CN111077301A - Micro-fluidic chip and method for detecting interaction between biomolecules - Google Patents
Micro-fluidic chip and method for detecting interaction between biomolecules Download PDFInfo
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a micro-fluidic chip for detecting the interaction between biomolecules, which belongs to the technical field of biomolecule detection and comprises a bottom plate, wherein a sample injection unit, a detection unit and a power unit which are sequentially communicated are arranged on the bottom plate; the power unit is a peristaltic pump, and a liquid inlet of the peristaltic pump is provided with a negative pressure interface; the sample introduction unit comprises a sample introduction box, and a sample introduction hole is formed in the sample introduction box; the detection unit comprises a detection box, a detection hole is formed in the detection box, a nitrocellulose membrane is arranged in the detection box, and avidin is coated outside the nitrocellulose membrane; the sampling box is communicated with the detection box through a first micro-tube, and the negative pressure interface is communicated with the detection box through a second micro-tube; the micro-fluidic chip has the advantages of simple structure, convenience in operation, short time for the whole detection process and suitability for popularization and application.
Description
Technical Field
The invention belongs to the technical field of biomolecule detection, and particularly relates to a micro-fluidic chip and a method for detecting interaction between biomolecules.
Background
In the field of immunology and biochemical research, Enzyme-Linked Immunosorbent Assays (ELISA) and immunochromatographic strip methods are mainly used for detection of antigens and antibodies. The ELISA is widely applied since the surface, the detection principle is based on the specific binding between antigen and antibody, the color reaction is carried out through enzyme-labeled antibody, and the detection signal is read by adopting an enzyme-labeled instrument. The ELISA has high sensitivity and good specificity, but the detection process comprises the steps of coating, blocking, antibody incubation, developing and the like, so that the time consumption is long, generally more than 10 minutes, and the requirement of rapid detection is difficult to achieve.
With the increasingly strict requirement on the detection time, the colloidal gold chromatography detection method in the immunochromatographic test strip method is developed rapidly in recent years, the most common method is the early pregnancy test strip, the sampling is simple, the detection result can be judged within a few minutes, the detection time of the colloidal gold chromatography test strip is short, but the optimization and preparation process are complex, and even if a general unit has a related antigen-antibody, the colloidal gold chromatography detection method is difficult to prepare easily.
Therefore, in order to solve the problems of long ELISA time consumption and complex preparation of the immunochromatographic test strip, the invention provides a scheme: a micro-fluidic chip and a method for detecting the interaction between biological molecules.
Disclosure of Invention
In order to solve the problems of long ELISA time consumption and complex preparation of an immunochromatographic test strip, the invention provides a micro-fluidic chip for detecting the interaction between biomolecules.
The technical scheme adopted by the invention is as follows: a micro-fluidic chip for detecting the interaction between biomolecules comprises a bottom plate, wherein a sample introduction unit, a detection unit and a power unit which are sequentially communicated are arranged on the bottom plate;
the power unit is a peristaltic pump, and a liquid inlet of the peristaltic pump is provided with a negative pressure interface; the sample introduction unit comprises a sample introduction box, and a sample introduction hole is formed in the sample introduction box; the detection unit comprises a detection box, a detection hole is formed in the detection box, a nitrocellulose membrane is arranged in the detection box, and avidin is coated outside the nitrocellulose membrane; the sampling box is communicated with the detection box through a first micro-tube, and the negative pressure interface is communicated with the detection box through a second micro-tube.
The invention has the beneficial effects that: the method is based on a biotin-labeled macromolecular substance strategy, biotin is combined with a first biological macromolecule, and then an object to be detected and a second biological macromolecule carrying a specific label are added; wherein, the object to be detected can be specifically combined with the first biological macromolecule and the second biological macromolecule of biotin at the same time, thereby forming a complex; when the complex flows through the detection hole, biotin in the complex is specifically captured by avidin coated on the nitrocellulose membrane, and signal detection is carried out by naked eyes or related detection equipment. Only 5 minutes are needed from the dropwise adding of the object to be detected, the first biomacromolecule of biotin and the second biomacromolecule carrying the specific marker until a detection result appears, the time consumption is short, the detection efficiency is improved, and the preparation of avidin is simple; it is suitable for all biological molecules based on immune principle detection.
Further limiting, the pipe diameters of the first micro pipe and the second micro pipe are both 1.5-2.5 mm.
Further defined, the bottom plate, the first microtube and the second microtube are all made of polystyrene.
Further defined, the avidin has a coating thickness of 0.4 mm.
Further limiting, the volume of the sample injection box is 50-200 microliter.
The invention also provides a method for detecting the interaction between the biomolecules, which is realized based on the micro-fluidic chip.
Further defined, comprising the steps of:
s1: obtaining a biotin first biological macromolecule by adopting the biotin first biological macromolecule;
s2: mixing the first biomacromolecule of biotin, the second biomacromolecule with the specific mark and the object to be detected and then adding the mixture into the sample inlet, or sequentially adding the first biomacromolecule of biotin, the object to be detected and the second biomacromolecule with the specific mark into the sample inlet;
s3: the peristaltic pump is started and the signal intensity of the avidin is detected.
The method is simple and rapid, the whole process only needs 5 minutes, and the method is suitable for popularization.
Further defined, the first biological macromolecule is any one of a protein, a peptide fragment, or a nucleic acid.
Further defined, the second biological macromolecule is any one of a protein, a peptide fragment, a small molecule peptide or a nucleic acid.
Further defined, the specific marker of the second biomacromolecule is any one of colloidal gold, fluorescent microspheres or quantum dots.
Drawings
FIG. 1 is a schematic diagram of a microfluidic chip;
FIG. 2 is a schematic view of the structure of the detecting unit;
in the figure: 1-a bottom plate; 2-sample introduction unit; 3-a detection unit; 31-a detection box; 32-detection wells; 33-nitrocellulose membrane; 34-avidin; 4-a power unit; 5-a first microtube; 6-a second microtube; 7-sample injection hole.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
In the description of the present invention, some structures or devices are not specifically described, and it is understood that there are structures or devices that can be implemented in the prior art.
A method for detecting the interaction between biomolecules based on a microfluidic chip comprises the following steps:
s1: obtaining a biotin first biological macromolecule by adopting the biotin first biological macromolecule;
s2: mixing the first biomacromolecule of biotin, the second biomacromolecule with the specific mark and the object to be detected and then adding the mixture into the sample inlet, or sequentially adding the first biomacromolecule of biotin, the object to be detected and the second biomacromolecule with the specific mark into the sample inlet;
s3: starting a peristaltic pump, and detecting the signal intensity of the avidin;
wherein: the first biological macromolecule is any one of protein, peptide fragment or nucleic acid. The second biological macromolecule is any one of protein, peptide fragment, small molecule peptide or nucleic acid, the second biological macromolecule is usually a commercial product, the specific reaction concentration is determined according to the recommended concentration of a manufacturer, and the recommended data of different manufacturers are slightly changed. The special mark of the second biomacromolecule is any one of colloidal gold, fluorescent microspheres or quantum dots.
Example 1
As shown in fig. 1 and 2, a micro-fluidic chip for detecting the interaction between biomolecules comprises a bottom plate, wherein a sample injection unit, a detection unit and a power unit which are sequentially communicated are arranged on the bottom plate;
the power unit is a peristaltic pump, and a liquid inlet of the peristaltic pump is provided with a negative pressure interface; the sample introduction unit comprises a sample introduction box, and a sample introduction hole is formed in the sample introduction box; the detection unit comprises a detection box, a detection hole is formed in the detection box, a nitrocellulose membrane is arranged in the detection box, and avidin is coated outside the nitrocellulose membrane; the sampling box is communicated with the detection box through a first micro-tube, and the negative pressure interface is communicated with the detection box through a second micro-tube.
The pipe diameters of the first micro pipe and the second micro pipe are 1.5-2.5 mm. The bottom plate, the first micro-pipe and the second micro-pipe are all made of polystyrene. The coating thickness of the avidin is 0.4 mm. The volume of the sample injection box is 50-200 microliters; the sampling hole, the detection hole and the negative pressure interface are communicated.
The working principle is as follows: adding a substance to be detected and other auxiliary reagents into the sample inlet, starting the peristaltic pump, enabling the substance to be detected and other auxiliary reagents to reach the detection box through the first micro-tube, capturing the substance to be detected and other auxiliary reagents by avidin on the nitrocellulose membrane, detecting the signal intensity of the avidin to obtain a detection result, enabling a positive reaction to present a color reaction in the detection hole, and enabling the position to be a point coated with the avidin; the peristaltic pump is used for promoting the liquid to smoothly flow through the detection hole, and other devices with the same function can be used for replacing the peristaltic pump.
Example 2
1. Micro-fluidic chip for manufacturing and detecting interaction between biological molecules
The mold was customized according to the structure of the microfluidic chip of fig. 1, dimensions: length x width 13 x 3 cm; the material is Polystyrene (PS). Sample introduction box size: circular, 1.5 cm in diameter, for sample application. The detection case is the same with advance kind case size, the inside fixed rectangular nitrocellulose membrane of detection case, the size of rectangular nitrocellulose membrane: length × width is 1.5 × 0.7 cm; the detection box is connected with the sample injection box through a first micro-pipeline, and the diameter of the first micro-pipeline is 2 mm. The detection box is provided with a detection hole, a second micro pipeline is arranged in the detection hole, the second micro pipeline extends outwards to form a negative pressure interface, and the negative pressure interface is connected with the peristaltic pump; the first micro-pipeline and the second micro-pipeline are manufactured by a mould and are formed in one step.
2. Application of microfluidic chip
The microfluidic chip can be applied to all reactions based on immunological reactions. The current commercialized biotin labeling kit can be used for labeling protein and antibody, and the synthesized aptamer can also be labeled by biotin. The biotin-labeled proteins and antibodies were performed as described in the specification. The following procedures illustrate the process and detection method of labeling CPS protein with biotin, taking the example of labeling the Streptococcus suis type 2 capsular polysaccharide protein CPS with biotin (kit: purchased from Piece, Inc., Cat. No. 21435).
Taking prokaryotic expression to purify to obtain 3mg (1.5mg/mL) of CPS protein, according to the requirements of the specification, the mole number of Biotin labeling is about 20 times of that of the molecule to be labeled, and the specific steps of calculating the dosage of Biotin (Sulfo-NHS-LC-Biotin) to be about 73 mu L (6 mg/mL): 1mL of protein CPS to be marked is taken, the concentration of the protein CPS is 3mg/mL (the solvent is 50mM phosphate buffer solution, and the pH value is 7.4); weighing 3mg of Sulfo-NHS-LC-Biotin, dissolving in 500 mu L of ultrapure water, and preparing a Biotin solution with the concentration of 6 mg/mL; adding 73 mu L of prepared Sulfo-NHS-LC-Biotin solution into the CPS protein to be marked, slightly reversing the solution up and down, uniformly mixing the solution and reacting the solution at room temperature for 1 h; after the reaction is finished, the mixture is centrifuged for 3min by 1000 Xg desalting column, the biotin molecules of the reaction are removed, and the labeled product is collected.
Diluting the CPS protein marked by biotin to 2 mu g/mL by using a phosphate buffer solution (20mM, pH8.0), placing 25 mu L into a 2mL EP tube, then adding 25 mu L of streptococcus suis type 2 antibody to be detected, simultaneously adding 25 mu L of SPA (other markers can be adopted) marked by quantum dots, uniformly mixing and reacting for 3 minutes, dripping 125 mu L of reaction liquid into a sample injection hole of a microfluidic chip, starting a connection peristaltic pump to enable the liquid to flow, and finally enabling the reaction liquid to flow into a waste liquid pool, wherein the process only needs 5 minutes.
And detecting and judging the detection result in different modes according to different markers. If the second biological macromolecule marked by quantum dots or colloidal gold is adopted, the result can be judged by naked eyes; if HRP is adopted for marking, color reaction is needed to be carried out, and whether a reaction point appears or not is observed.
In the reaction samples dropped into the wells, the concentration of biotin-CPS protein conjugate was 2. mu.g/mL, the lower limit line concentration of detection of Streptococcus suis 2 antibody to be detected was 0.003. mu.g/mL, and the commercial second biomacromolecule carrying a label was used at a concentration of 0.0001. mu.g/mL for the quantum dot labeled SPA according to the manufacturer's recommendations.
Example 3
The difference from example 1 is: application of microfluidic chip
2. Application of microfluidic chip
Taking biotin-labeled nucleic acid as an example, the detection method is described as follows: diluting the synthesized biotin-nucleic acid fragment (Youkang bioscience, Hangzhou Co., Ltd.) to 0.2. mu.g/mL by using a phosphate buffer solution (20mM, pH8.0), putting 50. mu.L into a 2mL centrifuge tube, adding 50. mu.L of a small molecular toxin (OTA) to be detected to the concentration of 0.1ng/mL, simultaneously adding 50. mu.L of an OTA specific antibody (monoclonal antibody with the dilution multiple of 1:20000), adding 50. mu.L of a quantum dot-labeled secondary antibody (QDs-anti-mouse antibody with the dilution multiple of 1:10000), reacting for 3 minutes at room temperature, and then dropping 150. mu.L of reaction solution into a sample inlet hole in a microfluidic chip; and (3) opening the peristaltic pump, enabling the reaction liquid to flow to the detection hole from the sample inlet hole, and finally enabling the reaction liquid to flow into the waste liquid pool completely, wherein the process is simple to operate.
In the reaction solution dropped into the sample injection hole, the concentration of biotin-labeled nucleic acid was 0.2. mu.g/mL, the lower limit concentration of OTA detection was 0.1ng/mL, the concentration of OTA monoclonal antibody was 0.1ng/mL, and the concentration of commercial QDs anti-mouse antibody was 0.5. mu.g/mL.
Example 4
The difference from example 1 is: application of microfluidic chip
2. Application of microfluidic chip
The detection method is illustrated by taking a biotin labeled aflatoxin AFB1 monoclonal antibody as an example. The following procedures illustrate the procedures and detection methods for biotin labeling of AFB1 monoclonal antibody using the biotin labeled AFB1 monoclonal antibody (kit: available from Piece, Inc., cat # 21435).
Taking 3mg (1.5mg/mL) of the purified AFB1 monoclonal antibody, and according to the requirements of the specification, the mole number of Biotin labeling is about 20 times of that of a molecule to be labeled, and calculating the specific steps of the usage amount of the Biotin (Sulfo-NHS-LC-Biotin) to be about 73 μ L (6 mg/mL): taking 1mL of the antibody to be marked, wherein the concentration of the antibody is 3mg/mL (the solvent is 50mM phosphate buffer solution, pH 7.4); weighing 3mg of Sulfo-NHS-LC-Biotin, dissolving in 500 mu L of ultrapure water, and preparing a Biotin solution with the concentration of 6 mg/mL; adding 73 mu L of prepared Sulfo-NHS-LC-Biotin solution into the antibody protein to be marked, slightly reversing the solution up and down, uniformly mixing the solution and reacting the solution at room temperature for 1 h; after the reaction is finished, the mixture is centrifuged for 3min by 1000 Xg desalting column, the biotin molecules of the reaction are removed, and the labeled product is collected.
The biotin-labeled antibody was diluted to 0.1. mu.g/mL with phosphate buffer (20mM, pH8.0), 50. mu.L was placed in a 2mL LP tube, 50. mu.L of AFBI small molecule toxin (concentration 0.05ng/mL) was added, and FITC-labeled aptamer capable of reacting with the toxin (concentration 0.05. mu.g/mL) was added and reacted at room temperature for 3 minutes.
And dripping 125 mu L of reaction sample into a sample inlet of the microfluidic chip. And starting the peristaltic pump, enabling the reaction liquid to flow to the detection hole from the sample inlet hole, and finally enabling the reaction liquid to completely flow out of the waste liquid pool.
In the reaction solution to be detected, the concentration of the biotin-labeled antibody is 0.1. mu.g/mL, the lower detection limit of AFB1 to be detected is 0.001ng/mL, and the concentration of the FITC-labeled aptamer is 0.05. mu.g/mL.
The invention is not limited to the above alternative embodiments, and any other various forms of products can be obtained by anyone in the light of the present invention, but any changes in shape or structure thereof, which fall within the scope of the present invention as defined in the claims, fall within the scope of the present invention.
Claims (10)
1. A micro-fluidic chip for detecting the interaction between biomolecules is characterized by comprising a bottom plate, wherein a sample injection unit, a detection unit and a power unit which are sequentially communicated are arranged on the bottom plate;
the power unit is a peristaltic pump, and a liquid inlet of the peristaltic pump is provided with a negative pressure interface; the sample introduction unit comprises a sample introduction box, and a sample introduction hole is formed in the sample introduction box; the detection unit comprises a detection box, a detection hole is formed in the detection box, a nitrocellulose membrane is arranged in the detection box, and avidin is coated outside the nitrocellulose membrane; the sampling box is communicated with the detection box through a first micro-tube, and the negative pressure interface is communicated with the detection box through a second micro-tube.
2. The microfluidic chip for detecting the interaction between the biomolecules according to claim 1, wherein the first microtube and the second microtube have a tube diameter of 1.5-2.5 mm.
3. The microfluidic chip for detecting intermolecular interaction of claim 1, wherein the base plate, the first microtube and the second microtube are made of polystyrene.
4. The microfluidic chip for detecting intermolecular interactions of claim 1, wherein the coating thickness of the avidin is 0.4 mm.
5. The microfluidic chip for detecting intermolecular interactions according to claim 1, wherein the volume of the sample chamber is 50 to 200. mu.l.
6. A method for detecting an interaction between biomolecules, which is carried out based on the microfluidic chip of any one of claims 1 to 5.
7. The method for detecting interaction between biomolecules according to claim 6, comprising the steps of:
s1: obtaining a biotin first biological macromolecule by adopting the biotin first biological macromolecule;
s2: mixing the first biomacromolecule of biotin, the second biomacromolecule with the specific mark and the object to be detected and then adding the mixture into the sample inlet, or sequentially adding the first biomacromolecule of biotin, the object to be detected and the second biomacromolecule with the specific mark into the sample inlet;
s3: the peristaltic pump is started and the signal intensity of the avidin is detected.
8. The method of claim 7, wherein the first biological macromolecule is any one of a protein, a peptide fragment, or a nucleic acid.
9. The method of claim 7, wherein the second biomacromolecule is any one of a protein, a peptide fragment, a small molecule peptide or a nucleic acid.
10. The method of claim 7, wherein the specific label of the second biomacromolecule is any one of colloidal gold, fluorescent microspheres, or quantum dots.
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