CN102478582A - Multi-channel micro-fluidic chip for simultaneously detecting various subtype swine influenza viruses - Google Patents

Abstract

The present invention relates to the field of analysis and testing, and in particular to a multi-channel microfluidic chip for simultaneously detecting multiple subtypes of swine influenza virus (SIV). Rapid and inexpensive diagnosis of SIV is one of the goals of the advancement of related diagnostic and treatment technologies, and the present invention provides a diagnostic device directed to the above goal. The key points of this case are that the device, i.e., the microfluidic chip, contains a pipeline in a parallel structure, and the parallel structure contains three mutually parallel branch pipelines. There are three beaded working electrodes respectively installed in the three branch pipelines, and the beaded working electrodes are composed of a conductive electrode and a gold colloid sensitive film attached to the conductive electrode and embedded with different subtypes of SIV specific _antibodies . The gold colloid sensitive films on the surface of the three beaded working electrodes are respectively embedded with three different subtypes _of SIV specific antibody substances, _and the three different subtypes of SIV specific antibody substances are subtypes _H1N1 , _H3N2 and _H1N2 .

Description

Multi-channel microfluidic chip for simultaneous detection of multiple subtypes of swine influenza virus

technical field

The invention relates to a multi-channel microfluidic chip for simultaneous detection of multiple subtypes of swine influenza virus. The swine influenza virus diagnostic device is a special microfluidic chip for diagnosing swine influenza virus antigens based on antigen/antibody specific reactions, belonging to Analyze test areas.

Background technique

Swine influenza virus (Swine influenza vires, SIV) belongs to Orthomyxoviridae Influenza virus A (Influenza virus A), is a single-stranded negative-sense RNA virus, and its genome consists of 8 independent segments of varying sizes. The subtypes of influenza A are classified according to the difference of hemagglutinin and neuraminidase, which can be divided into 15 different H antigen subtypes and 9 different N antigen subtypes. Currently, there are three main subtypes of SIV serotypes that are popular in the world: H ₁ N ₁ , H ₃ N ₂ and H ₁ N ₂ . The recent global outbreak of influenza A H ₁ N ₁ mainly in Mexico and the United States has strengthened the research and prevention of SIV. Therefore, it is very urgent to develop a method for rapid diagnosis and diagnosis of SIV, which has far-reaching significance in reducing world economic losses and improving human health.

At present, the traditional swine flu diagnosis methods can be roughly divided into four types: one is virus isolation, which is regarded as the standard of laboratory diagnosis, but the disadvantage is that it takes a long time and can only be carried out in specialized laboratories. It is often impossible to provide evidence for timely prevention and control measures; the second is to detect viral nucleic acid, the most commonly used is reverse transcription PCR. If multiple pairs of specific primers are used for multiple PCRs, different subtypes of SIV can be detected simultaneously, but there are many influencing factors. The operation is complicated and time-consuming, and requires professional and technical personnel to operate; the third is to detect viral proteins, such as fluorescent antibody methods and immunohistochemical methods. Although these methods can meet the needs of rapid diagnosis, they have low sensitivity and low specificity. Strong; the fourth is to use immunological methods to detect specific antibodies in serum, such as enzyme-linked immunosorbent assay (ELISA); although it has high sensitivity and specificity, there are also a small number of false negatives and false positives.

Electrochemical immunosensor has unique advantages such as high degree of automation, low cost, easy preparation, fast sensitivity, etc., combined with the high efficiency and specificity of immune response, it is very suitable for the construction of an analyzer for multiple immune analysis and detection of major infectious diseases. The microfluidic chip (lab-on-chip), known as "life science integrated circuit", is an ideal technology for constructing miniaturized and integrated electrochemical immunosensors. Microfluidic chip technology is a cutting-edge technology that integrates sampling, pretreatment, reagent addition, reaction, separation, and detection on a single microchip. It has fast analysis speed, large amount of information, less reagent consumption, less pollution, It has the advantages of low operating cost and easy operation of the instrument. And it is very suitable for industrialized production. Microfluidic technology represents the development direction of miniaturization and integration of analytical instruments in the 21st century. The construction of a microfluidic amperometric detection chip suitable for the detection of major infectious diseases has the following advantages: (1) It is easy to integrate multiple detection channels and detection electrodes on the chip through micro-processing technology, so as to achieve simultaneous analysis of multiple targets, technical feasibility high. (2) Through the micro-fluid injection technology, the sample flows in the pipeline to ensure that the surface of the electrode is constantly updated, which overcomes the problems that the electrode is easy to be polluted, resulting in high false positive rate and poor parallelism of results. (3) The diameter of the pipeline on the microfluidic sensor is only at the μm level, and the volume of the reaction cell is only at the μL level. The amount of reagents required for analysis is very small, and the diffusion distance of the analyte to the electrode surface is short, so the incubation time can be greatly reduced. Achieve the goal of rapid joint inspection. (4) At present, most microfluidic sensors are based on optical detection systems. The instruments are expensive and bulky, and full integration is difficult. Moreover, the detection cost is high, which limits its promotion. The sensor adopts electrochemical detection, which is small in size, high in automation and low in cost.

At present, in the field of microfluidic chip technology, related technologies and methods for simultaneously detecting and rapidly diagnosing multiple subtypes of SIV antigens by using specific antibodies of multiple subtypes of swine influenza virus (SIV) have not been reported.

Contents of the invention

The technical problem to be solved by the present invention is to develop a specific antibody that can use multiple subtypes of SIV to simultaneously detect and rapidly diagnose corresponding SIV antigens within the general technical framework of microfluidic chip technology. dedicated microfluidic chip.

The present invention solves the technical problem through the following solution. The device provided by the solution is a multi-channel microfluidic chip for simultaneously detecting multiple subtypes of SIV. The structure of the microfluidic chip includes two A plate-shaped object, the two plates are the cover sheet of the microfluidic chip and the substrate of the microfluidic chip, and a pipeline is installed at the position where the two plates are attached to each other. , and three pools, one end of the pipeline communicates with two of the pools through a manifold-shaped fluid channel, and the other end of the pipeline communicates with the remaining pool, and is respectively installed in each of the pools in sequence. The working electrode, the counter electrode and the reference electrode at different positions in the pipeline, the working electrode is composed of a conductive electrode and a gold colloid sensitive film embedded in the SIV specific antibody attached to the conductive electrode, The special feature of this case is that the structure of the pipeline is a parallel structure, and the pipeline in the parallel structure is composed of three branch pipelines connected in parallel, and the number of the working electrodes is three, and the installation positions of the three working electrodes are: They are respectively located in the three branch pipelines, and the specific antibodies in the gold colloid sensitive membrane structure on the surface of the three working electrodes are three different subtypes of SIV antibody substances that can specifically bind to different subtypes of SIV antigens, The specific antibody substances of the three different subtypes are respectively H ₁ N ₁ , H ₃ N ₂ and H ₁ N ₂ , and the shape of each working electrode is beaded.

The gold colloid sensitive film is formed by fully mixing the chitosan gold colloid solution and the SIV specific antibody solution evenly, and drying them to form a film. The different subtypes of SIV in the gold colloid sensitive membrane are all horseradish peroxidase or glucose oxidase-labeled SIV antibodies, and the gold colloid sensitive membrane has been introduced into it for immobilizing each of the above SIV specific antibodies. Auxiliary medium, said auxiliary medium such as chitosan, cellulose acetate, gelatin one or their mixture.

The internal diameters of the pipelines, the branch pipelines and the manifold-shaped fluid channels in the microfluidic chip structure can be any selected size, but, for the sake of using as little liquid samples as possible and reducing the Considering the loss of reagents, etc., the pipeline, the branch pipeline and the manifold-like fluid channel are preferably capillary-level channels. The inner diameter is equivalent. The cross-sectional shape of the internal channel of the capillary can be any shape, such as a circle, an ellipse, a square, a rectangle, a bar, and of course any curved line, and the The internal shape of the capillary can also be different from the cross-sectional shape of different parts along with the extension of the pipeline. As far as the term capillary is concerned, its technical meaning is known.

The counter electrode and reference electrode involved in the structure are all micro-sized electrodes, and the shape of the electrode can be any selected shape, such as a square sheet shape, a rectangular sheet shape, a strip shape or a circular shape. Flaky and more.

The working electrode in the chip in this case is beaded, and its beaded electrode shape helps to greatly expand its effective working interface, and, further, the beaded shape also makes the working electrode have good flow disturbance, which Helps to enhance contact between the working electrode and the analyte flow around it.

The structure of the microfluidic chip in this case involves several pools. The pools are pool-shaped or capsule-shaped structures used for transitional liquid storage, and the shape of the inner cavity of each pool can be any shape. The selected shape, the shape of the inner cavity is, for example, a cylindrical cavity, a square column cavity, an elliptical cavity or a spherical cavity and the like. The size of the pool can be arbitrarily selected, but in order to minimize the use of liquid samples to be tested and reduce reagent loss, the pool is preferably a miniature pool that can match the capillary .

Of course, the device of this case may further include some accessories, such as a multi-channel electrochemical workstation and a micro-flow pump, etc. The technical meaning of the multi-channel electrochemical workstation and the micro-flow pump are well known. Each beaded working electrode, counter electrode, and reference electrode involved in the structure of the microfluidic chip in this case can be connected to the corresponding interfaces of the multi-channel electrochemical workstation via corresponding dedicated serial lines. The special serial line is a special cable for interconnecting each electrode and each corresponding interface of the multi-channel electrochemical workstation. The micro-flow pump is dedicated to driving a small amount of liquid flow, and the micro-flow pump can communicate with any one of the pools selected as required.

The working electrode whose appearance is beaded can be either an integrated press-molded beaded conductive electrode or a casted beaded conductive electrode; the structure of the working electrode can of course allow conductivity In this case, the material of the beaded working electrode is ferric oxide material with conductivity, and the beaded working electrode is composed of many It is a quasi-one-dimensional conductive electrode formed by splicing magnetic beads made of ferroferric oxide under the guidance of an external magnetic field.

The working electrode with a beaded appearance, that is, the working electrode of the magnetic bead string structure, the diameter of the magnetic beads in the structure is not limited, and the diameter of the magnetic beads can be any size set according to needs, but , the preferred diameter range of the magnetic beads is between 20 nanometers and 2000 nanometers, and the number of magnetic beads that are spliced together in the structure of each of the working electrodes can be set according to needs The number is arbitrary, and the number is not limited. However, the preferred value of the number of magnetic beads constituting one working electrode is between 100 and 100,000.

In view of the small width of the space occupied by each beaded working electrode, the working electrode can be directly placed in the capillary channel, and the relatively narrow fluid channel is more conducive to the disturbance of the beaded electrode to the surrounding flowing liquid capabilities, and is conducive to realizing the full potential of its effective working interface.

The gold colloid sensitive film installed on the surface layer of the working electrode is coated by spraying or sample spotting or other suitable processes, and its film thickness can be allowed to be arbitrarily set to generate electricity for the sample solution to be tested. The thickness of the signal response, however, is recommended or preferred thickness is between 10nm and 200nm.

The material of the cover sheet and the substrate in the chip structure can be any electrically insulating material, such as polypropylene, glass, etc., and the preferred material is polymethyl methacrylate.

The width of the cover sheet and base sheet in the structure can be allowed to be any set width value that can reach the relevant test purpose, but the preferred width value is between 2 cm and 4 cm; The length values of the cover sheet and the base sheet can also be arbitrarily set to achieve the relevant test purpose. However, the preferred length value or the recommended length value is between 3 cm and 6 cm.

The thickness of the cover sheet and the base sheet can be set arbitrarily to facilitate device assembly, and the recommended or preferred thickness is between 0.5 mm and 5 mm. Smaller thickness is beneficial to save material.

The method of using the microfluidic chip in this case:

An external micropump is used to drive the liquid to flow stably in the capillary channel of the three-channel microfluidic chip, and three-channel electrochemical analysis instruments are used to detect different subtypes of SIV diagnostic antigens.

The specific detection steps of the microfluidic chip in this case are as follows:

1. Serum sample solution is added to the micropipeline, driven by an external micropump, various subtypes of SIV antigen molecules are labeled by the corresponding horseradish peroxidase embedded in the gold glue sensitive membrane on the electrode surface in each channel SIV-specific antibody capture.

2. The SIV-specific antibody labeled with horseradish peroxidase forms an immune complex with the corresponding SIV antigen in the serum sample.

3. Use a multi-channel electrochemical analyzer, add catechol and other electronic mediators, and use the amperometric method to detect the current changes caused by the above reactions, thereby obtaining the types and contents of various analytes.

4. Comprehensively analyze the results to diagnose different subtypes of SIV antigens.

The advantage of the present invention is that three beaded working electrodes coated with three different subtypes of swine influenza virus-specific antibody substances are integrated on one physical device, that is, a microfluidic chip, and the three beaded working electrodes are respectively For the detection of characteristic antigens of three different subtypes of SIV, the microfluidic chip in this case is a microfluidic chip that can use multiple different subtypes of SIV-specific antibodies for simultaneous detection. The structural characteristics of its integrated structure and specific The morphology of the beaded working electrode is helpful to improve the diagnostic efficiency and reduce the diagnostic cost of swine influenza virus.

Description of drawings

Figure 1 is a schematic diagram of the structure of the embodiment of the microfluidic chip in this case. What is shown is the perspective form of the structure of this example. The detailed morphology of the electrode is enlarged and displayed.

In the figure, 1, 2, and 8 are three pools with different installation locations, 3 is a manifold-like fluid channel, and 4, 9, and 12 are three branch pipes with different installation locations but connected in parallel to form a parallel communication structure. , 5, the specific antibody substance in its surface gold colloid sensitive membrane structure installed in the branch pipeline 4 is the beaded working electrode of subtype H ₁ N ₁ swine influenza virus specific antibody, and 10 is installed in the branch pipeline The specific antibody substance in its surface gold colloid sensitive membrane structure in 9 is the beaded working electrode of the subtype _{H3N2 swine} influenza virus specific antibody, and 11 is its surface gold colloid sensitive electrode installed in the branch pipeline 12. The specific antibody substance in _the membrane structure is the beaded working electrode of the subtype _H1N2 swine influenza virus specific antibody, 6 is the counter electrode, and 7 is the reference electrode.

Detailed ways

In the embodiment of this case shown in Figure 1, the structure of the device, that is, the microfluidic chip, includes two plates that are mounted together, and the two plates are the cover sheet of the microfluidic chip and the microfluidic chip. The substrate of the microfluidic chip is provided with pipes at the position where the two plate-shaped objects are attached to each other, and three pools, the three pools are pool 1, pool 2 and pool 8, one end of the pipeline communicates with pool 1 and pool 2 via manifold fluid channel 3, and the other end of the pipeline communicates with the remaining pool 10, and, The beaded working electrode, the counter electrode 6 and the reference electrode 7 are installed in different positions in the pipeline in sequence, and the beaded working electrode is embedded by the conductive electrode and the electrode attached to the conductive electrode. The gold colloid sensitive membrane of swine influenza virus specific antibody antibody is composed, and the structure of the pipeline is in parallel structure, and the pipeline in parallel structure is composed of three branch pipelines connected in parallel, and the three branch pipelines are respectively branch pipeline 4 and branch pipeline 9 and Branch pipeline 12, and the number of beaded working electrodes is three, the three beaded working electrodes are respectively beaded working electrode 5, beaded working electrode 10 and beaded working electrode 11, wherein beaded working electrode 5 is The specific antibody substance in the surface gold colloid sensitive film structure installed in _the branch pipeline 4 is the beaded working electrode of swine influenza virus specific antibody _H1N1 , and the beaded working electrode 10 is installed in the branch pipeline 9 The specific antibody substance in its surface gold gel sensitive membrane structure is the beaded working electrode of swine influenza virus specific antibody H ₃ N ₂ , and the beaded working electrode 11 is the surface gold gel installed in the branch pipeline 12 The specific antibody substance in the sensitive membrane _structure is the beaded working electrode of swine influenza virus specific antibody _H1N2 . Fig. 1 does not draw accessories such as micro-flow pumps and multi-channel electrochemical workstations as accessories. Each pool in the structure of this example can communicate with the micro flow pump as an accessory in any way as required. Each bead-shaped working electrode, counter electrode and reference electrode in the structure of this example can be respectively connected to the corresponding cable interface or serial interface of the multi-channel electrochemical workstation as an accessory via their own dedicated cables or serial lines. It seems necessary to make a little additional explanation here: Antibodies and antigens are related but different substances. The relationship between them, in a popular metaphor, is like the relationship between the lock and the key in a set of locks .

Claims (8)

1. (structure of this micro-fluidic chip comprises that applying is installed in two plate objects together for Swineinfluenza vires, multichannel micro-fluidic chip SIV) to detect multiple hypotype swine influenza virus simultaneously; Said two plate objects are respectively the cover plate of micro-fluidic chip and the substrate of micro-fluidic chip; On the position of the mutual applying between said two plate objects, be equiped with pipeline, and, three pond shape things; One end of pipeline via the manifold shape fluid passage respectively with wherein two pond shape thing UNICOMs; The other end of pipeline and a remaining pond shape thing UNICOM, and, be installed in the said pipeline working electrode on the diverse location in regular turn respectively and to electrode and contrast electrode; Said working electrode by conductive electrode and be attached to embedding on the said conductive electrode gold size sensitive membrane of different subtype swine influenza virus antibody constitute; It is characterized in that the structure of said pipeline is the parallel connection structure, the said pipeline that is the parallel connection structure is made up of three lateral parallel connections; And; The quantity of said working electrode is three, and the installation position of these three working electrodes lays respectively in said three laterals, and; Specific antibody in its top layer gold size sensitive membrane structure of these three working electrodes is respectively three kinds of corresponding swine influenza virus antibody materials that different subtype swine influenza virus antigen ability specificity is combined, and these three kinds of antibody materials are respectively swine influenza virus specific antibody H ₁N ₁, H ₃N ₂And H ₁N ₂, and its pattern of each working electrode all is beading.

2. the multichannel micro-fluidic chip that detects multiple hypotype swine influenza virus simultaneously according to claim 1 is characterized in that, said pipeline and said lateral and said manifold shape fluid passage are capillary channel.

3. the multichannel micro-fluidic chip that detects multiple hypotype swine influenza virus simultaneously according to claim 1; It is characterized in that; It is the tri-iron tetroxide material with conductive capability that said pattern is its material of catenate working electrode; And it is to add the accurate one-dimensional electric property electrode that is spliced each other under the magnetic field condition guiding by the magnetic bead of many tri-iron tetroxide materials that said pattern is catenate working electrode.

4. the multichannel micro-fluidic chip that detects multiple hypotype swine influenza virus simultaneously according to claim 3; It is characterized in that; The diameter of said magnetic bead is between 20 nanometer to 2000 nanometers; And the number of the magnetic bead that is stitched together each other that is contained in the structure of each said working electrode is between 100 to 100000.

5. the multichannel micro-fluidic chip that detects multiple hypotype swine influenza virus simultaneously according to claim 1 is characterized in that, the thickness of said gold size sensitive membrane is between 10 nanometers and 200 nanometers.

6. the multichannel micro-fluidic chip that detects multiple hypotype swine influenza virus simultaneously according to claim 1 is characterized in that, the said cover plate in the structure and its material of substrate are polymethylmethacrylate.

7. the multichannel micro-fluidic chip that detects multiple hypotype swine influenza virus simultaneously according to claim 1 is characterized in that, all between 2 centimetres and 4 centimetres, its length is all between 3 centimetres and 6 centimetres for the said cover plate in the structure and its width of substrate.

8. the multichannel micro-fluidic chip that detects multiple hypotype swine influenza virus simultaneously according to claim 1 is characterized in that, the said cover plate in the structure and its thickness of substrate are all between 0.5 millimeter and 5 millimeters.

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