detection pressure of related detection personnel.
Description
Technical Field
The invention relates to the technical field of food safety rapid detection, and more particularly relates to a micro-fluidic chip immunoassay kit and a detection method thereof.
Background
Veterinary drug residues refer to drug protoforms, metabolites and drug impurities that accumulate or are stored in animal cells, tissues or organs after administration of a drug to an animal. Veterinary drugs play very important roles in preventing and treating animal diseases, improving production efficiency, improving the quality of animal products and the like, but due to the lack of scientific drug administration knowledge or the driving of economic benefits of breeding personnel, the problems of veterinary drug residue are very serious because of the use of forbidden drugs, no pharmacological drugs and fake drugs in the breeding process. The residue of harmful substances in animal-derived food not only causes direct harm to human life health, but also greatly harms the development and ecological environment of animal husbandry.
The veterinary drug residues mainly comprise growth-promoting veterinary drug residues, antibiotic residues and antibacterial veterinary drug residues, such as growth-promoting veterinary drug residues of ractopamine, clenbuterol hydrochloride and the like, and antibiotic drug residues of chloramphenicol, florfenicol, tetracyclines, gentamicin and the like; antibacterial drug residues such as furans, sulfonamides, quinolones, and the like.
At present, the detection of various veterinary drug residues in China mainly adopts an instrumental analysis method or an immune rapid detection method mainly based on an ELISA (enzyme-linked immunosorbent assay) method and a colloidal gold method. The traditional instrument analysis method and ELISA method need professional operation, are complex in process and long in detection time, and need to be matched with a large instrument or analysis software for detection; the colloidal gold immunochromatography rapid detection method needs multilayer material connection, variable flow rate control and poor detection repeatability. The methods have the defects that a plurality of indexes cannot be detected simultaneously, the field simple and rapid detection of the veterinary drug residue cannot be realized, and huge detection pressure is brought to relevant detection personnel of enterprises, detection institutions, government departments and the like.
The microfluidic chip technology has the capability of scaling down the basic functions of chemical and biological laboratories to a chip with the size of a few square centimeters, can realize the miniaturization, automation, integration and portability from sample processing to detection, is applied in a plurality of fields, and has strong development activity. Although the manufacturing technology of the microfluidic chip is developed vigorously, most of the current laboratory chips need to be driven by means of electroosmosis drive, hot gas pump drive or optical capture micropumps, and the manufacturing cost is high. In addition, various factors such as the material and the structure of the chip bring certain difficulties to the establishment of an immunoassay method, such as the fixation, release and flow of an immunoreagent on the chip, and a mature method for applying the microfluidic chip to veterinary drug multi-residue immune rapid detection does not exist at present.
Disclosure of Invention
The invention aims to provide a micro-fluidic chip immunoassay kit. The microfluidic chip immunoassay kit has the characteristics of high flux, less sample consumption, high detection speed, simple and convenient operation and the like in the detection process, does not need to use electrodes, voltage or an external magnetic field, does not need to be driven by electroosmosis drive, hot gas pump drive or optical capture micropump and the like, can simultaneously detect the residues of various veterinary drugs in various matrixes, is suitable for rapid screening and analysis on site, and can greatly relieve the detection pressure of related detection departments and detection personnel.
The invention also aims to provide the application of the micro-fluidic chip immunoassay kit in the detection of drug residues in edible animal tissues, animal urine and/or animal serum.
The invention further aims to provide a detection method of the microfluidic chip immunoassay kit.
The above object of the present invention is achieved by the following scheme:
a micro-fluidic chip immunoassay kit comprises a micro-fluidic chip and comprises a wafer cover plate and a wafer bottom plate; one or more detection units are arranged in the wafer bottom plate, and the detection units are arranged on a radius shaft of the wafer bottom plate and are divergently arranged according to the central points of the wafer bottom plate; the detection unit is provided with a liquid storage tank, a mixed reaction tank, a detection tank, a waste liquid tank and a first vent hole which are sequentially arranged from a round point to the edge and are sequentially connected through a micro-channel; wherein the mixed reaction pool is fixed with a fluorescent probe corresponding to the detection drug; a goat anti-mouse immunoglobulin G and a corresponding drug antigen are fixed in the detection pool;
the wafer cover plate is also provided with a sample inlet and a second vent hole; the position of the sample inlet hole corresponds to the position of the liquid storage tank on the bottom plate of the wafer; the first vent hole corresponds to the second vent hole in position;
the wafer cover plate and the wafer base plate are formed by bonding up and down.
Preferably, the bonding comprises ultrasonic bonding, thermocompression bonding, and/or laser bonding.
Preferably, the round points of the wafer cover plate and the wafer bottom plate are both provided with fixing holes.
Preferably, the wafer bottom plate is made of polydimethylsiloxane, polymethyl methacrylate, polyamide, polystyrene, polycarbonate, epoxy resin and/or polyurethane material; more preferably, the wafer base is made of polydimethylsiloxane material.
When the wafer bottom plate is prepared by adopting polydimethylsiloxane, the surface local modification is carried out, and the specific modification process is as follows: after the detection pool is cleaned by toluene, the surface of the detection pool is treated for 2 hours by 5 percent ethanol solution of 3-Aminopropyltriethoxysilane (APTES); washing the detection tank with ethanol solution and pure water in sequence, drying, and adding 2% glutaraldehyde PBS solution to treat the reaction tank for 1 h; and cleaning for 3 times by using pure water, and drying to finish the surface modification of the chip, thereby improving the protein fixation efficiency.
Preferably, the width of the micro-channel is 5-50 μm, and the depth is 5-150 μm.
Preferably, the volumes of the liquid storage tank, the mixed reaction tank, the detection tank and the waste liquid tank are respectively 10-40 mu L.
Preferably, only the sample inlet hole and the second vent hole of the microfluidic chip are communicated with the outside.
Preferably, the fluorescent probe is a polystyrene fluorescent probe antibody complex; more preferably, the preparation method of the polystyrene fluorescent probe antibody complex comprises the following steps: washing, ultrasonically treating and redissolving the fluorescent probe in MES (morpholine ethanesulfonic acid) buffer solution; sequentially adding EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) for activation for 30 min; after activation, washing, performing ultrasonic treatment, redissolving in BB (boric acid) buffer solution, then adding corresponding antibodies and the fluorescent probe, coupling for 1-2 h at room temperature, adding BSA (bovine serum albumin) buffer solution after washing, and sealing the fluorescent probe for 30min at room temperature; and (4) re-dissolving the washed product by using a re-dissolving solution to obtain the polystyrene fluorescent probe antibody complex.
Preferably, the labeling quantity ratio of the fluorescent probe to the antibody is 1: 6-40.
Preferably, the formula of the complex solution is as follows by mass percent: trehalose 2.5%, sucrose 2.5%, Bovine Serum Albumin (BSA) 0.5%, polyvinylpyrrolidone (PVP) 0.5%, Tween-20 0.5%, ProClin-300 0.03%, and phosphate buffer (PB, 0.02M, pH 7.4) the remainder. Wherein, the trehalose and the sucrose belong to small molecules, and the BSA is inert protein and plays an important role in protecting the activity of target protein and maintaining stability; PVP belongs to macromolecules and plays an important role in promoting the release of the fluorescent probe antibody; tween-20 is a surfactant and plays an important role in promoting dispersion of the fluorescent probe antibody, maintaining the stability of the fluorescent probe antibody and the like; procline-300 is a preservative.
The components of the complex solution have important influence on the uniform dispersibility and stability of the fluorescent probe antibody in the solution and the release of the fluorescent probe antibody in the reaction.
The method for fixing the polystyrene fluorescent probe antibody complex on the mixed reaction tank comprises the following steps: and (3) sucking the polystyrene fluorescent probe antibody complex by using a microfluidic pipette, spotting and coating the polystyrene fluorescent probe antibody complex in a mixing reaction tank, and fixing for 0.5h at 4 ℃.
The method for fixing the goat anti-mouse immunoglobulin G and the corresponding drug antigen on the detection pool comprises the following steps: using a microflow pipettor to suck the goat anti-mouse immunoglobulin G and the corresponding drug antigen to be spotted on a detection pool, and fixing for 1h at 4 ℃.
The invention also protects the application of the micro-fluidic chip immunoassay kit in the detection of drug residues in edible animal tissues, animal urine and/or animal serum.
Preferably, the drug is β -receptor agonist, chloramphenicol, florfenicol, malachite green, amantadine, olaquindox, macrolides, furans, sulfonamides, fluoroquinolones, tetracyclines, aminoglycosides and/or penicillins or metabolites thereof.
Preferably, the β -receptor agonist includes ractopamine, clenbuterol hydrochloride, salbutamol, and the like.
Preferably, the macrolides include tilmicosin, tylosin, and the like.
Preferably, the furans include furazolidone, nitrofurantoin, nitrofurazone, furaltadone, and the like.
Preferably, the sulfonamides include sulfadiazine, sulfamethazine, sulfamethoxazole, and the like.
Preferably, the fluoroquinolones include norfloxacin, ofloxacin, enrofloxacin, ciprofloxacin and the like.
Preferably, the tetracyclines include chlortetracycline, oxytetracycline, and the like.
Preferably, the aminoglycosides include gentamicin, neomycin, kanamycin, streptomycin, and the like.
Preferably, the penicillins include amoxicillin, ampicillin, and the like.
The detection method of the microfluidic chip immunoassay kit is also within the protection scope of the invention, and comprises the following steps:
s1, preparing a micro-fluidic chip with a corresponding fluorescent probe and a corresponding drug antigen according to the type of a drug to be detected;
s2, fixing the prepared microfluidic chip in a centrifuge through a fixing hole; then adding the sample to be detected into a liquid storage tank of the microfluidic chip through the sample inlet hole; then, centrifuging, driving the liquid in the liquid storage tank to enter a mixed reaction tank, and mixing and reacting with the fluorescent probe fixed in the mixed reaction tank for 3 min; driving the mixed solution into a detection pool by secondary centrifugation, and reacting with the goat anti-mouse immunoglobulin G and the drug antigen for 5 min; centrifugally driving again to enable redundant liquid to enter the waste liquid pool;
and S3, detecting, namely detecting the fluorescence intensity in the detection, and converting the detection signal value to obtain the detection concentration of the object to be detected.
Preferably, the centrifugation is carried out at a constant temperature of 37 ℃ throughout the detection.
Preferably, the detection process uses a fluorescence detector for detection.
The principle of applying the microfluidic chip immunoassay kit to detect is as follows:
(1) in the detection process, the liquid in the liquid storage tank of the matched fluorescent detector with the functions of centrifugation and constant temperature maintenance (37 ℃) centrifugally drives the chip to enter a mixed reaction tank to be mixed and reacted with a fluorescent probe antibody (first antibody) fixed in the tank;
(2) when the liquid to be detected contains a target detection object (antigen), a fluorescent probe-first antibody-antigen complex is formed in the reaction tank;
(3) driving the mixed solution into a detection pool by secondary centrifugation, wherein the fluorescent probe-first antibody complex which is not combined with the target detection object in the solution to be detected is combined with the antigen fixed in the detection pool, and the fluorescent probe-first antibody-antigen complex is formed in an antigen fixing area; reacting the excessive fluorescent probe-first antibody complex or the flowing fluorescent probe-first antibody-antigen complex which is not combined by the antigen with the goat anti-mouse immunoglobulin G (second antibody) fixed in the detection pool to form a fluorescent probe-first antibody-second antibody complex or a fluorescent probe-first antibody-antigen-second antibody complex;
(4) driving the redundant liquid into a waste liquid pool by centrifugation again; irradiating the detection cell with light of a certain wavelength by the instrument, detecting the luminous intensity of goat anti-mouse immunoglobulin G and an antigen fixed position, and converting the luminous intensity into a signal value; when the content of the target detection object in the liquid to be detected is higher, the number of the fluorescent probe-first antibody complexes combined with the antigen fixing area is less, the fluorescence intensity is weaker, and the signal value is lower; the goat anti-mouse immunoglobulin G fixed zone is used as a quality control zone to play a role in quality control, and when the zone has no fluorescence signal value, the detection result is invalid. The signal value is related to the concentration of the detected object, and the detector outputs a detection result according to the signal value.
Compared with the prior art, the invention has the following beneficial effects:
the veterinary drug multi-residue immunodetection method based on the microfluidic chip immunodetection kit provided by the invention has the characteristics of high flux, less sample consumption, high detection speed, simplicity and convenience in operation and the like, is suitable for rapid screening and analysis on site, and can greatly relieve the detection pressure of relevant detection departments and detection personnel.
From the technical aspect, compared with the traditional instrument analysis method and ELISA method, the method does not need professional operation and does not need to be matched with a large-scale instrument or other analysis software for detection; compared with a colloidal gold immunochromatographic rapid detection method, the method has better detection repeatability and higher sensitivity, and avoids human errors caused by judging only by naked eyes by the colloidal gold method; the traditional instrument analysis method, ELISA method and colloidal gold method can not detect multiple indexes simultaneously, and can not realize the on-site simple and rapid detection of multiple veterinary drug residues; compared with the existing microfluidic technology, the preparation of related immunological reagents and the establishment of immunological methods are mature, and the aim of rapidly detecting the multiple residues of the veterinary drugs on site can be achieved.
From economic benefits, compared with the traditional detection method or most of current laboratory chips, the method has the advantages that the reagent consumption is low, electrodes, voltage or external magnetic fields are not needed, the electroosmosis drive, the hot gas pump drive or the optical capture micropump drive is not needed, and the manufacturing cost is low; can detect the residues of various veterinary drugs in various matrixes simultaneously, thereby greatly saving time and labor cost.
Drawings
Fig. 1 is a schematic structural diagram of a wafer substrate of the microfluidic chip immunoassay kit in example 1.
Fig. 2 is a schematic structural diagram of a disk cover plate of the microfluidic chip immunoassay kit in example 1.
FIG. 3 is a schematic view of the structure sequence of a detection unit in the immunodetection kit with a microfluidic chip in example 1.
Detailed Description
The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1
A microfluidic chip immunoassay kit is shown in the structural schematic diagrams of figures 1 to 3, and comprises a microfluidic chip, a chip cover plate 1 and a chip bottom plate 2; the structure of the wafer cover plate 1 is shown in fig. 2, and the structure of the wafer base plate 2 is shown in fig. 1.
One or more detection units are arranged in the wafer bottom plate 2, and the structural sequence of a single detection unit is schematically shown in FIG. 3; the detection unit is arranged on a radius shaft of the wafer base plate 2 and is divergently arranged by the center point of the wafer base plate 2; the detection unit is provided with a liquid storage tank 21, a mixing reaction tank 22, a detection tank 23, a waste liquid tank 24 and a first vent hole 25 which are sequentially arranged from a dot to the edge and are connected through a micro-channel in sequence; wherein, the mixed reaction tank 22 is fixed with a corresponding fluorescent probe for detecting the drug; a sheep anti-mouse immunoglobulin G and a corresponding drug antigen are fixed in the detection pool 23;
the wafer cover plate 1 is also provided with a second vent hole 12 of a sample inlet hole 11; the position of the sample inlet hole 11 corresponds to the position of the liquid storage tank 21 on the wafer bottom plate 2; the first vent hole 25 corresponds to the second vent hole 12;
the wafer cover plate 1 and the wafer base plate 2 are formed by bonding up and down; commonly used bonding means include ultrasonic bonding, thermocompression bonding, and/or laser bonding.
The round points of the wafer cover plate 1 and the wafer base plate 2 are both provided with fixing holes 3 for fixing the microfluidic chip in the centrifuge.
The wafer base plate 2 can be prepared by adopting polydimethylsiloxane, polymethyl methacrylate, polyamide, polystyrene, polycarbonate, epoxy resin and/or polyurethane material; the wafer base 2 in this example is made of polydimethylsiloxane material.
When the wafer base plate 2 is prepared by adopting polydimethylsiloxane, the surface is locally modified, and the specific modification process is as follows: after the detection pool is cleaned by toluene, the surface of the detection pool is treated for 2 hours by 5 percent ethanol solution of 3-Aminopropyltriethoxysilane (APTES); washing the detection tank with ethanol solution and pure water in sequence, drying, and adding 2% glutaraldehyde PBS solution to treat the reaction tank for 1 h; and cleaning for 3 times by using pure water, and drying to finish the surface modification of the chip, thereby improving the protein fixation efficiency.
The width of the micro-channel is 5-50 μm and the depth is 5-150 μm.
The volumes of the liquid storage tank 21, the mixed reaction tank 22, the detection tank 23 and the waste liquid tank 24 are respectively 10-40 mu L.
The micro-fluidic chip is only provided with the sample inlet hole 11 and the second vent hole 12 which are communicated with the outside.
The fluorescent probe is a polystyrene fluorescent probe antibody compound; the preparation method comprises the following steps: washing and ultrasonically treating 10 mu L of fluorescent probe, and dissolving in 1mL of MES (morpholine ethanesulfonic acid) buffer solution; sequentially adding 10 μ L of 0.8mg/mL EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) and 10 μ L of 1.6mg/mL NHS (N-hydroxysuccinimide) for activation for 30 min; after activation, washing, performing ultrasonic treatment, dissolving in 1mL of BB (boric acid) buffer solution again, then adding a corresponding antibody and the fluorescent probe, coupling for 1-2 h at room temperature, adding 15 mu L of 20% BSA (bovine serum albumin) buffer solution after washing, and sealing the fluorescent probe for 30min at room temperature; and (4) re-dissolving the washed product by using a re-dissolving solution to obtain the polystyrene fluorescent probe antibody complex.
The labeling quantity ratio of the fluorescent probe to the antibody is 1: 6-40.
The formula of the redissolution comprises the following components in percentage by mass: trehalose 2.5%, sucrose 2.5%, Bovine Serum Albumin (BSA) 0.5%, polyvinylpyrrolidone (PVP) 0.5%, Tween-20 0.5%, ProClin-300 0.03%, and phosphate buffer (PB, 0.02M, pH 7.4) the remainder. Wherein, the trehalose and the sucrose belong to small molecules, and the BSA is inert protein and plays an important role in protecting the activity of target protein and maintaining stability; PVP belongs to macromolecules and plays an important role in promoting the release of the fluorescent probe antibody; tween-20 is a surfactant and plays an important role in promoting dispersion of the fluorescent probe antibody, maintaining the stability of the fluorescent probe antibody and the like; procline-300 is a preservative.
The components of the complex solution have important influence on the uniform dispersibility and stability of the fluorescent probe antibody in the solution and the release of the fluorescent probe antibody in the reaction.
The method for fixing the polystyrene fluorescent probe antibody complex on the mixed reaction tank 22 comprises the following steps: the polystyrene fluorescent probe antibody complex is sucked by a microflow pipettor, spotted and smeared on the mixing reaction pool 22, and fixed for 0.5h at 4 ℃.
The method for fixing the goat anti-mouse immunoglobulin G and the corresponding drug antigen on the detection pool 23 comprises the following steps: using a microflow pipette to suck the goat anti-mouse immunoglobulin G and the corresponding drug antigen to be spotted on the detection pool 23, and fixing for 1h at 4 ℃.
Example 2
The microfluidic chip immunoassay kit prepared in example 1 is applied to detection, and in this example, swine urine is taken as a sample to be detected, and the content of ractopamine in the swine urine is detected, and the specific detection process is as follows:
1. a plurality of microfluidic chips for preparing and detecting ractopamine before detection
2. Sample treatment:
(1) selecting 3 different negative pig urine, and adding ractopamine medicine at three concentrations in the concentration range of 0.5-15 ng/mL.
(2) And (4) analyzing fresh and clear pig urine after the medicine is added. If the urine sample is turbid, the sample must be centrifuged at 4000r/min at room temperature (20 ℃ -25 ℃) for more than 5min, and the supernatant is taken for detection.
3. Detection of
And placing the prepared micro-fluidic chip for detecting ractopamine in a centrifuge of a corresponding detector, so that the chip is firmly fixed on a centrifugal shaft. Sucking 30 mu L of liquid (supernatant) to be detected, adding the liquid through the chip sample inlet hole to enable the liquid to enter the liquid storage tank, starting the instrument after the sample adding of each detection unit of the chip is finished, and sequentially operating the instrument by the following steps: centrifugally driving liquid in the liquid storage tank to enter a mixed reaction tank, and mixing and reacting the liquid with the fluorescent microsphere antibody fixed in the tank for 3 min; driving the mixed solution into a detection pool by secondary centrifugation, and reacting with goat anti-mouse immunoglobulin G and antigen in the detection pool for 5 min; driving the redundant liquid into a waste liquid pool by centrifugation again; the instrument irradiates the detection cell with light with a certain wavelength to obtain the fluorescence intensity in the detection cell, the detection signal value is related to the concentration of the object to be detected, and the detection result is output.
The same drug-added sample was tested using 3 different batches of chips, each sample being tested in duplicate 3 times. And calculating the addition recovery rate and the variation coefficient. The results are shown in Table 1.
TABLE 1
From the above results, the addition recovery rate in the test is between 80% and 120%, and the variation coefficient is less than 10%, which indicates that the test method of the present embodiment has good accuracy and stability.
Example 3
The detection is carried out by applying the microfluidic chip immunoassay kit prepared in the embodiment 1, pork is taken as a sample to be detected, and the content of chloramphenicol in the sample is detected, wherein the specific detection process is as follows:
1. a plurality of micro-fluidic chips for detecting chloramphenicol are prepared before detection.
2. Sample treatment:
(1) selecting 3 different negative pork samples, and adding chloramphenicol medicines at three concentrations in the concentration range of 0.5-15 ng/mL.
(2) Weighing 2 +/-0.05 g of homogenized pork sample added with the medicine into a 50mL centrifuge tube, adding 6mL ethyl acetate, and fully shaking and uniformly mixing for 3 min; centrifuging at 4000r for 5min at room temperature (20-25 ℃); taking 3mL of supernatant, and drying the supernatant in water bath nitrogen at 60-70 ℃; taking 1mL of n-hexane for redissolving, adding 1mL of PBS solution, and fully shaking for 1 min; centrifuging at 4000r for 5min at room temperature (20-25 ℃); 30 μ L of the supernatant was taken for detection.
3. Detection of
The prepared micro-fluidic chip for detecting chloramphenicol is placed in a centrifuge of a corresponding detector, so that the chip is firmly fixed on a centrifugal shaft. The remaining steps were the same as the detection step in example 2.
The same drug-added sample was tested using 3 different batches of chips, each sample being tested in duplicate 3 times. And calculating the addition recovery rate and the variation coefficient. The results are shown in Table 2.
TABLE 2
From the above results, the addition recovery rate in the test is between 80% and 120%, and the variation coefficient is less than 10%, which indicates that the test method of the present embodiment has good accuracy and stability.
Example 4
The detection is carried out by applying the microfluidic chip immunoassay kit prepared in the embodiment 1, in the embodiment, chicken is taken as a sample to be detected, and the content of amantadine in the chicken is detected, and the specific detection process is as follows:
1. before detection, a plurality of microfluidic chips for detecting amantadine are prepared.
2. Sample treatment:
(1) selecting 3 different negative chicken samples, and adding amantadine drugs at three concentrations in the concentration range of 1-20 ng/mL.
(2) Weighing 2 +/-0.05 g of homogenized chicken sample added with the medicine into a 50mL centrifuge tube, adding 6mL of acetonitrile, and fully shaking and uniformly mixing for 3 min; centrifuging at 4000r for 5min at room temperature (20-25 ℃); taking 3mL of supernatant, and drying the supernatant in water bath nitrogen at 60-70 ℃; adding 1mL of PBS solution, and fully shaking for 1 min; centrifuging at 4000r for 5min at room temperature (20-25 ℃); 30 μ L of the supernatant was taken for detection.
3. Detection of
The prepared microfluidic chip for detecting amantadine is placed in a centrifuge of a corresponding detector, so that the chip is firmly fixed on a centrifugal shaft. The remaining steps were the same as the detection step in example 2.
The same drug-added sample was tested using 3 different batches of chips, each sample being tested in duplicate 3 times. And calculating the addition recovery rate and the variation coefficient. The results are shown in Table 3.
TABLE 3
From the above results, the addition recovery rate in the test is between 80% and 120%, and the variation coefficient is less than 10%, which indicates that the test method of the present embodiment has good accuracy and stability.
The embodiment shows that the microfluidic chip immunoassay kit can be used for detecting various samples and medicines, the detection method has good accuracy, stability and repeatability, can be widely applied to the actual detection of medicine residues in multipurpose animal samples, and has the advantages of simple and convenient detection process and strong operability.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. The micro-fluidic chip immunoassay kit is characterized by comprising a micro-fluidic chip, wherein the micro-fluidic chip comprises a wafer cover plate (1) and a wafer bottom plate (2); one or more detection units are arranged in the wafer bottom plate (2), and the detection units are arranged on a radius axis of the wafer bottom plate (2) and are divergently arranged according to the central point of the wafer bottom plate (2); the detection unit is provided with a liquid storage tank (21), a mixed reaction tank (22), a detection tank (23), a waste liquid tank (24) and a first vent hole (25) which are sequentially arranged from a dot to the edge and are sequentially connected through a micro-channel; wherein the mixed reaction pool (22) is fixed with a fluorescent probe corresponding to the detection medicine; a sheep anti-mouse immunoglobulin G and a corresponding drug antigen are fixed in the detection pool (23);
the wafer cover plate (1) is also provided with a sample inlet hole (11) and a second vent hole (12); the position of the sample inlet hole (11) corresponds to the position of the liquid storage tank (21) on the wafer bottom plate (2); the first vent hole (25) corresponds to the second vent hole (12);
the wafer cover plate (1) and the wafer base plate (2) are formed by bonding up and down.
2. The microfluidic chip immunoassay kit according to claim 1, wherein the circular dots of the wafer cover plate (1) and the wafer bottom plate (2) are provided with fixing holes (3).
3. The microfluidic chip immunoassay kit according to claim 1, wherein the wafer substrate (2) is made of polydimethylsiloxane, polymethyl methacrylate, polyamide, polystyrene, polycarbonate, epoxy resin and/or polyurethane material.
4. The microfluidic chip immunoassay kit according to claim 1, wherein the microchannel has a width of 5 to 50 μm and a depth of 5 to 150 μm.
5. The microfluidic chip immunoassay kit according to claim 1, wherein the volumes of the liquid reservoir (21), the mixing reaction reservoir (22), the detection reservoir (23) and the waste liquid reservoir (24) are each 10 to 40 μ L.
6. The immunodetection kit of the microfluidic chip according to claim 1, wherein the microfluidic chip only has the sample inlet (11) and the second vent (12) to communicate with the outside.
7. The microfluidic chip immunoassay kit of claim 1, wherein the drug-related fluorescent probes are applied to the mixing reaction chamber (22) by spotting and fixed at 4 ℃ for 0.5 h; the goat anti-mouse immunoglobulin G and the corresponding drug antigen are spotted in a detection pool (23) and fixed for 1h at 4 ℃.
8. Use of the microfluidic chip immunoassay kit of claims 1 to 7 in the detection of drug residues in edible animal tissues, animal urine and/or animal serum.
9. Use according to claim 8, wherein the drug is β -receptor agonist, chloramphenicol, florfenicol, malachite green, amantadine, olaquindox, macrolides, furans, sulfonamides, fluoroquinolones, tetracyclines, aminoglycosides and/or penicillins or metabolites thereof.
10. The detection method of the microfluidic chip immunoassay kit according to claims 1 to 7, characterized by comprising the steps of:
s1, preparing a micro-fluidic chip with a corresponding fluorescent probe and a corresponding drug antigen according to the type of a drug to be detected;
s2, fixing the prepared microfluidic chip in a centrifuge through a fixing hole; then adding a sample to be detected into a liquid storage tank (21) of the microfluidic chip through the sample inlet hole (11); then, centrifuging, driving the liquid in the liquid storage tank (21) to enter a mixed reaction tank (22), and mixing and reacting with a fluorescent probe fixed in the mixed reaction tank for 3 min; the mixed solution is driven to enter a detection pool (23) by secondary centrifugation and reacts with the goat anti-mouse immunoglobulin G and the drug antigen for 5 min; centrifugally driving again to make the redundant liquid enter a waste liquid pool (24);
and S3, detecting the fluorescence intensity in the step (23), and converting the detection signal value to obtain the detection concentration of the object to be detected.
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