TWI384218B - An improved biological chip macro channel divergent structure - Google Patents

Description

Improved biochip microchannel divergence structure

The present invention discloses an improved biochip microchannel divergence structure, in particular a biochip microchannel system comprising at least one main channel and two shunt channels, the two shunts being at an equal angle to the main channel The same end point is connected, and the intersection point of the main flow channel and the two branch flow paths is a divergent structure, and the divergent structure is a circular arc structure between each flow path.

According to traditional biomedical detection techniques, there are many methods for testing proteins, such as SDS gel electrophoresis, Western blot, immunoprecipitation, and enzyme-linked immunosorbent assay (enzyme). -link immunosorbent assay, hereinafter referred to as ELISA). Among them, ELISA is a very popular technique for immunoassay, and is widely used to detect and quantify chemical and biological molecules (especially antigens, mainly including proteins and polypeptide chains). Therefore, ELISA is a clinical test and food safety test. And applications such as environmental monitoring are becoming more and more important.

The principle of ELISA is to use the specificity of an antigen (antigen) to bind to an antibody, plus the coloration of an enzyme (or a fluorescent reaction) to show the presence or absence of a particular protein. Please refer to the ELISA step diagram as shown in the first figure. The basic methods and steps of ELISA are as follows: First, the primary antibody (antibody specific against a specific antigen) is fixedly attached to a detection slot. On the wall (step A02); then adding a masking agent (usually protein) (step A03) for binding to the wall of the cell not bound by the primary antibody to reduce non-specific binding of the antigen or the antibody A sample or standard containing the antigen is then added to the detection well (step A04), and reacted for a sufficient time (step A05) to cause the antigen to react with the primary antibody (ie, antigen-antibody binding); antigen and primary After the antibody has generated sufficient reaction, a first cleaning agent is added to wash away the excess sample (step A06); then a secondary antibody combined with a specific enzyme is added (step A07), and the secondary antibody and the antigen are also allowed to perform for a sufficient time. After the reaction (step A08), a second cleaning agent is added to wash off the excess secondary antibody (step A09); finally, a specific substrate (step A10) is added, and the substrate can be converted into an enzyme-catalyzed reaction on the secondary antibody. A detectable substance, such as a substance containing a specific color or fluorescence, can be used to determine the amount of antigen contained in the sample or the amount of antigen contained in the sample by detecting the yield of the substance (step A11).

The enzymes used in the ELISA include horseradish peroxidase, alkaline phosphatase, lysozyme, and glucose 6-phosphate dehydrogenase. These enzymes often use glutaldehyde or dimaleimide to attach them to antigens or antibody molecules. Their properties are chemically stable, soluble in water, and low in tissue, so as not to interfere with the results. ELISA is often used to detect antibodies against CEA (carcinoembryonic antigen), steroid hormones, immunoglobulin, DNA, bacteria, and viruses. Its sensitivity is high (ng/ml range), it is easy to operate, reagents are stable, the instrument is inexpensive, and it is not necessary to use The advantages of high-risk radioactive materials make it increasingly applicable in the future. In the ELISA experiments, the sensitivity of commonly used substrates ranged from low to high, respectively Colorimetric<Fluorescent<Chemiluminescent. The selection of substances depends on the enzyme reporter system; Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are examples of substances commonly used in immune reactions; and the two enzymes have various substances; and their molecular weight is small and can be quickly reacted. The signal is generated and the required price is low and the system is stable. The substrate currently used in ELISA is QuantaBlu, Fluorogenic, Luminol, and the required enzymes are HRP, PNPP, and Alkaline phosphatase (AP).

The clinical diagnostic applications of ELISA include: determination of German measles antibodies; detection of viral antibodies such as CMV, HSV, measles, mumps, VZV, etc. HIV antibody assay (Rapid ELAVIA, SERODIA-HIV) and hepatitis C virus antibody assay (hepatitis C virus can also be determined by PCR). The product is detected by enzyme-linked immunosorbent assay (ELISA), and the stool sample of the suspected infected food or food poisoning patient is cultured, and the strain is qualitatively and quantitatively detected by enzyme immunosorbent assay (ELISA). The product is suitable for use in sanitary units when testing in laboratory or confirming poisonous bacteria. Food microbiology test series, Listeria monocytogenes ELISA, Vibrio parahaemolyticus ELISA, Bacillus cereus ELISA. Plant virus diagnostic reagent; three kinds of virus diagnostic reagents such as yellowing inlay, phthalocyanine inlay and courgette inlay, using rabbit to produce virus antiserum, and separating it to make a virus diagnostic reagent, which is determined by the color of the diagnostic reagent. The virus is strong and weak. In addition, bacterial testing, Lyme disease, leptospirosis infection can also be detected by ELISA principle; rapid detection of cow's milk adulteration in goat milk, isolation and purification of casein from milk as antigen, used to immunize rabbits and goats immune rabbits After a large amount of blood was collected and the serum was collected, the serum white matter was precipitated with ammonium sulfate, and the bovine cheese was purified by a DEAE-Sephacel anion exchange column and a CNBr-activated Sepharose 4B affinity column coupled with goat casein. The protein has a highly specific antibody. The purified antibody was detected by an indirect enzyme-linked immunosorbent assay (indirect ELISA), and the result showed that the antibody has specificity and reactivity against bovine cheese protein, and has the ability to detect 2.5% milk incorporation in goat milk. That is, the purified antibody can be applied to the detection method of indirect ELISA to distinguish bovine and felin protein.

However, such an ELISA test procedure is very lengthy and it takes a lot of time to perform a test. In addition, ELISA assays are typically performed using 96-well plates, but often produce large amounts of error and experimental instability, and 96-well plates, antibodies, and agents are very expensive. Therefore, if the detection speed of the ELISA can be improved and the cost of the test can be reduced, there will be a great progress in the detection of biomedicine.

Then, referring to bio-micro-electromechanical technology, bio-micro-electromechanical technology is an emerging technology that emerged in the twentieth century. It is an advanced country, and all of them are listed as national key technologies and actively developed. In the first seven years of the Republic of China, the government promulgated the "Science and Technology Development Program", which clearly defines biotechnology as one of the eight key technologies. For more than a decade, there has been a considerable foundation. At present, in the areas of medical testing, agricultural food testing and environmental protection, there is an increasing demand for biochemical testing of different types of samples. However, based on the consumption of instruments, techniques, and pharmaceuticals, although the traditional analysis has good sensitivity, there are still limitations in the detection technology. For example, in medical testing, many subtle biological information cannot be further explored, so a new detection platform must be developed to reduce the problem of instrument and reagent consumption.

With the completion of the genetic map sequencing, the next step is to understand the meaning and interrelationship of tens of thousands of genes, as well as related issues such as protein tissue function research and drug development, so to improve the efficiency of analyzing genes or detection by new methods. Is a very important study.

Research shows that the instrumentation equipment can be miniaturized, which can increase the analysis speed and simplify the operation procedure. Therefore, the micro-electromechanical system process technology can be used to miniaturize the traditional large-scale instruments on a single or multiple wafers, which can greatly reduce the detection. Cost, increase the speed of detection and analysis, and also greatly increase the sensitivity of detection. The biochips that will be developed now need to miniaturize all the testing instruments. This will not only reduce the problem of instrument and reagent consumption, but also speed up the detection, shortening the detection time in the past few days to 1 to 2 hours, even in It can be done in a few minutes.

On the other hand, using micro-electromechanical system process technology to make micro-components, in addition to its advantages of lightness, thinness and shortness, its high frequency response and good spatial resolution make it more widely used and have the potential for parallel arraying. The goal of accomplishing multiple inspections and analyses on a single wafer has been achieved.

Microelectromechanical systems that have been combined with biomedical detection technologies include: micro-electro-mechanical-systems (MEMS), micro-Optical-Electro-Mechanical-Systems (MOEME), and biological Wafer system (Bio-micro-Electro-Mechanical-Systems, BioMEMS) and the like. A micro-electromechanical system that utilizes semiconductor process technology and integrates electronic and mechanical functions to create a miniature device; it is defined as a smart, miniaturized system that contains sensing, processing, or actuation functions, including two or more electrons. , mechanical, optical, chemical, biological, magnetic or other properties integrated into a single or multiple wafers; for optical MEMS electro-mechanical systems, the most common component is a micro-mirror, which uses a driven micro-mirror to make the beam Deflection, to achieve the purpose of modulating the beam. The general micromirrors are manufactured by the surface micromachining technology with flexible, easy and microelectronic circuit integration, but studies have shown that the inherent properties of thin film deposition - residual stress, will bend the micro mirror .

Bioarray System Microarray is one of the most mature biochip technologies in development and application. This wafer technology has been widely used in biomedical fields such as cancer, pharmacology, toxicology, infection, cell differentiation, development and Reproductive medicine, etc., will also be directly or indirectly applied to the diagnosis, classification, prognosis assessment of diseases, and to improve the treatment of diseases. The plant genome project has completed the model plant Arabidopsis thaliana genome in 2000, and completed the economic crop rice (Oryza sativa L. ssp. Indica) genome project in 2002, using biochip technology. The research and application in agriculture is still relatively lacking. The scale of research and the number of genes to be explored are also smaller and less than those in the biomedical field.

Biochips are used in a wide range of applications, such as cell biochemistry research and disease diagnosis (eg clinical diagnosis of cancer oncology or diagnosis of various viral infections, such as AIDS and enterovirus diagnosis). In order to get a quick and correct diagnosis, a lot of manpower and material resources can be saved, and the patient can be rescued at the first time point in order to achieve early detection and early treatment. In addition to DNA, proteins and receptors of drugs in some cells can also be placed on the wafer, so these wafers are collectively referred to as biochips.

Biochips contain at least ten different uses:

(1) Gene expression profiling: Some diseases usually involve changes in most genes, and in order to understand the difference in protein synthesis between patients and normal humans, it is necessary to observe these at different time points. The performance of most genes. And through these forms of gene expression at many points in time, researchers can learn how our complex human body produces different types of proteins. Biochips for this purpose are useful for current-like oscilloscopes. In order to complete the blueprint for this gene expression, researchers must prepare many fresh cell samples, so this type of research requires the use of very high-density DNA wafers, similar to the Affymetrix Gene Chip system.

(2) Toxicology Analysis: DNA wafers can also be used to detect the performance of organic viruses on certain genes, such as those associated with liver toxicity. Affymetrix spokesperson also stated that the wafers used in this area have become a new focus for their company. They have assembled a number of experts to collect genes that are most likely to represent the toxins of certain human organs, in order to quickly analyze the effects of some toxic substances on the human body.

(3) Gene Sequencing: One day the wafer can be used to do a lot of work on gene sequencing and discovery. Hyseq is the first company to apply DNA chips to genetic sequencing. The principle is to place all possible ribonucleic acid arrangements on the wafer, and then place the unknown genes on the wafer. Only the probes with the exact same order can be complemented, so that the order of the unknown genes is known. But the biggest problem is the length of the order. According to common sense, it is necessary to determine four mers (A, T, C, G), and two mers are 16 combinations, and so on to determine five mers requires 45 (ie 1024) combinations. Probe. Hyseq's strategy is to use a two-step strategy in which a long, unknown piece of DNA is first placed on the first DNA wafer, and the complement of the five mers is first identified and then exposed to the second set. The solution of the five mers will be complementary to other DNA fragments that are not complementary, and then a computer program will be used to construct a long series of DNA sequences for a segment of 10mer.

(4) SNP identification of single ribonucleic acid: It is necessary to find the genotype of an individual in order to know that individual polymorphisms are the target of many wafer companies. But in order to do this type of work, the first step is to do a lot of work on genetic sequencing. So using the DNA sequencing wafer developed by Hyseq, we are building our own database of genetic polymorphisms. The company is also working with the University of California, San Francisco to target individuals with cardiovascular disease. They expect to bring this wafer for polymorphism determination of single RNA to market within one or two years.

(5) Forensics: As DNA chips are fast, accurate and easy to carry, they may become one of the tools for forensic on-site handling in the near future.

(6) Immunoassay (Immunoassays): Some wafer companies have developed technologies that can place things other than DNA on the wafer, for example, by using tight binding between antigens and antibodies, in order to analyze some immune reactions. . It is currently known that the aforementioned Illumina company and Nanogen Company have a high degree of interest in the application of this product.

(7) Protein Chips: Ciphergen is using the protein wafers they have developed to perform a wide range of protein biology research.

(8) Combat Biowarfare: In the past few years, the US Department of Defense has provided millions of dollars to some biotech companies, hoping to find some tools to deal with biological weapons. But the first thing that must be done is how to detect and verify it. Therefore, Nanogen has received more than seven million US dollars from the Ministry of Defense to develop a portable system (such as DNA chips), in order to quickly and accurately detect harmful biological weapons on the battlefield.

(9) Drug screening: Illumina believes that the combination between the drug and its receptor can also be applied to the wafer, similar to the close combination between DNA and complementary probes. After the introduction of this kind of wafer, it is expected to save a lot of time and money for saving drug screening.

(10) Hard drives and microprocessors: Nanotronics, a subsidiary of Nanogen, already owns the patent to apply this DNA wafer technology to computers. Because of the use of DNA self-assembly (Self-Assembles), it is a computer-like language. Therefore, the synthesized fragment DNA can be bound to some non-biological substances, such as some light sources or some microelectronic parts, in order to take advantage of this property of DNA and bring these substances to specific locations to achieve the goal.

With the rapid advancement and widespread use of biochips, the previously mentioned shortcomings of ELISA can be solved. A variety of biochip-based ELISA assays have been developed. The principle is to have a microchannel system on the biochip to achieve the ELISA test procedure. The biochip microchannel system can be used for ELISA. Reduce the time of each test.

Among them, BioLOC has combined the micro-channel system with centrifugal fluid technology to develop a test platform and method called Compact-Disk Based ELISA (hereinafter referred to as CD-ELISA). The platform uses a CD-shaped substrate as a tool for inspection, and micro-channel systems, storage tanks, and valves are fabricated on the substrate. Multiple sets of experiments can be performed in parallel and simultaneously using the CD-ELISA platform, and have higher precision than conventional ELISA. At present, CD-ELISA can simultaneously perform 24 sets of samples on one CD substrate. The platform incorporates several microchannel functions, including pump and capillary valve control, and mixes or reacts with antibodies, reagents, and buffers in different reservoirs via the microchannel system. By applying a centrifugal force to the substrate, the centrifugal force overcomes the capillary resistance possessed by each valve, allowing liquid in the reservoir to pass through the valve into the microchannel system. The order of each liquid entering the microchannel system can be arranged according to the traditional steps of the ELISA. When each liquid sequentially enters the microchannel system and interacts with the sample, the steps of the ELISA can be achieved.

Although the CD-ELISA platform can achieve rapid detection at a lower cost, there are still some problems to be overcome and solved. For example, the traditional ELISA test usually performs two to three repeated tests on one sample. Although the CD-ELISA can add the reaction detection area of each sample to achieve the purpose of repeated experiments, the sample or other liquid is split into multiple numbers. Before the detection zone, it must flow through a branch point. Because the flow channel itself also has surface tension, the fluid will flow into the detection zone on the other side because the edge first reaches the pressure point at the branching point, so that the other side of the flow channel Unable to break through the pressure and produce uneven fluid distribution, even if only one side has reagents flowing through it. In order to solve such bottlenecks, it is necessary to develop a solution to improve the accuracy and practicality of the CD-ELISA detection technology.

Therefore, in view of the aforementioned problems and deficiencies, the inventors have accumulated years of experience, and exerted imagination and creativity. After continuous trial and modification, there has been an improved biochip microchannel divergence structure of the present invention.

A first object of the present invention is to provide an improved biochip microchannel divergence structure, the microchannel system comprising at least one main channel and two shunts, the two shunts being at an equal angle to the main channel Connected to form a left-right symmetric structure with the main channel as the central axis. The angle between the two shunts and the main flow channel is equal, so that the flow rate of the liquid into the shunt is equal, so as to overcome the conventional CD-ELISA. The situation where the flow distribution is uneven.

A second object of the present invention is to provide an improved biochip microchannel divergence structure, the microchannel system having at least one main channel and two shunts, and the main channel and the two shunts are divergent The structure, the divergent structure is a circular arc-shaped structure, which can reduce the impact force generated by the liquid flowing through the divergent structure on the wall surface of the flow channel, so that the flow rate of the liquid into the branching channel is equal, Overcoming the conventional CD-ELISA is prone to uneven distribution of flow in divergent structures.

A third object of the present invention is to provide an improved biochip microchannel divergence structure, the microchannel system comprising at least one main channel, two shunt channels, the same local node region and two detection regions, and the sample node region is available. After injecting and storing the sample, the sample can flow through the main channel, the two shunt passages through the valve after applying the centrifugal force, and enter the two detection areas to perform the double-cover detection.

A fourth object of the present invention is to provide an improved biochip microchannel divergence structure, the microchannel system comprising at least one main channel, two shunt channels, the same local node region, a first cleaning agent node region, and an antibody a node area, a second cleaning agent node area, a substrate node area, two detection areas and at least one waste liquid storage area, wherein each node area can be respectively injected with the same, a first cleaning agent, an antibody, and a first The second cleaning agent and a substrate are respectively stored in the storage tanks of the respective node zones. After applying different centrifugal forces, the liquids can sequentially enter the main channel and the two bypass channels by overcoming the different resistances of the valves of the respective nodes. And performing the ELISA detection step in the two detection zones, and the liquid after the reaction finally flows into the waste liquid storage area for storage.

A fifth object of the present invention is to provide an improved biochip microchannel divergence structure, the microchannel system comprising at least one main flow channel, two primary flow channels, four secondary flow channels, two sample node regions, and one a first cleaning agent node region, an antibody node region, a second cleaning agent node region, a matrix node region, two detection regions, and at least one waste liquid storage region, wherein the two sample node regions can inject the same sample For the quadruple test, different samples may be separately injected to perform two replicates of the two samples simultaneously, and the other node regions may be separately injected with a first cleaning agent, an antibody, a second cleaning agent and a substrate, and Stored in the storage tanks of each node zone, respectively, after applying different centrifugal forces, the liquids can sequentially enter the main channel, the two primary shunts and the four secondary shunts by overcoming the different resistances of the valves of the respective nodes. The ELISA test step is performed in the four detection zones, and the liquid after the reaction finally flows into the waste storage area for storage.

The invention provides an improved biochip microchannel divergence structure, which is formed on a substrate to form a series of detection tank chambers, comprising: a main flow channel; two branch channels, the two branch channels are respectively at equal angles The angle is connected with the main channel, forming a left-right symmetric structure with the main channel as the central axis. The main channel and the two shunts are separated by a substructure, and the divergent structure is a circle between the two shunts. Curved structure; the same node area, the sample node area is connected to the opposite end of the main channel combined with the divergent structure end, the sample node area contains at least the same storage tank and the same node valve, the sample node valve is set in the sample storage tank Between the main channel and the main channel, the sample is randomly inserted into the main channel; and the two detection regions are respectively connected to the opposite ends of the two shunts and the divergent structure end; wherein the micro flow is A centrifugal force is applied to the sample storage tank of the channel system in the direction of the valve of the sample node. When the centrifugal force reaches a certain threshold value of the valve resistance, the sample storage tank is in the sample storage tank. Sample Sample node will break resistance of the valve of the valve flows into the sprue, runners and into two-branched structure via shunt average, and finally into the two sample detection zone is detected.

In order to achieve the aforementioned purposes and effects, the inventors combined the biochip microchannel system with ELISA technology and improved the structure of the microchannel system to obtain the improved biochip microchannel divergence structure of the present invention. The improved biochip microchannel system structure of a first preferred embodiment of the present invention, the improved biochip microchannel system structure of a second preferred embodiment, and the improvement of a third preferred embodiment The structure of the biochip microchannel system is described in detail in the system structure and implementation principle of the present invention.

Referring first to the second embodiment, the improved biochip microchannel system structure of the first preferred embodiment of the present invention is formed on a substrate 10 to form a series of detection chambers, including a main channel 100; two sub-channels 110a, 110b, which are connected to the main channel 100 at equal angles 106a, 106b, respectively, forming a main axis 100 as a central axis The left and right symmetrical structure, the main channel 100 and the two branch channels 110a, 110b are a divergent structure 105, the bifurcated structure 105 is a circular arc structure between the two branch channels 110; the same node area 120, the sample The node area 120 is connected to the opposite end of the main channel 100 and the end of the branch structure 105. The sample node area 120 includes at least the same injection hole 121, the same storage tank 122 and the same node valve 123. The sample injection hole 121 can be used. A specific sample is injected, and the injected sample can flow into the sample storage tank 122 via the sample injection hole 121. The sample node valve 123 is disposed between the sample storage tank 122 and the main flow channel 100 to block the sample flow. Into the main channel 100; and two detection areas 130a, 130b, the two detection areas 130a, 130b are respectively connected to the opposite ends of the two branch channels 110a, 110b combined with the end of the divergent structure 105; wherein, the micro flow can be A centrifugal force in the direction of the sample node valve 123 is applied to the sample storage tank 122 of the channel system. When the centrifugal force reaches a certain threshold value of the valve resistance, the sample in the sample storage tank 122 will break the valve resistance of the sample node valve 123. And flowing into the main channel 100, and equally splitting into the two shunts 110a, 110b via the divergent structure 105, the sample finally enters the two detecting areas 130a, 130b for detection.

Referring to the third embodiment, the improved biochip micro-channel system structure of the second preferred embodiment of the present invention is formed on a circular substrate 20 to form a series of detection chambers. The main channel 200 is radially disposed on the circular substrate 20, and the main channel 200 is disposed between the center 21 and the circumference 22 of the circular substrate 20; two branch channels 210a, 210b, the two shunts 210a, 210b are connected to the end of the main channel 200 near the circumference 22 of the circular substrate 20 at an angle 206a, 206b of equal angles, forming a main axis 200 as a central axis The symmetrical structure has a divergent structure 205 between the main flow channel 200 and the two sub-flow channels 210a, 210b. The divergent structure 205 has a circular arc structure between the two sub-flow channels 210a, 210b; The node area 220 includes at least the same injection hole 221, the same storage tank 222, the same node valve 223 and the same node junction channel 224. The sample injection hole 221 can be used to inject a specific sample, and the injected sample is injected through the sample. Hole 221 can flow The sample storage tank 222 is reserved. The sample node valve 223 is disposed between the sample storage tank 222 and the sample node manifold 224 for blocking the sample from flowing into the sample node manifold 224, the sample node manifold 224 and the main channel. a first cleaning agent node region 230, the first cleaning agent node region 230 includes at least a first cleaning agent injection hole 231, a first cleaning agent storage tank 232, a first cleaning agent node valve 233, and a first cleaning agent node 234, the first cleaning agent injection hole 231 can be used to inject a specific first cleaning agent, and the injected first cleaning agent can flow into the first cleaning through the first cleaning agent injection hole 231. The first cleaning agent node valve 233 is disposed between the first cleaning agent storage tank 232 and the first cleaning agent node confluence channel 234 for blocking the first cleaning agent from flowing into the first cleaning agent at will. In the node bus 234, the first cleaning agent node bus 234 is connected to the main channel 200; an antibody node area 240, the antibody node area 240 includes at least one antibody injection hole 241, an antibody storage tank 242, The antibody node valve 243 and an antibody node confluence channel 244 can be used to inject a specific antibody, and the injected antibody can flow into the antibody storage tank 242 via the antibody injection hole 241 for use. The antibody node valve 243 is set. The antibody storage tank 242 and the antibody node confluence channel 244 are configured to block the antibody from flowing into the antibody node confluence channel 244, and the antibody node confluence channel 244 is connected to the main channel 200; a second cleaning agent node region 250, The second cleaning agent node area 250 includes at least a second cleaning agent injection hole 251, a second cleaning agent storage tank 252, a second cleaning agent node valve 253, and a second cleaning agent node manifold 254. The second cleaning The agent injection hole 251 can be used to inject a specific second cleaning agent, and the injected second cleaning agent can flow into the second cleaning agent storage tank 252 via the second cleaning agent injection hole 251 for standby, the second cleaning agent node valve 253 is disposed between the second cleaning agent storage tank 252 and the second cleaning agent node confluence channel 254, for blocking the second cleaning agent from flowing into the second cleaning agent node confluence channel 254, the second The cleaning agent node manifold 254 is connected to the main flow channel 200; a substrate node region 260 including at least one substrate injection hole 261, a substrate storage tank 262, a substrate node valve 263, and a substrate node manifold 264. The substrate injection hole 261 can be used to inject a specific substrate, and the injected substrate can flow into the substrate storage tank 262 via the substrate injection hole 261, and the substrate node valve 263 is disposed in the substrate storage groove 262 and the substrate node. Between 264, the barrier matrix is randomly flown into the matrix node manifold 264, and the matrix node manifold 264 is connected to the main channel 200. The two detection zones 270a, 270b are respectively connected to the two detection zones 270a, 270b. The splitting passages 210a, 210b are combined with opposite ends of the branching structure 205, and the two detecting areas 270a, 270b are respectively provided with a detecting area collecting passage 271a, 271b; and at least one waste liquid storage area 280, and the second detecting areas 270a, 270b respectively The detection zone confluence channels 271a, 271b are connected to the waste liquid storage area 280; wherein a centrifugal force from the center 21 to the circumference 22 can be applied to the circular substrate 20, The sample node valve 223, the first cleaning agent node valve 233, the antibody node valve 243, the second cleaning agent node valve 253, and the substrate node valve 263 respectively have different valve resistances, so the sample can be controlled by adjusting the centrifugal force. The first cleaning agent, the antibody, the second cleaning agent and the matrix break through the valve to enter the main channel 200. After entering the main channel 200, the liquid is evenly split into the two branch channels 210a, 210b via the diverging structure 205, and enters two The detection zone 270a, 270b is subjected to ELISA detection, and the liquid that has been reacted finally flows into the waste storage area 280 for storage.

In the modified biochip microchannel system structure diagram of the second preferred embodiment, the sample node region 220, the first cleaning agent node region 230, the antibody node region 240, the second cleaning agent node region 250, and the matrix node region The way of connecting 260 to the main channel 200 is not limited by the second figure, and the relationship between the upstream and the downstream can be randomly arranged and combined, as long as the valve resistance between the valves of each node has a hierarchical relationship, so that the liquid of each node is in accordance with the sequence of the ELISA detection steps. It is sufficient to enter the detection area 270.

Finally, please refer to the improved biochip micro-channel system structure diagram of the third preferred embodiment of the present invention, which is formed on a circular substrate 30 to form a series of detection chambers. The main channel 300 is radially disposed on the circular substrate 30, and the main channel 300 is disposed between the center 31 and the circumference 32 of the circular substrate 30; The two primary shunts 310a, 310b are connected to the end of the main channel 300 near the circumference 32 of the circular substrate 30 at first angles 306a, 306b of equal angles, respectively, to form a main channel 300. The left and right symmetrical structure of the central axis has a first diverging structure 305 between the main flow channel 300 and the two primary shunts 310a, 310b. The first divergent structure 305 is a circular arc between the two shunting channels 310a, 310b. Structure; four secondary shunts 320a, 320b, 320c, 320d, which are respectively connected in two groups to two primary shunts 310a, 310b combined with a divergent structure The opposite end of the 305 end, the detailed link is The secondary shunts 320a, 320b are connected to the primary shunt 310a at second angles 316a, 316b of equal angles to form a left-right symmetric structure with the primary shunt 310a as a central axis, the primary shunt 310a and the secondary shunt. There is a second diverging structure 315a between 320a and 320b. The second diverging structure 315a has a circular arc configuration between the two secondary shunts 320a and 320b. The secondary shunts 320c and 320d are at an equal angle. The two angles 316c, 316d are connected to the primary shunt channel 310b to form a left-right symmetric structure with the primary shunt channel 310b as a central axis, and a second diverging structure 315b between the primary shunt channel 310b and the secondary shunt channel 320c, 320d. The second branch structure 315b has a circular arc structure between the two secondary shunts 320c, 320d; two sample node regions 330a, 330b, and the two sample node regions 330a, 330b respectively include at least the same injection hole 331a. 331b, the same storage tank 332a, 332b, the same node valve 333a, 333b and the same node junction 334a, 334b, the sample injection hole 331a, 331b can be used to inject a specific sample, the injected sample The samples are stored in the sample storage tanks 332a, 332b via the sample injection holes 331a, 331b. The sample node valves 333a, 333b are disposed between the sample storage tanks 332a, 332b and the sample node junctions 334a, 334b for blocking the sample. Flowing into the sample node sinks 334a, 334b, the sample node buss 334a, 334b of the two sample node regions 330a, 330b are respectively coupled to two primary shunts 310a, 310b, wherein the two sample node regions 330a, 330b One of the same specific samples may be injected to perform multiple repeated detections of the specific sample, or different samples may be separately injected to simultaneously detect two different samples; a first cleaning agent node area 340, the first cleaning The agent node area 340 includes at least a first cleaning agent injection hole 341, a first cleaning agent storage tank 342, a first cleaning agent node valve 343 and a first cleaning agent node manifold 344, the first cleaning agent injection hole The 341 series can be used to inject a specific first cleaning agent, and the injected first cleaning agent flows into the first cleaning agent storage tank 342 via the first cleaning agent injection hole 341 for use, the first The detergent node valve 343 is disposed between the first cleaning agent storage tank 342 and the first cleaning agent node confluence channel 344 for blocking the first cleaning agent from flowing into the first cleaning agent node confluence channel 344, the first cleaning agent. The node junction channel 344 is connected to the main channel 300. The antibody node region 350 includes at least an antibody injection hole 351, an antibody storage tank 352, an antibody node valve 353, and an antibody node manifold 354. The antibody injection hole 351 can be used to inject a specific antibody, and the injected antibody flows into the antibody storage tank 352 via the antibody injection hole 351. The antibody node valve 353 is disposed between the antibody storage tank 352 and the antibody node confluence channel 354. For blocking the antibody to flow into the antibody node confluence channel 354, the antibody node confluence channel 354 is connected to the main flow channel 300; a second cleaning agent node region 360, the second cleaning agent node region 360 includes at least a second cleaning agent The injection hole 361, a second cleaning agent storage tank 362, a second cleaning agent node valve 363 and a second cleaning agent node manifold 364, the second cleaning agent injection hole 361 is available Injecting a specific second cleaning agent, the injected second cleaning agent flows into the second cleaning agent storage tank 362 via the second cleaning agent injection hole 361, and the second cleaning agent node valve 363 is disposed in the second cleaning agent. Between the storage tank 362 and the second cleaning agent node 364, the second cleaning agent is prevented from flowing into the second cleaning agent node 364, and the second cleaning agent node 364 is connected to the main flow channel 300; a substrate node region 370 comprising at least one substrate injection hole 371, a substrate storage tank 372, a substrate node valve 373, and a substrate node manifold 374, which can be used to inject a specific substrate The implanted substrate flows into the substrate storage tank 372 via the substrate injection hole 371. The substrate node valve 373 is disposed between the substrate storage tank 372 and the substrate node manifold 374 to block the matrix from flowing into the matrix node manifold. In 374, the substrate node bus passage 374 is connected to the main flow channel 300; the four detection regions 380a, 380b, 380c, and 380d are respectively connected to the four detection regions 380a, 380b, 380c, and 380d. The four secondary shunts 320a, 320b, 320c, 320d are combined with opposite end points of the primary shunts 310a, 310b, and the four detecting areas 380a, 380b, 380c, 380d are respectively provided with a detecting area confluence channel 381a, 381b, 381c, 381d; and at least one waste liquid storage area 390, wherein the four detection areas 380a, 380b, 380c, 380d are respectively connected to the waste liquid storage area 390 by the detection area collecting channels 381a, 381b, 381c, 381d; A centrifugal force from the center 31 to the circumference 32 is applied to the circular substrate 30 due to the two sample node valves 333, the first cleaning agent node valve 343, the antibody node valve 353, the second cleaning agent node valve 363, and the substrate node. The valves 373 respectively have different valve resistances, so the two samples, the first cleaning agent, the antibody, the second cleaning agent and the matrix can be controlled to enter the main channel 300 by adjusting the centrifugal force, and the liquid enters the main channel. After 300, the first divergent structure 305 and the second divergent structure 315 are evenly split into the two primary shunts 310a, 310b and the four secondary shunts 320a, 320b, 320c, 320d, and enter four Measuring area 380a, 380b, 380c, 380d is performed by ELISA, the final completion of the reaction liquid into the waste storage region 390 in storage.

In the modified biochip microchannel system structure diagram of the third preferred embodiment, the first cleaning agent node region 340, the antibody node region 350, the second cleaning agent node region 360, and the matrix node region 370 and the main channel 300 The connection method is not limited by the third figure, and the relationship between the upstream and the downstream can be randomly arranged and combined. As long as the valve resistance between the valves of each node has a hierarchical relationship, the liquid of each node enters the detection area 380 according to the sequence of the ELISA detection steps. can.

In addition to the improved microchannel divergence structure of the present invention, the roughness of the microchannel may affect the frictional relationship between the centrifugal force generated by the fluid and the flow path, and the radius of the semicircular divergence structure may also be diverted to the fluid. The influence was made, so the inventors measured the roughness of the microchannel and the radius of the semicircular divergence structure to see if these factors would affect the shunting of the fluid in the microchannel. Firstly, we can see the measurement of roughness. It is found through experiments that if the roughness of the surface of each channel is close, the flow rate of the fluid is similar through the divergence structure, which is very close to 1:1. The ratio of the peaks of the first-class roads is large, the flow velocity of the fluid is slow and unstable, so the flow splitting ratio of the fluid through the divergent structure is large, and may reach a phenomenon of 1:1.4 or more. Therefore, the more stable the roughness of the flow path, the closer the split ratio to the fluid.

Then, the influence of the radius of the semi-circular divergence structure on the fluid shunt is carried out. The radius of the semi-circular divergence structure is 0.3 mm and the radius of the bifurcation structure is the same under the condition that the two roughness levels are very close to each other and the rotation speed, the flow channel width and other parameters are the same. The effect of 0.4mm on fluid shunting. The experimental results show that the radius of the semicircular divergence structure is 0.3 mm. The ratio data of the fluid split is better than the data with a radius of 0.4 mm. Therefore, it can be seen that the radius of the semicircular divergence structure is 0.3 mm, which can achieve a better split ratio.

The embodiments described above are merely illustrative of the technical spirit and characteristics of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the contents of the present invention and to implement the present invention. Equivalent variations or modifications in accordance with the spirit of the invention are still intended to be included within the scope of the invention.

The inventor has been continually conceived and modified to finally obtain the design of the present invention, and possesses the above-mentioned many advantages. It is an excellent invention, and should conform to the requirements of the invention patent, and the application is made, and the review committee can give the invention early. Patents to protect the rights of inventors.

A01. . . Step start

A02. . . A primary antibody (an antibody that specifically targets a specific antigen) is attached to the wall of the detection slot

A03. . . Add masking agent (usually protein)

A04. . . Add a sample or standard containing the antigen to the test well

A05. . . React for a sufficient time

A06. . . Add a first cleaning agent to wash away the excess sample

A07. . . Add a secondary antibody that binds to a specific enzyme

A08. . . Secondary antibody reacts with antigen for a sufficient time

A09. . . Add a second cleaning agent to wash off the excess secondary antibody

A10. . . Adding a specific matrix that can be converted into a detectable substance by an enzyme reaction on the secondary antibody, such as a substance containing a specific color or fluorescence

A11. . . By detecting the yield of the substance, it can be determined whether the sample contains antigen or the amount of antigen contained in the sample.

A12. . . End of step

10. . . Substrate

100. . . Mainstream road

105. . . Bifurcation structure

106a, 106b. . . Angle

110a, 110b. . . Split runner

120. . . Sample node area

121. . . Sample injection hole

122. . . Sample storage tank

123. . . Sample node valve

130a, 130b. . . Detection area

20. . . Circular substrate

200. . . Mainstream road

205. . . Bifurcation structure

206a, 206b. . . Angle

twenty one. . . Center of mind

210a, 210b. . . Split runner

twenty two. . . circumference

220. . . Sample node area

221. . . Sample injection hole

222. . . Sample storage tank

223. . . Sample node valve

224. . . Sample node manifold

230. . . First cleaning agent node area

231. . . First cleaning agent injection hole

232. . . First cleaning agent storage tank

233. . . First cleaning agent node valve

234. . . First cleaning agent node manifold

240. . . Antibody node region

241. . . Antibody injection hole

242. . . Antibody storage tank

243. . . Antibody node valve

244. . . Antibody node confluence channel

250. . . Second cleaning agent node area

251. . . Second cleaning agent injection hole

252. . . Second cleaning agent storage tank

253. . . Second cleaning agent node valve

254. . . Second cleaning agent node manifold

260. . . Matrix node region

261. . . Matrix injection hole

262. . . Matrix storage tank

263. . . Matrix node valve

264. . . Matrix node manifold

270a, 270b. . . Detection area

271a, 271b. . . Detection zone

280. . . Waste storage area

30. . . Circular substrate

300. . . Mainstream road

305. . . First divergent structure

306a, 306b. . . First angle

31. . . Center of mind

310a, 310b. . . Primary runner

315a, 315b. . . Second divergent structure

316a, 316b, 316c, 316d. . . Second angle

32. . . circumference

320a, 320b, 320c, 320d. . . Secondary runner

330a, 330b. . . Sample node area

331a, 331b. . . Sample injection hole

332a, 332b. . . Sample storage tank

333a, 333b. . . Sample node valve

334a, 334b. . . Sample node manifold

340. . . First cleaning agent node area

341. . . First cleaning agent injection hole

342. . . First cleaning agent storage tank

343. . . First cleaning agent node valve

344. . . First cleaning agent node manifold

350. . . Antibody node region

351. . . Antibody injection hole

352. . . Antibody storage tank

353. . . Antibody node valve

354. . . Antibody node confluence channel

360. . . Second cleaning agent node area

361. . . Second cleaning agent injection hole

362. . . Second cleaning agent storage tank

363. . . Second cleaning agent node valve

364. . . Second cleaning agent node manifold

370. . . Matrix node region

371. . . Matrix injection hole

372. . . Matrix storage tank

373. . . Matrix node valve

374. . . Matrix node manifold

380a, 380b, 380c, 380d. . . Detection area

381a, 381b, 381c, 381d. . . Detection zone

390. . . Waste storage area

The first figure is a conventional ELISA step chart;

2 is a structural diagram of an improved biochip microchannel system according to a first preferred embodiment of the present invention;

3 is a structural diagram of an improved biochip microchannel system according to a second preferred embodiment of the present invention;

Figure 4 is a structural diagram of an improved biochip microchannel system according to a third preferred embodiment of the present invention;

10. . . Substrate

100. . . Mainstream road

105. . . Bifurcation structure

106a, 106b. . . Angle

110a, 110b. . . Split runner

120. . . Sample node area

121. . . Sample storage tank

122. . . Sample node valve

123. . . Sample injection hole

130a, 130b. . . Detection area

Claims (16)

An improved biochip microchannel divergence structure is formed on a substrate to form a series of detection chambers, including: a main flow channel; and two distribution channels, the two sub-flow channels respectively at an angle of an equal angle The main channel is connected to form a left-right symmetric structure with the main channel as the central axis, and a divergent structure between the main channel and the two shunts, the bifurcated structure is a circular arc structure between the two shunts; In the node area, the sample node area is connected to the opposite end of the main channel and the divergent structure end, and the sample node area includes at least the same storage tank and the same node valve, and the sample node valve is disposed between the sample storage tank and the main flow channel. , for blocking the sample to flow into the main channel at random; and two detection areas respectively connected to the opposite ends of the two shunts combined with the divergent structure end; wherein the sample storage tank of the micro flow channel system Applying a centrifugal force in the direction of the valve of the sample node, when the centrifugal force reaches a certain threshold value of the valve resistance, the sample in the sample storage tank will break through. This node of the resistance of the valve flows into the valve in the main passage, and into two in the shunt via a branch structure where the mean shunt, and finally into the two sample detection zone is detected. The improved biochip microchannel divergence structure of claim 1, wherein the sample node region further comprises a same injection hole, the sample injection hole system can be used to inject a specific sample, the injected sample is via The sample injection hole can flow into the sample storage tank for use. An improved biochip microchannel divergence structure is formed on a circular substrate to form a series of detection chambers, comprising: a main channel, the main channel is radially disposed on the circular substrate, and is mainstream The position of the track is disposed between the center of the circular substrate and the circumference; and the two branch channels are respectively connected at an equal angle to the end of the main channel near the circumference of the circular substrate, forming a The main channel is a left-right symmetric structure of the central axis, and a divergent structure is formed between the main channel and the two branch channels, and the divergent structure is a circular arc structure between the two branch channels; the same node region, the sample node region At least the same storage tank, the same node valve and the same node manifold, the sample node valve is disposed between the sample storage tank and the sample node convergence channel to block the sample from flowing into the sample node convergence channel at random, the sample node convergence The channel is connected to the main flow channel; a first cleaning agent node zone, the first cleaning agent node zone comprises at least a first cleaning agent storage tank and a first cleaning agent node valve a first cleaning agent node is disposed between the first cleaning agent storage tank and the first cleaning agent node confluence channel to block the first cleaning agent from flowing into the first cleaning agent node at random In the manifold, the first cleaning agent node is connected to the main channel; an antibody node region, the antibody node region comprises at least an antibody storage tank, an antibody node valve and an antibody node confluence channel, and the antibody node valve is disposed on the antibody The storage tank and the antibody node confluence channel are configured to block the antibody from flowing into the antibody node confluence channel at random, and the antibody node confluence channel is connected to the main flow channel; and a second cleaning agent node region, the second cleaning agent node region includes at least one a second cleaning agent storage tank, a second cleaning agent node valve and a second cleaning agent node manifold, the second cleaning agent node valve is disposed between the second cleaning agent storage tank and the second cleaning agent node confluence channel , the second cleaning agent is blocked from flowing into the second cleaning agent node convergence channel, and the second cleaning agent node is connected to the main flow channel; a substrate node region comprising at least a substrate storage tank, a substrate node valve and a substrate node manifold, the substrate node valve being disposed between the substrate storage tank and the substrate node confluence channel for blocking the matrix from flowing into the substrate node at random In the manifold, the matrix node is connected to the main channel; in the two detection areas, the two detection areas are respectively connected to the opposite ends of the two main channels, and the two detection areas are respectively provided with a detection area. And at least one waste liquid storage area, wherein the two detection areas are respectively connected to the waste liquid storage area by the detection area manifold; wherein a centrifugal force from the center of the circle to the circumferential direction can be applied to the circular substrate, The node valve, the first cleaning agent node valve, the antibody node valve, the second cleaning agent node valve and the substrate node valve respectively have different valve resistances, so the sample, the first cleaning agent, the antibody, and the like can be controlled by adjusting the centrifugal force. The second cleaning agent and the matrix break through the valve to enter the mainstream channel, and the liquid enters the main channel and is evenly divided by the divergent structure. The flow enters the two branch channels and enters the two detection zones for detection. The liquid finally flows into the waste storage area for storage. The improved biochip microchannel divergence structure of claim 3, wherein the sample node region further comprises a same injection hole, the sample injection hole system can be used to inject a specific sample, the injected sample is via The sample injection hole can flow into the sample storage tank for use. The improved biochip microchannel divergence structure of claim 3, wherein the first cleaning agent node region further comprises a first cleaning agent injection hole, and the first cleaning agent injection hole can be used for injection. a specific first cleaning agent, the injected first cleaning agent can flow into the first cleaning agent storage tank via the first cleaning agent injection hole for use. The improved biochip microchannel divergent structure according to claim 3, wherein the antibody node region further comprises an antibody injection hole, and the antibody injection hole system can be used to inject a specific antibody, and the injected antibody is passed through The antibody injection well can flow into the antibody storage tank for use. The improved biochip microchannel divergence structure of claim 3, wherein the second cleaning agent node region further comprises a second cleaning agent injection hole, and the second cleaning agent injection hole can be used for injection. A specific second cleaning agent, the injected second cleaning agent can flow into the second cleaning agent storage tank via the second cleaning agent injection hole for use. The improved biochip microchannel divergence structure of claim 3, wherein the matrix node region further comprises a matrix injection hole, the matrix injection hole system can be used to inject a specific matrix, and the implanted substrate is via the substrate The matrix injection holes can flow into the substrate storage tank for use. An improved biochip microchannel divergence structure is formed on a circular substrate to form a series of detection chambers, comprising: a main channel, the main channel is radially disposed on the circular substrate, and is mainstream The track is disposed between the center of the circular substrate and the circumference; and the two primary shunts are respectively connected to the end of the main channel adjacent to the circumference of the circular substrate at a first angle of equal angle, The system forms a left-right symmetric structure with the main channel as the central axis, and the main channel and the two primary shunts are a first divergent structure, and the first divergent structure is a circular arc between the two primary shunts. Constructed; four secondary runners, the two secondary runners are respectively connected in two groups, the two primary runners are combined with the opposite ends of the first branching structure end, and the two secondary runners of each group Connected to the primary shunt at a second angle of equal angle, respectively, forming a left-right symmetric structure with the primary shunt as a central axis, and a second divergent structure between the primary shunt and the secondary shunt, the second Disagree Between the two secondary shunts is a circular arc structure; two sample node regions, the two sample node regions respectively contain at least the same storage tank, the same node valve and the same node junction channel, the sample The node valve is disposed between the sample storage tank and the sample node convergence channel to block the sample from flowing into the sample node convergence channel at random, and the sample node convergence channel of the two sample node regions is respectively connected with the two primary bypass channels; a cleaning agent node zone, the first cleaning agent node zone comprises at least a first cleaning agent storage tank, a first cleaning agent node valve and a first cleaning agent node manifold, the first cleaning agent node valve is disposed at the first The cleaning agent storage tank and the first cleaning agent node confluence channel are configured to block the first cleaning agent from flowing into the first cleaning agent node confluence channel at random, the first cleaning agent node confluence channel is connected with the main flow channel; and an antibody node region The antibody node region comprises at least an antibody storage tank, an antibody node valve and an antibody node manifold, and the antibody node valve is disposed in the antibody storage tank and The antibody node is connected between the flow channels to block the free flow of the antibody into the antibody node confluence channel, and the antibody node confluence channel is connected to the main flow channel; and a second cleaning agent node region, the second cleaning agent node region includes at least a second cleaning agent a storage tank, a second cleaning agent node valve and a second cleaning agent node manifold, the second cleaning agent node valve is disposed between the second cleaning agent storage tank and the second cleaning agent node confluence channel for blocking The second cleaning agent flows into the second cleaning agent node manifold, and the second cleaning agent node is connected to the main channel; a substrate node region, the matrix node region includes at least one substrate storage tank, a substrate node valve and a substrate a node manifold, the substrate node valve is disposed between the substrate storage tank and the substrate node convergence channel for blocking the matrix from flowing into the matrix node convergence channel, and the matrix node convergence channel is connected to the main channel; the four detection zones, the four The detection zones are respectively connected to the opposite ends of the four secondary runners combined with the primary runners, and the four detection zones are respectively provided with a detection zone sink. a flow channel; and at least one waste liquid storage area, wherein the four detection areas are respectively connected to the waste liquid storage area by the flow area of the detection area; wherein a centrifugal force from the center of the circle to the circumferential direction can be applied to the circular substrate Since the two sample node valves, the first cleaning agent node valve, the antibody node valve, the second cleaning agent node valve, and the substrate node valve respectively have different valve resistances, the two samples can be controlled by adjusting the centrifugal force. The first cleaning agent, the antibody, the second cleaning agent and the matrix break through the valve to enter the main channel. After the liquid enters the main channel, it is evenly split into two primary and four secondary channels through the divergent structure, and enters four The detection is carried out in the detection zone, and the liquid finally flows into the waste storage area for storage. The improved biochip microchannel divergence structure of claim 9, wherein the two sample node regions further comprise the same injection hole, the sample injection hole system can be used to inject a specific sample, the injection The sample can flow into the sample storage tank via the sample injection hole for use. The improved biochip microchannel divergence structure of claim 9, wherein the two sample node regions are capable of injecting one of the same specific samples for performing multiple iterations of the particular sample. The improved biochip microchannel divergence structure according to claim 9, wherein the two sample node regions can respectively inject different samples to simultaneously detect two different samples. The modified biochip microchannel divergence structure of claim 9, wherein the first cleaning agent node region further comprises a first cleaning agent injection hole, and the first cleaning agent injection hole can be used for injection. a specific first cleaning agent, the injected first cleaning agent can flow into the first cleaning agent storage tank via the first cleaning agent injection hole for use. The improved biochip microchannel divergence structure according to claim 9, wherein the antibody node region further comprises an antibody injection hole, and the antibody injection hole system can be used to inject a specific antibody, and the injected antibody is passed through The antibody injection well can flow into the antibody storage tank for use. The modified biochip microchannel divergence structure of claim 9, wherein the second cleaning agent node region further comprises a second cleaning agent injection hole, and the second cleaning agent injection hole can be used for injection. A specific second cleaning agent, the injected second cleaning agent can flow into the second cleaning agent storage tank via the second cleaning agent injection hole for use. The improved biochip microchannel divergence structure of claim 9, wherein the matrix node region further comprises a matrix injection hole, the matrix injection hole system can be used to inject a specific matrix, and the implanted substrate is via the substrate The matrix injection holes can flow into the substrate storage tank for use.