KR101066598B1 - Method of Multiplex Detecting on Microfluidic Chip - Google Patents

Abstract

Multiple detection methods in microfluidic chips are provided. The method provides fluorescent particles comprising fluorescent molecules that are adjusted to such that the fluorescence sensitivity of the antigen having the highest concentration among the plurality of antigens present in the fluid sample is not saturated, and the fluorescence is caused by the movement of the fluid sample. Reacting the particles with the plurality of antigens respectively form a plurality of antigen-fluorescent particle conjugates, and reacting the plurality of antibodies and the plurality of antigen-fluorescent particle conjugates immobilized in each of the plurality of reaction regions, respectively. Forming a plurality of antibody-antigen-fluorescent particle conjugates, and irradiating the same excitation wavelength to a plurality of antibody-antigen-fluorescent particle conjugates immobilized in each of the plurality of reaction regions to simultaneously measure the respective fluorescence sensitivities. It includes.

Microfluidic, Fluorescent, Antigen, Antibody, Multiple Detection

Description

Method of Multiplex Detecting on Microfluidic Chip

The present invention relates to a detection method in a microfluidic chip, and more particularly to a multiple detection method in a microfluidic chip.

The present invention is derived from a study performed as part of the IT source technology development project of the Ministry of Information and Communication and the Ministry of Information and Communication Research and Development (Task Management Number: 2005-S-070-03, Task name: Ubiquitous Health Management Module System).

The detection of diseases using the immune response is excellent in the specificity of the immune response, which is relatively low in the presence of prostate specific antigen (PSA), human chorionic gonadone protein (hCG), alpha fetus It is useful for detecting diseases such as protein (Alpha Feto Protein: AFP).

Such disease detection includes a labeling method using a tagging fluorescent material, and a method of detecting a disease in an unlabeled form such as an electrochemical reaction or surface plasmon resonance (SPR).

Labeling using fluorescent materials is advantageous for low concentration detection. Therefore, since disease factor antigens are generally present at low concentrations, the labeling method using fluorescent materials is mainly used for the detection of antigens at low concentrations.

Recent developments in nanoparticle technology have led to the presence of a large number of fluorescent molecules in particles, thereby increasing the sensitivity of fluorescence. In particular, research on techniques for further improving the sensitivity of fluorescence is increasing to be used in samples containing low concentrations of antigens. For example, fluorescent particles such as latex beads or silica beads in which a plurality of fluorescent molecules are present have been applied to an immune response. These fluorescent particles increase the sensitivity of the measured fluorescence, thereby making the detection of low concentration more effective. In addition, by allowing fluorescent molecules to be present in the beads, the disadvantages for photo-bleaching, which are generally inevitable in fluorescent molecules, can be compensated for. The improvement of low concentration detection and the detection of an immune response using fluorescent particles that can overcome the disadvantages of photo-bleaching, many studies have been conducted due to this advantage. In addition, biochips capable of detecting diseases using the same are commercially available.

In recent years, disease detection in microfluidic has increased interest in a system for detecting multiple disease agents at once in detecting a single disease. In particular, since detection using fluorescence has excellent performance in detecting low concentrations, researches on multiplex detection techniques for simultaneously detecting a plurality of disease agents on one microfluidic chip have been conducted.

Basically, the fluorescence detection system consists of a fluorescence material, a wavelength for fluorescence excitation, and a device for reading the generated fluorescence. That is, the basic mechanism is to irradiate a fluorescent material with an excitation wavelength, excite the fluorescent material, and then read the fluorescent wavelength generated from the fluorescent material. Therefore, in order to detect a plurality of disease factors, an excitation wavelength is required as many as the number of disease factors for which detection is required. Many complex optical devices are needed.

However, since it is difficult to implement a large number of complex optical devices on a single chip, systems have been developed that can simplify many complex optical devices. An example is a detection system using quantum dots (Q dots). After the quantum dot is excited according to the size, the wavelength of fluorescence generated is different. Accordingly, quantum dots of different sizes can be excited at the same excitation wavelength at the same time, which is a very advantageous material for multiple detection. That is, after immobilizing different antibodies on the surfaces of quantum dots of different sizes, and reacting antigens of a plurality of disease agents with them, quantum dot-antibody-antigen conjugates having a plurality of different sizes are produced. Is formed. Then, by irradiating the same excitation wavelength to a plurality of different sizes of quantum dot-antibody-antigen conjugates, by measuring the fluorescence of each of the wavelengths generated from the plurality of quantum dot-antibody-antigen conjugates, Multiple disease agents are detected simultaneously.

Although quantum dots are physically stable compared to general fluorescent labeling materials, the quantum dots have low binding properties with biological materials to be labeled, and have many limitations on the surface processing of quantum dots.

Another example is the immobilization of different antibodies to one type of fluorescent particles, followed by the reaction of antigens of a plurality of disease agents with them to form a plurality of antigen-fluorescent particle conjugates. Subsequently, combining the plurality of antigen-fluorescent particle conjugates with antibodies capable of reacting with the plurality of antigen-fluorescent particle conjugates provided at different positions of the microfluidic chip, respectively, results in the plurality of antibody-antigen-fluorescent particle conjugates. Are formed. The plurality of antibody-antigen-fluorescent particle conjugates are then irradiated with the same excitation wavelength to determine the degree of fluorescence generated from the plurality of antibody-antigen-fluorescent particle conjugates formed at different positions. Disease factors are detected at the same time.

In this case, since one type of fluorescent material is used, the number of excitation wavelengths can be fixed to one, while the different disease agents can be read separately from each other at different positions of the microfluidic chip. The antibodies are fixed and distinguished from each other.

In the above case, if the concentration of one type of antigen among the antigens of different disease factors is extremely high, one type of antigen will first reach a saturation concentration within the fluorescence sensitivity concentration range in which the other types of antigens are detected. Can be. As such, when one kind of antigen first reaches a saturation concentration, detection of this antigen becomes impossible. Accordingly, there is a disadvantage in that multiple detection is not complete for samples containing different concentrations of antigens.

An object of the present invention is to provide a multiple detection method in a microfluidic chip capable of simultaneously detecting a plurality of antigens using fluorescent materials.

In order to achieve the above object, the present invention provides a multiple detection method in a microfluidic chip. The method comprises the steps of providing fluorescent particles comprising fluorescent molecules that have been adjusted such that the fluorescence sensitivity is not saturated for antigens of the species having the highest concentration among the plurality of antigens present in the fluid sample. Reacting the fluorescent particles with a plurality of antigens, respectively, to form a plurality of antigen-fluorescent particle conjugates, wherein the plurality of antibodies and the plurality of antigen-fluorescent particle conjugates immobilized in each of the plurality of reaction regions are formed. Each reacting to form a plurality of antibody-antigen-fluorescent particle conjugates, and irradiating the same excitation wavelength to the plurality of antibody-antigen-fluorescent particle conjugates immobilized in each of the plurality of reaction regions, Simultaneously measuring the fluorescence sensitivities so that the fluorescence sensitivities are not saturated.

The fluid sample may comprise at least one selected from blood, plasma or urine.

Movement of the fluid sample may be using capillary force.

Fluorescence sensitivity may be proportional to the number of antibody-antigen-fluorescent particles.

As described above, according to the problem solving means of the present invention, by controlling the number of fluorescent molecules in the fluorescent particles, all of the plurality of antigens may not be saturated within the fluorescence sensitivity concentration range. Accordingly, it may be possible to simultaneously detect multiple multiple types of antigens simultaneously in a microfluidic chip using fluorescent materials.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the embodiments disclosed herein are provided so that the disclosure can be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In addition, since they are in accordance with the preferred embodiment, the reference numerals presented in the order of description are not necessarily limited to the order. In the drawings, the thicknesses of films and regions are exaggerated for clarity. In addition, if it is mentioned that the film is on another film or substrate, it may be formed directly on the other film or substrate, or a third film may be interposed therebetween.

1 is a conceptual plan view illustrating a multiple detection method in a microfluidic chip according to an embodiment of the present invention.

Referring to FIG. 1, the microfluidic chip 100 may include a fluid sample chamber 110, a fluorescent particle chamber 120, and a reaction unit 150 connected to each other by a flow path.

The fluid sample chamber 110 may be a place where a fluid sample (not shown) is injected from the outside. The fluid sample may comprise a plurality of different antigens (see 115 of FIG. 3). The fluid sample may comprise at least one selected from blood, plasma or urine. The fluid sample injected into the fluid sample chamber 110 may be transferred to the fluorescent particle chamber 120 by capillary force.

The fluorescent particle chamber 120 may be a place where the plurality of antigens included in the fluid sample transferred from the fluid sample chamber 110 and the fluorescent particles 125 provided in the fluorescent particle chamber 120 react, respectively. . The reaction between the plurality of antigens contained in the fluid sample and the fluorescent particles 125 is an antigen-antibody reaction between the first antibodies and the plurality of antigens immobilized on the surface of the fluorescent particles 125. Can be. Accordingly, a plurality of antigen-fluorescent particle conjugates (see 135 of FIG. 3) may be formed in the fluorescent particle chamber 120. The plurality of antigen-fluorescent particle conjugates may be transferred to the reaction unit 150 by the flow of the fluid sample by capillary force.

In order to increase the degree of reaction between the plurality of antigens contained in the fluid sample and the fluorescent particles 125, a time gate 140 is provided in the flow path between the fluorescent particle chamber 120 and the reaction part 150. It may be further provided. The time gate 140 may serve to delay the flow of the fluid sample transferred by the capillary force. The time gate 140 may change the shape of the hydrophobic grooves whose surfaces are treated with a hydrophobic material or the flow path through which the fluid sample flows. Accordingly, the degree of reaction between the plurality of antigens and the fluorescent particles 125 included in the fluid sample may be increased.

The fluorescent particles 125 provided in the fluorescent particle chamber 120 may include fluorescent molecules therein. The fluorescent particles 125 according to the embodiment of the present invention may include fluorescent molecules such that the fluorescence sensitivity of the antigen having the highest concentration among the plurality of antigens is not saturated. Accordingly, even if the concentration of one type of antigen among the plurality of antigens is extremely high, one type of antigen cannot reach a saturation concentration within the fluorescence sensitivity concentration range in which the other types of antigens are detected. In other words, the plurality of antigens cannot all reach saturation concentrations. As a result, it is possible to simultaneously detect multiple fluid samples containing a plurality of antigens of different concentrations simultaneously.

The reaction unit 150 may include a plurality of reaction regions. The reaction unit 150 includes a plurality of antigen-fluorescent particle conjugates included in the fluid sample transferred from the fluorescent particle chamber 120 and the plurality of reaction regions of the reaction unit 150 (156a, 156b of FIG. 4, and 156c) may be where the plurality of second antibodies immobilized on each (see 157a, 157b and 157c of FIG. 4) react, respectively. The reaction between the plurality of antigen-fluorescent particle conjugates contained in the fluid sample and the plurality of second antibodies is characterized by the plurality of antigens of the plurality of antigen-fluorescent particle conjugates and the corresponding plurality of second kinds of antigen. It may be an antigen-antibody reaction between antibodies. Accordingly, a plurality of antibody-antigen-fluorescent particle conjugates (see 155 of FIG. 5) corresponding to the plurality of reaction regions of the reaction unit 150 may be formed and fixed, respectively.

Antigens, fluorescent particles 125 and / or antigen-fluorescent particle conjugates that are not bound in the reaction unit 150 may be cleaned by a fluid sample or / and a washing buffer solution. Accordingly, only a plurality of antibody-antigen-fluorescent particle conjugates corresponding to the plurality of reaction regions of the reaction unit 150 may exist.

The same excitation wavelength is irradiated to the plurality of antibody-antigen-fluorescent particle conjugates fixed to each of the plurality of reaction regions of the reaction unit 150, thereby generating each of the plurality of antibody-antigen-fluorescent particle conjugates. The fluorescence sensitivities of can be measured simultaneously. In this case, the degree of each fluorescence sensitivity may be proportional to the number of the plurality of antibody-antigen-fluorescent particle conjugates fixed in each of the plurality of reaction regions of the reaction unit 150. That is, since the respective fluorescence sensitivities are proportional to the concentrations of the plurality of antigens, the type and extent of the disease can be known according to the binding positions of the plural antigens and the degree of the fluorescence sensitivities.

Since the fluorescent particles 125 contain fluorescent molecules such that the fluorescence sensitivity of the antigen having the highest concentration among the plurality of antigens is not saturated, the concentration of one type of antigen among the plurality of antigens is increased. Even higher, one type of antigen cannot reach a saturation concentration within the fluorescence sensitivity concentration range at which other types of antigens are detected. In other words, the plurality of antigens cannot all reach saturation concentrations. Accordingly, even the fluid sample containing the plurality of antigens of different concentrations, both the type and extent of the disease can be known at the same time.

2 is a conceptual diagram for describing fluorescent particles used in the microfluidic chip according to an embodiment of the present invention.

At least one first antibody 127 capable of binding to an antigen (see 115 in FIG. 3) contained in the fluid sample may be fixed to the surface of the fluorescent particle 125. The fixing of the plurality of first antibodies 127 on the surface of the fluorescent particle 125 may be for facilitating an antigen-antibody reaction between the antigen and the antibody. The fluorescent particles 125 provided in the fluorescent particle chamber (see 120 of FIG. 1) of the micro-use chip (see 100 of FIG. 1) may include a plurality of antigens and a plurality of antigen-fluorescent particles contained in a fluid sample. In order to form the conjugates (see 135 of FIG. 3), the plurality of first antibodies 127 corresponding to the plurality of antigens may be fixed.

The fluorescent particles 125 may have a size of about several tens of nm to several μm. Fluorescent molecules 126 may be present in the fluorescent particle 125. The fluorescent particle 125 according to the exemplary embodiment of the present invention may include fluorescent molecules 126 such that the fluorescence sensitivity of the antigen having the highest concentration among the plurality of antigens is not saturated. Accordingly, even if the concentration of one type of antigen among the plurality of antigens is extremely high, one type of antigen cannot reach a saturation concentration within the fluorescence sensitivity concentration range in which the other types of antigens are detected. In other words, the plurality of antigens cannot all reach saturation concentrations. As a result, it is possible to simultaneously detect multiple fluid samples containing a plurality of antigens of different concentrations simultaneously.

3 is a conceptual diagram illustrating a reaction and bonding between a sample and fluorescent particles in a microfluidic chip according to an embodiment of the present invention.

In the fluorescent particle chamber (see 120 in FIG. 1) of the microfluidic chip (see 100 in FIG. 1), the antigen 115 and the fluorescent particles 125 included in the fluid sample may react. The reaction between the antigen 115 contained in the fluid sample and the fluorescent particle 125 is performed between the first antibody 127 immobilized on the surface of the fluorescent particle 125 and the antigen 115 contained in the fluid sample. May be an antigen-antibody response. Accordingly, the antigen-fluorescent particle conjugate 135 may be formed in the fluorescent particle chamber.

4 is a conceptual diagram illustrating a reaction unit of a microfluidic chip according to an embodiment of the present invention.

The reaction unit (see 150 of FIG. 1) of the microfluidic chip (see 110 of FIG. 1) may include a plurality of reaction regions 156a, 156b, and 156c. The plurality of reaction zones 156a, 156b, and 156c may be provided to each of the plurality of antigen-fluorescent particle conjugates (see 135 in FIG. 3) and the plurality of reaction zones 156a, 156b, and 156c included in the fluid sample. The fixed plurality of second antibodies 157a, 157b and 157c may be reacted with each other.

5 is a conceptual diagram illustrating a reaction and binding between a sample and an antibody coupled with fluorescent particles in a reaction unit of a microfluidic chip according to an embodiment of the present invention.

In the reaction zone 156 of the reaction section (see 150 in FIG. 1) of the microfluidic chip (see 100 in FIG. 1), the antigen-fluorescent particle conjugate (see 135 in FIG. 4) and the second antibodies contained in the fluid sample are shown. The reaction between 157 may be an antigen-antibody reaction between the antigen 115 of the antigen-fluorescent particle conjugate and the corresponding second antibody 157. Accordingly, the antibody-antigen-fluorescent particle conjugate 155 corresponding thereto may be formed and fixed in the reaction region 156.

Antigens 115, fluorescent particles (see 125 in FIG. 2) and / or antigen-fluorescent particle conjugates (135 in FIG. 3) in the fluid sample that are not bound in the reaction zone 156 may be flushed with the fluid sample or / and washed. Can be washed by a buffer solution. Accordingly, only the antibody-antigen-fluorescent particle conjugate 155 corresponding thereto may exist in the reaction region 156.

The antibody-antigen-fluorescent particle conjugate immobilized in the reaction region 156 may be irradiated with the same excitation wavelength to determine the fluorescence sensitivity generated by the antibody-antigen-fluorescent particle conjugate. That is, since the fluorescence sensitivity is proportional to the concentration of the antigen 115, the degree of disease can be known according to the degree of fluorescence sensitivity of the antigen.

By controlling the number of fluorescent molecules in the fluorescent particles by the method according to the embodiment of the present invention described above, all of the plurality of antigens may not be saturated within the fluorescent sensitivity concentration range. Accordingly, a multiple detection method in a microfluidic chip capable of simultaneously detecting a plurality of antigens using fluorescent materials can be provided.

1 is a conceptual plan view illustrating a multiple detection method in a microfluidic chip according to an embodiment of the present invention;

2 is a conceptual diagram for explaining fluorescent particles used in the microfluidic chip according to an embodiment of the present invention;

3 is a conceptual diagram illustrating a reaction and binding between a sample and fluorescent particles in a microfluidic chip according to an embodiment of the present invention;

4 is a conceptual diagram illustrating a reaction unit of a microfluidic chip according to an embodiment of the present invention;

5 is a conceptual diagram for explaining the reaction and binding between the sample and the antibody coupled to the fluorescent particles in the reaction unit of the microfluidic chip according to an embodiment of the present invention.

* Description of the symbols for the main parts of the drawings *

100: microfluidic chip 110: fluid sample chamber

115: antigen 120: fluorescent particle chamber

125 fluorescent particle 126 fluorescent molecule

127: first antibody 135: antigen-fluorescent particle conjugate

140: time gate 150: reaction unit

155: antibody-antigen-fluorescent particle conjugate 156, 156a, 156b, 156c: reaction region

157, 157a, 157b, 157c: second antibody

Claims (4)

Providing fluorescent particles in which the number of fluorescent molecules is adjusted such that fluorescence sensitivity is not saturated for antigens of the species having the highest concentration among the plurality of antigens present in the fluid sample; Reacting the fluorescent particles with the plurality of antigens by moving the fluid sample, respectively, to form a plurality of antigen-fluorescent particle conjugates; Reacting the plurality of antibodies immobilized on each of the plurality of reaction regions with the plurality of antigen-fluorescent particle conjugates, respectively, to form a plurality of antibody-antigen-fluorescent particle conjugates; And Irradiating the same excitation wavelength to the plurality of antibody-antigen-fluorescent particle conjugates immobilized in each of the plurality of reaction regions to simultaneously measure the respective fluorescence sensitivities so that the respective fluorescence sensitivities are not saturated. Multiple detection method in a microfluidic chip. The method of claim 1, And the fluid sample comprises at least one selected from blood, plasma or urine. The method of claim 1, The method of multiple detection in the microfluidic chip, characterized in that the movement of the fluid sample using a capillary force. The method of claim 1, And said fluorescence sensitivity is proportional to the number of said antibody-antigen-fluorescent particle conjugates.

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