KR20170032951A - Antigen-Antibody Detector and Antigen-Antibody Detecting Method Using Ultra-Violet Light Emitting Diode and Fluorescent Particles - Google Patents

Antigen-Antibody Detector and Antigen-Antibody Detecting Method Using Ultra-Violet Light Emitting Diode and Fluorescent Particles Download PDF

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KR20170032951A
KR20170032951A KR1020150130578A KR20150130578A KR20170032951A KR 20170032951 A KR20170032951 A KR 20170032951A KR 1020150130578 A KR1020150130578 A KR 1020150130578A KR 20150130578 A KR20150130578 A KR 20150130578A KR 20170032951 A KR20170032951 A KR 20170032951A
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light
wavelength
particles
particle
antigen
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KR1020150130578A
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Korean (ko)
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최규진
정상욱
윤정열
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서울바이오시스 주식회사
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Publication of KR20170032951A publication Critical patent/KR20170032951A/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
    • G01N15/0211—Investigating a scatter or diffraction pattern
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1468—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle
    • G01N15/1475—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle using image analysis for extracting features of the particle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N2015/0065—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials biological, e.g. blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
    • G01N2015/0238—Single particle scatter
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N2015/0693—Investigating concentration of particle suspensions by optical means, e.g. by integrated nephelometry

Abstract

The present invention relates to a method for detecting existence of an antigen and existence concentration by using a principle of making difference in interaction between particles and light radiated to the particles as an actual particle diameter of a fluorescent particle is changed since the fluorescent particle connected to an antibody aggregates around the antigen, and as the actual particle diameter is changed in a Mie scattering area. According to the present invention, the method for detecting the existence of a target antigen in a body fluid and the concentration of the target antigen includes: a step of preparing a particle emitting light of a second wavelength longer than a first wavelength after attaching the antibody reacting with the target antigen and absorbing the light of the first wavelength; a step of supplying the body fluid to the particle; and a step of radiating at least the light of the first wavelength to the particle to which the body fluid is supplied, and detecting the light emitted from the particle.

Description

{Antigen-Antibody Detector and Antigen-Antibody Detection Method using Ultra-Violet Light Emitting Diode and Fluorescent Particles}

More particularly, the present invention relates to an apparatus for detecting an antigen-antibody, and more particularly, to an apparatus and method for detecting an antigen-antibody, and more particularly, Mie scattering The detection of the presence and concentration of an antigen by using the principle that the scattering cross-sectional area differs according to the wavelength and the particle size of the light irradiated to the particle as the particle diameter changes, And a device using the same.

Currently, the ingredients contained in body fluids are often used as indicators related to health. As an example, the sugar component contained in the blood can serve as an important marker in determining whether or not it is diabetes. Particularly, in the case of suffering from a chronic disease, the health condition can be checked by periodically monitoring the components contained in the body fluids.

On the other hand, the body fluid may contain an infectious component such as various microorganisms, and the health condition may be diagnosed by analyzing an infectious component contained in the body fluid. In the case of conventional immunoassays for screening for infectious agents, a body fluid suspected of microbial infection is collected, cultured to make a colony, and then the cultured colonies are analyzed by biological or biochemical observation The analysis proceeds in the order. However, in the immunological assays described above, only about 1% of the microorganisms which can be cultured are known to be cultivable in the colony, and the time required for culturing may be at least two to three days, There is a problem that it is difficult to detect and analyze the real-time unit of the component.

Thus, there is a need for analytical techniques for the components of body fluids that can increase detection efficiency while reducing the effort involved in conventional biological or biochemical observations.

Thus, the applicant of the present invention has proposed a method of detecting the difference of the scattered light due to the non-scattering phenomenon and confirming whether or not the antigen-antibody reaction and the degree of the reaction are confirmed. However, in this method, it is necessary to locate the photodetecting device at a position where it is possible to detect the scattered light from the light irradiated to the particle and the scattered light from the particle can not be distinguished by the wavelength, It is difficult to implement the device in the point of view. In addition, there was no problem in that it was necessary to use various filters to detect the change of the scattered light.

H. C. van de Hulst, Light Scattering by Small Particles. Dover Publications, Inc. New York, 1981.

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and it is an object of the present invention to provide a technique for analyzing constituent components of body fluids capable of increasing detection efficiency.

It is another object of the present invention to provide an antigen-antibody detection technique capable of promptly and visually confirming the presence and concentration of an antigen.

Another object of the present invention is to provide an antigen-antibody detection technique capable of simplifying the structure of the analyzer and simplifying the analysis.

According to an aspect of the present invention, there is provided a fluorescence device comprising: a fluorescent particle to which an antibody reactive with a target antigen is attached and emits light of a first wavelength and then emits light of a second wavelength longer than the first wavelength; And at least one of the size of the individual particles or the size of the particles formed by the reaction of the antibody with the target antigen and the size of the individual particles is not corresponding to the first wavelength, ) Region of the antigen-antibody.

The light of the first wavelength may be light having a peak wavelength in the ultraviolet region. Here, the ultraviolet ray region in which the peak wavelength exists may be from 340 nm to 400 nm.

The light of the second wavelength may be light having a peak wavelength in the visible light region. Here, the visible light region in which the peak wavelength exists may be 480 to 570 nm.

The specimen includes a channel inlet through which body fluids are supplied; A channel having one end connected to the channel inlet to move the body fluid; And a reaction region provided in at least a portion of the channel and in which the particles are provided. The absorption pad may be connected to the other end of the channel.

A light source for irradiating the specimen with light of a first wavelength; And a photodetector that detects light of at least the second wavelength. And a data processing unit for calculating and processing the light amount of light of the second wavelength detected by the photodetector.

The present invention also provides a method for detecting whether or not a target antigen exists in a body fluid and a method for detecting the presence or absence of a target antigen in a body fluid, Preparing particles for releasing the particles; Supplying body fluids to the particles; And irradiating the particle supplied with the body fluid with light of at least a first wavelength and detecting light emitted from the particle.

The size of the particle may be determined so that at least one of the size of the individual particle or the size formed by aggregation of the particles due to the reaction of the target antigen with the antibody exists in the Mie scattering region corresponding to the first wavelength have.

The light of the first wavelength may be light having a peak wavelength in the ultraviolet region. Here, the ultraviolet ray region in which the peak wavelength exists may be from 340 nm to 400 nm.

The light of the second wavelength may be light having a peak wavelength in the visible light region. Here, the visible light region in which the peak wavelength exists may be 490 to 570 nm.

And confirming whether or not the target antigen is present in the body fluid based on the light amount of the second wavelength in the light detected from the particle and the concentration thereof. Here, the amount of light of the second wavelength is compared with the reference amount of light, and it is possible to confirm whether or not the target antigen exists in the body fluid and its concentration based on the increase / decrease of the reference amount.

Irradiating the particle with light of at least a first wavelength and detecting light emitted from the particle before supplying the body fluid to the particle. Here, it is determined whether or not the target antigen exists in the body fluid based on the light amount of the second wavelength detected before supplying the body fluid to the particle and the light amount of the second wavelength detected after supplying the body fluid to the particle, Step;

According to the present invention, it is very convenient to instantly and visually confirm the presence and concentration of the antigen.

Further, according to the present invention, the method of detecting an antigen is simple, so that a person who is not skilled can be carried out without difficulty.

Further, according to the present invention, the apparatus for detecting the antigen is not difficult to construct, and the equipment design and manufacture are convenient.

The above and other objects, features and advantages of the present invention will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: FIG.

FIG. 1 is a graph showing the intensity of scattered light for each scattering angle according to the condition of incident light of spherical particles in a wavelength-particle relationship in a non-scattering condition,
2 (a) to 2 (c) are graphs showing the radius of the particle and the transition of the cross-sectional area of scattering according to the wavelength of the light irradiated to the particle,
Figure 3 shows the individual particles used in the detection of the antigen-antibody of the present invention and the actual radii produced by the particles as they are aggregated around the antigen,
Figure 4 shows a specimen used for antigen-antibody detection according to the present invention,
5 is a schematic view showing a method for detecting an antigen-antibody according to the present invention,
FIG. 6 is a graph showing the spectrum of light detected from particles and light irradiated to particles in order to explain the principle of the antigen-antibody detection method according to the present invention,
7 is an enlarged view of a peak portion indicated by a dotted line in Fig. 6, and Fig.
FIG. 8 is a graph showing the intensity of light detected by the antigen-antibody detection method shown in FIG. 5 according to the concentration of the antigen present in the body fluid when the method of detecting an antigen-antibody of the present invention is used, In the graph of FIG.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

It is to be understood that the present invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, It is provided to inform.

FIG. 1 is a graph showing scattering intensity of scattered light according to the condition of scattered light generated by non-scattering of particles, FIG. 2 is a graph showing the intensity of scattered light according to the radius of particles and the wavelength of light irradiated on the particles FIG. 3 is a graph showing the actual radii of the individual particles used for the detection of the antigen-antibody of the present invention and the particles formed as they are concentrated around the antigen. FIG.

Mie scattering is a phenomenon in which scattering occurs when the wavelength of the light to be irradiated to the particle is almost equal to the particle size. 2, when the ratio of the wavelength (λ) the radius (r) of the contrast particles to be irradiated to the particles 1 and out, the cross-sectional area caused by the cross-sectional area according to the actual size of the particle (πr 2) compared to the scattering (σ) Is a region that varies greatly as r / λ value changes, and this region becomes a Mie scattering range.

1, the intensity of the scattered light differs depending on the angle at which scattering occurs with respect to the direction in which the light is incident on the particle, and the difference in intensity of the scattered light varies depending on the polarization direction of incident light and scattered light . ≪ / RTI >

Therefore, if the diameter of the particle is different for light of the same wavelength, the tendency of the scattered light also changes. Using this principle, it is possible to optically detect the antigen-antibody reaction.

As shown in Fig. 3, after the antibody 20 is attached to the particle 10 having the radius R1 near the wavelength of the light to be irradiated, a body fluid is applied to these particles 10 in a state where these particles are collected When the antigen 25 in the body fluid is located around the particle 10 and the antigen 25 is the target antigen 25 that reacts with the antibody 20 attached to the particle 10, The antibody 20 reacts around the antigen 25 to aggregate the particles 10 around the antigen 25. Thus, when a plurality of particles 10 are concentrated around the antigen 25, it becomes similar to one particle having a radius R2 at the position of the irradiated light.

Accordingly, since the diameter of the particles varies from R1 to R2 while the wavelength of the light to be irradiated is the same, the tendency of non-scattering varies. Therefore, if the scattering angle position capable of detecting the non-scattering tendency can be well selected and the intensity difference of the scattered light generated by the non-scattering at the position can be detected, , The concentration of the antigen can be confirmed.

However, as shown in FIGS. 1 and 2, the tendency of the scattering angle or the intensity of the scattered light to change is very sensitive to the change of the particle size, so it is necessary to carefully consider the design of the antigen-antibody detection device.

On the other hand, non-scattering is a scattering phenomenon taking place in a microscopic range. Therefore, it can be seen that scattering phenomenon caused by interaction of light energy and particles is not a scattering phenomenon caused by irregular reflection of light on particles. Therefore, it can be assumed that the phenomenon and tendency of the non-scattering as described above is caused by a difference in the interaction between the energy of the light and the particle as the diameter of the particle changes with respect to the wavelength (energy) of light.

However, as shown in Fig. 3, when the particles 10 include the fluorescent substance 12 in such a manner that the fluorescent substance 12 is adhered to the particles 10, or when the particles 10 are self-fluorescence It is possible to confirm that the degree of fluorescence of the particle 10 is different due to the energy absorbed by the particle 10 while interacting with the energy of the light. It is more advantageous to determine the presence or concentration of the target antigen in the body fluid by checking the difference in the non-scattering caused by the difference in the diameters of the particles 10, It is confirmed that there are advantages in terms of advantages.

First, unlike micro-scattering, when light is generated by a fluorescent material, there is no directionality. In other words, unlike the case where scattering light intensity varies according to the scattering angle (see FIG. 1), the fluorescent material absorbs energy from the irradiated light, absorbs energy, and excites the excited electrons to a stable orbit It is not greatly influenced by the direction in which such fluorescence is measured. Therefore, it is not necessary to carefully consider the detection direction of fluorescence when designing an antigen-antibody detection apparatus, thereby facilitating the design.

Secondly, since the fluorescent material that absorbs energy from the light of the first wavelength generates light of the second wavelength longer than the first wavelength, a difference occurs in the wavelength of the light emitted by the fluorescent material and the emitted light will be. Therefore, the process of detecting the fluorescence of the second wavelength, which is the visible light region, is facilitated after the light of the first wavelength, which is the ultraviolet ray, is irradiated. That is, if the light receiving element of the photodetector is insensitive to the first wavelength which is the ultraviolet ray and is sensitive to the second wavelength which is the visible light region, the light of the second wavelength is easily detected without being influenced by the first wavelength irradiated to the particle do. If the first wavelength and the second wavelength are configured in this way, it is possible to use a general CCD or CMOS camera as a photodetector.

Third, since there is a difference in the wavelengths of the light incident on the particles and the light emitted from the particles, it is possible to measure the light of the second wavelength with high precision by placing a filter capable of filtering only the first wavelength in front of the photodetector . Considering that the shorter the wavelength is, the lower the transmittance and the higher the absorptivity, the material of the specimen on which the particles are provided is selected as a material through which the light of the second wavelength is not transmitted but the light of the first wavelength is transmitted , The light of the second wavelength may be measured in the direction opposite to the direction in which the light is irradiated on the basis of the specimen. Of course, as described above, if the two wavelengths are located apart from each other in the spectrum, and are insensitive to the first wavelength and sensitive to the second wavelength, it is not necessary to use such a filter itself.

Hereinafter, an apparatus and method for detecting an antigen-antibody using the above-described principle will be described.

FIG. 4 is a view showing a specimen used for detecting an antigen-antibody according to the present invention, and FIG. 5 is a schematic view showing a method for detecting an antigen-antibody according to the present invention.

Referring to Fig. 4, particles 10 to be used in the antigen-antibody detection method of the present invention are provided on a test piece 30. The channel 33 is provided with a channel inlet 31 through which a body fluid is supplied to one end of the channel 33. A body fluid is supplied to the other end of the channel 33 Absorbing pad 35 is provided. The portion of the specimen other than the region of the channel 33, the channel inlet 31 and the absorbent pad 35 described above is made of a material that does not absorb body fluids.

There is a reaction zone 332 in which particles of FIG. 3 are provided in a predetermined area on the channel 33. The particle 10 is attached to the periphery of the antibody 20, and the antibody 20 can react with the target antigen 25. Further, the particles 10 may include the fluorescent substance 12 or the particles themselves may be made of a fluorescent substance. As an example, the material of the particles 10 may be polystyrene having a refractive index n of up to 1.59.

The radius of the individual particles is R1, and when they are concentrated around the antigen 25, the effective radius is R2. At this time, R1 and R2 can all be in the non-scattering region as shown in Fig. 2 (a) in contrast to the first wavelength?. R1 may be selected so that the difference in scattering area generated by R1 and R2 is maximized. For example, when the intensity of fluorescence generated by non-scattering generated by one individual particle having a radius of R1 is a, the intensity of fluorescence due to non-scattering generated by 3n individual particles is 3n * a And when the intensity of fluorescence generated by non-scattering becomes 6a when three individual particles aggregate and have a substantial radius of R2, 3n individual particles are generated by n collecting particles of three each The intensity of fluorescence due to non-scattering is n * 6a. When R1 and R2 are selected so that the intensity of fluorescence generated from the aggregate particles when the individual particles are aggregated is significantly larger than the intensity of fluorescence generated from the individual particles merely by arithmetic addition, The intensity of the fluorescence generated by the antigen-antibody reaction may vary greatly depending on whether or not the antigen-antibody reaction occurs. This difference can be detected by the photodetector. Conversely, even if R1 and R2 are selected so that the intensity of fluorescence generated in the aggregated particles when the individual particles are aggregated is significantly smaller than the intensity of fluorescence generated in the individual particles simply by arithmetic addition Results can be obtained. At this time, the scattering cross-sectional area of R1 and R2 can be determined in consideration of the number of particles constituting R2.

On the other hand, as shown in FIG. 2 (b), R1 may exist in a more fine region than the non-scattered region, and R2 may be located in a non-scattered region, in contrast to the first wavelength?. At this time, it is also possible to determine the size of the particles so that the intensity of fluorescence by the aggregation particles is greater than the intensity of fluorescence by the individual particles.

Also, as shown in FIG. 2 (c), R1 may exist in the non-scattering region in contrast to the first wavelength?, And R2 may exist in the macroscopic region rather than the non-scattering region. At this time, it is also possible to determine the size of the particles so that the intensity of fluorescence by the aggregation particles is greater than the intensity of fluorescence by the individual particles.

2 (a) to 2 (c), the sizes of the particles are selected so that the intensity of fluorescence by the aggregated particles is greater than the intensity of fluorescence by the individual particles, but the opposite case is of course also possible. That is, even if the size of the particles is selected so that the intensity of fluorescence becomes weaker when the particles are aggregated compared with the intensity of fluorescence by the individual particles, it is possible to determine whether the particles are aggregated or not by the intensity difference of the fluorescence. However, it is noteworthy that the higher the degree of aggregation of the particles and the more intense the fluorescence intensity, the more intuitively they can grasp the degree of particle aggregation, that is, the presence of the antigen, depending on the degree of fluorescence intensity be worth.

As shown in FIG. 5, a UV LED may be utilized as the light source 40 that generates light of the first wavelength to be irradiated on the specimen 30. Since the LED has a narrower full width at half maximum, the amount of light is concentrated at the peak wavelength and its surrounding wavelength. Accordingly, ultraviolet rays having the first wavelength as the peak wavelength are generated by UV LEDs and irradiated to the particles, whereby the fluorescence efficiency of the particles 10 can be increased.

As the light of the first wavelength, a light source having a peak wavelength in an ultraviolet region of 200 nm to 400 nm can be applied. Ultraviolet rays are generally perceived to be insensitive to camera sensors such as smart phones. Therefore, a conventional camera or a smart phone can be utilized as means for measuring light of the second wavelength to be described later. As an example, light of the first wavelength may be ultraviolet rays having a peak wavelength within a range of 340 to 400 nm, and more particularly, near-ultraviolet rays having a peak wavelength of about 395 nm may be used. Ultraviolet light of this wavelength induces fluorescence of particles made of polystyrene. The light of the first wavelength is irradiated to the specimen 30.

Body fluid is supplied to the channel inlet 31 of the test piece 30 to detect whether or not the antigen exists. When the body fluid is supplied to the channel inlet 31, the body fluid moves along the channel 33 and moves to the absorption pad 35. As the body fluid moves along the channel 33 and past the reaction zone 332 located on the channel, it is mixed with the particles 10 provided in the reaction zone. At this time, if there is a target antigen (25) reacting with the antibody in the body fluid, the particles in the reaction region (332) are aggregated with each other, so that the substantial radius of the particle changes from R1 to R2.

Meanwhile, a photodetector 50 is further provided for detecting the fluorescence of the second wavelength generated in the particle 10 by the light of the first wavelength irradiated to the specimen 30 and for measuring the intensity of the light. The photodetector 50 may be disposed at a position where it can receive light generated when the particles 10 in the specimen 30 are fluorescent. Fluorescence due to the fluorescent material is emitted irrespective of the angle of the incident light, unlike the simple scattering, so that there is no particular limitation on the position of the photodetector 50.

In one embodiment, such a photodetector 50 may be disposed at a position deviated from the direction in which the first wavelength is mainly reflected from the surface of the specimen. In another embodiment, the specimen is made of a material that can transmit light of a second wavelength and can not transmit light of a first wavelength, and the photodetector 50 is disposed on the opposite side of the light source 40 As shown in FIG. This can further minimize the influence of the light of the first wavelength on the photodetector 50.

The light of the second wavelength is composed of light in the visible light region, more specifically, green light in the visible light region of 490 to 570 nm. When the particles are composed of polystyrene and the peak value of the light of the first wavelength is near 395 nm, the peak value of the light of the second wavelength has a peak wavelength at approximately 525 nm. The light of the second wavelength is clearly distinguished from that of the light of the first wavelength having a blue color and is also visually distinguished from other elements included in the commercial sensors for sensing visible light Respectively, so that signal processing is easier. Further, due to such a difference in wavelength, a separate filter is not required in front of the photodetector 50. That is, the light of the second wavelength is sufficiently spaced apart from the light of the first wavelength in the spectrum so that bleaching phenomenon does not occur in the photodetector.

The electrical signal generated by the light measured by the photodetector 50 is processed by the data processing unit 60. For example, the data may be processed by an application installed on the smartphone. The main purpose of the data processing is to detect how the intensity of the fluorescence that occurs after the body fluid is provided to the specimen changes.

Detection of such light may be performed after bodily fluid is provided to the specimen 30, or both after the bodily fluid is provided to the specimen 30 and after the bodily fluid is provided to the specimen 30.

For example, if the fluorescence intensity standardized according to the concentration of the antigen present in the body fluid is data based on the reference value, the intensity of the fluorescence detected after the body fluid is supplied to the sample 30 can be compared with the reference value to confirm the concentration of the antigen .

Alternatively, it is possible to confirm the concentration of the antigen by comparing the intensity of fluorescence measured before the body fluid is supplied to the specimen 30 with the intensity of fluorescence measured after the body fluid is supplied to the specimen 30. If the intensity of fluorescence before and after the supply of body fluids has hardly changed, it can be assumed that the antigen does not exist. If the intensity of fluorescence before and after the delivery of the body fluid is large, it is also possible to estimate the concentration of the antigen according to the degree of difference.

Thus, deriving the concentration of the antigen can be performed automatically by the data processing unit 60.

The method of estimating the concentration of the antigen by the intensity difference of the fluorescence can be classified into a method of comparing the intensity of fluorescence corresponding to the absolute value and a method of comparing the intensity difference of the fluorescence before and after the body fluid is supplied to the sample, Can be used.

In order to estimate the concentration of the antigen by comparing the intensity of the fluorescence corresponding to the absolute value, first, a solution having already known concentration of the antigen is supplied to the sample, and the fluorescence level of each sample is measured and converted into a database. Then, the concentration of the antigen on the database corresponding to the fluorescence level is compared with the fluorescence level obtained in advance by database, and the intensity of the fluorescence measured after feeding the body fluid to which the concentration of the antigen is to be known is supplied to the specimen. The target antigen concentration of the body fluid can be grasped. This is the method that can be used mainly when the antigen-antibody detection device can be supplied with certain equipment.

However, in addition to the above, it is also possible to implement the database-based information corresponding to the features of the camera of each type of smartphone by installing the smartphone application for each model. In this case, only the UV LED and the specimen which can irradiate the ultraviolet ray of the first wavelength may be separately supplied. The use of a smartphone has various applications, for example, when it is necessary to individually check a certain component in body fluids, for example, when it is necessary to check pregnancy earlier than a commercially available pregnancy tester.

A method of comparing and comparing fluorescence intensities before and after the body fluid is provided to a specimen is to normalize the intensity of the light of the second wavelength after providing the body fluid based on the light intensity of the second wavelength before providing the body fluid ). ≪ / RTI > That is, the standardized increase / decrease amount of the light intensity according to the difference of the concentration of the target antigen can be databaseed, and the standardized increase / decrease amount of the measured light intensity can be compared with the database to confirm the concentration of the antigen.

FIG. 6 is a graph showing the spectrum of light detected from particles and light irradiated to particles in order to explain the principle of the antigen-antibody detection method according to the present invention, and FIG. 7 is an enlarged view And FIG. 8 is a graph showing the intensity of fluorescence generated in particles according to the concentration of antigen present in the body fluid.

FIGS. 6 and 7 are the results of measurement of the self fluorescence of polystyrene particles (Magsphere, CA, US S.A.) on paper microfluidics with a spectrometer (USB 4000, Ocean Optics, FL, U.S.A.). Microfluidics were prepared with SU-8 negative-photoresist (Microchem, MA, U.S.A) and chromatographic paper (GE Healthcare, Springfield Mill, U.K.). Deionized water and polystyrene particles of five different concentrations were provided in the channels of the microfluidic channels, and the spectrum was measured by irradiating the particles with UV-LED having a peak wavelength of 395 nm in a dark room. UV LED and specimen were measured at 45 degree angle, and spectrometer and specimen at 90 degree angle.

The spectrometer, which can measure a wide range of ultraviolet and visible light, is only used to measure the spectra of FIGS. 6 and 7. When constructing an actual antigen-antibody detection apparatus, the spectrometer is insensitive to the ultraviolet region and sensitive to the visible region It is possible to use a photodetector having a camera sensor which is a photodetector and a separate filter is not required in front of the photodetector.

In the spectrum shown, both a peak due to the light of the first wavelength and a peak due to the light of the second wavelength appear, and they are clearly distinguished by the difference in the peak wavelength and the color difference. The sharp peak at 395 nm is obtained by the ultraviolet light emitted from the UV LED reflected from the specimen and measured by the spectrometer, and the broad peak near 523 nm is fluoresced from the particles on the specimen. The distance between two peaks in the spectrum is considerably distant, and the color of the light of the second wavelength is clearly distinguished as green. Conventional camera sensors used in everyday life, such as CCDs used in smartphone cameras, are sensitive to green and hardly respond to ultraviolet light. Therefore, a conventional camera can be used as a photodetector without a separate filter.

As shown in FIG. 7, it can be seen that as the concentration of the particles increases, the peak value of the second wavelength becomes higher. Therefore, it can be seen that the change in the fluorescence particle concentration and the substantial size change depending on the aggregation influences the intensity of the fluorescent light (fluorescence in deionized water DI is presumed to result from fluorescence of the channel part of the sample itself, Which can be compensated by changing the material of the channel portion).

FIG. 8 is a graph showing the intensity of fluorescence generated in particles according to the concentration of an antigen present in body fluids, when using the antigen-antibody detection method according to the present invention.

E. coli was used as a target antigen, polystyrene particles having a diameter of 920 nm with an antibody reactive with the target antigen were used as fluorescent particles, and 0.5 μg of the particles were placed on the reaction region of the sample channel and dried.

The solution was made to have different concentrations of the target antigen and supplied to the channel inlet. The specimen was irradiated with light of the first wavelength having a peak of 395 nm through the UV-LED at an incident angle of 30, and the channel region before and after the supply of the solution was photographed by a camera of the smartphone. The image measured by the camera was converted into a green channel, and the intensity of the green was calculated.

Referring to FIG. 8, it can be seen that the intensity of the green light gradually increases when the concentration increases from the case where E. coli is absent.

Therefore, if the light intensity of the first wavelength irradiated by the light source is made constant, it is possible to detect the antigen-antibody reaction even if only the light of the second wavelength that fluoresces from the particle is measured.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the invention is not limited to the disclosed exemplary embodiments. It is obvious that a transformation can be made. Although the embodiments of the present invention have been described in detail above, the effects of the present invention are not explicitly described and described, but it is needless to say that the effects that can be predicted by the configurations should also be recognized.

10: particles
12: Fluorescent material
20: Antibody
25: target antigen
30: The Psalms
31: Channel entrance
33: channel
332: Reaction zone
35: Absorption pad
40: Light source
50: photodetector
60:

Claims (19)

A fluorescent particle to which an antibody reactive with the target antigen is attached and emits light of a first wavelength and then emits light of a second wavelength longer than the first wavelength; And
And a sample on which the particles are arranged,
Wherein at least one of the sizes of the individual particles or the size formed by the aggregation of particles due to the reaction of the target antigen with the antibody is present in a Mie scattering region corresponding to the first wavelength. Detection device.
The method according to claim 1,
Wherein the light of the first wavelength is light having a peak wavelength in an ultraviolet region.
The method of claim 2,
Wherein the ultraviolet region in which the peak wavelength exists is 340 nm to 400 nm.
The method according to claim 1,
Wherein the light of the second wavelength is light having a peak wavelength in the visible light region.
The method of claim 4,
And the visible light region in which the peak wavelength exists is 480 to 570 nm.
The method according to claim 1,
The specimen,
A channel inlet through which fluid is supplied;
A channel having one end connected to the channel inlet to move the body fluid; And
A reaction zone provided in at least a portion of the channel, the reaction zone being provided with the particles;
Antibody-antibody detection device.
The method of claim 6,
And an absorption pad is connected to the other end of the channel.
The method according to claim 1,
A light source for irradiating the specimen with light of a first wavelength; And
And a photodetector for detecting light of at least the second wavelength.
The method of claim 8,
And a data processing unit for calculating and processing the light amount of light of the second wavelength detected by the photodetector.
A method for detecting whether a target antigen is present in a body fluid and its concentration,
Preparing particles for attaching an antibody reactive with a target antigen and absorbing light of a first wavelength and emitting light of a second wavelength longer than the first wavelength;
Supplying body fluids to the particles; And
Irradiating the particles supplied with the body fluid with light of at least a first wavelength, and detecting light emitted from the particle.
The method of claim 10,
At least any one of the size of the individual particles or the size of the particles formed by the reaction of the antibody with the target antigen is determined to be in a Mie scattering region corresponding to the first wavelength ≪ / RTI >
The method of claim 10,
Wherein the light of the first wavelength is light having a peak wavelength in an ultraviolet region.
The method of claim 12,
Wherein the ultraviolet region in which the peak wavelength exists is 340 nm to 400 nm.
The method of claim 10,
Wherein the light of the second wavelength is light having a peak wavelength in a visible light region.
15. The method of claim 14,
Wherein the visible light region in which the peak wavelength exists is 490 to 570 nm.
The method of claim 10,
Detecting the presence or absence of the target antigen in the body fluid based on the light amount of the second wavelength in the light detected from the particle and the concentration thereof.
18. The method of claim 16,
Comparing the amount of light of the second wavelength with a reference amount of light, and determining whether the target antigen is present in the body fluid based on the increase / decrease of the reference amount and its concentration.
The method of claim 10,
Irradiating the particle with light of at least a first wavelength and detecting light emitted from the particle before supplying body fluid to the particle.
19. The method of claim 18,
Confirming whether or not the target antigen is present in the body fluid and its concentration based on the light amount of the second wavelength detected before supplying the body fluid to the particle and the light amount of the second wavelength detected after supplying the body fluid to the particle; ≪ / RTI >
KR1020150130578A 2015-09-15 2015-09-15 Antigen-Antibody Detector and Antigen-Antibody Detecting Method Using Ultra-Violet Light Emitting Diode and Fluorescent Particles KR20170032951A (en)

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
H. C. van de Hulst, Light Scattering by Small Particles. Dover Publications, Inc. New York, 1981.

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