KR20150139639A - Paper based biosensor - Google Patents

Abstract

The present invention relates to a paper-based biosensor, which has a gold combining line for combining an antibody of a specimen and gold nanoparticles, a control line (reference line) made of cortisol combined with bovine serum albumin, and a test line (inspection line) made of antibodies on paper made of a hydrophilic material, has a signal amplification effect by forming wax barriers on the underside of the paper, around the edges of the paper, and on both ends of the test line and the control line so as to reduce the length of the wax barriers to gather the specimen for an analysis on the central parts of the test line and the control line, and senses human emotions. The paper-based biosensor of the present invention includes the paper made of the hydrophilic material; the gold combining line formed on the paper to be spaced from one end of the paper and made of the gold nanoparticles to combine the antibody of the specimen for an analysis to the gold nanoparticles; the control line made of cortisol-BSA and formed on the paper to be spaced from the gold combining line; the test line made of immunoglobulin G (IgG) and formed on the paper to be spaced from the test line; and a wax unit forming the wax barriers by hardening wax on the underside of the paper and around the edge of the paper. In addition, the paper-based biosensor is formed to gather the specimen for an analysis on the central parts by forming the wax barriers on both sides of the test line and the control line.

Description

Paper based biosensor [0001]

The present invention is based on a paper made of hydrophilic material, comprising a gold binding line for binding gold nanoparticles to an antibody of a sample, a control line (baseline) consisting of cortisol-BSA combined with bovine serum albumin, (Test line), forming a wax barrier at the bottom and edges of the paper, and forming wax barriers at each end of each of the test and control lines to reduce the length thereof. To a paper-based biosensor capable of measuring human sensibility, wherein a sample is collected in the center, resulting in a signal amplification effect.

When stressed, the sympathetic nervous system becomes excited, the pulse and respiration accelerate, and the physiological phenomenon appears, and then the adaptation or resistance to the external stimulus becomes. However, even if you try to adapt, if the stimulus persists or fails to adapt, you will experience internal energy exhaustion and psychological depression or depression.

80% of suicide victims experience depression. In Korea, the number of depressed patients and suicide victims is increasing rapidly in the 2000s.

Generally, emotional biomarkers in body fluids are changed in real time by the feedback reaction to maintain the homogeneity of the body. Therefore, the sensor needs to be able to carry it so that it can measure emotions changing in real time, it has to be fast in measurement, and the price should be low for disposable use.

For this purpose, the present inventors filed a patent application in Korean Patent Laid-Open No. 10-2012-0057596. Korean Patent Laid-Open No. 10-2012-0057596 relates to an electric device-based biosensor for measuring stress, which detects cortisol, which is an index of stress in saliva, using a small microwave resonant element, To measure, a very high frequency measurement system is required. However, miniaturization of such a microwave measurement system is extremely difficult.

Paper-based sensors can be considered for this purpose. Paper is cheap, easy to use and store, and has a biocompatible advantage, but quantitative diagnosis is still difficult.

As a biosensor strip using paper, there is Korean Patent No. 10-1359750. Korean Patent No. 10-1359750 discloses a method of printing an electrode pattern through a carbon ink printing process using a screen printing or an ink jet printer on a lower plate of a paper material, A channel for inserting a living body sample is formed, and an upper plate made of a paper material formed on an upper portion of the channel by an air outlet is adhered to the adhesive film.

Although the biosensor of the Korean Patent No. 10-1359750 is made of paper, it has a specific structure including an air vent, and is complicated, difficult to manufacture, and low in cost. Also, it is not possible to amplify the signal for quantitative diagnosis to measure emotion.

A paper-based biosensor capable of quantitatively diagnosing and measuring human sensibility is desired as a biocompatible paper-based sensor, which is cheap, convenient to use, and easy to store.

In addition, a paper-based biosensor capable of measuring human sensibility with a simple structure, a simple manufacturing process, a portable measuring device capable of measuring emotions changing in real time, and a measuring speed is desired.

In addition, a paper-based biosensor having improved sensitivity to a paper-based microfluidic chip (μPAD) capable of signal amplification for quantitative diagnosis for measuring sensitivity is desired.

Disclosure of Invention Technical Problem [8] The present invention has been made to solve the above problems, and an object of the present invention is to provide a gold binding line (a gold nanoparticle-antibody conjugate) which binds a sample antibody with gold nanoparticles (AuNP-antibpdy, AuNPs-Ab) (Test line) consisting of a control line (baseline) consisting of cortisol (cortisol-BSA) bound to bovine serum albumin, an antibody (immunoglobulin g, immunoglobulin G, IgG) Forming a wax barrier on the bottom and edges of the paper and forming wax barriers at both ends of each of the test and control lines to reduce the length of the test and control lines, The present invention also provides a paper-based biosensor capable of measuring human sensibility, which causes a signal amplification effect.

Another object to be solved by the present invention is to provide a method and apparatus which can be easily carried out in a simple structure, simple in manufacturing process, capable of measuring emotions changing in real time, fast in measurement speed, A paper-based biosensor capable of measuring human sensibility, which is convenient and biocompatible paper-based sensor capable of quantitative diagnosis.

Another object of the present invention is to provide a paper-based biosensor capable of measuring human sensibility, which has improved sensitivity to a paper-based microfluidic chip (μPAD) capable of signal amplification for quantitative diagnosis of emotion.

According to an aspect of the present invention, there is provided a paper-based biosensor comprising: a paper made of a hydrophilic material; A gold binding line made of gold nanoparticles on the paper so that the antibody of the assay sample and the gold nanoparticles are bonded to each other; A control line spaced from the gold binding line on the paper, the control line consisting of cortisol-BSA; A test line spaced from the test line on the paper, the test line comprising immunoglobulin g (IgG); And a wax portion that forms a barrier by hardening wax on a bottom surface and an edge of the paper.

In addition, the paper-based biosensor of the present invention is characterized in that a fluid channel is formed by forming a barrier wall by hardening wax on a bottom surface and a rim of a paper made of a hydrophilic material.

A wax barrier is formed on both sides of the test line so that the test sample flows and collects in the middle test line.

A wax barrier is formed on both sides of the control line so that the analytical sample flows and collects in the control line in the middle.

The paper consists of paper used in Whatman Chromatography.

The length of the control line is 3 mm to 4 mm, and the length of the test line is 3 mm to 4 mm.

The method of manufacturing a paper-based biosensor according to the present invention comprises: a first step of printing with a solid wax print so as to form a barrier by solidifying wax on the bottom and edges of a paper made of a hydrophilic material; And a second step of baking the paper in an oven at 125 degrees for about 1 minute after the first step.

On the paper after the second step, a control line consisting of a gold binding line consisting of gold nanoparticles, a cortisol-BSA, a third line forming a test line consisting of immunoglobulin G (IgG) Further comprising:

In a first step, the method further comprises forming a test line wax barrier, which is a wax barrier formed on both sides of the test line, and a control line wax barrier, which is a wax barrier formed on both sides of the control line.

According to the present invention, on a paper made of a hydrophilic material, a gold-binding line for binding the antibody of the sample and gold nanoparticles, a control line (baseline) consisting of cortisol-BSA combined with bovine serum albumin, (Test line), forming a wax barrier at the bottom and edges of the paper, and forming wax barriers at each end of each of the test and control lines to reduce the length thereof. The present invention provides a paper-based biosensor capable of measuring human sensibility, wherein the sample is collected in the center, thereby causing a signal amplification effect.

The present invention also provides a paper-based sensor that is simple in structure, simple in manufacturing process, capable of measuring emotions changing in real time, fast in measurement speed, low in cost, easy to use and store, And quantitative diagnosis of human emotion is possible.

The present invention also provides a paper-based biosensor having improved sensitivity to a paper-based microfluidic chip (μPAD) capable of signal amplification for quantitative diagnosis of emotion.

1 is a schematic diagram of a paper-based biosensor capable of measuring emotion according to an embodiment of the present invention.
FIG. 2 is an explanatory view for explaining sensitivity improvement by adjusting the length (or width) of a test line and a control line in the paper-based biosensor of FIG. 1;
3 is an example of a channel section of the paper-based biosensor manufactured in the present invention.
4 is an example of the output intensity comparison of a paper-based biosensor with different channel widths.
5 is an example of light intensity detection results for comparing output intensities of paper-based biosensors with different channel widths.
FIG. 6 is an example of experimental results comparing fluid flow in a paper-based biosensor with different channel widths.
FIG. 7 is an example of an experimental result for comparing the flow of fluid according to the angle (?) At which the channel width decreases when the channel width is reduced to improve the sensitivity.
Figure 8 is an example of an experimental result comparing the flow of fluids when there is only a primary channel reduction and when there is a secondary channel reduction.
FIG. 9 is an example of an experimental result comparing the output strength when there is no channel reduction, only the first channel reduction, and the second channel reduction.
FIG. 10 shows the output intensity according to the cortisol concentration of the paper-based biosensor with secondary channel shrinkage.

Hereinafter, a paper-based biosensor according to the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 is a schematic diagram of a paper-based biosensor capable of measuring sensibility according to an exemplary embodiment of the present invention. FIG. 2 is a graph showing sensitivity (sensitivity) by adjusting the length Fig.

1, the biosensor of the present invention includes a gold binding line 120 for binding a sample antibody and gold nanoparticles (AuNP-antibpdy, AuNPs-Ab) on a hydrophilic paper 100, And a test line 160 (test line) composed of a control line 140 (baseline) composed of cortisol-BSA as cortisol bound to albumin and immunoglobulin G (IgG) as an antibody , The wax is hardened on the bottom surface and the edge of the paper 100 to form a barrier. The wax barriers on the underside of the paper 100 are referred to as bottom wax portions 220 and the wax barriers on the edges of the paper 100 are referred to as edge wax portions 240. [ Also, the gold binding line 120 may be referred to as a gold nanoparticle-antibody conjugate (AuNPs-Ab) line.

A control line 140 and a test line 160 are formed by forming a wax barrier on both sides of the control line 140 and the test line 160 to reduce the lengths of the control line 140 and the test line 160 So that the analytical sample flows and is collected in the center. The wax barrier formed on both sides of the test line 160 is referred to as a control line wax barrier 260. The wax barrier formed on both sides of the control line 140 is referred to as a test line wax barrier. In other words, by reducing the lengths of the control line 140 and the test line 160 in FIG. 2 (b) compared to FIG. 2 (a), the analytical samples are gathered together, Amplification effect. The biosensor of the present invention is a paper-based microfluidic chip (μPAD) capable of signal amplification, and the sensitivity can be adjusted by adjusting the test line wax barrier 250 and the control line wax barrier 260.

The paper 100 may be paper used for Whatman Chromatography. That is, it is possible to use wattmann paper or wattman filter paper (filter paper).

The paper 100 is provided with a first spacing part 115 which is not provided with any line on one side and a gold bonding line 120 is positioned beside the first spacing part 115, The second spacing portion 125 is located between the control line 140 and the test line 160 and the third spacing portion 145 is located between the control line 140 and the test line 160, The fourth spacing portion 165 is located. Although the test line 160 is shown next to the control line 140 in FIG. 1, these locations may be reversed. For example, the test line 160 may be located and then the control line 140 may be located.

The gold binding line 120 couples or binds gold nanoparticles (AuNP-antibpdy, AuNPs-Ab) to an antibody of a sample that has entered through the first spacing 115 of the paper 100 to form a dye indicator dye. As shown in Fig. That is, the antibody in the sample (i.e., cortisol) combines with the gold nanoparticles to form a complex, which moves along the paper 100.

The control line 140 (reference line) is a line on which the paper 100 is treated with cortisol-BSA. At the time of testing, the control line 140 proceeds to the competitive reaction principle of cortisol-BSA and the antigen (cortisol).

The test line 160 (test line) is a line on which the paper 100 is subjected to line treatment with an antibody, that is, immunoglobulin g (IgG). In testing, the complex formed in the gold binding line 120 proceeds in a manner that combines with the antibody of the test line 160. As a result, the color of the test line becomes darker as the concentration of cortisol increases.

In other words, when a sample such as saliva (that is, a biological sample) is dripped (loaded) on the first spacing part 115, which is one side of the paper 100, it flows to the other side of the paper 100 by capillary phenomenon, As the antibody passes through the gold binding line 120, the antibody (i. E., Cortisol) in the sample binds with gold nanoparticles (e. G., Gold-labeled protein A) or the like to form a complex, At this time, the test line 160 binds to the antibody immobilized on the test line 160 of the paper 100, and the test line 160 appears red.

In the case of Fig. 1, a wax barrier was formed to form a channel. It is to be understood, however, that this is not to be construed as an example, and thus the invention is not limited thereto. Generally, the paper 100 can be channel-formed by various methods and can control the flow of the fluid in the paper. In the case of Fig. 1, cortisol is used as the target, but it is not intended to limit the present invention.

<Channel formation method>

Paper can easily change its shape, can be easily cut, and is also hydrophilic, so the surface can be chemically treated.

There are two major types of channel shape fabrication: physical and chemical.

The physical method is literally a method of cutting paper, and in the case of a pattern that is difficult to cut by hand, it can be manufactured using a laser.

The chemical method can form a desired shape by making an obstacle with a hydrophobic material on paper made of hydrophilic material. Examples are printing methods using solvent materials, PDMS, and solid wax printers.

In the case of FIG. 1, a predetermined shape was designed using a computer, and then the channel was printed with a commercialized solid wax print. Methods for improving sensitivity for quantitative diagnosis have led to the concentration of the analytical sample in the middle by reducing the length or width of the middle of the channel. The paper used was paper used for Whatman Chromatography and baked in a 125 ° oven for about 1 minute to allow the desired shape to penetrate into the paper.

3 is an example of a channel section of the paper-based biosensor manufactured in the present invention.

The cross section of the channel of FIG. 3 is photographed with an optical microscope. In this case, the channel is set to have a limited area and the width thereof is reduced from 5 mm to 2 mm at intervals of 1 mm.

<Surface Treatment Process>

(For example, gold-labeled protein A) is formed on the paper 100 before or after the channel is formed to form the gold bonding line 120, and the control line 140 ) Is formed by line treatment with cortisol-BSA, and the test line 160 (test line) is formed by line treatment with immunoglobulin G (IgG). The line processing may be performed by a printer, manually or automatically using a separate line drawing tool such as a spencer, or may be processed by various other methods.

Here, cortisol among a variety of emotional biomarkers changed by external stimuli was targeted and induced a competitive reaction during the conventional antigen-antibody reaction. Cortisol is a mechanism that protects our body from the risks of mental and physical stress and changes its concentration in the body. First, the cortisol antibody and gold particles were synthesized to cortisol, and by using BSA-cortisol on the test line surface, the intensity of the color signal of the test line is lowered because the competitive reaction is induced according to the concentration of cortisol.

4 is an example of the output intensity comparison of a paper-based biosensor with different channel widths.

FIG. 4 is a graph showing the flow of the fluid through the channel surface while dropping 100 μl of ink on one side of the channel. When the width was 4mm, the arrival time was shortest, and it was confirmed that the speed was the fastest. In a channel width of 2 mm, the bottleneck was observed and the fluid could not spread to the end. When cortisol was loaded at 100 ng / ml, it was confirmed that the signal at 3 mm was stronger than the commercialized channel width of 5 mm. This allowed us to check the flow rate and signal amplification.

5 is an example of light intensity detection results for comparing output intensities of paper-based biosensors with different channel widths.

In order to compare the output intensity of the paper-based biosensor having different channel widths (the lengths between A and B in FIG. 5), an experiment was conducted as shown in FIG. 4 and then a 532 nm wavelength was projected. Respectively. FIG. 5 (a) shows paper-based biosensors having different channel widths, and FIG. 5 (b) shows output light intensities of paper-based biosensors having different channel widths. Also in this case, it can be seen that the signal at the channel width of 3 mm is stronger.

6 (a) and 6 (b) show paper-based biosensors having different channel widths, and FIG. 6 (b) shows an example of a paper-based biosensor having different channel widths. FIG. 2 is a graph showing the arrival time according to the channel width in the paper-based biosensor. FIG.

As shown in FIG. 4, the time passing through the proposed channel surface was measured while dropping 100 μl of ink on one side of the channel. When the width was 4 mm, the fastest arrival time was shortest. Therefore, the speed was the fastest when the width was 4 mm.

7A and 7B illustrate experimental results for comparing the flow of the fluid according to the angle? At which the channel width decreases when the channel width is reduced to improve the sensitivity. In FIG. 7A, the angle? FIG. 7 (b) is a graph showing the arrival time according to the channel width in the paper-based biosensor of FIG. 7 (a). FIG.

An experiment was conducted as shown in Fig. 4, and the time passing through the channel surface was measured. It can be seen that the reaching time is the shortest when the angle (θ) at which the channel width decreases is 90 degrees.

8 shows an example of an experimental result comparing the flow of the fluid when only the primary channel reduction is performed and when the secondary channel reduction is performed. FIG. 8 (a) is a paper based biosensor (primarily) FIG. 8 (b) is a paper-based architecture modification u-PAD with secondary channel reduction, and FIG. 8 (c) is a graph showing the arrival time in these paper-based biosensors.

It can be seen that the architecture modification u-PAD has a faster reach time than the primary channel reduction only (primarily).

9 shows the results of an experiment comparing the output intensities of the architecture modification u-PAD 2 when there is no channel reduction, only the first channel reduction (architecture modifications u-PAD 1) and the second channel reduction (architecture modifications u-PAD 2) For example, FIG. 9A shows paper-based biosensors having different channel widths, and FIG. 9B shows output strengths of paper-based biosensors having different channel widths.

In FIG. 9, it can be seen that the signal when the secondary channel reduction is performed is the strongest.

FIG. 10 shows the output intensity according to the cortisol concentration of the paper-based biosensor with secondary channel shrinkage. 10 (a) is a paper-based biosensor in which cortisol concentrations are differently applied, and FIG. 10 (b) is a graph showing output intensities of paper-based biosensors in FIG. 10 (a).

It can be seen from FIG. 10 that the intensity increases with cortisol concentration.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, Modification is possible. Accordingly, it is intended that the scope of the invention be defined by the claims appended hereto, and that all equivalent or equivalent variations thereof fall within the scope of the present invention.

100: paper 115: first separator
120: gold bonding line 125: second separation portion
140: Control line 145: Third separation part
160: Test line 165: Fourth separation part
220: bottom wax part 240: rim wax part
250: test line wax barrier 260: control line wax barrier

Claims (13)

Paper made of a hydrophilic material;
A gold binding line made of gold nanoparticles on the paper so that the antibody of the assay sample and the gold nanoparticles are bonded to each other;
A control line spaced from the gold binding line on the paper, the control line consisting of cortisol-BSA;
A test line spaced from the control line on the paper, the test line comprising immunoglobulin G (IgG);
A wax portion that forms a barrier by solidifying wax on a bottom surface and an edge of the paper;
Based biosensor. Wherein a fluid channel is formed by forming a barrier by solidifying wax on a bottom surface and a rim of a paper made of a hydrophilic material. 3. The method of claim 2,
And a gold binding line made of gold nanoparticles is provided on the paper so that the antibody of the assay sample and the gold nanoparticles are bound to the paper. 3. The method of claim 2,
And a control line made of cortisol-BSA on the paper. 3. The method of claim 2,
And a test line made of an antibody on the paper. 5. The method according to any one of claims 1 to 4,
Wherein a wax barrier is formed on both sides of the control line so that the analytical sample flows and collects in the control line at the center. 6. The method according to any one of claims 1 to 5,
Wherein a wax barrier is formed on both sides of the test line so that the analytical sample flows and collects in the middle test line. 3. The method according to any one of claims 1 to 3,
Wherein the paper is a paper used for Whatman chromatography. 8. The method of claim 7,
Wherein the length of the test line is from 3 mm to 4 mm. The method according to claim 6,
Wherein the length of the control line is 3 mm to 4 mm. A first step of printing with a solid wax print so as to form a barrier by solidifying wax on the bottom and edges of a paper made of a hydrophilic material;
After the first step, the paper is baked in a 125 degree oven for about 1 minute;
Based biosensor according to any one of claims 1 to 3. 12. The method of claim 11,
On the paper after the second step, a control line consisting of a gold binding line consisting of gold nanoparticles, a cortisol-BSA, a third line forming a test line consisting of immunoglobulin G (IgG) step;
Further comprising the steps of: preparing a paper-based biosensor; 13. The method of claim 12,
Wherein a test line wax barrier, which is a wax barrier formed on both sides of the test line, and a control line wax barrier which is a wax barrier formed on both sides of the control line are also formed in the first step.

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