WO2013051890A2 - Method for manufacturing multiple-diagnosis membrane sensor by using screen printing - Google Patents

Abstract

The present invention relates to a method for manufacturing a multiple-diagnosis membrane sensor provided with multiple channels by using screen printing, and more specifically, to a method for manufacturing a membrane sensor capable of multiple diagnosis by means of forming multiple channels by screen-printing a hydrophobic ink on top of a membrane. The membrane sensor according to the present invention can allow mass-production of sensors and secure reliability of detection by forming the plurality of channels on top of the membrane by means of a simple method.

Description

Manufacturing Method of Multi-Diagnostic Membrane Sensor Using Screen Printing

The present invention relates to a method for manufacturing a multi-diagnosis membrane sensor in which multiple channels are formed by using screen printing. More particularly, the present invention relates to a method for manufacturing a multi-diagnosis membrane sensor in which multiple channels are formed by screen printing on a membrane. It relates to a manufacturing method.

The strip diagnostic sensor has been widely used since the pregnancy kit in 1976, and is now widely used as a technology for early diagnosis of various diseases. The most widely used strip sensor is an immunochromatography method using a nitrocellulose membrane, and has advantages of easy mass production, convenient use, and low manufacturing cost. In general, in addition to sensitivity and reproducibility, diagnostic biosensors are known to be important for manufacturing cost, productivity, and easy measurement methods. In addition, the necessity of a technique for simultaneously diagnosing various diseases with a single measurement is increasing. Therefore, if a method of easily printing and manufacturing multiple patterns on a membrane widely used in diagnostic biosensors is devised, it is possible to mass-produce strip sensors capable of multiple diagnosis in various diagnostic biosensors including lateral flow assay (LFA). will be.

Recently, researches on manufacturing sensors by printing microfluidic channels using wax or paraffin on paper or membranes have been actively conducted (Diagnostics for the Developing World: Microfluidic Paper-Based Analytical Devices, Anal.Chem. , 2010, 82 (1), pp 3-10; Inkjet-printed microfluidic multianalyte chemical sensing paper, Anal Chem, 2008, 80, pp 6928-6934; FLASH:.. A rapid method for prototyping paper-based microfluidic devices, Lab Chip , 2008, 8, pp 2146-2150; Simple telemedicine for developing regions: Camera phones and paper-based microfluidic devices for real-time, off-site diagnosis, Anal.Chem., 2008, 80, 3699-3707; Fabrication and characterization of paper-based microfluidics prepared in nitrocellulose membrane by wax printing, Anal.Chem., 2010, 82, pp 329-335)

Such microchannel channel sensors have advantages in that they are simple to use, inexpensive, and can be mass-produced, but there is a difficulty in optimizing the composition of the ink to be used according to the inkjet printing technology.

Therefore, if the method of manufacturing a multi-channel by applying a micro euro channel manufacturing technique to a strip sensor is easy and mass-produced, it will be possible to manufacture a multi-channel strip sensor that can be easily measured at a low price.

SUMMARY OF THE INVENTION The present invention is derived from this background, and an object thereof is to provide a method for manufacturing a multi-diagnostic membrane sensor that can be mass-produced through screen printing on a membrane.

In order to solve the above problems, the present invention provides a method of manufacturing a multi-channel membrane sensor comprising the step of forming a multi-channel by screen printing (hydrophobic ink) on the membrane through which a water-soluble sample can flow.

In one embodiment, the membrane is characterized in that made of one or more of nitrocellulose, nylon, polysulfone, polyethersulfone and PVDF (Polyvinylidene fluoride).

In one embodiment, the hydrophobic ink is one or more selected from silver paste, carbon paste, platinum paste, ceramic paste, and wax, alcohol having 1 to 6 carbon atoms, dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and It is characterized by comprising one or two or more organic solvents selected from acetone.

The present invention also provides a membrane sensor using a lateral flow including multiple channels formed by screen printing hydrophobic ink on the membrane surface.

In one embodiment the membrane sensor, characterized in that it comprises a sample pad, a conjugation pad and an absorbent pad on the membrane.

In one embodiment of the membrane sensor, the sample pad or absorbent pad is characterized in that consisting of at least one selected from cellulose, polyester, polypropylene and glass fibers.

In one embodiment of the membrane sensor, the conjugation pad is made of at least one selected from cellulose, polyester, polypropylene, fiberglass, nitrocellulose, nylon, polysulfone, polyethersulfone and polyvinylidene fluoride (PVDF). do.

In one embodiment of the membrane sensor, the signaling material is a metal nanoparticle, quantum dot (dot), nanoparticles, magnetic nanoparticles, enzymes, enzyme substrate, enzyme reaction generating material, absorbing material, fluorescent material or luminescent material It features.

The present invention also provides a method for analyzing a biological sample injecting a sample into the membrane sensor to measure the analyte.

In one embodiment, the measuring method comprises measuring an electrochemical signal between multiple channels of the membrane sensor.

The membrane sensor of the present invention can form a plurality of channels on the membrane by a simple method, it is possible to mass-produce the sensor, and also to ensure the reliability of the detection.

Figure 1 illustrates the process of forming multiple channels on a membrane using screen printing of the present invention.

2 shows a configuration of a membrane sensor according to an embodiment of the present invention.

Figure 3 is a photograph confirming the flow spread around the pattern when the phosphate buffered saline (PBS) solution on the screen printed membrane in accordance with Examples 1 to 3 (Example 1: Ethanol free, Example 2: Ethanol 5 Weight percent, Example 3: 10 weight percent ethanol).

Figure 4 is a photograph showing the sensing results according to the concentration of the CRP antigen in Example 4.

5 is a graph comparing the absorbance of each channel by the concentration in Example 4 by concentration.

Figure 6 shows a schematic diagram and a photograph of a sensor for measuring the electrochemiluminescence signal in the membrane sensor screen-printed in Example 5.

FIG. 7 is a photograph of a chemiluminescent signal measured according to an applied voltage in Example 5. FIG.

FIG. 8 is a graph comparing measurement of a current change according to an applied voltage with time in Example 5. FIG.

[Description of the code]

10: membrane 11: sample pad

12: conjugation pad 13: absorption pad

14: lower substrate 15: anti-CRP polyclonal antibody

16: anti-mouse IgG 20: hydrophobic ink

30: paraffin ink

The present invention relates to a method for manufacturing a multi-channel membrane sensor, characterized in that to form a multi-channel on the membrane using a screen printing (Screen Printing) method.

1 illustrates a process of a multi-channel membrane sensor according to an embodiment of the present invention.

First, the membrane 10 includes all materials in the form of a membrane capable of horizontal flow that can act as a passage through which a biological sample is transferred and at the same time confirm a desired chemical or biological reaction result. More preferably, a membrane made of a material selected from nitrocellulose, nylon, polysulfone, polyethersulfone, and polyvinylidene fluoride (PVDF) may be used, but is not limited thereto. May be appropriately selected.

Hydrophobic ink 20 is printed on the membrane by using a screen printing method. The screen printing may be applied by a method well known in the art. For example, the printer for performing the screen printing may include a membrane formed under the mask and a squeegee for applying a fine mesh and a hydrophobic ink having an opening forming a predetermined pattern. It may be composed of a supporting stage module. The screen printed ink is applied on the membrane to a thickness of 5 to 10 μm, and the applied ink is partially deposited through the pores of the membrane.

The hydrophobic ink is not limited as long as the component is not mixed with the injected sample and can serve as a channel for inducing the flow of the sample, but is preferably silver paste, carbon paste, platinum paste, ceramic paste or wax. Etc. may be used, and most preferably, silver paste is used.

After printing in a desired form through the screen printing, and drying it to form a multi-channel that enables the movement of the sample.

In addition, it is preferable that the hydrophobic ink further includes an organic solvent. Since the hydrophobic ink is printed with an appropriate organic solvent because the membrane has a property of dissolving in an organic solvent, the hydrophobic property of the ink is increased, so that the sample around the pattern Can prevent the spread. In addition, when the organic solvent is used, the efficiency of penetration of the printed ink through the pores of the membrane is increased, thereby increasing the hydrophobic property of the formed channel.

The organic solvent may be an alcohol having 1 to 6 carbon atoms, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, or the like. However, the organic solvent may be appropriately controlled according to the material of the membrane used. In addition, the organic solvent is preferably 1 to 80 parts by weight, more preferably 5 to 50 parts by weight based on 100 parts by weight of hydrophobic solvent.

In addition, when drying the screen printed membrane, it is preferable to dry for 10 minutes to 3 hours in an oven at 50 ~ 200 ℃. Meanwhile, the membrane sensor of the present invention is a component of a typical strip biosensor as shown in FIG. The sample pad 11 may include a conjugation pad 12, an absorbent pad 13, and a lower substrate 14. In the present invention, the sample pad is a liquid sample is introduced into the reaction membrane, the absorption pad serves to absorb the sample developed into the reaction membrane. The sample pad or absorbent pad is not limited as long as it is a material capable of absorbing a liquid sample, but is preferably made of a material such as cellulose, polyester, polypropylene or glass fiber.

In the present invention, the conjugation pad may include a signal generating material; Alternatively, a conjugate of a substance that selectively binds to an analyte and a signal generating agent, such as a detection antibody, may be used.

When the signaling material is applied to the conjugation pad and then dried, for example, when the signaling material is an enzyme, an enzyme substrate or a chemiluminescent material, the reaction material reacts with the receptor on the membrane. By injecting or adsorbing an enzyme substrate or an enzyme in advance, and then injecting a sample, the analyte may be measured by a signal of a signal generating material by an enzyme reaction. If the conjugate pad is coated with a substance that selectively binds to the analyte and a signal generating material, and then dried on the conjugation pad, the analyte as a signal of the signal generating material by the selective reaction between the receptor and the analyte. Can be measured.

In the present invention, after the conjugate pad is coated and dried, the conjugate pad may be used as long as the conjugate pad is easily separated from the conjugate pad when the conjugate pad is wet with liquid, and is commonly used in LFA systems. Any gating pad can be used, as well as materials such as cellulose, polyester, polypropylene or fiberglass, as well as membrane materials such as nitrocellulose, nylon, polysulfone, polyethersulfone or polyvinylidene fluoride (PVDF). .

The signal generating material may be a metal nanoparticle, a quantum dot nanoparticle, a magnetic nanoparticle, an enzyme, an enzyme substrate, an enzyme reaction generating material, an absorbing material, a fluorescent material, or a light emitting material. When the signal generating material is a metal nanoparticle, the analyte may be detected through the color change of the metal nanoparticle by a selective reaction between the receptor and the analyte, and the analyte selectively bound to the receptor on the membrane. The analyte can be quantitatively analyzed by measuring the absorbance and electrical conductivity of the conjugate of the metal nanoparticles. Such metal nanoparticles may be, for example, gold nanoparticles, silver nanoparticles, copper nanoparticles, and the like, but are not limited thereto.

When the signal generating material is an enzyme, an enzyme substrate or an enzyme reaction generating material, a redox reaction is performed by reacting the analyte or receptor with the enzyme, enzyme substrate or enzyme generating product by a selective reaction between a receptor and an analyte. Enzymatic reactions such as, etc. may be caused, and the analyte may be detected by measuring absorption, fluorescence, and luminescence of the product by the enzyme reaction. Such enzymes may be, for example, but not limited to, glucose oxidase, glucose dehydrogenase, alkaline phosphatase, peroxidase, and the like, and the enzyme substrate may be, for example, but not limited to, glucose, hydrogen peroxide, and the like.

In addition, as the signal generating material, a light absorbing material, a fluorescent material, or a light emitting material known in the art may be used, and a specific kind thereof may be appropriately selected by those skilled in the art.

In the present invention, the sample injected through the sample pad may be any sample including or without an analyte, and means a fluid that may flow from the sample pad to the absorption pad using the reaction membrane as a medium. Specifically, it means a sample in liquid form containing blood, serum, or a specific analyte (DNA, protein, chemical, toxic substance, etc.).

In another aspect, the present invention is characterized by a method of measuring the analyte through an immune response by injecting a sample into a multi-channel membrane sensor formed by printing a hydrophobic ink as described above.

By detecting a detection antibody or the like in each of the multiple channels, independent measurement in each channel is possible. That is, for example, in the case of a typical LFA strip, the unreacted gold nanoparticle antibody complex passes first through the immobilized portion of the detection antibody, and then binds with anti-mouse IgG to display a control signal. As a result, when the concentration of antigen is high, the result of the anti-mouse IgG region is low, but in the case of the sensor of the present invention, the antigen-antibody reaction is independently performed in each of the multiple channels, and the detection antibody is fixed to the channel. Otherwise, the control channel is not affected by the concentration of the antigen, thereby obtaining more reliable results.

In addition, by using the electrical properties of the screen-printed hydrophobic ink to measure the electrochemical signal between the multi-channel of the membrane sensor can be analyzed the sample, more specifically injecting the test solution containing the sample in the multi-channel between the electrodes After applying a constant voltage or current between the opposite electrodes, the concentration of the detection substance in the injected sample can be measured by measuring the current or resistance reflecting the electrical properties of the test solution existing between the electrodes. For example, when using a test solution with a higher resistivity compared to the detection material, a lower current flows through the test solution due to the higher resistance as the concentration of the detection material increases. Therefore, it is possible to measure the concentration of the detection substance by measuring the current flowing between the two electrodes.

The foregoing and further aspects of the present invention will be more clearly described through the preferred embodiments described with reference to the accompanying drawings. Hereinafter, the present invention will be described in detail so that those skilled in the art can easily understand and reproduce the present invention through these embodiments.

 However, the following examples are only intended to illustrate the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation of Multichannel Membrane by Screen Printing

As a hydrophobic ink Silver paste was screen printed on a nitrocellulose membrane (Millipore, 180 sec Nitrocellulose, 4 mm × 25 mm) with a thickness of 5-10 μm to form four channels as shown in FIG. 1. Then, after drying for 15 minutes at room temperature, and treated for 1 hour at 100 ℃ drying oven to finally obtain a multi-channel membrane.

Examples 2-3: Screen Printing Using Organic Solvents

The membrane was prepared in the same manner as in Example 1 except for mixing ethanol to 5% by weight and 10% by weight to the silver paste, and then screen printing the same.

A phosphate buffered saline (PBS, Gibco, USA) solution containing 1% (w / v) protease free Bovine serum albumin (Fitzerald) was flowed to the prepared multi-channel membrane, and the flow spread around the pattern. It confirmed and shown in FIG.

As shown in FIG. 3, in the case of the printed pattern of silver paste to which ethanol was added, the solution spread around the channel was greatly reduced compared to the pattern without addition, especially when 10 wt% of ethanol was included. It can be seen that there is almost no flow spread around. In general, nitrocellulose membranes are soluble in ethanol, so when the concentration of ethanol is properly adjusted, silver paste is invaded and its hydrophobicity is increased.

Example 4 Measurement of C-Reactive Protein in Human Serum Using a Biosensor

4-1. Gold nanoparticle-antibody conjugate synthesis

0.1 mL of 0.1 M boric acid buffer (pH 8.5) was added to 1 mL of gold nanoparticle colloid solution (20 nm, BBInternational, GB), and 10 μl of 1 mg / mL anti-CRP antibody (Abcam) was added for 30 minutes. I was. After the reaction, 1% (w / v) of BSA (protease free Bovine serum albumin, Fitzerald) dissolved in phosphate buffered saline (PBS, Gibco, USA) was added and reacted at 4 ° C. for 60 minutes. After the reaction, centrifuged at 10,000 rpm and 4 ° C. for 20 minutes, 1 mL of 1 mg / mL BSA solution dissolved in 10 mM PBS was added and purified and recovered three times to synthesize gold nanoparticle-antibody conjugates. The synthesized gold nanoparticle-antibody conjugate was concentrated 2.5 times, and dried by injecting 10 μL into a conjugation pad (fusion 5, whatman) cut to about 7 × 4 mm.

4-2. Preparation of the Membrane

As a hydrophobic ink Silver paste was screen printed on a nitrocellulose membrane (Millipore, 180 sec Nitrocellulose, 7 mm × 25 mm) to a thickness of about 10 μm so that two channels were formed as shown in FIG. 2.

Then, after drying for 15 minutes at room temperature, and treated for 1 hour at 100 ℃ drying oven to finally obtain a multi-channel membrane.

The membrane and the sample pad (glass fiber, 7 mm × 7 mm), the absorption pad (glass fiber 7 mm × 10 mm) and the prepared conjugation pad was used to configure a biosensor as shown in FIG.

One of the two channels formed was fixed with 1 μL of anti-CRP polyclonal antibody (15) as a detection antibody and anti-mouse IgG (16) on the other at a concentration of 1 mg / ml.

Next, 50 μl of human plasma (CRP free serum) in which CRP was dissolved at concentrations of 0, 1, 10, 100, and 1000 ng / mL was injected into the sample pad, and absorbance in each channel was measured after 3 minutes.

A photograph of the injection result of the sample is shown in FIG. 4, and the measured absorbance at each channel was compared in FIG. 5.

As shown in FIGS. 4 and 5, the control region (right, anti-mouse IgG is fixed) shows high response regardless of CRP concentration, whereas the test region (left, anti-CRP polyclonal). In the case where the antibody is immobilized, the signal was increased as the CRP concentration was increased.

Example 5: Measurement of electrochemiluminescence signal using ruthenium tris-bipyridine in strip sensor with silver paste screen printing

5-1. Sensor manufacturing

Print a 1.1 mm channel on a nitrocellulose membrane (Millipore, 90 sec Nitrocellulose, 12 mm × 10 mm) using a printer using a paraffin solid ink, and treat it for 1 minute at 100 ° C to allow the paraffin to penetrate into the membrane. It was. As shown in FIG. 6, the electrode was printed with silver paste on the screen 500 μm by the method of Example 1, and dried at room temperature for 3 hours. After drying was completed, copper wires were bonded to each electrode surface using DOTITE D-500 conductive silver paint (Fujikura Kasei Co. Ltd) between the two channels, through which each electrode was connected to CompactStat (Ivium Technologies). .

5-2. Electrochemiluminescence Signal Measurement

After dissolving 1 mg of ruthenium tris-bipyridine in 100 μl of DMSO, 5 M tripropyl amine dissolved in PBS buffer solution was diluted 100-fold in 10 mM PBS buffer, respectively, and each solution was mixed in a volume ratio of 1: 1. To prepare a reaction solution. 20 μl of the prepared reaction solution was injected into the lower end of the membrane of the sensor, and passed through the channels, and then 1.7V, 2.5V, and 4V were applied for 160 seconds, and current was measured in units of 1 second. It was. The chemiluminescence signal was measured using LAS3000 (Fujifilm).

7 is a photograph showing a chemiluminescence signal measured through the above process, and FIG. 8 is a graph illustrating a comparison of measured current changes according to an applied voltage over time. As shown in FIG. 7 and FIG. 8, it can be seen that as the applied voltage is higher, the chemiluminescence signal and the measurement current increase between the channels, which can be measured using the electrical characteristics of the silver paste pattern manufactured through the present invention. To support it.

As a result, it was confirmed that the antigen concentration can be easily measured through multiple immunochromatography by the biosensor of the present invention. In addition, it was confirmed that it is possible to measure the electrochemiluminescence signal using the electrical properties of the screen printed paste.

Having described the specific parts of the present invention in detail, it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

1. A method of manufacturing a multi-channel membrane sensor comprising the step of screen printing a hydrophobic ink on a membrane through which a water-soluble sample can flow.
2. The method of claim 1,

   The membrane is a nitrocellulose, nylon, polysulfone, polyethersulfone and PVDF (Polyvinylidene fluoride) a method for producing a multi-channel membrane sensor, characterized in that made of one or more.
3. The method of claim 1,

   The hydrophobic ink is a method of manufacturing a multi-channel membrane sensor, characterized in that at least one selected from silver paste, carbon paste, platinum paste, ceramic paste and wax is used.
4. The method of claim 1,

   The hydrophobic ink is a method for producing a multi-channel membrane sensor, characterized in that it comprises one or two or more organic solvents selected from alcohols of 1 to 6 carbon atoms, dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and acetone.
5. A membrane sensor using a lateral flow, characterized in that it comprises a multi-channel formed by screen printing the hydrophobic ink on the membrane surface.
6. The method of claim 5,

   A membrane sensor comprising a sample pad, a conjugation pad and an absorbent pad on a membrane.
7. The method of claim 6,

   The sample pad or absorbent pad is a membrane sensor, characterized in that consisting of at least one selected from cellulose, polyester, polypropylene and glass fibers.
8. The method of claim 6,

   The conjugation pad is a membrane sensor comprising at least one selected from cellulose, polyester, polypropylene, glass fiber, nitrocellulose, nylon, polysulfone, polyethersulfone and polyvinylidene fluoride (PVDF).
9. The method of claim 6,

   The conjugation pad is a membrane sensor, characterized in that a conjugate of a signal generating material or a material selectively binding to the analyte and a coral generating material is processed.
10. The method of claim 9,

    The signal generating material is a metal nanoparticle, quantum dot (quantum dot), nanoparticles, magnetic nanoparticles, enzymes, enzyme substrates, enzyme reaction generating material, light absorbing material, fluorescent material or light emitting material, characterized in that the membrane sensor.
11. A method of analyzing a biological sample, wherein the analyte is measured by injecting a sample into the membrane sensor of any one of claims 5 to 10.
12. The method of claim 11,

    And measuring an electrochemical signal between the multiple channels of the membrane sensor.

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