WO2016144093A1

WO2016144093A1 - Method for manufacturing membrane sensor

Info

Publication number: WO2016144093A1
Application number: PCT/KR2016/002345
Authority: WO
Inventors: 김민곤; 송문범; 정효암
Original assignee: 주식회사 인지바이오
Priority date: 2015-03-11
Filing date: 2016-03-09
Publication date: 2016-09-15
Also published as: CN106796220A

Abstract

An embodiment of the present invention relates to a method for manufacturing a membrane sensor, wherein the method may comprise the steps of: using a craft printer to manufacture a paper plate in which a pattern is formed on paper; soaking the paper plate in an organic solvent; attaching the paper plate to a membrane through which a water-soluble sample can flow, thereby stamping the paper plate to the membrane; removing the paper plate from the membrane; and drying the membrane for a period of time. Therefore, a bio-sensor can be manufactured by forming a plurality of various design patterns on a membrane using a simple method of stamping, on a membrane, a paper plate soaked with an organic solvent.

Description

Manufacturing method of membrane sensor

The present invention relates to a method of manufacturing a membrane sensor, and more particularly, to a method of manufacturing a biosensor by forming a pattern on the membrane using an organic solvent.

With the recent development of medicine and medical engineering, various causes and treatments of diseases are introduced, as well as more accurate diagnosis of various diseases, more effective and safe treatment methods have been developed, and treatment rates of diseases are increasing. It is a trend.

Nevertheless, the diagnosis and treatment of such diseases are mainly made in hospitals, and thus, in the case of a diagnosis subject who wants to be diagnosed, there is a problem that it is impossible to diagnose and diagnose the disease at all without visiting a hospital. As a result, it was difficult for the diagnosis subject or the patient to bear the costly examination and treatment costs.

Recently, due to the development of the electronics industry and wired and wireless communication network, a telemedicin system has been introduced as an effort to solve the above problems and difficulties. Such a remote medical system can be diagnosed by a patient or a patient at a relatively low cost in a remote place without directly visiting a hospital, etc., but most of them are only diagnosed for external abnormalities. Inevitably very limited, many difficulties were involved in making an accurate diagnosis.

In particular, diseases that can be life-threatening (eg, various cancers, AIDS, etc.) are usually infected and developed by antigens such as pathogens. There is an urgent need to develop a device capable of diagnosing biomaterials including various antigens. Accordingly, biosensors using membranes have been used as immunoassay devices that do not require specialized knowledge or complicated processes and are simple to use and have a short execution time. In the analysis apparatus using such a biosensor, an immunochromatographic method using a pore membrane as an immobilized parent of a sensitive protein is generally applicable. Absorption of the sample containing the analyte from the bottom of the membrane strip causes the analyte to be transported to the immobilized sensitized protein layer through pores of capillary action, causing adhesion between the antigen and the antibody on the solid surface and unbound components Separated by. Membrane strip immunochromatography techniques based on this principle accelerate the mass transfer of reactive components using the lateral flow of the fluid, so that diagnostics can be completed with rapid measurement of the analyte and only sample addition.

Recently, such biosensors have been researched to produce channels by printing wax, paraffin, etc. on the membrane. However, this biosensor is detected by the influence of temperature and pressure and flow of fluid when forming multiple patterns on the membrane. There is a problem of low reliability.

In LATERAL FLOW AND FLOW-THROUGH BIOASSAY BASED ON PATTERNED POROUS MEDIA, METHODS OF MAKING SAME, AND METHODS OF USING SAME In addition, a patterning technique is described in which a polymer (eg, PDMS) is imbedded in a stamp made for patterning, taken on paper, and the polymer is cured to form a hydrophobic barrier. However, the prior document 1 has to be preceded by a stamp production for patterning, which requires a number of stamp production according to the pattern, excessive time and cost for producing a stamp when the initial pattern is established. In the case of stamps made of plastic and rubber, stamps should be newly produced if the stamps are broken or the patterns are deformed due to impacts during storage. In addition, if the pressing force is not constant during the paper-printing process, the polymer is not evenly distributed, and it is necessary to adjust the pressing force according to the viscosity or property of the polymer. In addition, the phenomenon of spreading on the paper may appear depending on the viscosity or property of the polymer, which may affect channel formation, and thus it is difficult to improve detection reliability by uniform channel formation.

In the 'Flexible Microfluidic Device with Interconnected Porous Network' of U.S. Patent Application Publication No. US2012-0052250 (published on Jan. 1, 2012; hereafter referred to as 'Previous Document 2'), a polymeric sheet capable of forming a microporous network and A patterning technique using a solvent is presented. In the prior document 2, a method of using double-sided adhesive vinyl as a plate for patterning, attaching the double-sided adhesive vinyl to a polymer sheet (eg, polystyrene) forming a microporous network, and then immersing it in a solvent. At this time, the polymer sheet is formed except a portion where the double-sided adhesive vinyl is attached to the reaction by contact with the solvent to swell while forming a microporous network, thereby forming a pattern. However, Prior Art 2 discloses that when the double-sided adhesive vinyl is not properly adhered to the polymer sheet, the double-sided adhesive vinyl is formed in the polymer sheet in the process of dipping the polymer sheet in the solvent, and the reaction is performed by introducing a solvent into the gap. There is a problem that the desired pattern is not formed properly. In addition, in order to form the microporous network only on the surface where the pattern is required during patterning, an adhesive sheet is attached to the other surfaces to prevent the reaction with the solvent. In addition, in the case of forming a microporous network, since the surface is hydrophobic, a process of forming a pattern is complicated and difficult to form a uniform pattern, such as a step of converting the surface to hydrophilicity in order to use a hydrophilic sample. There is a problem that it is difficult to improve the detection reliability.

The present invention is a membrane sensor manufacturing method,

Multi biosensor using membranes to solve the problem of low detection reliability due to difficulty in uniform formation of channels or patterns, which is a common problem of prior art documents such as International Publication No. WO2008-049083 and US Publication No. US2012-0052250 Providing a method of manufacturing a membrane sensor that can improve the detection reliability by forming a plurality of patterns desired by the user on the membrane in a simple manner without being affected by temperature, pressure or fluid flow in manufacturing For the purpose of

Embodiments of the present invention provide a method of manufacturing a membrane sensor, comprising the steps of: producing a plate-shaped plate formed on paper using a kraft printer; Soaking the plate in an organic solvent; Attaching and stamping the plate to a membrane through which a water-soluble sample can flow; Removing the plate from the membrane; And drying the membrane for a predetermined time.

The organic solvent may be selected from any one of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and 1.4-Dioxan. Preferably, the organic solvent may be DMSO having a concentration of 100%.

In addition, the step of attaching and stamping the plate to the membrane through which the water-soluble sample may flow may include: bonding a second fixing plate to one surface of the plate; The first and second fixing plates may be pressed to contact the membrane and the plate, and a predetermined pressure may be applied to the membrane and the plate.

Further, in the embodiment, the step of drying the membrane for a predetermined time, characterized in that for drying for a predetermined time at a temperature of 37 ℃ after removing the plate making from the membrane.

An embodiment may include forming a biosensor without removing the organic film present in the portion dissolved by the organic solvent on the membrane, and may include forming the biosensor by removing the organic film. This makes it possible to manufacture membrane sensors with different applications.

Embodiments of the present invention use a simple method of stamping paper making with organic solvents onto the membrane, thereby forming a plurality of patterns of various designs on the membrane in a single process.

That is, by using paper as a plate for patterning, plate making can be made only through the printing and cutting process of the pattern, and plate making having various patterns according to the conditions of the pattern can be easily produced and tested in one step. In addition, since the solvent absorption amount forms a plate with a constant paper, only the desired portion can be contacted with the solvent, thereby preventing a phenomenon in which the solvent absorbed by the excessive solvent supply is diffused. In addition, the thickness of the pattern can be controlled by controlling the solvent content using the material or thickness of the paper constituting the plate making, and the pattern formed on the membrane can be formed accurately and uniformly in the same shape as the pattern formed on the paper. Can be greatly improved.

Embodiments of the present invention can be used to distinguish the flow of the fluid according to the design of the pattern to be formed, it is possible to improve the reliability of the detection because the temperature and pressure and the flow of the fluid does not affect the membrane.

Embodiments of the present invention can be produced and used by fixing the antibody or antigen, it is possible to produce a multi-biosensor by a simple method.

1 is a view showing a process for patterning the membrane in accordance with an embodiment of the present invention

2 is a view showing a pattern formed by bonding a plurality of plates to the membrane at different intervals;

3 is a graph comparing the width of the actual plate making and the width of the pattern formed on the membrane for 13 patterns formed on the membrane

4 is a graph comparing the value obtained by subtracting the width of the pattern from the width of the plate making according to the width of the paper.

5A illustrates an example of plate making for patterning various designs on a membrane in accordance with an embodiment.

5B shows an actual membrane patterned according to an embodiment.

6 is a graph showing the results of biosensing using the patterned membrane

7 shows a membrane with dual channels formed using 'Tear off' patterning.

8 is a view illustrating a side flow analysis in a conventional membrane sensor, (a) is the case of CK-MB (b) is a view showing the case of myoglobin

9 is a graph measuring the strength for CK-MB in the membrane sensor of the dual channel structure formed by applying the embodiment

10 is a graph measuring the strength of Myoglobin in the membrane sensor of the dual channel structure formed by applying the embodiment

11 is a photograph showing a membrane sensor

12 to 14 are photographs and result graphs of fluid flow tests using the membrane sensors of FIG. 11.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, but are not limited or limited by the embodiments of the present invention. In describing the present invention, a detailed description of known functions or configurations may be omitted to clarify the gist of the present invention.

1 is a view showing a process for patterning the membrane in accordance with an embodiment of the present invention.

Membrane 10 may include any material in the form of a membrane capable of horizontal flow to confirm the desired chemical and biological reaction results while serving as a passage through which the biological sample is transferred. More preferably, a membrane of a material selected from nitrocellulose, nylon, polysulfone, polyethersulfone, and polyvinylidene fluoride (PVDF) may be used, and the present embodiment proposes a method for forming a pattern of a membrane composed of nitrocellulose.

In the method of manufacturing a membrane sensor according to the embodiment, using a craft (craft) printer to form a plate-shaped plate, the step of soaking the plate in an organic solvent, the plate to the membrane through which the water-soluble sample flows Bonding and stamping, removing the plate making from the membrane, and drying the membrane for a predetermined time, will be described below in the specific method of each step.

1 (a) shows a process of preparing a plate quenched with a membrane, a fixed plate and an organic solvent.

In an embodiment, the fabrication of the plate 20, which is first placed on the membrane 10, may be preceded. The plate 20 may be made of paper, but is not limited thereto. A member of a material capable of containing a predetermined level of a solvent in a liquid state may be used.

After the paper is cut to have a predetermined standard, a pattern of a design desired by a user may be formed through a printer. The printer may be a craft printer, and when a user transmits data on a pattern to be formed on a membrane to the craft printer, the plate forming 20 having a desired pattern is formed by processing the paper with a pattern corresponding to the data. Can be prepared.

In the embodiment, when the plate making 20 designed as described above is formed, a process of peeling the paper from the kraft printer machine and quenching the organic solvent 30 may be performed. At this time, any one of DMSO (dimethyl sulfoxide), 1.4-Dioxane, DMF may be used as the organic solvent 30. In the embodiment, DMSO was used as the organic solvent to form a desired pattern on the membrane. DMSO with a concentration of 100% was used as an example.

The organic solvent has a property of dissolving the membrane, and the DMSO, an organic solvent used in the embodiment, has a property of selectively dissolving only a portion in contact with the membrane, and thus may be used to form a pattern on the membrane as in this embodiment. The plate making 20 quenched in DMSO melts only the surface contacted with the membrane when attached to the membrane 10, so that a pattern may be formed on the membrane in the same shape as the shape designed in the Craft printer.

Subsequently, first and

second fixing plates

11 and 12 are provided on upper and lower portions of the membrane 10 and the plate 20, and the membrane 10 is fixed while being attached to an upper surface of the first fixing plate 11. The plate 20 with the solvent 30 may be adhered to the bottom surface of the second fixing plate 12. At this time, the plate 20 is to be bonded to the lower surface of the second fixing plate 12 to correspond to the position of the membrane (10).

If the organic solvent is completely wetted to the plate making 20, the step of taking out and fixing the organic solvent to the glass plate, and then stamping the glass plate in the upward direction of the NC membrane to contact the plate and the membrane can be performed.

FIG. 1 (b) shows a process of stamping a plate and a membrane quenched with the organic solvent DMSO and removing the plate on the membrane.

Bonding and stamping the plate to the membrane, the step of adhering the first fixing plate 11 to one surface of the membrane, adhering the second fixing plate 12 to one surface of the plate 20, the membrane and And compressing the first and second fixing plates so that the plate makes contact with the plate and applying a predetermined pressure to the membrane and the plate.

The second fixing plate to which the plate 20 is bonded is pressed to contact the upper surface of the first fixing plate 11 to which the membrane 10 is bonded, and the membrane 10 provided between the first and

second fixing plates

11 and 12. ), A predetermined pressure may be applied to the stamping process.

When the stamping step as described above is performed, the step of removing the second fixing plate 12 and drying the membrane 20 for a predetermined time may be performed.

After a predetermined time, a step of removing the plate 20 adhered to the membrane 10 may be performed, and a melting part is formed on the membrane by an organic solvent buried in the plate, and a design desired by the user. Pattern is formed. If this is dried for a predetermined time at a temperature of 37 ℃ it can be used as a multi-bio sensor, and the sample can be injected to measure the analyte through an immune response.

On the other hand, the organic film 21 by the organic solvent is formed on the molten portion on the membrane from which the plate making 20 is removed in the step (b), the organic film 21 formed on the molten portion of the membrane as in step (c) After the removal using the tweezers may be carried out a process of drying at a temperature of 37 ℃ for a certain time. According to the embodiment, a biosensor without or without the organic layer 21 may be manufactured, and a biosensor having a different utilization may be manufactured.

2 is a view showing a pattern formed by bonding a plurality of plates to the membrane at different intervals.

Referring to Figure 2, each plate is formed to be spaced apart by the interval of (0.5, 0.7, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10) mm, a total of 13 designs It was formed to have. When the platelets formed with different widths as described above are contacted with the membrane to form a membrane pattern, the width of the desired pattern can be derived by comparing the width of the originally designed plate with the width of the membrane pattern formed by the method of the embodiment.

Specifically, as shown in the following figure of FIG. 2, the widths of each of the membrane patterns designed with 13 different widths were identified by PSI-Stage MIC 10 mm / 0.1 mm DIV in units of 100 μm under a microscope (OLYMPUS uplan 10X 10.30). It was.

3 is a graph comparing the width of the actual plate making and the width of the pattern formed on the membrane for the 13 patterns formed on the membrane, Figure 4 compares the width of the plate minus the width of the pattern according to the width of the plate One graph.

Referring to Figure 3, it can be seen that the width of the plate making to have a different value between 0.5 ~ 10mm always appears larger than the width of the formed NC pattern. This can be interpreted as dissolving the membrane by digging inward to some extent when the organic solvent buried in the plate makes contact with the NC pattern.

Referring to FIG. 4, the trend of a value obtained by subtracting the width of the pattern from the width of the plate making according to the width of the plate making may be confirmed. In other words, when the plate is formed in the range of 0.5 to 1.3 mm, samples 1 to 4, the difference between the width of the plate and the width of the NC pattern is relatively large, but when the plate is formed to be 2 mm or more, It can be seen that the difference in the width of the NC pattern is significantly reduced.

Accordingly, embodiments of the present invention are preferred for designing a pattern aimed at forming the width of the plate making at least 2 mm to form a pattern for the NC membrane when forming a desired pattern. However, the present invention is not limited thereto, and the exemplary embodiment may form a pattern having a size of at least 0.5 mm or more on the membrane according to the design of the biosensor.

5A shows an example of plate making for patterning various designs on a membrane in accordance with an embodiment, and FIG. 5B shows an actual membrane patterned in accordance with an embodiment.

5A and 5B, various patterns having a shape desired by a user may be easily formed on a membrane through the design of a plate making using a craft printer as in the embodiment. The embodiment can easily set the size and separation distance of each pattern as shown, and can design the shape of the pattern according to the sample to be measured and a membrane sensor that can measure the various antigens and antibodies I can make it.

6 is a graph showing the results of biosensing using the patterned membrane. Referring to FIG. 6, a membrane sensor is fabricated by patterning two channels to form a membrane, and biosensing is performed by adding different antigens to each channel.

FIG. 7 is a diagram illustrating a membrane in which dual channels are formed using 'Tear off' patterning. Referring to FIG. 7, dual-channels were formed using the patterning technique of the present invention, and a response to CK-MB was confirmed in myoglobin in the left channel and CK-MB in the right channel. As shown in the figure, when the channels are different from each other, it can be seen that the color development is not affected.

Myoglobin and CK-MB are indicators of cardiac abnormalities such as heart failure and myocardial infarction, and can be fabricated as membrane sensors for use in emergencies.

8 is a view illustrating a side flow analysis in a conventional membrane sensor, (a) is the case of CK-MB (b) is a view showing the case of myoglobin.

9 is a graph measuring the strength for CK-MB in the membrane sensor of the dual channel structure formed by applying the embodiment, Figure 10 is a strength of the Myoglobin in the membrane sensor of the dual channel structure formed by applying the embodiment One graph.

9 and 10, when the membrane sensor of the dual channel structure is manufactured by using the patterning method of the membrane according to the embodiment, it is compared with the lateral flow of the conventional membrane sensor of FIG. 8. It was confirmed that the intensity was increased.

This can be interpreted that the activity of the reaction is high because the flow of the fluid proceeds relatively slowly in the dual channel.

That is, the embodiment of the present invention as described above, by using a simple method of stamping plate making moistened with an organic solvent on the membrane, a plurality of patterns of various designs can be formed on the membrane, depending on the design of the pattern The flows can be used separately, and the reliability of detection can be improved since temperature, pressure, and fluid flow do not affect the membrane.

In addition, embodiments of the present invention can be produced and used by fixing the antibody or antigen, it is possible to produce a multi-biosensor by a simple method.

Experimental Example

It was measured whether the membrane sensor subjected to the pattern forming process of the present invention has a change in fluid flow and time compared to the conventional membrane sensor.

11 is a photograph showing a membrane sensor. The membrane used NC180 sec, nitro cellulose. (A) was cut by the conventional LFA (Lateral flow assay) method was produced in 4mm width, (B) is carried out the pattern forming process of the present invention, but after drying the organic film formed by changing the properties by contact with the platen forceps It is a removed membrane sensor, (C) is a photograph which shows the membrane sensor which did not remove the organic film.

Use 1 X PBS (phosphate buffered saline) containing human 1% (v / v) surfactant (product name: surfactant 10G from fitzgerald) and human serum (product name: Sigma H4522) to confirm flow status and time. This is shown in Figures 12-14. At this time, the food coloring was added in an amount of 0.1 to 0.5% by weight based on the total weight to easily determine the fluid flow degree in the '1 X PBS'.

12 and 14, the flow time was measured using the solution of '1 X PBS'. As a result, A, B, and C were all 101-103 seconds in time, and the flow state was A. , B, and C are not significantly different from each other. Even though the patterning of the present invention is performed, the fluid flow is hardly changed.

13 and 14, when the flow time was measured using the solution of the 'Human serum', A, B, and C were all present in the range of 180 to 185 seconds. It can be seen that the fluid flow shows little change, so that the analytical performance is not degraded. In addition, it can be seen that the removal of the organic film does not affect the fluid flow.

The present invention has been described above with reference to the preferred embodiments, which are merely examples and are not intended to limit the present invention, and those skilled in the art to which the present invention pertains do not depart from the essential characteristics of the present invention. It will be appreciated that various modifications and applications are not possible that are not illustrated above. For example, each component specifically shown in the embodiment of the present invention can be modified. And differences relating to such modifications and applications will have to be construed as being included in the scope of the invention defined in the appended claims.

Claims

Manufacturing a plate-formed plate-making on paper using a craft printer;

Soaking the plate in an organic solvent;

Attaching and stamping the plate to a membrane through which a water-soluble sample can flow;

Removing the engraving from the membrane; And

Drying the membrane for a predetermined time;

Method of manufacturing a membrane sensor comprising a.
The method of claim 1,

The membrane is a method of manufacturing a membrane sensor made of nitrocellulose (Nitrocellulose).
The method of claim 1,

The organic solvent is a method of manufacturing a membrane sensor is selected from any one of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), 1.4-Dioxanw.
The method of claim 3, wherein

The organic solvent is a method of manufacturing a membrane sensor that the concentration of 100% DMSO is used.
The method of claim 1,

Bonding and stamping the plate to a membrane through which the water-soluble sample can flow,

Adhering a first fixing plate to one surface of the membrane;

Adhering a second fixing plate to one surface of the plate;

Pressing the first and second fixing plates to contact the membrane and the plate making, and applying a predetermined pressure to the membrane and the plate making;

Method of manufacturing a membrane sensor comprising a.
The method of claim 1,

Drying the membrane for a predetermined time,

Removing the plate from the membrane and drying the membrane at a temperature of 37 ° C. for a predetermined time.
The method of claim 1,

After the membrane is dried for a predetermined time,

And forming a biosensor without removing the organic film present in the portion melted by the organic solvent on the membrane.
The method of claim 1,

After the membrane is dried for a predetermined time,

And removing the organic film present in the portion dissolved by the organic solvent on the membrane to form a biosensor.
The method of claim 1,

The method of manufacturing a membrane sensor, characterized in that for transmitting the data for the pattern set by the user to the input unit of the craft (craft) printer patterning the paper or cloth.