KR100800436B1 - Bio sensor - Google Patents

Abstract

A bio-sensor is provided to be easy to manufacture and carry, have a simple structure, be mass-produced at low cost and quantitatively or qualitatively analyze an analyte without a washing step by moving particles using centrifugal force. A bio-sensor comprises at least one sample inlet(40) where a sample including an analyte is injected; at least one reaction chamber(50) which is communicated with the sample inlet and has a first binding material fixed at microparticles which recognize a specific portion of the analyte to be coupled to each other and a second binding material where a signal generation material recognizing the other portion of the analyte to be coupled thereto is fixed; at least one signal generation chamber(60) which is communicated with the reaction chamber and includes a signal inducing material generating a spectroscopic signal or an electrochemical signal by being reacted with the signal generation material; at least one particle capturing chamber(70) which is communicated with the signal generation chamber and captures the microparticles by centrifugal force; and at least one particle capturing chamber air outlet(78) which is communicated with the particle capturing chamber and discharges air existing in each of the chambers in accordance with the inflow of the sample, where the microparticle is an organic or an inorganic polymer particle such as latex bead, polystyrene bead, silica bead, agarose bead and dextrane bead; a metal particle such as magnet particle, god particle or silver particle; an organic or an inorganic polymer particle coated by the magnet, gold or silver particle; or a magnet, gold, or silver particle coated by the organic or inorganic polymer particle, the binding material is protein, protein A, protein G, DNA, RNA, peptide, antigen, antibody, avidin, biotin or a combination thereof, and the signal generation material is enzyme, organic or inorganic fluorescent material or a chelate compound binding to the fluorescent material.

Description

Bio sensor

1 is a side view of a biosensor according to an embodiment of the present invention.

2 is an overall configuration diagram of a biosensor according to an embodiment of the present invention.

3 is a cross-sectional view of the biosensor according to one embodiment of the present invention.

4A is a view showing the top surface of the upper substrate of the biosensor according to one embodiment of the present invention.

4B is a view showing the underside of the upper substrate of the biosensor according to one embodiment of the present invention.

4C is a view showing the side of the upper substrate of the biosensor according to one embodiment of the present invention.

5 is a view showing an intermediate substrate of a biosensor according to an embodiment of the present invention.

6 is a view showing the shape of the chambers in the biosensor according to an embodiment of the present invention.

7A is a view showing a lower substrate of a biosensor according to an embodiment of the present invention.

7B is a view showing a lower substrate of the biosensor according to one embodiment of the present invention.

7C is a diagram illustrating a lower substrate of a biosensor according to one embodiment of the present invention.

7D is a diagram illustrating a lower substrate of a biosensor according to one embodiment of the present invention.

8 is a view showing a reaction for generating a signal in a biosensor according to an embodiment of the present invention.

9 is a view showing a reaction for generating a signal in the biosensor according to an embodiment of the present invention.

10 is a view showing the operating principle of the biosensor according to an embodiment of the present invention.

11 is a view showing the operating principle of the biosensor according to an embodiment of the present invention.

<Description of the code for the main part>

10: upper substrate 20: intermediate substrate

30: lower substrate 40: sample inlet

45: sample inlet hole 50: reaction chamber

51: reaction chamber flow path 55: reaction chamber hole

60: sample generating chamber 61: signal generating chamber flow path

65: sample generating chamber hole 70: particle collection chamber

71: particle collection chamber flow path 75: particle collection chamber hole

77: air outlet 78: air outlet

80: waste storage chamber 81: waste storage chamber flow path

85: waste storage chamber hole 90: fixing groove

100: biosensor 200: working electrode

210: reference electrode 220: sample recognition electrode

230: sample recognition electrode 240: reference electrode lead wire

250: sample recognition lead wire 260: working electrode lead wire

270: sample recognition lead wire 280: biosensor recognition electrode

300: reaction layer 310: signal generating layer

601: fine particles 603: analyte

604: Specific region of the analyte 610: Similar analyte

The present invention relates to a biosensor using the movement of fine particles by centrifugal force.

A biosensor is a device capable of selectively detecting a substance to be analyzed by combining a biological receptor having a recognition function with a specific substance with an electrical or optical transducer to convert biological interaction and recognition reaction into an electrical or optical signal. Substances include common chemicals as well as biomaterials such as DNA, antibodies, antigens or blood sugar. Biological receptors are biomolecules that selectively recognize analytes and generate signals that can be measured by the transducer, and enzymes, proteins, DNA, cells, hormones, biofilms, tissues, and the like are used. Electrochemical, optical, magnetic, piezoelectric and electronic methods are used to convert the generated biosignals or recognition reactions into useful signals. Ultimately, electrical signals are obtained.

The structure of the biosensor is a form in which a biological detection element is coupled to the surface of a conventional physical and chemical sensor. That is, it is a structure for utilizing the characteristics of high selectivity and high sensitivity of the biological sensing element that selectively binds and reacts with analyte or substrate. Thus, in biosensors, three key elements are the biological sensing element used, the signal transducer, and the immobilization method for connecting the two. Biological sensing elements include enzyme / substrate of enzyme reaction, antibody / antigen of immune response, hydrogen bonding between complementary sequences of DNA, receptors, microorganisms, cells / Muscle / organ (cell / tissue / organ) and the like is used. Adsorption, microencapsulation, entrapment, cross-linking, covalent bonding, and the like are used for fixing the biological sensing element. Finally, as the signal converters used, electrochemical, optical, piezoelectric, surface acoustic wave, and thermal transducers are used.

The biosensor glucose oxidase to offer 1962 Clark first enzyme electrode (enzyme electrode); first known as blood glucose sensor (glucose sensor) that combines (glucoseoxidase below GOD) and an oxygen sensor (O ₂ sensor). Since then, blood glucose sensors have been widely used in clinical practice as the most successful biosensors.

The application fields of biosensors can be broadly divided into medical, environmental, and industrial applications. Medical biosensors, the largest market, include metabolic sensors such as blood sugar, microbial detection sensors, hormone sensors, and marker markers for various diseases. This is marketed / developed. In addition, environmental sensors include BOD (biological oxygen demand) sensors, various pollutant detection sensors, heavy metal and toxic substance detection sensors, biochemical weapon detection sensors, etc., and industrial food and bioprocess sensors as fermentation test and food Safety testing, animal and plant diseases and quality control sensors, and bioprocess measurement and control sensors.

To date, most clinical diagnostic analytical devices for detecting small amounts of analytes in fluids are designed for efficiency and economics by designing devices for multiple sample preparation and automated reagent addition and for analyzing large numbers of test samples in parallel or in series. Improvements were made. Often these automated reagent preparation devices and automated multiple analyzers are integrated into a single device. This type of clinical analyzer can accurately and automatically perform hundreds of analyzes with a small sample and reagents within an hour.

However, these analyzers were large and expensive, so they were purchased and used only in central laboratories and hospitals. This requires the transport of samples to laboratories and hospitals and often leads to the inability to quickly analyze urgent samples. Therefore, in order to overcome these problems, there is an urgent need for a clinical analysis device that can be easily handled by anyone.

Recently, a bio disc has been developed as such a small clinical analysis device.

In general, as a known technique, an analysis apparatus using an optical compact disc is known as 'Confocal compact scanning optical microscope based on compact disc technology' (1991, Vol. 30, No, 10, Applied Optics), and 'Gradient-index'. pobjectives for CD applications' (April, 1987, Vol26, Issue 7, Applied Optics) is published in the paper.

In addition, as a device for forming a fluid film on the surface of the disk using the centrifugal force of the sample injected into the injection hole on the disc, the centrifugal force of the 'Disc for centrifuge' and the fluid sample injected into the injection hole on the disc in EP 1075800 Separating disks for centrifuge is disclosed in European Patent No. 3,335,946 as an apparatus for separating a sample while moving to a channel and a chamber using the.

Furthermore, as a device for chemical analysis by centrifugal force and optical measurement on a disc, 'Disc centrifuge photosedimentometer' is disclosed in US Patent No. 4,311,039.

Korean Patent No. 590902 discloses a nucleic acid hybridization method and apparatus using a cleavage technique that specifically reacts with complementary double or single strands of nucleic acids and oligonucleotides. The analysis method includes the steps of: (a) dropping the sample into the sample inlet near the center of the disk installed in the nucleic acid hybrid analysis device; (b) rotating the disk to stop the rotation after the sample front reaches the outer edge of the disk; (c) a hybrid reaction step of culturing the disk at a room temperature at a steady state; (d) a first washing step of adding the buffer solution to the washing liquid while strongly rotating the disk; (e) a cutting step of placing a restriction enzyme solution that cuts only double strands or single strands of the nucleotide sequence and incubating the disk in a stagnant state; (f) a second washing process step of washing by adding a buffer while strongly rotating the disk, or by applying an external electric or magnetic field; And (e) drying the disk and reading by a detector comprising the optical, electrochemical or capacitance and impedance device, programmed to inspect a predetermined site on which a cleavable signal element is deposited.

Korean Patent Laid-Open Publication No. 2005-0118651 discloses a nucleic acid analysis device including a microvalve device using a microbead for controlling the flow or flow of a fluid. Specifically, the nucleic acid analysis device includes sample injection means for injecting a sample containing a nucleic acid; A preparation chamber for preparing DNA or RNA from the sample; A PCR chamber for amplifying the DNA or RNA; An array chamber for hybridizing the DNA or RNA with a capture probe; A trech chamber for collecting the unhybridized debris; A nucleic acid analysis apparatus including a plurality of chambers for storing various enzymes and buffer solutions required for each process, characterized in that to use a microvalve device to move the fluid between the chambers.

However, the prior arts have a disadvantage in that the biological sensing element is fixed to the substrate, which requires a cleaning process and the device is complicated.

Therefore, the present inventors are researching to develop a biosensor that is easy to manufacture, easy to carry, and has a simple structure, and can be mass-produced at low cost. Particles can be moved by centrifugal force, thereby confirming that the analyte can be qualitatively and quantitatively analyzed without washing, thereby completing the present invention.

An object of the present invention is to provide a biosensor that does not require a cleaning process by using the movement of fine particles by centrifugal force.

In order to achieve the above object, the present invention

At least one sample inlet located on a central axis or on a concentric circle, into which a sample containing the analyte is injected;

The primary binding material that is radially in communication with the sample inlet and is fixed to the fine particles that selectively recognize specific sites of the analyte in the sample and binds to each other, and is different from the recognition site of the primary binding material for the analyte. At least one reaction chamber formed with a secondary binding material having a fixed signal generating material for recognizing and binding a specific site;

At least one signal generating chamber in communication with the reaction chamber, the signal inducing material reacting with the signal generating material to generate a spectroscopic signal or an electrochemical signal;

At least one particle collecting chamber in communication with the signal generating chamber, wherein the fine particles are collected by centrifugal force; And

At least one particle collection chamber air outlet in communication with the particle collection chamber and configured to discharge air present in each chamber according to the inflow of the sample;

It provides a biosensor configured to include.

Alternatively, the reaction chamber is in communication with the sample inlet, a binding material fixed to the fine particles to selectively recognize and bind to a specific portion of the analyte in the sample, the analyte or the binding material selectively It may be at least one reaction chamber in which a signal generator is fixedly introduced into a similar analyte having a specific portion of the analyte that recognizes and binds to each other.

Further, alternatively, the reaction chamber is in communication with the sample inlet, the binding material is fixed to the signal generating material that selectively recognizes and binds to a specific portion of the analyte in the sample, the analyte or the binding material The selective analyte having a specific portion of the analyte that selectively recognizes and binds to each other may be at least one or more reaction chambers fixedly introduced into the microparticles.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

1 is a side view of a biosensor according to an embodiment of the present invention.

Biosensor according to the present invention is configured by loading two or more substrates, preferably two to three substrates may be loaded.

2 and 3 are an overall configuration diagram and a cross-sectional view of the biosensor according to an embodiment of the present invention, respectively.

In the biosensor according to the present invention, one or more sample injection holes 40 may be positioned on a central axis or concentric circles, and a sample containing analyte is injected. The injected sample moves to the reaction chamber 50, the signal generating chamber 60, and the particle collecting chamber 70 through the flow path. An air outlet 77 and an air outlet 78 are provided at the end of the particle collection chamber 70 to discharge the air inside the biosensor for easy injection of the sample. The particle collection chamber air outlet 78 is preferably located at least closer to the signal generation chamber flow path 61 from the sample inlet 40 to prevent the liquid sample from flowing out by the rotation of the biosensor. The signal generating chamber may be formed in various directions, such as an upper side, a lower side, a left side, and a right side.

In the biosensor according to the present invention, the reaction chamber 50 is in radial communication with the sample inlet 40, and the analysis of the analyte and the signal generating material in the sample is coupled to the inside of the reaction chamber 50. A substance and a binding substance that selectively recognizes at least one or more of the similar analytes having a structure similar to the analyte and bind to each other are fixed to the fine particles independently or mixed with each other.

At this time, the fine particles are organic or inorganic polymer particles including latex beads, polystyrene beads, silica beads, agarose beads, dextrin beads; Metal particles including magnet particles, gold particles, and silver particles; Particles in which gold or silver is partially or entirely coated on the organic or inorganic polymer particles; Magnetic particles, gold particles, particles coated with a polymer on the surface of the silver particles may be used, the size of the fine particles is 0.001 to 3 mm in diameter, preferably 0.01 to 2 mm, more preferably 0.05 to 1.1 mm. . When the size of the microparticles is less than 0.001 mm in diameter, it is difficult to move the microparticles by centrifugal force, and when the size of the microparticles exceeds 3 mm, the density of the binding material fixed to the surface of the microparticles is lowered, thereby lowering the reactivity. There is.

The binding agent may be protein, protein A, protein G, DNA, RNA, peptide, antigen, antibody, avidin, biotin, or a combination thereof.

The signaling material may be an enzyme, an organic or inorganic fluorescent material, or a chelating compound, a chemiluminescent material, an electrochemical light emitting material, etc. which bind to the fluorescent material, wherein the enzyme is glucose oxidase, glucose dehydrogenase, alkali phosphatase, can be used for peroxidase, etc., in the fluorescent material is Sm ^{3 +} ion, Eu ^{3 +} ions, Tb ^{3 +} of the lanthanide series of ions, such as a fluorescent material or a fluorescence signal in combination with the phosphor of the lanthanide series The substance which amplifies can be used preferably.

The fine particles to which the binder is fixed may be prepared using a known method or commercially available, and in general, a solution containing the fine particles to which the binder is fixed may be applied to the reaction layer and dried to form a reaction layer. Can be formed.

In addition, it is preferable to use a polymer binder in order to stably form a reaction layer made of fine particles in which a binding material is fixed in the reaction chamber 50. The reaction layer is formed by external physical impact even after drying. This is because it prevents destruction.

At this time, the polymer used may be polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), cellulose acetate, poly Water-soluble polymers such as amide, polyethylene glycol (PEG), gum, gelatin, agarose, dextran, polyacrylic acid can be used, and the content of the polymer is preferably 0.02-5%. If the content of the polymer is less than 0.02%, the binding capacity of the polymer is too low to destroy the reaction layer in the external impact. If it exceeds 5%, the binding capacity of the polymer is so strong that the binder does not loosen when the sample solution is injected. There is a problem of low reactivity.

When the sample is introduced into the reaction chamber 50, the analyte present in the sample and the binding material fixed to the microparticles and other signal generating materials that cause signal generation combine to form a complex (compelx), In the formation of the complex, the reaction mechanism used in the conventionally used biosensor may be used, for example, as shown in FIG . 8 .

8A illustrates that a primary binding material 602 that selectively reacts with and binds to a specific portion 604 of an analyte 603 in the reaction chamber is subjected to chemical or physical adsorption on the surface of the fine particles 601. When a second particle that is fixed by the microparticles and selectively reacts with other specific portions of the analyte and binds therein and is coupled with a signal generating material is introduced, the two binders are separated from each other when the sample is introduced. It is shown that other parts are selectively recognized and combined to form a complex.

8B illustrates that a binding material 602 that selectively recognizes and binds a specific portion 604 of the analyte 603 in the reaction chamber is fixed to the surface of the fine particles 601 by chemical or physical adsorption. When the signal generating material 607 is introduced into the analogous material 610 having the fine particles 601 and the specific region 604 of the analyte, when the sample is introduced, the analyte 603 is Reacts competitively with the analogous analyte 610 introduced into the reaction chamber to form a complex.

(C) of FIG. 8 selectively shows the fine particles 601 in which the analog analyte 610 is fixed inside the reaction chamber, and the specific region 604 of the analyte 603 or the analog analyte 610. In the case where the binding material 602 in which the reaction material binds to the signal generating material 607 is introduced, the analyte 603 and the analogous analyte 610 fixed to the microparticles 601 at the time of the sample inflow are introduced. Competitively reacts with the binding material 602 to form a complex.

The microparticle-binding material-analyte-signal complex formed in the reaction chamber 50 is moved to the signal generating chamber 60 by the rotation of the biosensor.

In the biosensor according to the present invention, the signal generating chamber 60 is in communication with the reaction chamber, the signal generating material activated in the microparticle-binding material-analyte-signaling complex formed in the reaction chamber 50. The spectroscopic signal or the electrochemical signal is generated by reacting with the signal generating material in the signal generating chamber. The signal generated can thus be measured spectroscopically or electrochemically.

The spectroscopic measurement refers to a method of measuring a change in concentration of a coloring reagent by absorbance, reflected light, chemiluminescence, or transmittance, or measuring a fluorescence signal generated by a chemiluminescence, electrochemiluminescence, or fluorescent material. It refers to a method of measuring the oxidation or reduction current of a material capable of oxidation / reduction produced or consumed by a reaction between a signal generating material introduced from the reaction chamber 50 and a signal generating material present in the signal generating chamber 60. .

For example, when the signal is spectroscopically measured, at least one of the surfaces provided with the signal inducing material or the opposite surfaces in the signal generating chamber is preferably made of a material having a visible light transmittance of 50% or more. , The material preferably contains glass, polycarbonate, polyethylene terephthalate, polystyrene polymer and the like.

When the signal is measured electrochemically, the signal generating chamber is generated by the reaction of the signal generating material and the signal generating material on at least one or both surfaces of the signal generating material or the surface facing each other in the signal generating chamber. It is provided with a working electrode, a reference electrode and a lead wire which induce an oxidation or reduction reaction of a substance or cause an oxidation or reduction reaction of a signal inducing substance that is consumed by the reaction between the signal generating substance and the signal inducing substance. desirable.

In addition, the biosensor according to the present invention comprises a biosensor recognition electrode made of a conductive material for measuring position recognition and rotation speed; And at least one sample recognition electrode or lead wire on at least one substrate surface for detecting whether the sample flowing into the biosensor is normally introduced.

The sensor recognition electrode 280 is an electrode made of a conductive material to measure the type, location recognition, and rotation speed of the biosensor, and may be provided on at least one substrate surface. The number is not limited.

The sample recognition electrodes 220 and 230 are preferably used as electrode pairs, and as shown in FIG . 7D , one is preferably formed in the reaction chamber flow path 51, and the other is the particle collecting chamber 70. It is preferably formed at one end of the) or the air discharge passage (77). After the sample is injected, the resistance between the sample recognition electrodes 220 and 230 may be measured or the conductivity may be measured to determine whether sufficient sample is introduced or bubbles are present in the sample. The number of sample recognition electrodes 220 and 230 is not limited.

The electrodes or lead wires may be thick film printed carbon, graphite, silver, silver chloride; Thin film coated gold, silver chloride, palladium, titanium oxide, a conductive material, or a material composed of a polymer containing the conductive material, etc. may be used, and the number or formation position of the electrode or lead wire is not limited.

It is preferable that the thick film uses a screen printing technique, and the thin film coating may be formed by a sputtering technique.

As shown in FIG . 7C , a portion of the electrode and the lead wire may be coated with the insulating layer 290, wherein the insulating layer 290 is made of a water-insoluble material. The insulating film 290 protects the electrode or lead wire from insulation, chemical corrosion or physical shock.

The signal inducing material includes an enzyme substrate capable of oxidation / reduction by reacting with an enzyme that is a signal generating material; Alternatively, a material for amplifying a fluorescent signal by reacting with a fluorescent material, a chemiluminescent material, or the like may be used. For example, glucose, hydrogen peroxide (H ₂ O ₂ ), ferricyanide (Fe (CN) ₆ ^3- ) ions, hexamine ruthenium (III), Ru (NH ₃ ) ₆ ³⁺ ) , Ferrocene, 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidine, TMB), o-phenylenediamine dihydrochloride (OPD), 2 , 2'-azino-di- [3-ethyl-benzothiazoline-6-sulfonic acid] diammonium salt (2,2'-azino-di- [3-ethyl-benzothiazoline-6-sulfonic acid] diammonium salt, ABTS) , 3-methyl-2-benzothiazolinone hydrazone (MBTH), p-nitrophenyl-phosphate (pNPP), adamantly methoxy phosphoryloxyphenyl dioxetane (AMPPD) Mediators including the like; A coloring reagent, etc. can be used.

The material for amplifying the fluorescent signal may be used a lanthanide-based fluorescent material such as Sm ^{3 +} ions, Eu ^{3 +} ions, Tb ^{3 +} ions or a material that amplifies the fluorescent signal by combining with a lanthanide-based fluorescent material. For example, a material that reacts with a fluorescent material to amplify a fluorescent signal is 2-naphthoyltrifluoroacetone (β-NTA), trioctylphosphineoxide (TOPO), 3,4-bis [4- (4,4,5,5,6,6,6, -heptafluoro-1,3-dioxohexyl) phenyl] benzenesulfonyl chloride (3,4-Bis [4- (4,4 , 5,5,6,6,6, -heptafluoro-1,3-dioxohexyl) phenyl] benzenesulfonyl choride (BHHCT), N, N, N ', N' [2,6-bis (3'-aminomethyl- 1′-pyrazolyl) -4-phenylpyridine] tetrakis (acetic acid) (N, N, N ′, N ′ [2,6-bis (3′-aminomethyl-1′-pyrazolyl) -4-phenylpyridine] tetrakis -(acetic acid), BPTA), 5- (4'-chlorosulfo-1 ', 1'-diphenyl-4'-yl) -1,1,1,2,2-pentafluoro-3,5 ′ -Pentanedione ( 5- (4'-chlorosulfo-1 ', 1'-diphenyl-4'-yl) -1,1,1,2,2-pentafluoro-3,5'-pentanedione, CDPP), 4-methyl-umbeli Peryl phosphate (4-methyl-umbelliferyl phosphate (4-MUP), 8-anilino-1-naphthalenesulfonic acid (ANS), benzidine, prussian blue, 4-aminophenazone, 4-aminophenazone, 2,4-dichlorophenol, 4-aminoantipyrin (AAP), hydroquinone, and the like can be used.

The chemiluminescent material is luminol (luminol), phenol, p-iodophenol (p-iodophenol), acridan (acridan), acyl hydrazide, imidazole, acridinium ester ( acridinium ester), peroxyoxalate, tris (2,2'-bipyridine) ruthenium, and the like can be used.

In the biosensor according to the present invention, the particle collecting chamber 70 communicates with the signal generating chamber and is a space in which the fine particles are collected by centrifugal force, and the rotation of the biosensor in the signal generating chamber 60 is performed. As a result, the fine particles are moved to the particle collection chamber 70.

In the biosensor according to the present invention, the shapes of the chambers are circular, semicircular, elliptical; Polygon shapes including triangles and squares; Alternatively, the polygonal shape may include a shape in which curves or bends are added to the polygonal shape, and the chambers may communicate with each other by a microchannel, or may communicate with each other without a microchannel. For example, as shown in FIG . 6 , the particle collecting chamber 70 is connected to the signal generating chamber 60 by the particle collecting chamber flow path 71 or directly generates a signal without the particle collecting chamber flow path 71. The particle collection chamber 70 may be formed in a portion of the signal generating chamber 60 or the chamber 60. In addition, the reaction chamber, the signal generation chamber and the particle collection chamber may be formed in direct communication without a micro-channel.

In the biosensor according to the present invention, the waste storage chamber 80 collects a sample remaining after the sample is injected in an excessive amount to fill the reaction chamber 50, the signal generating chamber 60, the particle collection chamber 70, and the like. The remaining sample is introduced into the waste storage chamber 80 along the waste storage chamber flow path 81, and at a predetermined position of the waste storage chamber 80 to facilitate the inflow of the remaining sample. A waste storage chamber air outlet 82 is formed, the number and location of which are not limited.

The fixing groove 90 is a groove formed for fixing the biosensor to the signal measuring device rotor.

In the biosensor according to the present invention, the shape of the biosensor is circular; Polygon shapes including triangles and squares; Or it may be configured as a shape in which a curve or bend is added to the polygonal shape.

4A, 4B and 4C illustrate an upper substrate of a biosensor in accordance with one embodiment of the present invention. 4A shows the top surface of the upper substrate, FIG. 4B shows the bottom surface of the upper substrate, and FIG. 4C shows the side surface of the upper substrate.

As shown in Figures 4a, 4b and 4c , the upper substrate 10 is a circular sample inlet 40 located in the center of the substrate, a reaction chamber flow path 51 radially located from the sample inlet, The signal generating chamber flow path 61 spaced apart from the reaction chamber flow path by a predetermined distance, the waste storage chamber 80 formed by combining with the intermediate substrate 20 and the lower substrate 30 at the substrate end portion, the waste storage chamber 80 The waste storage chamber air outlet 82 formed at a predetermined position, the waste storage chamber flow path 81 formed to be integrally connected with the waste storage chamber 80 from one side of the sample inlet, the intermediate substrate 20 and the lower substrate ( It comprises an air discharge passage 77 formed in the end portion of the particle collection chamber 70 formed in combination with 30) and an air outlet 78 formed at the end of the air discharge passage (77).

The material of the upper substrate 10 is preferably transparent or opaque plastic or glass, it can be produced by injection by a method widely used in the art.

5 shows an intermediate substrate 20 of a biosensor in accordance with one embodiment of the present invention.

As shown in FIG . 5 , the intermediate substrate 20 is combined with the upper substrate 10 and the lower substrate 30 to form a sample inlet hole 45 and the upper substrate 10. And a reaction chamber hole 55 for coupling with the lower substrate 30 to form the reaction chamber 50, the signal generating chamber 60 in combination with the upper substrate 10, and the lower substrate 30. Particle collection chamber hole 75 and the upper substrate to form a particle collection chamber 70 in combination with the signal generating chamber hole 65, the upper substrate 10 and the lower substrate 30 to form a And a waste storage chamber hole 85 for combining with the lower substrate 30 to form a waste storage chamber 80.

The reaction chamber hole 55, the signal generating chamber hole 65, or the particle collection chamber hole 75 formed in the intermediate substrate 20 may have a polygonal shape such as a triangle or a quadrangle, or a curved or curved shape may be added to the polygonal shape. Shape, or circular semicircular oval or the like. In addition, as shown in FIG . 6 , the particle collecting chamber hole 75 may be formed independently of the signal generating chamber hole 65 or may be connected to the signal generating chamber hole 65. In FIG. 6 , (a) shows an embodiment in which the particle collecting chamber 70 is connected to the signal generating chamber 60 by the particle collecting chamber flow path 71, and (b) shows directly without the particle collecting chamber flow path 51. (C) is an embodiment in which a particle collecting chamber 70 is formed in a part of the signal generating chamber 60, and (d) is a reaction chamber 50, a signal. The generating chamber 60 and the particle collecting chamber 70 are configured without a connecting flow path.

The material of the intermediate substrate 20 is preferably a transparent or opaque polymer, and an adhesive layer for attaching the upper substrate 10 and an adhesive layer for attaching the lower substrate 30 and for fixing the two adhesive layers. It consists of an adhesive substrate.

The thickness of the intermediate substrate 20 is preferably 0.1 to 5 mm, more preferably 0.1 to 0.3 mm.

Figures 7a-7d shows a lower quality group 30 of the biosensor in accordance with one embodiment of the invention.

As shown in Figure 7a , the lower substrate 30 comprises a fixing groove 90 for fixing to the signal measuring device rotating body on one side of the substrate. Preferably, the fixing groove is provided with at least one, and the forming position may be formed in the lower substrate as well as the other substrate. As further shown in FIG . 7B , A reference electrode 210, a reference electrode lead wire 240, a working electrode 200, and a working electrode lead wire for measuring an electrical signal by oxidizing or reducing a signal material generated or consumed in the signal generation chamber 60. 260, sample recognition electrodes 220 and 230 and sample recognition electrode lead lines 250 and 270 for recognizing a sample flowing into the biosensor may be formed.

The lower substrate 30 may be made of transparent or opaque plastic or glass. In particular, the material of the lower substrate 30 in the case of measuring spectroscopic signals such as fluorescence, phosphorescence, chemiluminescence, absorption, reflection, and transmission is made of glass, polycarbonate polymer or polyethylene having a light transmittance of 50% or more at 450 nm wavelength. It is preferable to use a polymer made of a transparent material such as terephthalate, polystyrene, and polyvinyl chloride.

In the case of measuring the electrical signal when measuring the signal, as shown in FIG . 7B , the lower substrate 30 is used to measure the current generated by the oxidation or reduction reaction of the signal measuring material. It may be composed of a working electrode 200, a reference electrode 210, a plurality of working electrode lead wires 260 and the reference electrode lead wires 240 to the substrate 30 for connection with the electrode and the signal measuring device It is provided on one side of the, the reference electrode 210 and the working electrode 200 is formed in the signal generating chamber (60). Taking amperometry as an example, a constant voltage is applied to the working electrode 200 with reference to the reference electrode 210 through the reference electrode lead wire 240 and the working electrode lead wire 260 in the measuring instrument. The signal measuring material is oxidized at the surface of the working electrode to produce an oxidizing current or to reduce a producing current. The amount of oxidation current or reduction current generated depends on the concentration of the analyte, so the concentration can be determined by measuring the current. It is common to form one reference electrode 210 in one working electrode 200, but the number and position of forming the reference electrode 210 are not limited in this specification.

In addition, as shown in FIG . 7C or 7D , a plurality of sample recognition electrodes 220 and 230 and sample recognition electrodes connected thereto are additionally used to detect whether the sample is normally introduced into the biosensor into the lower substrate 30. Lead wires 250 and 270 may be provided. It is preferable to use the sample recognition electrodes 220 and 230 as electrode pairs, in which one is preferably formed in the reaction chamber flow path 51, and the other is one end or air of the particle collection chamber 70. It is preferable that it is formed in the discharge path 77. After the sample is injected, the resistance between the sample recognition electrodes 220 and 230 may be measured or the conductivity may be measured to determine whether sufficient sample is introduced or bubbles are present in the sample. The number and formation positions of the sample recognition electrodes 220 and 230 are not limited.

The material constituting the electrode and the lead wire is composed of a conductive material such as thick film printed carbon, graphite, silver, thin film coated gold, silver, titania, or a polymer containing the conductive material. The thick film is preferably a screen printing technique, and the thin film coating may be formed on the substrate 30 by the sputtering technique.

In addition, a portion of the electrode and the lead wire may be coated with the insulating film 290, wherein the insulating film 290 is made of a water-insoluble material. The insulating film 290 protects the electrode or lead wire from insulation, chemical corrosion or physical shock.

The operating principle of the biosensor is as follows.

When the sample is injected through the sample inlet, the sample is reacted to the reaction chamber flow path 51, the reaction chamber 50, the signal generation chamber flow path 61, the signal generation chamber 60, the particle collection chamber flow path 71, and the particle collection chamber. 70 is filled, and the excess sample moves to the waste storage chamber 80 through the waste storage chamber flow path 81. In the reaction chamber 50 for a predetermined time, a binding reaction occurs between the analyte and the binding agent and the signal generator fixed to the microparticles, either directly or through a competition reaction. After the binding reaction for a predetermined time, the microparticles in which the binding substance in the reaction chamber 50 is fixed by the rotation of the biosensor are moved to the signal generating chamber 60 by centrifugal force. The signaling material bound to the analyte also moves together, and the unbound signaling or analyte remains in the reaction chamber 50. The signaling material fixed to the binding material that has moved to the signaling chamber 60 reacts with the signaling material to produce a product. When the biosensor rotates after a predetermined time, the microparticles existing in the signal generating chamber 60 move to the particle collecting chamber 70, and the signal inducing substance and the reaction product remain in the signal generating chamber 60. The product or signal-producing material absorbs light of a specific wavelength, is oxidized or reduced upon application of a constant voltage, or emits light of a specific wavelength. By measuring, the analyte present in the sample can be determined qualitatively or quantitatively.

Various reactions known in the biosensor may be used as a reaction for generating a signal. For example, the following reaction may be used.

An example of the reaction of Figure 8 generates a signal in the biosensor, and the reaction is specific portion 604 of, the fine particles 601 on the surface of the analyte 603, as illustrated in (a) of FIG. 8 The primary binding material 602 that selectively reacts and binds is introduced into the reaction chamber by being fixed chemically or by physical adsorption, and the secondary that selectively reacts and binds with other specific portions 605 of the analyte 603. The binding material 606 is coupled to the enzyme 607, which is a signal generating material, is introduced into the reaction chamber, and when the sample is introduced, the two binding materials selectively recognize and bind different portions of the analyte 603, or FIG. As shown in (b) of FIG. 8 , a binding material 602 that selectively recognizes and binds a specific portion 604 of the analyte 603 to the surface of the fine particles 601 is fixed by chemical or physical adsorption. Been in the reaction chamber The enzyme 607, which is a signaling material, is fixed to the analogous material 610 having the same material as the analyte 603 or a specific site 604 of the analyte, and is introduced into the reaction chamber. fixed Similar analyte 610 analyte 603 and competitively can be combined with a binding material (602), or, wherein the microparticles (601) analysis of the surface material as shown in (c) of FIG. 8 603 or a similar analyte 610 having a specific site 604 of the analyte is introduced into the reaction chamber fixed by chemical or physical adsorption, and the analyte 603 or similar analyte 610 The binding material 602 that selectively recognizes and binds to a specific site 604 is fixed to the enzyme 607 as a signal generating material and introduced into the reaction chamber, and the binding material 602 is an analyte 603 present in the sample. ) And the similar analyte 610 fixed to the fine particle 601 It may be bonded to the reactions. An enzyme substrate (608), which is a signal inducing substance that reacts with the enzyme (607), which is a signal generating material, is introduced into a signal generating chamber, and reacts with the enzyme (607) to produce a product (609). The consumption of 608 or reaction product 609 is measured to qualitatively or quantitatively determine analyte 603 present in the sample. The product 609 or the enzyme substrate 608 has a characteristic of absorbing light of a specific wavelength, oxidizing or reducing when a constant voltage is applied, or emitting light of a specific wavelength. In order to more efficiently obtain a large measurement signal, instead of directly fixing the enzyme 607 to the secondary binding material 606 or similar analyte 610 or binding material 602, biotin is fixed respectively. If biotin is fixed to the enzyme 607 and then avidin is added to the reaction chamber, the avidin-biotin bond causes a greater number of enzymes 607 to be bound to the secondary binding material 606 and similar analyte 610. Or fixed to each of the binding materials 602. In general, a large number of biotin may be immobilized to the enzyme 607, and one avidin may bind to four biotin, so that a large number of enzymes are secondary binding material 606 by an avidin-biotin complex. 610 or can be combined with the binding material 602

The enzyme may be used for glucose oxidase, glucose dehydrogenase, alkaline phosphatase, peroxidase, etc. The enzyme substrate is glucose, hydrogen peroxide, ferricyanide ion, ferrocyanide ion, hexaaminerucenium, ferrocene and ferrocene derivatives, Quinones and quinone derivatives, 3,3 ', 5,5'-tetramethylbenzidine, o-phenylenediamine dihydrochloride, 2,2'-azino-di- [3-ethyl-benzothiazoline-6-sulfonic acid] Diammonium salt, 3-methyl-2-benzothiazolinone hydrazone, p-nitrophenyl-phosphate, 4-methyl-umbelliferyl phosphate, 8-anilino-1-naphthalenesulfonic acid, benzidine, prussian blue, 4 -Aminophenazone, 2,4-dichlorophenol, 4-aminoantipyrine, hydroquinone and the like can be used.

FIG. 9 illustrates an example of a signal generating reaction in the biosensor, including fine particles 601, a primary binding agent 602, an analyte 603, specific regions 604 and 605, and a secondary of an analyte. The binding material 606 is the same as described in FIG. 8 , and the fluorescent material 651 is the secondary binding material 606 or the analyte 603 or similar analyte instead of the enzyme 607 used in FIG. 8 . It is coupled via a non-fluorescent chelate 650 to 610 or a binding material (602), and the enzyme substrate 608, instead of fluorescent amplification chelating 652 in FIG. 8 are used. The non-fluorescent chelate compound 650 is generally a material having no fluorescence, and binds to the fluorescent material 651 at pH 7 or higher, but dissociates the bound fluorescent material 651 at pH 4 or lower. Have The fluorescent material 651 uses lanthanide-based metal ions such as Sm ^{3 +} ions, Eu ^{3 +} ions, and Tb ^{3 +} ions, and the metal ions absorb excitation light in the UV region. It emits light in the visible region and shows a large stroke's shift. The long lifetime of fluorescence has the advantage that it can be measured by the time-resolved fluorometry (TRF) method.

In order to measure the fluorescence signal, the phosphor 651 is dissociated from the non-fluorescence chelate 650 under acidic conditions, and then the fluorescence signal is amplified using the fluorescence amplification chelate 652. The fluorescence amplification chelate 652 combines with a fluorescent material to form a stable complex 653 even under acidic conditions to generate a greater fluorescence signal than the combination of non-fluorescence chelate.

10 shows an example of the operating principle of the biosensor of the present invention using the signaling reaction of FIG. 8 .

Figure 10 (a) is according to one embodiment of the biosensor according to the present invention, represents the end face of the before sample injection biosensor, in the reaction chamber, the first binding material 602. The microparticles (601 fixed ) And the second binding material 606 combined with the enzyme 607 forms the reaction layer 300 on the lower substrate 30 in a state where the secondary binding material 606 is dried in the reaction chamber 50, and the enzyme reacts with the enzyme 607. The substrate 608 is formed on the lower substrate 30 in a dry state in the signal generating chamber 60.

Of Figure 10 (b) is a sample injection conditions, the sample reaction chamber flow path 51, the reaction chamber 50, a signal generating chamber flow path 61, a signal generating chamber 60, a particle collecting chamber passage (71 ) And the particle collection chamber 70, and the reaction chamber 50 binds between the analyte 603 and the primary binder 602 and the secondary binder 606 fixed to the fine particles for a predetermined time. Reaction takes place. As shown in (c) of FIG . 10 by the rotation of the biosensor 100 after a predetermined time binding reaction, the fine particles 601 to which the primary binding material 602 fixed in the reaction chamber 50 is fixed are centrifugal force. By moving to the signal generating chamber 60, wherein the secondary binding material 606 coupled to the analyte 603 bound to the primary binding material 602 also moves together, the secondary binding material that is not bound It remains in the reaction chamber 50. The enzyme 607 immobilized on the secondary binding material that has moved to the signaling chamber 60 reacts with the enzyme substrate 608 to produce the product 609. When the biosensor is rotated after the enzyme reaction for a predetermined time, as shown in FIG . 10 (d) , the fine particles 601 and the enzyme 607 to which the primary binding material 602 existing in the signal generating chamber 60 is fixed are fixed. Is moved to the particle collection chamber 70, the enzyme substrate 608 and the reaction product 609 remains in the signal generating chamber 60, the product 609 or enzyme substrate 608 is a light of a specific wavelength Absorbs, oxidizes or reduces when a certain voltage is applied, or emits light having a specific wavelength. Therefore, the consumption of the enzyme substrate 608 or the reaction product 609 is measured to determine the analyte present in the sample ( 603) can be measured qualitatively or quantitatively.

FIG. 11 shows an example of the operating principle of the biosensor of the present invention using the signaling reaction of FIG. 9 .

Of Figure 11 (a) is according to one embodiment of the biosensor according to the present invention, represents the end face of the before sample injection biosensor, in the reaction chamber, the first binding material 602. The microparticles (601 fixed ) And the secondary binding material 606, to which the fluorescent material 651 is coupled, forms the reaction layer 300 on the lower substrate 30 in a state where the secondary binding material 606 is dried in the reaction chamber 50, and reacts with the fluorescent material 651. The fluorescent amplification chelate 652 forms a signal generating layer 310 on the lower substrate 30 in a dried state in the signal generating chamber 60.

Of Figure 11 (b) is a sample injection conditions, the sample reaction chamber flow path 51, the reaction chamber 50, a signal generating chamber flow path 61, a signal generating chamber 60, a particle collecting chamber passage (71 ) And the particle collection chamber 70, and the reaction chamber 50 binds between the analyte 603 and the primary binder 602 and the secondary binder 606 fixed to the fine particles for a predetermined time. Reaction takes place. As shown in (c) of FIG . 11 by the rotation of the biosensor 100 after a certain time binding reaction, the fine particles 601 to which the primary binding material 602 fixed in the reaction chamber 50 is fixed are centrifugal force. Moves to the signal generating chamber 60 by the secondary binding material 606 coupled to the analyte 603, which is bound to the primary binding material 602, and moves together with the secondary binding material that is not bound. Remains in the reaction chamber 50. The fluorescent material 651 fixed to the secondary binding material that has moved to the signal generation chamber 60 is dissociated to react with the fluorescent amplification chelate 652 to form a fluorescent amplification complex 653. When the biosensor is rotated after a complex compound formation reaction for a predetermined time, as shown in FIG . 11 (d) , the fine particles 601 and the fluorescent material having the primary binding material 602 fixed in the signal generating chamber 60 are fixed. The secondary binding material 606 in which 651 is dissociated moves to the particle collection chamber 70, and the fluorescent amplification complex 653 remains in the signal generating chamber 60. By measuring the fluorescence of the remaining complex compound 653, the analyte can be qualitatively or quantitatively analyzed.

The present invention also provides a method of manufacturing the biosensor.

The biosensor attaches an intermediate substrate to a lower substrate to form chambers including a reaction chamber and a signal generating chamber, and then injects a solution containing a reactant and a signal generating substance into the formed reaction chamber and the signal generating chamber, respectively. After drying to produce a reaction layer and a signal generating layer, by combining the upper substrate to produce a biosensor.

At this time, the method of attaching the intermediate substrate to the lower substrate or the upper substrate may use a method widely used in the art.

Since the biosensor according to the present invention uses particles in a reaction chamber in a disk-type device, it can be moved by centrifugal force by rotation after the reaction, and thus does not require a cleaning process, is simple to manufacture, easy to carry, and simple in structure. It is economical because it can be mass produced inexpensively. In addition, it can be read even in low-cost equipment, and can be easily used for various diagnostics.

Hereinafter, the present invention will be described in detail through examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the following examples.

< Example  1> Manufacture of biosensors 1

<1-1> anti-alpha Chorionic gonadotropin  Alkaline phosphatase (ALP) Enzyme Fixation in Antibodies

Anti-alpha chorionic gonadotropin (HCG) antibody 1 in 1 ml of 0.1 M 2- [N-morpholino] ethane sulfonic acid (hereinafter referred to as MES, pH 6) buffer containing 0.5 M NaCl. 1 mg of mg and ALP enzyme were added and then completely dissolved. 0.4 mg of EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) and 1.1 mg of sulfo-NHS (N-hydroxysulfosuccinimide) were added to the solution, followed by reaction at room temperature for 2 hours, followed by 10 mM. To this, hydroxylamine was added and reacted at room temperature for 20 minutes. Subsequently, centrifugation was carried out for 10 minutes at 14,000 gravity using a Nanosep (Pall corporation, MI, USA) tube, which is a molecular weight cut-off (MWCO) 100 KDa for centrifugation, to remove molecules having a molecular weight of less than 100 KDa. ALP-bound anti-alpha chorionic stimulating hormone antibody solution was prepared by adding 1 ml of 0.1 M MES buffer solution containing 0.5 M NaCl to the remaining ALP enzyme-immobilized anti-alpha chorionic stimulating hormone antibody.

<1-2> Anti-beta chorionic stimulating hormone antibody immobilized on fine particles

5 ml of the MES buffer used in <1-1> was added to 1 ml of polystyrene carboxyl (diameter: about 9 µm, 10%, bangs laboratories, Inc., USA), followed by centrifugation to remove the supernatant. The buffer was added so that the volume was 1 ml. EDC and sulfo-NHS of the same concentration used in <1-1> were added to the solution, followed by reaction at room temperature for 30 minutes, 5 ml of MES buffer was added, and then centrifuged to remove the supernatant. After adding MES buffer to the precipitate to a volume of 1 ml, 1 mg of anti-beta-HCG antibody was added and reacted at room temperature for 2 hours. Thereafter, 0.1% bovine serum albumin (BSA) was added thereto, followed by reaction at room temperature for 30 minutes.

<1-3> reaction layer fabrication

0.5 ml of each of the solutions prepared in <1-1> and <1-2> was aliquoted and mixed, followed by addition of 0.05% PVP (polyvinylpyrrolidone K15) and 0.1% Tritone X-100 (New England Nuclear Corp.). After the complete dissolution, 15 μl was dispensed into the reaction chamber formed by attaching the intermediate substrate to the transparent lower substrate, and dried for 30 minutes in an oven at 30 ° C.

<1-4> Manufacture of signal generating layer and manufacture of bio sensor

After completely dissolving 0.1% PVP and 0.1% Triton X-100 in 20 ml of 1 M, pH 9.8, diethanolamine buffer solution containing 0.5 mM MgCl ₂ , p-nitrophenyl phosphate, PNPP, disodium salt was dissolved to 5 mM. Thereafter, 10 μl of the solution was dispensed into a signal generation chamber formed by attaching an intermediate substrate to a transparent lower substrate, and then dried in an oven at 30 ° C. for 30 minutes. After drying, a biosensor was manufactured by combining a transparent upper substrate thereto.

< Example  2> Manufacture of biosensor 2

<2-1> anti-alpha Chorionic gonadotropin  Antibody to glucose oxidase ( GOx A) fixed

The anti-alpha chorionic stimulating hormone antibody solution was prepared in the same manner as in Example 1-1, and the anti-alpha chorionic stimulating hormone antibody solution in place of ALP enzyme glucose oxidase ( After the addition of 1 mg glucose oxidase) was reacted for 2 hours at room temperature. Thereafter, hydroxylamine was added to a final concentration of 10 mM, followed by reaction at room temperature for 20 minutes to fix glucose oxidase on the anti-alpha chorionic gonadotropin antibody.

<2-2> reaction layer preparation

<1-3> of Example 1 except for using the anti-alpha chorionic stimulating hormone antibody conjugated with the glucose oxidase in place of the anti-alpha chorionic stimulating hormone antibody conjugated to the alkaline phosphatase enzyme It was carried out in the same way as.

<2-3> Manufacture of signal generating layer and manufacture of biosensor

Complete dissolution of 0.05% PVP, 0.1% Triton X-100 in 20 ml of 100 mM, pH 7.4 Tris buffer solution containing 200 mM glucose and 250 mM potassium ferricyanide (K ₃ Fe (CN) ₆ ) Then, 20 μl of the solution was dispensed into a signal generating chamber into which the working electrode and the reference electrode were introduced, and then dried in a 30 ° C. oven for 30 minutes. After drying, a biosensor was manufactured by combining a transparent upper substrate thereto.

< Example  3> Manufacture of biosensor 3

<3-1> anti-alpha Chorionic gonadotropin  On antibodies Biotin ( biotin A) fixed

After complete dissolution of 1 mg anti-alpha chorionic gonadotropin antibody in 50 mM phosphate buffer (pH 7.3), the concentration of Sulfo-NHS-LC-Biotin (PIERCE, USA) was added to 10 mM. After reacting at room temperature for 1 hour, using a Nanosep (Pall corporation, MI, USA) tube, which is a molecular weight cut-off (MWCO) 100 KDa for centrifugation, centrifuged at 14,000 gravity for 10 minutes to obtain a molecular weight of 100 KDa. Less than molecules were removed. Thereafter, 1 ml of 0.1 M MES buffer solution containing 0.5 M NaCl was added to the biotin-immobilized anti-alpha chorionic stimulating hormone antibody remaining in the upper layer to prepare a biotin-binding anti-alpha chorionic stimulating hormone antibody solution. It was.

<3-2> Glucose  Oxidase ( glucose oxidase )on Biotin  fixing

The procedure was performed in the same manner as in <1-2> of Example 1, except that the biotin was immobilized to the enzyme using glucose oxidase instead of the anti-alpha chorionic stimulating hormone antibody.

<3-3> Reaction layer  making

0.5 ml each of the solutions prepared in the above <3-1>, <3-2> and <1-2> was aliquoted and mixed, followed by addition of 0.05% PVP (polyvinylpyrrolidone K15) and 0.1% Tritone X-100. After complete dissolution, 10 μl was dispensed on one side of the reaction chamber, and dried in a 30 ° C. oven for 30 minutes. Also, after completely dissolving 0.1 mg of strap avidin (Streptavidin, PIERCE, USA) in 2 ml of 50 mM phosphate buffer (pH 7.3) containing 0.05% PVP and 0.1%, 3 μl of the reaction chamber was The other side was dispensed and dried for 30 minutes in an oven at 30 ℃.

<3-4> Manufacture of Signal Generation Layer and Manufacture of Biosensor

Fabrication of the signal generating layer and fabrication of the biosensor were performed in the same manner as in <2-3> of Example 2.

< Experimental Example > Signal Generation chamber  Signal measurement influence due to fine particles present in

(1) Effect of absorbance measurement by fine particles present in the signal generating chamber

In the biosensor of the present invention, the following experiment was performed to determine the effect of measuring the absorbance by the fine particles present in the signal generating chamber.

A urine sample in which villi gonadotropin is dissolved at a certain concentration is injected into a sample inlet of the biosensor prepared in Example 1 using a pipet, and then rotated at 60 rpm (rotation per minute) for 10 seconds. After the sample is completely filled in the reaction chamber, the signal generating chamber, and the particle collecting chamber, the sample is maintained for 10 minutes and then the biosensor is rotated at 550 rpm for 20 seconds to move the fine particles in the reaction chamber to the signal generating chamber. I was. After stopping for 10 minutes, and then rotated at 2000 rpm for 30 seconds to move the fine particles to the particle collection chamber, the absorbance of the signal generating chamber was measured. In the absorbance measurement, a light source used a light emitting diode (LED) of 405 nm and a photodiode as a detector (experimental group).

As a comparison group, all processes except the step of moving the fine particles to the particle collection chamber were performed under the same conditions as the experimental group, and the absorbance of the signal generation chamber was measured after stopping the biosensor for 30 seconds.

The experiment was conducted ten times for each of the five hCG concentrations, and the average and CV% were obtained, and the results are shown in Table 1.

hCG concentration (IU / ml) Measured value with fine particles present in the signal generating chamber Measured value after fine particles are removed from signal generation chamber Digit CV% Digit CV% 20 1.41 8.8 0.42 3.3 40 1.54 6.4 0.51 4.1 80 1.67 3.4 0.66 5.1 160 1.82 4.5 0.92 3.1 320 1.98 10.1 1.4 2.4

As shown in Table 1, when the microparticles are present in the signal generating chamber, the scattering of light or the transmission of light by the microparticles is disturbed, resulting in a high CV% of 3.4 to 10.1 at the measurement of each hCG concentration. Therefore, when quantitative analysis is difficult, when the fine particles are moved to the particle collection chamber, the CV% is relatively low with a value of 2.4 to 5.1, and thus the reproducibility is excellent, and the linearity of the calibration curve for the hCG concentration is fine. It was found to be good when the particles were transferred to the particle collection chamber.

(2) signal generation chamber  Current measurement effect due to fine particles present in

In the biosensor of the present invention, the following experiment was conducted to see how the microparticles present in the signal generation chamber affect the current measurement.

After injecting a urine sample with a certain concentration of chorionic gonadotropin using a pipette into the sample inlet of the biosensor prepared in Example 2, the fine particles were moved to the particle collection chamber by the method (1). Thereafter, a voltage of +0.5 V is applied to the working electrode based on the reference electrode to oxidize the reduced ferrocyanide (Fe (CN) ₆ ²⁺ ) ions generated by reacting with glucose oxidase at the working electrode. The oxidation current was measured to quantify the chorionic stimulating hormone present in the sample (experimental group).

All the procedures except the step of moving the fine particles to the particle collection chamber as a comparison group were performed under the same conditions as the above experimental group, and the oxidation current of the signal generation chamber was measured after stopping the biosensor for 30 seconds.

The experiment was conducted ten times for each of the five hCG concentrations, and the average and CV% were obtained, and the results are shown in Table 2.

hCG concentration (IU / ml) Measurement current in the presence of fine particles in the signal generating chamber Measurement current after fine particles are removed from the signaling chamber Digit CV% Digit CV% 20 37 4.3 48 3.4 40 48 3.5 57 4.1 80 72 4.4 72 3.3 160 85 3.2 99 4.1 320 151 5.0 161 3.7

As shown in Table 2, when microparticles are present in the signal generating chamber, the microparticles interfere with diffusion of the ferrocyanide produced by the enzymatic reaction to the electrode surface, so that the CV% is relatively high, but the difference is not large. It can be seen that the influence of the fine particles is less than the absorbance measurement measured in (1).

(3) Avidin - Biotin  Signal amplification effect by combining

In the biosensor of the present invention, the following experiment was performed to investigate the effect on the measurement signal amplification by the binding of avidin-biotin.

After the injection into the sample inlet of the biosensor prepared in Example 2, by moving the fine particles to the particle collection chamber by the method of (1), by applying a voltage of +0.5 V to the working electrode based on the reference electrode Determination of chorionic stimulating hormone present in the sample by measuring the oxidation current generated by oxidizing the reduced ferrocyanide (Fe (CN) ₆ ²⁺ ) ions produced by the reaction with glucose oxidase at the working electrode The results are shown in Table 3.

hCG concentration (IU / ml) Measured Current Without Avidin-Biotin Bond Current measured using avidin-biotin bond Digit CV% Digit CV% 20 48 3.4 66 3.3 40 57 4.1 90 2.4 80 72 3.3 130 3.7 160 99 4.1 177 4.5 320 161 3.7 280 3.8

As shown in Table 3, it was confirmed that the use of avidin-biotin binding (66 ~ 280 Digit), the electrical signal is amplified so that the electrical signal is measured larger than without the use of avidin-biotin binding (48 ~ 161 Digit) .

As described above, since the biosensor according to the present invention uses particles in which a biological sensing element is fixed in a reaction chamber in a disk-type device, it is possible to move by centrifugal force after the reaction, and thus a washing process is required. It is economical because it can be mass-produced at low cost because it is simple to manufacture, easy to carry and simple in structure. In addition, it can be read even in low-cost equipment, and can be easily used for various diagnostics.

Claims (17)

At least one sample injection port into which a sample containing the analyte is injected; A primary binding material in communication with the sample inlet and fixed to fine particles that selectively recognize a specific site of the analyte in the sample and bind to each other; and a specific site that is different from the recognition site of the primary binding material for the analyte At least one or more reaction chambers formed with a secondary binding material having a fixed signal generating material for recognizing and binding the same; At least one signal generating chamber in communication with the reaction chamber, the signal inducing material reacting with the signal generating material to generate a spectroscopic signal or an electrochemical signal; At least one particle collecting chamber in communication with the signal generating chamber, wherein the fine particles are collected by centrifugal force; And At least one particle collection chamber air outlet in communication with the particle collection chamber and configured to discharge air present in each chamber according to the inflow of the sample; Biosensor comprising a. At least one sample injection port into which a sample containing the analyte is injected; A binding material that is in communication with the sample inlet and is fixed to fine particles that selectively recognize a specific portion of the analyte in the sample and binds to each other, and the analyte or the analyte selectively recognizes and binds to each other. At least one reaction chamber into which a signaling material is immobilized and introduced into the analogous analyte having a specific site; At least one signal generating chamber in communication with the reaction chamber, the signal inducing material reacting with the signal generating material to generate a spectroscopic signal or an electrochemical signal; At least one particle collecting chamber in communication with the signal generating chamber, wherein the fine particles are collected by centrifugal force; And At least one particle collection chamber air outlet in communication with the particle collection chamber and configured to discharge air present in each chamber according to the inflow of the sample; Biosensor comprising a. At least one sample injection port into which a sample containing the analyte is injected; A binding material in communication with the sample inlet, the binding material having a signal generating material that selectively recognizes and binds to a specific portion of the analyte in the sample, and the analyte or the binding material selectively recognize and bind to each other; At least one reaction chamber into which a pseudoanalyte having a specific portion of the substance is fixedly introduced into the microparticles; At least one signal generating chamber in communication with the reaction chamber, the signal inducing material reacting with the signal generating material to generate a spectroscopic signal or an electrochemical signal; At least one particle collecting chamber in communication with the signal generating chamber, wherein the fine particles are collected by centrifugal force; And At least one particle collection chamber air outlet in communication with the particle collection chamber and configured to discharge air present in each chamber according to the inflow of the sample; Biosensor comprising a. The biosensor according to any one of claims 1 to 3, wherein the particle collecting chamber air outlet is located closer to the signal generation chamber than the sample inlet. The method of claim 1, wherein the fine particles comprise organic or inorganic polymer particles including latex beads, polystyrene beads, silica beads, agarose beads, or dextrain beads; Metal particles including magnetic particles, gold particles or silver particles; Or bio-sensors in which the organic or inorganic polymer particles are coated with some or all of the magnetic particles, gold particles or silver particles, or the organic or inorganic polymer particles are coated on the magnetic particles, gold particles or silver particles. . The method of claim 1, wherein the binding agent is at least one selected from protein, protein A, protein G, DNA, RNA, peptide, antigen, antibody, avidin, biotin, or a combination thereof. Biosensor. The biosensor of claim 1, wherein the signaling material is an enzyme, an organic or inorganic fluorescent material, or a chelate compound that binds to the fluorescent material. The biosensor of claim 7, wherein the enzyme is one or more selected from glucose oxidase, glucose dehydrogenase, alkaline phosphatase, and peroxidase. The biosensor of claim 7, wherein the fluorescent material amplifies a fluorescent signal by combining with a lanthanide-based fluorescent material or a lanthanide-based fluorescent material. The method according to any one of claims 1 to 3, wherein the signal generating material reacts with the signal generating material on at least one surface or both surfaces of the signal generating material or at an opposite surface in the signal generating chamber. Bio-induced oxidizing or reducing reaction of a material produced by the oxidizing agent or oxidizing or reducing reaction of a signal-causing substance that is consumed by the reaction between the analyte and the signal-causing substance and a reference electrode. sensor. The bio-material according to claim 10, wherein the electrode is a material composed of a conductive material including carbon, graphite, silver, silver chloride, thin film coated gold, silver chloride, palladium, titanium oxide, or a polymer containing the conductive material. sensor. The biosensor according to any one of claims 1 to 3, wherein the signal inducing substance is an enzyme substrate capable of reacting with an enzyme and capable of oxidation / reduction, or a material that amplifies a fluorescent signal by reacting with a fluorescent substance. The method of claim 12, wherein the enzyme substrate is glucose, hydrogen peroxide, ferricyanide ion, ferrocyanide ion, hexaaminerucenium, ferrocene and ferrocene derivatives, quinones and quinone derivatives, 3,3 ', 5,5'-tetra Methylbenzidine, o-phenylenediamine dihydrochloride, 2,2′-azino-di- [3-ethyl-benzothiazoline-6-sulfonic acid] diammonium salt, 3-methyl-2-benzothiazolinone hydrazone , p-nitrophenyl-phosphate, 4-methyl-umelliferyl phosphate, 8-anilino-1-naphthalenesulfonic acid, benzidine, prussian blue, 4-aminophenazone, 2,4-dichlorophenol, 4-aminoantipyrine Mediators including hydroquinone, luminol, phenol, p-iodophenol, acridan, acyl hydrazide, imidazole, acridinium ester, peroxyoxalate, tris (2,2'- A biosensor that is a chemiluminescent material or a coloring reagent comprising bipyridine) rudenium . The biosensor of claim 12, wherein the material for amplifying the fluorescent signal is to amplify the fluorescent signal by combining with a lanthanide-based fluorescent material or a lanthanide-based fluorescent material. The chamber of claim 1, wherein the chambers are circular, semicircular, elliptical; Polygon shapes including triangles and squares; Biosensor that is a shape in which the curved or curved is added to the polygonal shape. The biosensor according to any one of claims 1 to 3, wherein the chambers are in communication with each other by a microchannel, or in communication with each other without a microchannel. The biosensor according to claim 1, wherein the biosensor is circular; Polygon shapes including triangles and squares; Or a shape in which a curve or a bend is added to the polygonal shape.

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