KR20120046616A - Bio-sensing chip for convenient measurement of biochemical-binding and method of measuring bio-marker using it - Google Patents

Abstract

PURPOSE: A biosensing chip having a channel part and a method for optically measuring the biomarker using the same are provided to measure biomarker concentration without a special program. CONSTITUTION: A biosensing chip for bio sample analysis comprises: a sensing unit(100) having patterned gold thin film(112) of intaglio shape on a lower substrate; and a channel part(200) having a bio sample inlet(212b) of an upper substrate and a channel(212a) for providing moving pathway. A self assembly monolayer is formed on the surface of the gold thin film. The self assembly monolayer contains 3,3'-dithio-bis-propionic acid N-hydroxysuccinimide ester(DTSP).

Description

Bio-sensing chip for convenient measurement of biochemical-binding and method of measuring bio-marker using it}

The present invention relates to a biosensing chip in which a gold thin film is patterned in an intaglio form and a channel portion capable of moving a biological sample in a layered structure, and an optical measuring method of a biomarker using the same.

Biosensing devices use proteins, nucleic acids, or other biological elements, such as enzyme antibodies, as targets for measurement, and the recognition elements that recognize these targets and their quantification. It is a biochemical analysis device composed of a signal transducer element for converting an easy signal. In particular, a multi-step process of measurement and analysis is performed on a single chip by combining the transport, delivery, reaction, and washing reactions of a sample to a biosensing device. Bio-sensing chips of either a-chip or micro-total analysis systems have the advantages of miniaturization, high sensitivity, stability, speed and high reliability. Bio-sensing chips are widely used for commercialization in the fields of biotechnology such as diagnosis, medicine, environment, and food. [Yun-Chul Yoon, Hyung-Gil Choi, "Application of Nano-Bio Technology for Advanced Immunity Sensing", Journal of the Institute of Electronics Engineers, Vol. 32, No. 8, pp.52-61, 2005.].

Until now, the antigen-antibody reaction is the most widely used method for the diagnosis of disease markers, and the ELISA (Enzyme linked immunosorbent assay) method is the most widely used measurement method using the antigen-antibody reaction. The ELISA method measures the concentration of a disease marker, that is, a target, through a color reaction due to an enzymatic reaction on a 96-well plate. In order to measure this, an expensive measuring device capable of measuring absorbance, emission, or fluorescence in a well plate Should be used.

Therefore, there is a continuing need for research on the development of a biosensing chip and a biosensing method capable of easily and accurately measuring the concentration of a disease marker target in a specimen sample.

Accordingly, the present inventors have conducted research and efforts to develop a simple and accurate optical measurement method of a biomarker, and as a result, a channel including a sensing unit including a gold thin film patterned in a negative shape on a substrate surface, an inlet for a biological sample, and a moving channel The present invention has been completed by discovering that an additionally bound biosensing chip can be prepared to simply quantify the desired biomarker.

Accordingly, an object of the present invention is to provide a bio-sensing chip in which a sensing unit including a gold thin film patterned in a negative shape on a surface of a substrate and a channel unit providing a moving passage of a biological sample are combined.

The present invention also provides an optical measuring method of a biomarker for contacting a biological sample and a fluorescent bead to the biosensing chip and measuring the number of the fixed fluorescent beads, thereby patterning the bio to be simultaneously patterned for observing a plurality of sensing surfaces. The purpose of the sensing chip is to enable simultaneous identification and accurate quantification of nonspecific binding.

The present invention

A sensing unit in which a gold thin film is patterned in a negative shape on a lower substrate; And

Channel portion including a biological sample inlet of the upper substrate and a channel providing a moving passage for contacting the biological sample to the sensing unit

Characterized by the biosensing chip for biological sample analysis comprising a.

The present invention also provides a method for measuring a biomarker in a biological sample, comprising the step of injecting a biological sample, an antigen-specific antibody, and a fluorescent bead containing an antigen as a biomarker into a sample inlet of the biosensing chip. do.

The fluorescence measurement using a conventional fluorescence microscope could be made only through an expensive program for measuring the intensity of fluorescence of an image recorded by observing the surface of the solution and the chip to be measured by the fluorescence microscope, but the biosensing chip of the present invention may not perform a specific program. The biomarker concentration can be measured accurately and simply by quantifying the number of fluorescent beads coupled to the pattern portion of the chip and the outside of the pattern through an image measured using a fluorescence microscope even if not used. In addition, the images observed through the fluorescence microscope can simultaneously see the inside and outside of the patterned thin film where the reaction between the antigen and the antibody occurs, it is possible to accurately and easily distinguish the non-specific binding appearing outside the thin film. The number of fluorescence beads immobilized in the plurality of patterned thin films can be more accurately quantified by using simple statistical processing such as average value and dispersion.

1 is a perspective view illustrating a bio sensing chip in which a sensing unit and a channel unit are combined.
2 is a plan view illustrating a sensing unit and a channel unit, respectively.
FIG. 3 is a schematic diagram illustrating a sensing process of a bio sensing chip in Example 1. FIG.
4 is a photograph of a result of inducing optical signal detection according to Example 1. FIG.
FIG. 5 is a graph showing the number of beads immobilized inside and outside the thin film patterned through the fluorescence microscope according to the concentration of the antigen in Example 1. FIG.
FIG. 6 is a schematic diagram illustrating a sensing process of a bio sensing chip in Example 2. FIG.
7 is a photograph of the results of inducing optical signal detection according to the second embodiment.
FIG. 8 is a graph showing the number of beads immobilized inside and outside the thin film patterned through a fluorescence microscope according to the concentration of antigen.

Hereinafter, the present invention will be described in detail.

According to an embodiment of the present invention, a sensing unit in which a gold thin film is patterned in a negative shape on a lower substrate; And a channel unit including a biological sample inlet of the upper substrate and a channel providing a moving passage through which the biological sample may be in contact with the sensing unit.

Conventional bio-sensing chips are made of an embossed method in which a metal thin film is coated on a substrate, but the chip is characterized by an intaglio pattern in which a gold thin film is patterned beneath the substrate. This can prevent the phenomenon of gathering. The material that can be used as the substrate is not particularly limited as long as it does not react with a biological sample while maintaining a certain strength, preferably silicon, silicon oxide, glass, or the like.

The gold thin film is preferably patterned in a rectangular shape such that at least two portions in contact with one biological sample in each channel part exist, and the size of the pattern is such that the difference in signal according to the concentration of the antigen can be seen. Fluorescent beads may be fixed and patterned in a range of 100 to 800 μm and 10 to 200 μm long enough to be observed at a glance with a fluorescence microscope. In addition, it is preferable for the precise measurement that the three to ten metal patterns are arranged closely so that the measured values in each thin film are averaged.

A self assembly monolayer (SAM) is formed on the gold thin film to generate a reactor that can react with the amine. In order to produce the self-assembled monolayer film, an alkane thiol or a disulfide-based compound whose one end is modified with a functional group such as amine, succinimide, carboxyamide, and aldehyde may be used. For example, 3,3'-dithio- Bis-propionic acid N-hydroxysuccinimide ester (3,3'-dithio-bis-propionic acid N-hydroxysuccinimide ester, DTSP) can be used.

The biosensing chip of the present invention includes a channel portion including a biological sample inlet on an upper substrate and a channel providing a moving passage through which the biological sample may contact the self-assembled monolayer film.

The channel unit includes a biological sample inlet and a channel. Preferably, the biological sample inlet is formed at both ends of the channel. In addition, the biological sample may be brought into contact with the self-assembled monolayer film through a moving passage formed by the channel, and the height of the channel is preferably in the range of 0.01 to 1.0 mm. In addition, the channel portion is preferably made of a transparent material to facilitate the confirmation of the amount and contact of the biological sample used.

In another aspect, the present invention provides a method for measuring a biomarker in a biological sample comprising the step of injecting a biological sample, antigen-specific antibody and fluorescent beads containing the antigen as a biomarker in the sample inlet of the bio-sensing chip In addition, the antigen-antibody binding reaction can be measured by fluorescence label immunoassay.

The antigen-antibody binding reaction of the antigen and antibody can be performed by enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich immunoassay, western blot on polyacrylamide gel or Although there is an immunoblot analysis method, it is preferable to proceed by the sandwich measurement method.

According to the antigen-antibody binding, the number of fluorescent beads immobilized on the gold thin film may be measured by a fluorescence microscope to measure the concentration of the antigen, which is a biomarker. In this case, by comparing the measured values of the fluorescent beads with a standard curve The concentration of the biomarker can be quantified.

In addition, by comparing the inside and the outside of the patterned thin film observed through the fluorescence microscope can be observed whether the non-specific binding at a glance, by comparing the number of beads fixed inside and outside the thin film, specificity of the antigen-antibody reaction The signal by combining can be measured.

In particular, the biomarker is simultaneously reacted with a plurality of patterned thin films on the lower substrate to compare the number of beads bound to the inside and outside of the thin film, thereby distinguishing the non-specific binding from the specific binding, and simultaneously determining the number of bound beads. Quantification using the mean value and the variance can reduce the error than the quantification method by the response in one pattern, thereby ensuring reproducibility and accuracy in the quantification of the signal.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited thereto.

Preparation Example: Preparation of Bio Sensing Chip

1 illustrates a biosensing chip according to an embodiment of the present invention, wherein the biosensing chip includes a sensing unit 100 including an intaglio patterned gold thin film 112 and a silicon substrate, a biological sample inlet 212b, and a channel ( And a channel unit 200 including 212a. At this time, four channels (7.5 × 2 × 0.12 mm ³ ) exist in one chip, and the gold thin film contacting the biological sample in each channel is patterned into five rectangular shapes (800 μm × 180 μm). do.

2 shows the sensing unit and the channel unit separately.

Example 1 Measurement of Biomarkers Using Biosensing Chips

A 3,3'-dithio-bis-propionic acid N-hydroxysuccinimide ester (DTSP) self-assembled monolayer was formed to immobilize the antibody on the gold thin film surface of the bio-sensing chip. Prior to forming the monomolecular film, the sensing part of the biosensing chip may be washed with a piranha solution (30 wt% H ₂ O ₂ and H ₂ SO ₄ 1: 4, v / v) or 150 W, O ₂ 50 sccm Washing by exposure to plasma for 5 minutes. The washed sensing part was immersed in a DMSO solution in which 5 mM DTSP was dissolved for 2 hours to form a DTSP monomolecular film on the patterned gold thin film to expose the succinimide group.

After forming the monomolecular film, the sensing unit is washed with DMSO and ethanol solution and tertiary distilled water and combined with the channel unit. 8 μl of 0.1 mg / ml cTnI (Cardiac Troponin I, 625 clone from Biodesign) was flowed into one sample inlet of the channel part, and reacted for 1 hour. Immobilize the first antibody.

Excess PBS (phosphate buffered saline) was flowed into the channel to wash the remaining antibody, and 10 mM ethanol amine solution was flowed for 10 minutes to minimize non-specific binding at the succinimide group of unreacted residues in the self-assembled monolayer. The succinimide groups were blocked, and washed by pouring a bicarbonate buffer and a PBS solution. Thereafter, 8 μl of a PBS solution added with cTnI, a myocardial infarction marker at a concentration of 0, 0.1, 1, 10, and 100 ng / ml, was injected into the channel through a sample inlet, and reacted for 30 minutes. Excess PBS was flushed to wash out cTnI that remained unreacted.

Meanwhile, in order to optically measure cTnI immobilized on the chip surface, a composite of fluorescent beads and cTnI secondary antibody was prepared. Carbohydrates were prepared in 200 nm polystyrene fluorescent beads (Invitrogen F8809, containing 4.6 × 10 ¹³ beads per ml) loaded with orange fluorescent material which was excited at 540 nm and emitted at 560 nm. Vodaimide and hydroxysuccinic acid were added to activate the carboxylic acid on the surface of the beads for 15 minutes to react with the amine groups of the antibody. Then, cTnI secondary antibody (detecting antibody, 19C7 clone of Biodesign Co., Ltd.) was added and reacted for 2 hours, and then L-lysine (L-lysine) was used to minimize nonspecific binding other than specific binding according to antigen-antibody binding. ) And PEG reacted with the antibody and blocked the remaining amine reactive residues to obtain a composite of fluorescent beads and cTnI secondary antibody.

In order to measure the concentration of cTnI immobilized on the sensing unit, the prepared mixture of fluorescent beads and cTnI secondary antibody was diluted 100-fold with PBST (PBS-Triton X 100) buffer containing 0.1% of BSA, and the dilution 8 μl was injected into the channel and allowed to react for 10 minutes.

3 illustrates a sensing process of the sensing unit.

Afterwards, the beads bound to the patterned thin film of the chip were counted and quantified using the Image J program, and the specific reaction and the electrostatic reaction by the antigen-antibody reaction were compared by comparing the pattern and the number of beads bound outside the pattern. The degree of nonspecific binding, such as binding and hydrophobic binding, was compared together. At this time, the number of beads outside the pattern (silicon substrate portion) was quantified by dividing it into 800 μm × 180 μm having the same size as the pattern, and the results are shown in FIGS. 4 and 5.

As shown in FIG. 4, the degree of binding of the fluorescent beads in and out of the patterned thin film was different depending on the concentration of the antibody from 0.1 ng / ml to 100 ng / ml, and the number of beads was quantified in FIG. 5. In it was confirmed that the measurement to 0.1 ng / ml.

Example 2 Measurement of Biomarkers Using Binding Between Biotin-Binding Antibodies and Avidin-Bound Fluorescent Beads

In order to introduce a biotin-avidin reaction in order to obtain a more amplified signal in Example 1 was tested as follows.

As a second antibody, 8 μl of a cTnI antibody (LF-EK0128 ELISA kit, Youngin Frontier Co., Ltd.) bound to the biotin was poured into the channel portion and allowed to react for 30 minutes.

Meanwhile, carbodiimide and hydroxysuccinic acid were added to the same polystyrene fluorescent beads (Invitrogen F8809, including 4.6 × 10 ¹³ beads per ml) as in Example 1, whereby the carboxylic acid on the surface of the beads reacted with the amine group of the antibody. It was activated for 15 minutes. Next, after adding avidin for 2 hours and reacting the antibody with L-lysine and PEG to minimize nonspecific binding other than the specific binding according to antigen-antibody binding, the remaining amine reactivity The residues were blocked to yield avidin binding fluorescent beads.

In order to measure the concentration of cTnI immobilized on the sensing unit, the prepared avidin-bound fluorescent beads were diluted 100-fold with PBST (PBS-Triton X 100) buffer containing 0.1% of BSA, and 8 μl of the diluted solution was added to the channel unit. It was added and reacted for 10 minutes.

6 illustrates a schematic diagram of a sensing process of the sensing unit.

In addition, the quantification process was performed in the same manner as in Example 1, and the morphology and quantification results of the pattern and the beads bound to the outside of the pattern are shown in FIGS. 7 and 8.

As shown in FIGS. 7 and 8, it was confirmed that the amount of fluorescent beads immobilized in the pattern was increased as compared with Example 1, thereby enabling more accurate measurement.

10: lower substrate 12: gold thin film
14 self-assembled monolayer 20: primary antibody
30: antigen 40: secondary antibody
42: Biotin 46: Avidin
50: fluorescent bead
100 sensing unit 110 lower substrate
112, 114, 116, 118: gold thin film
200: channel portion 210: upper substrate
212, 214, 216, 218: Channel

Claims (11)

A sensing unit in which a gold thin film is patterned in a negative shape on a lower substrate; And
Channel portion including a biological sample inlet of the upper substrate and a channel providing a moving passage for contacting the biological sample to the sensing unit
Biosensing chip for biological sample analysis, comprising a.
The biosensing chip for biological sample analysis according to claim 1, wherein a self-assembled monolayer film formed on the surface of the gold thin film is formed.
The method of claim 2, wherein the self-assembled monolayer film is 3,3'-dithio-bis-propionic acid N-hydroxysuccinimide ester (3,3'-dithio-bis-propionic acid N-hydroxysuccinimide ester, DTSP). Bio-sensing chip for biological sample analysis, characterized in that made.
The biosensing chip of claim 1, wherein at least two channels are present.
The biosensing chip for biological sample analysis according to claim 1, wherein at least two gold thin films contacting the biological sample in each channel are present.
Claims 1 to 5 in the biological sample comprising the step of injecting a biological sample, antigen-specific antibody and fluorescent beads containing the antigen as a biomarker in the sample inlet of the biosensing chip of any one of the selected. Method for measuring biomarkers.
The method of measuring a biomarker in a biological sample according to claim 6, wherein the antigen-antibody binding reaction is measured by a fluorescence label immunoassay.
The method of measuring biomarkers in a biological sample according to claim 6, wherein the antigen-antibody binding reaction is measured by a sandwich immunoassay.
The method of claim 6, wherein the amount of fluorescent beads immobilized on the inside and outside of the patterned gold thin film is compared.
The method of claim 6, wherein the measured values in each of the patterned gold thin films are averaged and quantified.
The method of claim 6, wherein the concentration of the biomarker is quantified by comparing the measured value of the fluorescent beads with a standard curve.

Images