KR101743749B1 - Microfluidics Chip for Immunoanalyzing - Google Patents

Abstract

The present invention relates to a microfluidic chip for immunoassay, wherein the microfluidic chip for immunoassay comprises: (a) a first region in which electrode drawings are printed using conductive ink and inkjet printing; And (b) an antibody against the detection target antigen in the assay sample, (ii) an antigen against the detection target antibody in the assay sample, or (iii) an antibody against the detection target antigen in the assay sample is immobilized, And a second region in which the second region is generated. Wherein the first portion is in contact with a fluid selected from the group consisting of an assay sample, a blocking buffer, an antibody, a substrate and a wash buffer, the first portion in contact with the second portion , The fluid in contact with the first portion is moved to the second portion by electrowetting.

Description

{Microfluidics Chip for Immunoanalyzing}

The present invention relates to a microfluidic chip for immunoassay.

Conventional digital microfluidic chips (microfluid chips) are fabricated on glass or silicon substrates by complicated processes such as photolithography and etching (RB Fair, Microfluid Nanofluid , 3: 245281 (2007)), the equipment and chemicals used are not only expensive but also very harmful to the human body and the environment. Most of the digital microfluidic chips have a closed system (Robert J. Linhardt et al., J. AM. CHEM . SOC ., 131: 11041-11048 (2009) A cover plate has to be removed. Since the open chip system (Abdelgawad, Park, and Wheeler J. Appl . Phys ., 105: 094506 (2009)) uses the existing methods in the process of fabricating the electrode pattern, Occurs.

The electrowetting can control the movement of the fluid by electricity and the digital micro fluidic chip is used. Electric wetting phenomenon is a phenomenon that surface tension of electric furnace is changed easily. This phenomenon was first discovered in 1870 by Gabriel Lippmann (1845-1921). When water is poured into a glass tube, the glass tube wall has a higher water height than the center, due to the surface tension between the water and the glass tube wall. However, if you use a metal tube instead of a glass tube and apply electricity, the height of the water rising along the wall becomes higher. This is because the surface tension of the electric furnace is further reduced. Lippmann called it the "electro-capillary" phenomenon, but for the past 100 years, this technology has not seen much light. Electro capillary phenomenon occurred only at low voltage of 1V or less, and when it was applied at higher voltage, water was decomposed into oxygen and hydrogen. In 1990, an electric wetting phenomenon was found that can control surface tension even at high voltage. Dr. Bruno Berge of France put a metal plate on a thin insulator and dropped a drop of water on it. Next, when the electricity was applied to the metal plate and the water droplet, the water droplet spread thinner as the voltage increased. This method makes it possible to change the shape of water drops even at a high voltage of several tens of volts.

Carbon nanotube (CNT) is a new material in which six hexagons of carbon are connected to each other to form a tubular shape. In 1991, Dr. Iimase Sumio of the Institute of Nuclear Power Plants (NEC) Method was used to analyze the carbon mass formed on the graphite cathode. The hexagon shapes of six carbon are connected to each other to form a tube. The diameter of the tube is only a few to tens of nanometers, and it is called carbon nanotube. The nanometer is 1/10 billionth of a meter, usually one-tenth the thickness of hair. Carbon nanotubes have an electrical conductivity similar to that of copper, with a thermal conductivity equal to that of natural diamond, with strength 100 times better than steel. Carbon fibers can be broken even if they are deformed by only 1%, while carbon nanotubes can withstand 15% deformation. Since the discovery of this material, scientists have been focused on synthesis and applications, and devices using carbon nanotubes, such as semiconductors and flat panel displays, batteries, super fibers, biosensors, and television cathode ray tubes, are being developed extensively.

Enzyme-linked immunosorbent assay (ELISA) is one of the most widely used immunoassay methods to detect the target protein present in a sample. The detection of the target protein is enabled by an antigen-antibody reaction. The principle of immunosorbent antibody or antigen is used. In the prior art, a microtiter plate made of polystyrene is used as the solid phase. ELISA can be divided into direct ELISA, indirect ELISA and sandwich ELISA depending on the method of use of the antibody. Currently, diagnostic techniques using ELISA are being used to detect various viruses, parasites and microorganisms.

Appropriate technology means the simplest level of technology that can achieve the desired results efficiently in a specific geographical area. Proper technology has features of low price, simple operability, small scale applicability, creativity and environmental preservation. For example, a portable water purifier straw "Life straw" made for the supply of drinking water to people living in developing countries and third countries where water resources are polluted, and a low-cost incubator that uses automobile parts and can be used as a power source 'NeoNuture' is a representative suitable technology.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a microfluidic chip for immunoassay that can be used as an appropriate technology as well as being easy to manufacture and use, which can replace the conventional high cost and time consuming immunoassay methods . As a result, a first region where the electrode drawing is printed using conductive ink and inkjet printing and (i) an antigen to be detected in the assay sample, (ii) an antigen to the detection target antibody in the assay sample, or (iii) ) An analytical microfluidic chip consisting of a second site in which the antibody against the target antigen in the assay sample is immobilized and the antigen-antibody immune response is generated is developed. The microfluidic chip for immunoassay according to the present invention is an immunoassay method which is less costly and consumes less time, and it is confirmed that the object to be detected in the analytical sample can be analyzed economically and quickly as compared with the conventional immunoassay method, Respectively.

Accordingly, it is an object of the present invention to provide a microfluidic chip for immunoassay.

Another object of the present invention is to provide an immunoassay method.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, there is provided an inkjet printhead comprising: (a) a first region in which an electrode drawing is printed using conductive ink and inkjet printing; Wherein the first site is contacted with a fluid selected from the group consisting of an analytical sample, a blocking buffer, an antibody, a substrate and a washing buffer, (b) (i) (Iii) a second region in which an antibody against the detection target antigen in the assay sample is immobilized and an antigen-antibody immune response is generated; Wherein the first portion is in contact with the second portion and the fluid in contact with the first portion is moved to the second portion by electrowetting.

The present inventors have sought to develop a microfluidic chip for immunoassay that can be used as an appropriate technology as well as being easy to manufacture and use, which can replace the conventional high cost and time consuming immunoassay methods . As a result, it was found that the first region where the electrode drawing was printed using conductive ink and inkjet printing and (i) the detection target antigen in the assay sample, (ii) the antigen for the detection target antibody in the assay sample, or (iii) A microfluidic chip for immunoassay comprising a second site in which an antibody against a detection target antigen is immobilized and an antigen-antibody immune response is generated has been developed. The microfluidic chip for immunoassay of the present invention is an immunoassay method which is less expensive and consumes less time, and it is confirmed that the object to be detected can be analyzed economically and quickly in comparison with the conventional immunoassay method.

The main features of the immunoassay microfluidic chip of the present invention are that the first region where the electrode diagram is printed using conductive ink and inkjet printing and (i) the detection target antigen in the assay sample, (ii) the detection target antibody in the assay sample (Iii) an antigen to be detected in the assay sample immobilized on the second site to a second site to which the antibody against the antigen to be detected in the assay sample is immobilized, (ii) an antibody to be detected in the assay sample Or (iii) a fluid selected from the group consisting of an analytical sample, a blocking buffer, an antibody, a substrate and a washing buffer in contact with the first site for the antigen to be detected in the analytical sample is subjected to electrophoresis And then an antigen-antibody immune response is generated and detected.

The microfluidic chip for immunoassay of the present invention includes a first region on which electrode drawings are printed using conductive ink and inkjet printing.

The inkjet printing used in the present invention is a printer using a piezo electric head among printer apparatuses, which is a typical output apparatus of a computer, and forms characters by ejecting ink onto the paper. That is to say, in a method in which charged ink particles are diverted and deflected according to a character type and a deflecting plate to which a controlled voltage is applied in accordance with the character position, and are printed on the paper surface Printer. The ink is initially injected into the ink head and is emitted in the form of particles according to the action of the crystal oscillator and is gradually stuck to the paper surface to express a dot matrix shape.

The term " conductive ink " in the context of the present invention encompasses conductive materials such as silver and carbon in the form of powder or grains and can be printed or printed on various solid substrates such as paper or film, ≪ / RTI > Conductive inks include, for example, metal inks such as gold, silver, copper, nickel, gold, platinum, copper and palladium, ceramic inks such as metal oxides and carbon oxides (or carbon nanotubes) Organic materials, and polymeric materials).

The conductive ink that can be used in the method of the present invention requires a certain amount or more of conductivity to be used as an electrode. According to one embodiment, the conductive ink is carbon nanotube (CNT). The carbon nanotubes have superior electrical properties such as aluminum and copper, compared with metal materials having relatively good electrical conductivity or resistivity. Accordingly, when such a carbon nanotube is used as a conductive material, the electrical resistance can be reduced and the thermal conductivity is also excellent, so that heat inside the printed circuit board can be effectively radiated to the outside.

The carbon nanotubes include, for example, single-walled carbon nanotubes, double-walled carbon nanotubes, multi-walled carbon nanotubes, and multi-walled carbon nanotubes. The multi-walled carbon nanotube electrode exhibits a resistance of 1 k? / Sq (square) on plain A4 paper and a resistance value of 200? / Sq on photographic paper.

The conductive ink may be prepared using a suitable solvent, a conductive material, an adhesive improver, a reducing agent, or a coupling agent. The solvent may be glycidyl ethers, glycol ethers, vegetable oils, alpha-terpineol, or NMP (N-methyl-2-pyrrolidone pyrrolidone). The adhesion promoter may be an acrylic resin or a vinyl resin. When a vinyl resin is used, a mixture containing a silane compound may be used. The reducing agent serves to reduce the electrical conductivity by reducing the conductive material when it is oxidized, and includes, for example, a hydrazine-based reducing agent or an aldehyde-based reducing agent. The hydrazine-based reducing agent includes hydrazine, hydrazine hydrate, hydrazine sulfate, hydrazine carbonate, and hydrazine hydrochloride, and the aldehyde-based reducing agent includes, but is not limited to, formaldehyde, acetaldehyde, and propionaldehyde.

According to a specific embodiment of the present invention, in order to print various CNT electrode patterns on an ink-jet printer, a dispersion agent is put in a multiwall CNT and ball milling is performed to make CNT ink evenly dispersed in water have. Print the electrode drawings using a program such as Photoshop or CAD, and print the electrode drawings of the desired shape on plain A4 or photo print paper.

The first part of the invention comprises an active zone comprising printed electrodes. The active region refers to a region in contact with a fluid selected from the group consisting of an analytical sample, a blocking buffer, an antibody, a substrate and a washing buffer.

According to an embodiment of the present invention, the active region includes a plurality of parallel electrodes for moving the fluid, and the fluid moves toward the second portion in a direction perpendicular to the plurality of parallel electrodes.

According to another embodiment of the present invention, electricity is applied to a plurality of electrodes of the active region to move the fluid to the second region through the electrowetting method. The technique of electro-controlling the movement of water droplets by the electro-wetting method is a technique of changing the solid-electrolyte connection angle by the applied potential difference between the solid and the electrolyte. The principles of the electrowetting method are well known in the art (Chang, HC, Yeo, L. (2009). Electrokinetically Driven Microfluidics and Nanofluidics. Cambridge University Press. Kirby, BJ 2010. Micro- and Nanoscale Fluid Mechanics : Transport in Microfluidic Devices .. Cambridge University Press ISBN 978-0-521-11903-0; A Model of Electrowetting, Reversed Electrowetting and Contact Angle Saturation. Dan Klarman, David Andelman, Michael Urbakh and Experimental Validation of the Invariance of Electrowetting Contact Angle Saturation).

The microfluidic chip for immunoassay of the present invention comprises an active zone containing printed electrodes of a first site and an antigen to be detected in an assay sample of (i) the second site, (ii) Or (iii) single or multiple test zones in which the antibody against the antigen to be detected in the assay sample is immobilized.

According to an embodiment of the present invention, a number of active regions and / or test regions corresponding to the number of fluids applied according to a target object to be detected are included.

According to another embodiment of the present invention, a single active area and / or a test area to which various fluids can be applied is included.

An example of the above is shown in Figs. 6A and 6B.

The microfluidic chip for immunoassay of the present invention is immobilized on (i) an antigen to be detected in an assay sample, (ii) an antigen to an antibody to be detected in the assay sample, or (iii) , And a second site where an antigen-antibody immune response is generated. The second portion is in contact with the first portion, and the fluid contacted to the first portion by the electro-wetting method moves to the second portion.

According to an embodiment of the present invention,

(I) the target antigen is immobilized in the assay sample, the antibody against the antigen is transferred through the active site of the first site, and the target antigen in the assay sample is detected by applying a washing buffer and a substrate Direct ELISA).

(Ii) an analyte is immobilized on an antibody to be detected in the assay sample, the analyte is moved through the active region of the first site, and a secondary antibody, a washing buffer and a substrate are applied to the antibody to be detected in the assay sample (Indirect ELISA).

(Iii) an antibody against an antigen to be detected in the assay sample is immobilized, the analytical sample is moved through the active region of the first site, and the primary antibody, the washing buffer, the secondary antibody, the washing buffer, To detect the target antigen in the assay sample (Sandwich ELISA).

The microfluidic chip for immunoassay of the present invention is characterized in that (i) an antigen to be detected in the assay sample, (ii) an antigen to the detection target antibody in the assay sample, or (iii) And has a second site.

The analytical sample includes, but is not limited to, a specific protein, blood, serum, plasma, urine, body fluids including ascites, tissue extracts, and cell extracts.

According to one embodiment of the present invention, the second site develops an antigen-antibody immune response.

"Antibody" in the present invention means a specific protein molecule directed against an antigen. For purposes of the present invention, an antibody refers to an antibody that specifically binds to an antigen immobilized on a first site, and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

The production of the antibody can be easily carried out using techniques well known in the art. Polyclonal antibodies can be produced by methods well known in the art for obtaining sera containing antibodies by injecting the above-mentioned antigen into an animal and collecting it from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like. Monoclonal antibodies can be obtained from the hybridoma method (see Kohler and Milstein (1976) European Journal of Immunology 6: 511-519), or the phage antibody library (Clackson et al, Nature, 352: 624 Biol., 222: 58, 1-597, 1991) techniques. The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

In the present invention, "antigen" means a specific protein molecule indicated for an antibody. For purposes of the present invention, an antigen refers to an antigen that specifically binds to an antibody in contact with a second site.

The antigens and antibodies are antigens and antibodies to proteins, bacteria or viruses. For example, the protein includes, but is not limited to, Human Chorionic Gonadotropin (HCG), Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), and Brain Natriuretic Peptide (BNP). The bacteria may be Escherichia coli coli), Pseudomonas her labor Rouge (Pseudomonas aeruginosa), Pseudomonas footage is (Pseudomonas putida , Yersinia pestis , Shigella flexnerii , Treponema denticola , Vibrio cholerae , Vibrio vulnificus , Vibrio parahemolyticus), Chlamydia pneumoniae (Chlamydia pneumoniae , Chlamydia trachomatis , Staphylococcus aureus , Staphylococcus epidermidis , Streptococcus pneumoniae , Streptococcus agaractiae, Streptococcus pneumoniae , Chlamydia trachomatis , Staphylococcus aureus , Staphylococcus epidermidis , (Streptococcus agalactiae), Streptococcus mutans (Streptococcus mutans), Streptococcus Pio jeneseu (Streptococcus pyogenes), Enterococcus faecalis (Enterococcus faecalis , Bordetella bronchiseptica , Bordetella pertussis), Peer-to-Bode telra Para Systems (Bordetella parapertussis), H. pylori party Caicos (Helicobacter hepaticus , Helicobacter pylori , Salmonella typhimurium , Ralstonia < RTI ID = 0.0 > solanacearum ), Santomonas campestris ( Xanthomonas campestris , Pseudomonas sp. syringae , Pasteurella multocida, Brucella ( Brucella) melitensis , Brucella suis , Porphyromonas gingivalis , Toxoplasma gondii , Listeria monocytogenes , Propionibacterium < RTI ID = 0.0 > acnes ), Haemophilus ducreyi , Haemophilus influenzae , Neisseria neisseria , gonorrhoeae , Neisseria meningitidis), morak Cellar Kata LAL-less (Moraxella catarrhalis , Agrobacterium < RTI ID = 0.0 > tumefaciens), Helicobacter species (Helicobacter spp.), Campylobacter your Jeju (Campylobacter jejuni), Mycobacterium Tudor beokyul tuberculosis (Mycobacterium tuberculosis), Mycobacterium para tuba beokyul tuberculosis (Mycobacterium paratuberculosis), Mycobacterium Lev below (Mycobacterium leprae), Mycobacterium Oh Away (Mycobacterium avium), koksi Ella bee Connecticut (Coxiella burnetti), Reggie ohnel La pneumophila (Legionella pneumophila , Salmonella typhi , Gardnerella < RTI ID = 0.0 > vaginalis , Plasmodium falciparum , Plasmodium ovale , Giardia < RTI ID = 0.0 > lamblia , or Plasmodium yoelii , but are not limited thereto. The virus may be selected from the group consisting of Severe Acute Respiratory Syndrome (SARS), SARS coronavirus , Respiratory Syncytial Virus, Hepatitis A virus , Hepatitis B virus , HBV (hepatitis C virus), Lou Bella viruses (Rubella virus), herpes virus (herpes Simplex virus), influenza virus (influenza virus), cytomegalovirus (cytomegalovirus), papilloma virus (papilloma virus), Dengue virus (Dengue virus), hand, foot and mouth disease virus (Foot and Mouth disease virus), West Nile virus (West Nile virus), bird flu (avian influenza virus), rotavirus (rotavirjs), para myxovirus (paramyxovirus) or HIV (Human immunodeficiency virus), but is not limited thereto.

The detection or measurement of the antigen-antibody complex formation by the antigen-antibody immunoreaction of the second site of the present invention can be carried out by directly detecting the formed antigen-antibody complex, but preferably the detection of the label . According to one embodiment of the present invention, the detection antibody is labeled with an antibody capable of producing a detectable signal or an affinity substance is bound thereto. Such labels include enzymes (e.g., alkaline phosphatase,? -Galactosidase, horseradish peroxidase,? -Glucosidase and cytochrome P ₄₅₀ ), radioactive materials (such as C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET).

When an enzyme that catalyzes the color reaction is used as a label binding to the detection antibody, an enzyme-antibody complex can be detected by measuring the reaction product by adding a substrate used for the enzyme reaction to the measurement system of the present invention . For example, when alkaline phosphatase is used as a label, it is possible to use bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate chromophore and ECF (enhanced chemifluorescence) may be used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis (10-acetyl-3,7-dihydroxyphenoxaphone), TMB (3,3,5,5-tetramethylbenzidine), N-methylpyrrolidinone nitrate , ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]) and o -phenylenediamine (OPD) can be used.

According to another embodiment of the present invention, the antibody in the fluid contacting the first region of the microfluidic chip for immunoassay is bound to an enzyme catalyzing a chromogenic reaction.

One of the features of the microfluidic chip for immunoassay of the present invention is that a variety of electronic drawings can be easily obtained using a solid-phase substrate which is easy to handle and cut. The first portion may include various solid-state substrates used in the art and include, for example, paper (e.g., plain A4 paper, photographic printing paper), film (e.g., OHP film) According to one embodiment, the first portion is paper or film, and according to another embodiment, the first portion is paper.

(I) an antigen to be detected in the assay sample, (ii) an antigen to the antibody to be detected in the assay sample, or (iii) an antibody to the antibody to be detected in the assay sample is immobilized on the microfluid chip for immunoassay of the present invention The two sites can use various solid substrates used in the art and include, for example, easy-to-handle paper (such as plain A4 paper, photographic paper), film (e.g., OHP film) According to an example, the second portion is paper or film, and according to another embodiment, the second portion is paper.

The first portion of the microfluidic chip for immunoassay of the present invention may further comprise depositing an insulator to form an insulating layer on the electrodes.

The insulator may be a glass, porcelain or polymer composition, according to another embodiment, a polymer composition. When a polymer composition is used as an insulator, parylene can be used. For example, parylene N, parylene C, parylene D, parylene F can be used. According to a specific embodiment, parylene C is used.

According to another embodiment of the present invention, it is possible to additionally coat the reinforcing material on the electrode drawing. The coating may be formed in at least a portion of the surface of the plurality of chips by spin coating, spray coating or printing. The coating may also be formed in at least a portion of the surface of the plurality of chips by physical vapor deposition, chemical vapor deposition or electroplating.

The reinforcing material functions to withstand heat, pressure, etc. during the process of using the microfluidic chip of the present invention, and various materials known in the art can be used. According to a particular embodiment of the invention, it is polytetrafluoroethylene.

The analytical sample of the present invention is blood, plasma, serum, lymph, saliva, urine, feces, ocular fluid, semen, ascites or amniotic fluid.

The second portion of the microfluidic chip for immunoassay of the present invention may further include a filtration membrane to increase the fluidity of the fluid at the upper portion.

According to an embodiment of the present invention, the filtration film is a glass filtration film.

The second portion of the microfluidic chip for immunoassay of the present invention may further include a spacer and an adsorption pad at the lower end thereof.

The spacer is included for a gap between the second portion of the microfluidic chip and the adsorption pad. The spacer may be made of tape, paper, film or nonwoven fabric, but is not limited thereto. According to an embodiment of the present invention, the spacer is a tape, and according to another embodiment of the present invention, the spacer is a double-sided tape.

The adsorption pad is included for removal thereof when a large amount of fluid such as wash buffer is applied. The adsorption pad may be made of any material capable of absorbing a liquid such as a fiber pad, a gauze sponge or cotton, but is not limited thereto. According to an embodiment of the present invention, the adsorption pad is a fiber pad, and according to another embodiment of the present invention, the adsorption pad is a cellulose fiber pad.

According to another aspect of the present invention, there is provided a diagnostic kit comprising the microfluidic chip for immunoassay.

According to one embodiment of the present invention, the diagnostic kit detects an object to be detected in an analytical sample by an antibody-antigen reaction.

(I) a detection target antigen in the assay sample, (ii) an analyte sample in the assay sample, (ii) an analyte sample in the second portion of the microfluidic chip for immunoassay according to any one of (Iii) immobilizing an antibody against the antigen to be detected in the sample to be assayed; (b) a fluid selected from the group consisting of an analytical sample, a blocking buffer, an antibody, a substrate and a wash buffer in the first portion of the microfluidic chip for immunoassay according to any one of claims 1 to 14 (ii) the antigen for the detection target antibody in the assay sample, or (iii) the antibody for the detection target antigen in the assay sample in the step (a) Generating an antigen-antibody immune response; And (c) detecting the antigen-antibody immune response.

Since the immunoassay method of the present invention uses the microfluidic chip for immunoassay, the description common to the two is omitted in order to avoid the excessive complexity of the present specification.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a microfluidic chip for immunoassay.

(b) In the case of a conventional chip, most substrates are glass, but the present invention can easily be cut using paper as a substrate and easily assembled with a filter paper used in a test zone through cutting It is possible.

(c) The microfluidic chip for immunoassay of the present invention can control the movement time and position of the sample using an automation program.

1 shows a paper chip diagram printed with carbon nanotube (CNT) ink.
Fig. 2 schematically shows the movement of water droplets on the electrodes arranged.
Figure 3 shows the anti-rabbit IgG antibody, wash buffer and chromogenic reagent (BCIP / NBT) located on a paper chip.
FIG. 4 shows the color development of a test zone in which various concentrations of rabbit IgG were fixed.
Figure 5 graphically shows the Rabbit IgG concentration-dependent color development response as measured by Image J.
Figures 6A and 6B illustrate examples of microfluidic chips comprising a single active area and / or a test area to which various fluids may be applied.
7 shows a paper chip on which an electronic pattern is printed. The black part is printed by the carbon nanotube ink. The black part is designed largely because the hole is made in the part. Since the droplet needs to apply a voltage to enter the hole well, Respectively.
8 shows a schematic view and an image showing a glass filter membrane and an absorbent pad attached to a test area of a paper chip.
FIG. 9 is a schematic diagram showing the absorption of the absorbent pad additionally attached to the paper chip according to the amount of the reaction solution.
FIG. 10 shows the result of checking the sensitivity of a pregnancy diagnostic test using the paper chip of the present invention and the paper chip used.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1: Paper chip manufacturing

Carbon nanotubes ( CNT ) Production of ink

Ink was produced using CNT (Carbon Nano Tube) to print the electrode pattern, printed on paper, and then used as an electrode. In order to print the CNT electrode with an ink jet printer, a dispersion agent was first added to multiwall CNTs and ball milling was performed to produce CNT ink evenly dispersed in water. In the present invention, the CNT electrode has a resistivity of 1 k? / Sq on a plain A4 paper and a resistance of 200? / Sq on a photographic printing paper to obtain a conductivity sufficient for use as an electrode. Respectively.

Carbon nanotubes ( CNT ) Production of digital microfluidic paper chip using ink

In order to fabricate an electrode pattern for use in the production of a digital microfluidic paper chip, a drawing is made by using a program such as Photoshop or CAD, and an electrode of a desired shape is printed on an ordinary A4 paper or photo print paper using an inkjet printer and the CNT ink And printed at a resolution of several tens of 탆 (Fig. 1).

Then, in the printed drawing, a hole was made in a test zone, parylene C was deposited thereon, and a Teflon AF1600 was spin coated to cover the insulating layer. It was confirmed that the water droplet can be controlled on the chip surface as shown in FIG. 2 when water droplets are dropped on the electrode pattern of the paper chip on which the parylene C and Teflon are deposited and voltage (V) is applied.

Example  2: ELISA

Experimental Method

Rabbit IgG (I5006 and I8140, Sigma aldrich) was diluted with various concentrations (0, 7.6, 13, 37 and 77 fmol) using PBS (Phosphate buffered saline). 3 μl of rabbit IgG was dropped on a test area of 6 mm in diameter made of filter paper (Whatman No. 1). Rabbit IgG was fixed on paper by reaction at room temperature for 10 minutes. 3 ㎕ of blocking buffer (0.05% (v / v) Tween and 5% BSA in PBS) was added to the test area and reacted at room temperature for 10 minutes.

Rabbit IgG was fixed at the test area of the paper chip and the blocked paper was placed. Each sample (anti-rabbit IgG antibody (A9919, Sigma aldrich), wash buffer (79383, Sigma aldrich) and BCIP / NBT (B5655, Sigma aldrich)] was placed in each position of the paper chip prepared in Example 1 I dropped it.

An anti-rabbit IgG sample bound with ALP (alkaline phosphatase) was transferred to the test area. The anti-rabbit IgG antibody located on the paper chip is transferred to the second site (test area) by electrowetting followed by a fluid capillary in the filter paper in which the fluid, anti-rabbit IgG, - The rabbit fluid is transferred to the entire paper area. After 1 minute, wash buffer (PBS) was transferred to the test area to remove unbound anti-rabbit IgG (Figure 3). The substrate reaction solution, BCIP / NBP, was transferred to the test zone and reacted at room temperature for 7 minutes to induce color change (FIG. 3). Table 1 shows the reagent volume and reaction time of each treatment.

process Volume ([mu] l) Time (minutes) 1) antigen fixation 3 10 2) Blocking 3 10 3) Antigen-antibody reaction 3 One 4) Signal amplification 3 7 Sum 12 28

After photographs were taken, the intensity of color development was measured using the Image J program (Figure 4).

Specificity and Sensitivity

Rabbit IgG (0, 7.6, 13, 37 and 77 fmol) was immobilized on the test area and then blocked. The ALP was then transferred to the test zone to react with the anti-goto sample to which it was bound. One minute later, washing buffer was sent to the test area to remove unbound antibody. The substrate solution was transferred to induce color change.

To measure the sensitivity of paper chips, the following method was used:

(1) Rabbit IgG was diluted 1: 100 with PBS (maintained at the same concentration as the antigen, measured by adjusting the primary antibody concentration).

(2) Drop 3 μl of diluted IgG onto the test area made using Whatman No. 1 filter paper. At this time, the filter paper was produced by using a punch to a diameter of 6 ㎜.

(3) Rabbit IgG was fixed on paper by reaction at room temperature for 10 minutes.

(4) 3 μl of blocking buffer was added to the test area and reacted at room temperature for 10 minutes.

(5) A filter paper used as a test area was assembled on a paper chip.

(6) Before each sample was immobilized, the antigen was fixed and blocked, and each sample was dropped to its position. Each sample is an anti-goto IgG in rabbit (0, 7.6, 13, 37 and 77 fmol), anti-rabbit IgG in goat (no ALP), wash buffer and substrate reaction solution.

(7) Each sample (anti-goto IgG, anti-rabbit IgG, wash buffer and BCIP / NBP) was loaded into their respective positions.

(8) ALP unbound anti-Rabbit IgG samples were transferred to the test area

(9) After 3 minutes, wash buffer (PBS) was transferred to the test area to remove unbound anti-rabbit IgG.

(10) The anti-goto IgG sample to which ALP was bound was transferred to the test region.

(11) After 2 minutes, wash buffer (PBS) was transferred to the test area to remove unbound anti-goto IgG.

(13) Substrate The BCIP / NBP reaction solution was transferred to the test zone, and color change was induced after reaction at room temperature for 7 minutes.

(14) The photograph was taken using a mobile phone, and the intensity of the color developed was measured using the Image J program.

result

In order to perform an antigen-antibody reaction on a paper chip, the sample was divided into a moving region and a test zone. In order to use it as a test region, it is necessary to easily remove the sample after the reaction. In the present invention, since the paper chip is made of paper, the sample can be easily removed using the capillary phenomenon.

The solution used for the antigen-antibody reaction migrated according to the reaction sequence and reacted in the test area. Finally, when the substrate reaction solution moved to the test region, the degree of color development according to the antigen concentration could be distinguished.

Specificity and sensitivity

The change in color was insignificant with the substrate solution, and the change was consistent with the background color. (In fact, even if the antigen is not immobilized and only the blocking is done, the color appears finely because the nature of the paper does not completely remove the antibody that binds ALP.) This result shows that only the anti- .

In addition, the standard deviation of the control group was obtained, and the lowest detectable concentration of 56 fmol / zone was determined.

Example  3: Applying pregnancy test

Experimental material

Anti-hCG antibody (ab400) and HRP-conjugated anti-hCG antibody (ab30451) were purchased from abcam and BSA (Bovine serum albumin, A7906) and hCG kit (SE120063) were purchased from Sigma. Tween 20 (p1754) and TMB (T0440) were purchased from Sigma, and PBS buffer (10010-023 PBS pH 7.4 1x) was purchased from gibco.

Experimental Method

A hole (test area) was made in the chip made of the paper on which the electronic pattern was printed, and a glass filter membrane was inserted into the hole so that the liquid sample could move well (FIG. 7). It was also devised to allow a small amount of sample to remain in the test area with a blank space between the test area and the absorbent pad and to remove it from the test area by touching the absorbent pad in the case of large amounts of wash buffer (Figures 8 and 9) . Double - sided tape was used to give space. The applicability of the manufactured paper chip as a pregnancy diagnostic test was examined.

(1) Dilute hCG to various concentrations using PBS.

(2) Whatman No. 1 filter paper was prepared using a wax printer. The wax printer is a solid printer, and solid type wax is inserted instead of a liquid type ink into a general ink jet printer to heat the liquid to form a liquid state. Http://www.xeroxprinters.co.kr/ product / solidprinter.asp ).

(3) 0.1% (w / v) chitosan (448877, sigma) was dissolved in 0.5% acetic acid and 4 ㎕ was dropped in the test area (filter paper having a diameter of 6 mm).

(4) 20 占 퐇 of PBS containing 0.25% of glutaraldehyde was dropped to crosslink the chitosan. Treatment of the chitosan was performed to chemically immobilize the antibody. Antibodies in which the carboxyl group of the antibody binds to the -NH and -OH of chitosan and are immobilized are less lost during the washing process.

(5) washed three times with 20 μl of ultrapure water, and then dried.

(6) The filter paper used as the test area was assembled at the position of the test area of the paper chip. Each sample (anti-hCG antibody, hCG, HRP conjugated anti-hCG antibody, BSA in PBS, wash buffer (PBS) and substrate reaction solution (TMB)) was dropped at each location in the migration zone.

(7) When the test area was dry, it was placed on the electron pattern, and a voltage was applied to a moving area of 6 μl of anti-hCG antibody to move it to the test area and fixed at room temperature for 30 minutes.

(8) 3 μl of 0.025% Pluronic L64 Blocking Buffer containing 5% BSA in the test area was transferred to the test region by applying voltage to the transfer region and reacted at room temperature for 5 minutes.

(9) To remove unbound anti-hCG antibody and BSA, 10 μl of PBS containing 0.05% Tween 20 was transferred to the test area by applying voltage to the moving area and washed three times.

(10) 3 μl of hCG diluted to various concentrations was transferred to the test region by applying a voltage to the transfer region and reacted for 5 minutes.

(11) To remove unbound hCG, 10 μl of PBS containing 0.05% Tween 20 was washed three times in the test area by applying voltage to the moving area.

(12) After HRP-conjugated anti-hCG antibody was diluted 1,000-fold, 3 μl of the test region was transferred to the test region by applying voltage to the transfer region and reacted at room temperature for 5 minutes.

(13) To remove unbound HRP-conjugated anti-hCG antibody, 10 μl of PBS containing 0.05% Tween 20 was transferred to the test area by applying voltage to the moving area and washed three times.

(14) Substrate 3 μl of the reaction solution, TMB, was transferred to the test region by applying voltage to the moving region and reacted at room temperature for 10 minutes to induce color change.

(15) After photographs were taken using a mobile phone, the intensity of color developed was measured using an Image J program.

Experiment result

The solution used for the antigen-antibody reaction migrated according to the reaction sequence and reacted in the test area. Finally, when the substrate reaction solution moved to the test region, the degree of color development according to the antigen concentration could be distinguished. When the hCG concentration was plotted as a graph, a linear line was obtained, from which a limit of detection (LOD) of 5.81743 mIU / ml was obtained (FIG. 10).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (18)

Microfluidic chip for immunoassay of analytical samples comprising:
(a) a first region on which electrode drawings are printed using conductive ink and ink-jet printing; Wherein the first portion comprises an active zone in contact with a fluid selected from the group consisting of an assay sample, a blocking buffer, an antibody, a substrate and a wash buffer, The first portion moving in a direction perpendicular to the plurality of parallel electrodes; and a second portion moving in the second direction. And
(b) an antigen to be detected in the assay sample, (ii) an antigen to be detected in the assay sample, (iii) an antibody to the detection target antigen in the assay sample is immobilized, and the antigen- A second region to be generated; The first portion is in contact with the second portion and the fluid in contact with the first portion is moved to the second portion by electrowetting.
delete delete delete The microfluidic chip for immunoassay according to claim 1, wherein the electrode is electrically energized to move the fluid to a second site.
The microfluidic chip for immunoassay according to claim 1, wherein the conductive ink is a metal ink, a ceramic ink, or a molecular ink.
7. The microfluidic chip for immunoassay according to claim 6, wherein the ceramic ink is a carbon nanotube (CNT) ink.
The microfluidic chip for immunoassay according to claim 1, wherein the first portion or the second portion is paper or a film.
The microfluidic chip for immunoassay according to claim 1, wherein said first portion further deposits an insulator to form an insulating layer on said electrode figure.
10. The microfluidic chip for immunoassay according to claim 9, wherein the insulator is a glass, porcelain or polymer composition.
11. The microfluidic chip for immunoassay according to claim 10, wherein the polymer composition is parylene.
The microfluidic chip of claim 1, wherein the first portion further comprises a reinforcing agent on the electrode.
The microfluidic chip according to claim 12, wherein the reinforcing agent is polytetrafluoroethylene.
The microfluidic chip of claim 1, wherein the second portion further comprises a filtration membrane at the top.
15. The microfluidic chip according to claim 14, wherein the filtration membrane is a glass filtration membrane.
The microfluidic chip of claim 1, wherein the second portion further comprises a spacer and an adsorption pad at the bottom.
17. A diagnostic kit comprising the microfluidic chip for immunoassay according to any one of claims 1 to 16.
An immunoassay method comprising the steps of:
(a) a second portion of the microfluidic chip for immunoassay according to any one of (1) to (16), wherein (i) the target antigen in the assay sample, (ii) Antigen, or (iii) immobilizing the antibody against the antigen to be detected in the assay sample;
(b) selecting from the group consisting of an analytical sample, a blocking buffer, an antibody, a substrate and a washing buffer in the first portion of the microfluidic chip for immunoassay according to any one of claims 1 and 5 to 16 (Ii) the antigen for the detection target antibody in the assay sample, or (iii) the target antigen for the detection target in the assay sample by contacting the fluid to be detected in step (a) Generating an antigen-antibody immune response to the antibody; And
(c) detecting the antigen-antibody immune response.

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