CN111077303A - Vertical channel array microchip and imaging device and method for immunodetection - Google Patents
Vertical channel array microchip and imaging device and method for immunodetection 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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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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- 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
- B01L3/502707—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 characterised by the manufacture of the container or its components
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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
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
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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
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
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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
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
- B01L2300/168—Specific optical properties, e.g. reflective coatings
Abstract
The invention discloses a vertical channel array microchip for immunodetection, an imaging device and a method. The vertical channel array microchip has a three-layer structure, wherein the lower layer is a cleaning channel, the middle layer is a detection channel and is used for fixing an antibody, and the upper layer is a sample adding channel; the visible light signal imaging device comprises a self-made flat light source, a detection box and an intelligent mobile device. The invention also provides methods of operating vertical channel array microchips and imaging devices for use in immunoassays. The invention combines the advantages of the microfluidic technology, the immunoassay and the intelligent mobile device, has the advantages of small used sample, simple operation, low cost, portability and the like, and can be used in the fields of clinical diagnosis, drug screening, pollutant tracking, food safety and the like.
Description
Technical Field
The invention belongs to the technical field of biological detection, relates to a vertical channel array microchip for immunodetection, an imaging device and a method, and particularly relates to a visible light signal image immunodetection device and a method based on the vertical channel array microchip and intelligent mobile equipment.
Background
Immunoassay is a powerful biological analysis tool and is widely used in the fields of clinical diagnosis, pathogen determination, drug screening, pollutant tracing and the like. As a typical immunoassay method, enzyme-linked immunosorbent assay has been widely used in routine assays in various laboratories, including basic research and practical applications. In recent years, with the increasing use, some disadvantages have gradually emerged. For example, in a commercial 96-well plate, a very large volume of sample is typically consumed per well, about 20-200 μ L; the incubation takes a longer time; the microplate reader has large volume, is expensive and is complex to operate, so that the field timely detection of experiments is inconvenient to carry out at present.
The micro-fluidic chip is a rapid and instant detection analytical device which is widely concerned in recent years, and has wide development prospect. The micro-fluidic chip is made of very wide materials including glass, PDMS, COC, PC, PS and the like, so that different materials can be selected to be combined with different reactions, and various operation units such as sample introduction, reaction, detection and the like can be integrated. The micro-fluidic chip has the advantages of small volume, good biocompatibility, low cost and the like, and is widely used in the fields of clinical diagnosis, environmental monitoring, food safety and the like; the on-site instant detection technology refers to clinical detection carried out by a patient, has the characteristics of high speed, simple and convenient operation and low cost, and has wide application prospect; at present, the popularity of smart mobile devices is increasing, including smart phones, tablet computers, smart cameras and the like, which can obtain images with high definition, and simultaneously can share data to various platforms such as a cloud terminal in real time, thereby realizing rapid processing of image information.
The current microchips for immunoassay use a horizontal detection channel in many cases, and although the specific surface area is large, the optical path through the microchannel is short, which is disadvantageous for obtaining high sensitivity. The vertical channel has high specific surface area and larger depth, which is helpful for obtaining higher light absorption signal and can improve the sensitivity of the related detection method. To sum up, use vertical channel array microchip as immunoassay's reaction platform to intelligent mobile device image device is portable signal acquisition instrument, and the immunoassay platform that the two combined formation has advantages such as low cost, detects fast, portablely, possess fine using value.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides an immunodetection device and method based on a visible light signal image, which are used for on-site instant immunodetection. The technology combines the advantages of the microfluidic chip and the intelligent mobile devices, realizes the detection marker with multiple channels, low cost and high accuracy, and can be widely applied to the fields of clinical diagnosis, life science, food safety, environmental monitoring and the like.
The specific technical scheme is as follows:
the vertical channel array microchip and the imaging device for immunodetection comprise the vertical channel array microchip, a self-made flat light source and an intelligent mobile device imaging device, wherein the vertical channel array microchip is divided into three layers, the first layer comprises a sample adding hole and a sample adding channel, the second layer comprises a vertical channel, the third layer comprises a cleaning channel and a cleaning hole, and an antibody is fixed in each vertical channel through physical adsorption; the intelligent mobile device imaging device comprises a self-made flat light source, a shading box and an intelligent mobile device.
Furthermore, the vertical channel array microchip is made of Polycarbonate (PC), and other high polymer materials such as PDMS, COC and PS can be used as chip materials.
Further, each well of the vertical channel array microchip contains no more than 1 μ L of solution.
Further, the vertical channel array microchip in the vertical channel through physical adsorption fixed protein. The surface of the chip can also be modified by chemical reagents to covalently fix proteins.
Further, the manufacturing method of the vertical channel array microchip comprises the steps of designing a pattern, carving channels and holes and sealing chips.
Further, the self-made flat light source comprises an LED lamp, a 3D printing base and a shell; the light shading box covers the flat light source to form an imaging device, the intelligent mobile device takes pictures, the images are shared to the computer, the computer processes image data, and a result is output; the shooting box can be printed into any shape by a 3D printer and is used for shielding an external light source and fixing the intelligent mobile device.
Furthermore, the intelligent mobile device is any intelligent mobile phone, tablet computer and camera capable of shooting images, and the recommended camera pixel for shooting is not less than 800 thousands.
The immunodetection method on the visible light signal imaging device comprises the following steps:
(1) chip manufacturing: and engraving corresponding channels on the three PC substrates by a numerical control engraving machine, and manufacturing the three substrates into a chip by a thermal sealing method.
(2) Immunoassay: immune sandwich assay: adsorbing the antibody in a vertical channel, adding the antigen for reaction, then adding a biotinylated recognition antibody, and then adding streptavidin-labeled horseradish peroxidase.
(3) Color development: horseradish peroxidase substrate was added to the vertical channel for color development.
(4) And (4) recording the result: the vertical channel array microchip is placed on a self-made visible light signal imaging device, an external light source is shielded by a shooting box, and the intelligent mobile device is fixed above the shooting box and used for shooting and recording the result obtained by the color reaction.
(5) And (4) processing a result: and sharing the image to a computer, converting the color presented by the vertical channel into gray scale through image processing software, reading the gray scale value, and performing data analysis and result output.
(6) And (4) analyzing results: and analyzing the relation between the gray value and the concentration, making a correction curve, and finally obtaining a detection limit.
Further, the catalytic substrate is TMB or DAB and the like.
Further, the image processing software is ImageJ or photoshop, etc.
Compared with the prior art, the invention has the beneficial effects that:
the invention combines the advantages of the vertical channel array microchip technology and the intelligent mobile device, utilizes the 3D printing technology, has the advantages of low cost, quick detection, simple and convenient operation, good portability and the like, can realize on-site instant detection, and can be widely applied to the fields of clinical diagnosis, food safety, environmental detection and the like.
Drawings
FIG. 1 is a schematic diagram of a vertical channel array microchip prepared by the method of example 1.
Fig. 2 is a schematic diagram of the imaging apparatus using visible light signals according to embodiment 2.
FIG. 3 is a real object diagram and a result diagram of the detection result of example 3 on HIV p24 antigen using the method and apparatus, wherein A is the real object diagram and B is the result diagram.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
Example 1: construction of vertical channel array microchips
As shown in fig. 1, the vertical channel array microchip is made of PC, different channels are carved on three substrates by a carving machine, and the first layer is a sample adding hole 1 and a sample adding channel 2; the middle layer is a vertical channel 3; the third layer is a cleaning channel 4 and a cleaning hole 5, and then the third layer is sealed in an oven into a chip by using a heat sealing method, as shown in fig. 1, the chip has the advantages of low price and easier adoption of physical adsorption for fixing protein compared with other chips.
Example 2: construction of visible light signal imaging device
As shown in fig. 2, the visible light signal imaging device includes a homemade flat light source 6, a shooting box 7 and an intelligent mobile device 8, wherein the flat light source 6 includes an LED lamp, a 3D printed housing, a power supply and a light adjusting device, and can provide a uniform and stable light source for shooting; the shooting box 7 is a 3D printed shell with a specific shape, covers a flat light source when in use, shields an external light source, and is fixed with the intelligent shooting equipment 8 at the upper side to obtain a clear picture; when the intelligent mobile device is used, the shooting box is opened, the vertical channel array microchip 9 is placed, the chip is opposite to the small hole above the shooting box, the intelligent mobile device 8 is placed on the fixing groove of the shooting box 7, and the camera shoots the microchip through the small hole; the pictures are shared to the computer and then processed by software.
Taking a smart phone as an example of a shooting device, firstly measuring the size of the smart phone and the optimal shooting distance of the smart phone, drawing a three-dimensional model capable of fixing the smart phone by using 3D (three-dimensional) drawing software, and printing the model by using a 3D printer to be used as a shooting box; then, the smart phone is fixed on the flat light source in a buckling mode, the chip is placed on the flat light source of the shooting box, the flat light source is adjusted to be proper in brightness, and then a camera of the smart phone is used for shooting to obtain a picture with color information.
Taking Image software as an example, opening a picture with color information through the Image software, removing background and color conversion through the software, selecting a color information area by utilizing the shape of the software, reading the gray value of the area, reading all the gray values and then exporting data; and analyzing and plotting the derived data to obtain the result.
Example 3:
in the example, the detection object is HIV p24 antigen, and the p24 antigen is detected by using the detection technology to obtain the effect result between the HIV p24 antigen and the antibody fixed on the vertical channel microarray.
Specifically, the results were processed by software by visualization as shown in fig. 3A to obtain a signal-concentration log plot as shown in fig. 3B, which was determined in 100% human serum.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Claims (10)
1. The vertical channel array microchip and the imaging device are used for immunodetection and are characterized by comprising a vertical channel array microchip, a self-made flat light source and an intelligent mobile device imaging device, wherein the vertical channel array microchip is divided into three layers, the first layer comprises a sample adding hole and a sample adding channel, the second layer comprises a vertical channel, the third layer comprises a cleaning channel and a cleaning hole, and an antibody is fixed in each vertical channel through physical adsorption; the intelligent mobile device imaging device comprises a self-made flat light source, a detection box and an intelligent mobile device.
2. The vertical channel array microchip and imaging device for immunoassay of claim 1, wherein the vertical channel array microchip is made of polycarbonate.
3. The vertical channel array microchip and imaging device for immunoassay of claim 1, wherein each microwell of the vertical channel array microchip contains only no more than 1 μ L of solution.
4. The vertical channel array microchip and imaging device for immunoassay of claim 1, wherein the vertical channel of the vertical channel array microchip is immobilized with a protein by physical adsorption.
5. The vertical channel array microchip and imaging device for immunoassay of claim 1, wherein the vertical channel array microchip manufacturing method comprises carving of vertical channel, sealing of three layers of chips, and fixing of protein.
6. The vertical channel array microchip and imaging device for immunodetection of claim 1, wherein the homemade flat panel light source is made of LED lights, 3D printed housing; the shooting box covers the flat light source to form a shooting device, the intelligent mobile device shoots, the image is shared to the computer, the computer processes the image data, and the result is output; the shooting box can be printed into any shape by a 3D printer and is used for shielding an external light source and fixing the intelligent mobile device.
7. The vertical channel array microchip and imaging device for immunodetection of claim 1, wherein the smart mobile device is a smart phone, a tablet computer and a camera capable of taking images, and the recommended number of camera pixels for taking is not less than 800 ten thousand.
8. A method of operating the vertical channel array microchip and imaging device for immunoassay of claim 1, comprising: the method comprises the following steps:
(1) chip manufacturing: carving different channels on the three polycarbonate substrates by a carving machine, and then manufacturing the three substrates into a chip by a heat sealing method;
(2) color development: detecting antigen by a sandwich immunoassay method, then combining with horseradish peroxidase, and developing by a catalytic substrate;
(3) and (4) recording the result: the vertical channel array microchip is placed on a self-made flat light source of the photographing device, an external light source is shielded by a photographing box, and the intelligent mobile device is fixed on the photographing box and used for photographing and recording a result obtained by the color reaction;
(4) and (4) processing a result: sharing the image to a computer, converting the color presented by the vertical channel into gray scale through image processing software, reading the gray scale value, and performing data analysis and result output;
(5) and (4) analyzing results: and analyzing the relation between the gray value and the concentration, making a correction curve, and finally obtaining the detection limit.
9. The method of claim 8, wherein the vertical channel array microchip and imaging device comprises: the catalytic substrate is TMB or DAB.
10. The method of claim 8, wherein the vertical channel array microchip and imaging device comprises: the image processing software is ImageJ or photoshop.
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Cited By (1)
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| CN112240884A (en) * | 2020-09-30 | 2021-01-19 | 兰州大学 | Enzyme activity detection device and detection method based on smart phone and microchip |
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