JPH1176765A - Porous membrane - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a porous membrane capable of simultaneously accepting many kinds of inspection sample liquids with a single base material and useful as a base material for hybridization or immunity inspection sample. SOLUTION: In this porous membrane, divided porous areas are formed by providing a non-permeable part 2 in a membrane filter 1. The porous membrane is used as the base material for southern northern or western blotting hybridization, a base material for immunity inspection, an inspection sample holding part for produced, a cell holding part of cell incubation plate, a base plate for cell incubation or a base plate for immunochromato assay. The porous membrane is produced by forming the nonpermeable part 2 in the membrane filter 1 by a method that the pore of the specific part of the membrane filter is, for example, clogged by thermocompression bonding, fusion by ultrasonic wave, dissolution with a solvent or by bringing a coating solution containing a filler into contact with screen printing, roll coating, non impact printing.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a porous membrane filter partitioned by densification and useful for applications utilizing functions such as filtration and adsorption.

[0002]

2. Description of the Related Art A polymer membrane filter having a large number of pores having a uniform diameter is widely used as a separation filtration of a liquid, a separation of a biological substance, an adsorption fixation, and a chromatographic base material. And so on. Although there are many uses for the membrane filter, the main uses are as follows. When used for filtration applications, fix the membrane filter with a jig, apply liquid to one side of the membrane, apply a physical force to the membrane surface by hydrostatic pressure, pressurization, vacuum suction, etc., and force the liquid Is allowed to pass through the membrane filter to be separated into substances larger and smaller than the pores of the membrane filter. Alternatively, as in Southern blotting, Northern blotting, Western blotting, and dot blotting, a hybridization assay that transfers, adsorbs, and fixes nucleic acids and proteins to a membrane and searches for the presence of interaction with a substance labeled with RI, fluorescence, etc. Used for In a similar manner, an antibody, which is a kind of protein, is immobilized on a membrane and used for immunoassay. Further, it is also used as a chromatographic substrate in which a sample solution is supplied to one end of a membrane filter filled with an electrolyte solution, a voltage is applied to both ends of the membrane, and a substance is separated by interaction with the substrate. Recently, an in vitro test agent that tests the presence or absence of substances contained in body fluids by simply donating urine or blood, etc., using immunochromatography, taking advantage of the properties of both the chromatographic substrate and the blotting substrate of the membrane filter Has been developed, pregnancy tests,
It is used for applications such as fecal occult blood and drug tests.

[0003] However, when used as a substrate for filtration, hybridization, or immunochromatography, basically, only one kind of sample solution is applied to one substrate, so that it can be used for multiple samples. Unsuitable for In the case of a hybridization substrate, an arbitrary number of samples can be applied to the sample on the side to be fixed, but only one kind of the sample solution to be provided can be selected.
In the case of dot blotting including immunoassay, the sample liquid is dropped with a dispenser, pipette, etc., dried, and absorbed and fixed by heat treatment.However, due to the nature of the membrane, the dropped pattern bleeds or a certain amount of sample liquid is supplied. However, there are problems such as the size and area of the pattern being inconsistent,
There is a problem that the reliability of quantitative measurement in hybridization and immunoassay is poor.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a porous membrane which can simultaneously supply multiple types of sample solutions with a single substrate and is useful as a substrate for hybridization or immunoassay.

[0005]

SUMMARY OF THE INVENTION The present invention relates to a porous membrane having a porous region defined by providing an impermeable portion in a membrane filter. The present invention also provides
Southern, Northern, or Western blotting hybridization substrate, immunoassay substrate, biosensor specimen holding portion, cell culture plate cell holding portion, cell culture substrate or immunochromatographic assay substrate using the porous membrane About. The present invention also provides a method of applying ultrasonic waves in a state in which a jig is in contact with a portion to be densified by thermocompression bonding a hole of a membrane filter at a temperature equal to or higher than a melting point of a polymer resin constituting the membrane filter. By heating the portion of the membrane filter that is in contact with the jig, a solvent capable of dissolving the polymer resin constituting the membrane filter is brought into contact with a specific region of the membrane filter by a dispenser, screen printing, roll coating, inkjet, or the like. The present invention relates to a method for producing the porous membrane, wherein an impervious portion is formed in the membrane filter by closing a pore of a specific portion of the membrane filter by impregnating with a coating solution containing a solid material such as a resin or a filler.

The present invention provides a porous membrane having a plurality of partitioned porous regions by forming a sample liquid impermeable portion on the porous membrane. When used as an immunoassay or a chromatographic assay, it is characterized in that sample liquids can be prevented from mixing through a continuous porous membrane.
When used in a hybridization assay, the area of the donor protein to be adsorbed and immobilized is clearly limited, thereby preventing bleeding and area unevenness of the adsorption immobilization pattern and improving the reliability of the quantitative assay. Another feature of the porous membrane of the present invention is that by using a plurality of partitioned porous regions, it is possible to simultaneously process or assay many types of samples or specimens, so that high efficiency and high accuracy are achieved. Is to be obtained.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The porous membrane used in the present invention is a polymer filter generally called a membrane filter, and has a shape in which pores having the same pore diameter are continuously connected. Although the polymer material constituting the porous membrane is not particularly limited, examples thereof include nitrocellulose, regenerated cellulose, polyvinylidene fluoride (PVDF), nylon, polycarbonate, and cellulose acetate. These porous membranes are not particularly limited, and may be manufactured by any of the methods used for manufacturing a commercially available membrane filter. The preferred pore size of the porous membrane usable in the present invention is 0.002 to 40 μm as an average pore size, and the particularly preferred average pore size is 0.1 to 10 μm. In addition to the average pore size, the membrane filter used in the present invention preferably has a uniform pore size. Although not a membrane filter, a filter medium such as a cellulose filter paper or a glass filter may be used by forming a non-water-absorbing region by applying a solid component to partition a water-absorbing portion.

The shape and size of each partitioned porous region can be arbitrarily determined according to the method of use. The shape is generally circular, square, rectangular, or the like, for example. Although the size of each section is not limited at all, for example, a circle is typically 1 to 50 mm in diameter, and a square or rectangle is typically 1 to 50 mm on a side. In addition, the width of the impermeable portion that partitions the partitioned porous region is
Depends on how the membrane filter is used.

The following two typical methods are used to form an impermeable portion in a porous membrane. One is a method in which a resin portion is melted or dissolved by heating or an organic solvent to partially densify pores of a porous film to form an impermeable portion. Heating is performed at a temperature equal to or higher than the melting point of the polymer resin to improve the fluidity of the resin, and by further applying pressure, the pores collapse and disappear. As a heating method, there are a method of heating and pressing with a mold having a desired pattern shape, and a method of heating and melting by applying ultrasonic waves using a jig having a desired pattern shape. The heating temperature may be basically any number of times as long as the temperature is equal to or higher than the melting point of the polymer resin. Pressing is performed to assist the heating effect. The range of the pressurizing pressure is not particularly limited. However, if the pressurizing is performed at an excessively high pressure, the pressurizing is preferably performed at ² to 10 kgf / cm ² because the resin component may be broken. When bonding other substrates simultaneously with impermeable treatment of the membrane, pressurization is essential. Also in this case, the pressure condition is suitably ² to 10 kgf / cm ² . For example, in the case of a PVDF membrane (trade name “Durapore HV”; manufactured by Nippon Millipore), 230 to 250 is used.
° C, ² to 6 kgf / cm ² , 1 to 6 seconds. In the case of nylon membrane, 280 to 300 ° C, ² to 6 kgf / c
m ^2, preferably performed opaque processing by thermocompression bonding 1 to 3 seconds.

[0010] When a desired portion of the membrane filter is subjected to impermeability treatment using a solvent, thick film printing such as screen printing, non-impact printing such as dispenser, ink jet, and roll coating such as gravure printing are used. Although patterning is conceivable, a dispenser and an ink jet are preferable in that patterning can be performed using only an organic solvent, and a dispenser is preferable in terms of a coating amount. In the case of performing gravure printing or roll coating, the ink component may be an organic solvent alone, but considering the viscosity adjustment and the adjustment of the volatilization time, the addition of a polymer component and a viscosity modifier improves the patterning suitability. As the viscosity adjusting agent, a silica-based viscosity adjusting agent such as Aerosil (manufactured by Nippon Aerosil Co., Ltd.) is preferable because it does not inhibit the penetration of the ink component into the membrane. The solvent is not particularly limited as long as it is a substance that dissolves the membrane.
Alcohols, esters, aldehydes, ketones,
Organic solvents such as ethers and amines, sulfuric acid, nitric acid,
Although there are acids such as hydrochloric acid and alkalis such as sodium hydroxide, potassium hydroxide and ammonium, it is preferable to use an organic solvent in view of drying after solvent coating, post-treatment and the like.

Another method for densifying the pores of the porous membrane is a method of closing the pores of a required portion with a solid substance. In this method, fine particles having a smaller particle size than the pore size are used as the solid material. The size of the fine particles is 0.001 to 2.0.
Although a particle size in the range of μm can be applied, it is preferable that the particle size is small in consideration of the fluidity of the ink composition, the permeability to the membrane, and the like. The preferred particle size is 0.1.
01-0.2 μm. As the components of the fine particles,
Silica, mica, calcium carbonate, alumina, titanium oxide and the like can be used. These fine particles are mixed with an organic solvent and a binder to form an ink composition. The composition of the organic solvent, binder, and the like is not particularly limited as long as printability is not impaired.

The resin component alone, for example, an ink component containing no fine particles such as an ultraviolet curable resin, a thermosetting resin or an electron beam curable resin is patterned on a porous film, and then subjected to ultraviolet irradiation, electron beam irradiation, heat treatment or the like. There is a method in which a resin component is cured to form an impermeable portion. Both the fine particle-containing ink composition and the curable ink composition are patterned by screen printing, roll coating,
Dispenser application or the like is possible.

The thus formed porous membrane containing an impermeable portion can be used as a substrate for filtration, hybridization, biosensor, chromatography, and cell culture. Substrates for filtration, biosensors, chromatography, and cell culture are applicable to multiple sample applications.
In use, the area bounded by the opaque portion forms the application area of the individual sample.

Hereinafter, examples of individual applications used as a substrate will be described. When used as a substrate for filtration,
As shown in (1), in the membrane filter 1, an opaque area 2 is formed corresponding to a part other than the hole 9 of the molded body 3 having a hole 9 for a plurality of filtrate holders (which can also be used as a well when performing culture). I do. If the impervious part is not formed, different sample solutions may mix in the filter because the filter part communicates through the hole, but the impermeable part prevents mixing and contamination between solutions. it can.

FIG. 5 shows a case where the substrate is used as a substrate for hybridization, biosensor, and chromatography.
This will be described below with reference to FIG. In each of the figures, 1 'is a transmitting portion and 2 is a non-transmitting portion. Like a filter, contamination between different samples can be prevented, so that multiple samples can be measured simultaneously. FIG. 5 shows a patterning membrane having an impermeable portion of the present invention formed for hybridization. FIG. 5B shows a conventional method of applying a blotting solution to the hybridization substrate 25 to form a hybridization sheet. In dot blot hybridization, as shown in the figure, a normal sample solution is immobilized by infiltrating into a membrane with a dispenser or pipette. Therefore, even if a certain amount of sample solution is used, the size of the pattern 26 differs. And the pattern 26 becomes non-uniform, and in optical reading such as ELISA detection and fluorescence detection, there is a problem that the quantitative reliability is poor. FIG. 5 (a) shows a hybridization sheet of the present invention which can solve such a disadvantage. In FIG. 5A, reference numeral 2 denotes an opaque portion of the membrane filter formed by a method such as heat treatment or coating as described above. By this processing, the transmission portion 1 'having a determined shape and area is clearly defined as the blotting region 1'. The blotting solution added to the permeable portion 1 'can be fixed to that fixed area.

Similarly, the sensing site is clearly defined for the ligand-immobilized portion of the biosensor. 6 to 8 illustrate a substrate for measuring a blood glucose level using the porous membrane of the present invention. FIG. 9 shows an example of the use of the porous membrane of the present invention as an immunochromatographic substrate in which a plurality of chromatography zones are formed by forming an impermeable portion.

The substance, solution, and sample solution to be fixed to the filtration, hybridization, chromatography, biosensor are not particularly limited as long as they do not destroy the impermeable part. Nucleic acids, proteins, saccharides and the like can be considered as the fixing substance. As the detection means, electrochemical detection, fluorescence, reflected light, radioactivity, magnetic detection and the like can be used.

[0018] Membrane filters are often used for cell culture substrates. For example, when elucidating the function of a cell secretory component or the physiological function between cells, there is a method of contacting a sample liquid or cells via a membrane filter. Such analysis is often used for cell screening and often requires multi-sample analysis.

[0019]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1: Utilization as a substrate for filtration An example in which different strains of Escherichia coli are cultured on a substrate for filtration, then Escherichia coli is lysed, and a plurality of types of DNA are filtered. An example of preparing a membrane filter for hybridization of Escherichia coli DNA immobilized on a membrane filter will be described. PV
A DF membrane filter (pore diameter 0.4 μm; manufactured by Millipore) was used at 240 ° C., 4 kgf /
It was heated at cm ² 3 seconds leaving at intervals 1mm porous portion 1 'as a circular diameter 3mm to form an opaque portion 2. Separately, a 10 mm-thick polypropylene plate-shaped molded body 3 that has been subjected to a 3 mm-diameter circular punching process at 1 mm intervals,
Attached onto the PVDF membrane filter 1 via a silicone-based double-sided pressure-sensitive adhesive sheet 4 which has been circularly punched into the same shape.
A plus-charged nylon membrane (trade name “Hybond-N +”; manufactured by Amersham) as a membrane filter 6 having a DNA adsorbing property is bonded to the lower surface of the above through a silicone-based double-sided pressure-sensitive adhesive sheet 5 punched in the same manner as described above. A filtration device was prepared. An unstretched polypropylene film was adhered to the bottom of the filtration device as a waterproof material 7 coated with an acrylic pressure-sensitive adhesive 8 so as not to generate air bubbles (FIG. 1A).

LB containing different strains of Escherichia coli in the respective filtration holders / wells (9 in FIG. 1 (b)) whose bottoms formed by bonding are filter materials.
60 μl of each medium was added. As shown in FIG. 2, the top surface was sealed with a cover sheet 10 made of a PET film with an acrylic adhesive, and cultured at 37 ° C. for 24 hours (see FIG. 3).
(A)). After the culture, a buffer containing guanidine, surfactant, protease K, and RNAase was added to each well to adjust the pH to 7.3, and Escherichia coli was lysed (FIG. 3 (b)). Was peeled off. Instead of a polypropylene film, a water-absorbent sheet is attached to the bottom, and filtration is performed while also removing the Escherichia coli residue 14 and adsorbing and fixing the Escherichia coli chromosomal DNA 15 to the membrane filter (FIG. 3C). By the operation, a membrane for hybridization of Escherichia coli chromosomal DNA was prepared (FIG. 3 (d) and FIG. 4).

Example 2: As a substrate for a biosensor
Example 2 A regenerated cellulose membrane (manufactured by Schleicher and Shell Co., Ltd.) 1 having a size of 50 mm × 50 mm was heated at 350 ° C. for 3 seconds using a brass heating jig to form a porous portion 1 ′ having a circular shape with a diameter of 10 mm. An opaque portion 2 was formed leaving an interval of 1 mm. The electrodes 16 and the wiring portions 17 were formed on the membrane by screen printing using gold paste ink (FIG. 6). Next, an ink containing ferrocene-modified glucose oxidase was applied onto one of the electrodes by gravure printing to prepare an enzyme electrode 18 for glucose measurement. This configuration is shown in FIG.
This was adhered to a plastic sheet support 19, cut, and a substrate for blood glucose level measurement (FIG. 8) was prepared by a simple method.

By contacting the blood glucose level measurement substrate prepared above with the bleeding part of the finger using a lancet, the sample liquid was stably supplied to the substrate by capillary action of the membrane filter. Since the amount of the sample solution was defined by the absorption capacity of a fixed area of the membrane filter, the sample amount could always be kept constant, and the reproducibility of the measurement was obtained.

Example 3: As a substrate for immunochromatography
An example will be described with reference to FIG. Screen printing of nitrocellulose membrane (Millipore) 1 obtained by adding 10% V / V of cyclohexanone to sericol medium (manufactured by Teikoku Ink Co., Ltd.), and an impermeable portion so that many chromatographic zones are defined 2 was formed. An anti-human myoglobin antibody, an anti-human troponin T antibody, an anti-human creatine kinase antibody, and an anti-human myosin light chain antibody were immobilized on the respective detection regions 21 of the porous portion of the membrane, and bonded to a support 20 made of a plastic sheet. Polyester impregnated with colloidal gold-labeled anti-myoglobin antibody, colloidal gold-labeled anti-human troponin T antibody, colloidal gold-labeled anti-human creatine kinase antibody, and colloidal gold-labeled anti-human myosin light chain antibody in the upstream portion 23 of each of the partitioned chromatography zones A nonwoven fabric 23 was placed, and a test strip for testing for myocardial injury was easily prepared. The sample was dropped on a water absorbing body (for example, a nonwoven fabric made of synthetic fiber) of the sample donating portion 24, and the presence or absence of four items of myocardial injury markers was simultaneously measured for the same sample.

[0024]

As described above, the porous membrane of the present invention having a plurality of porous regions partitioned by providing an impermeable portion in a membrane filter can simultaneously supply various types of detection solutions with a single material, and perform hybridization. And a wide range of applications as a material for immunoassays.

[Brief description of the drawings]

FIG. 1 shows a filtration substrate using the porous membrane of the present invention.
(A) Configuration sectional view, (b) External view.

FIG. 2 is a schematic diagram showing the cultivation of Escherichia coli on a filtration substrate using the porous membrane of the present invention.

FIG. 3 is a schematic diagram per well illustrating the steps of culturing E. coli on a filtration substrate using the porous membrane of the present invention and the subsequent step of fixing E. coli DNA to a membrane filter. (A) Escherichia coli culture step, (b) Escherichia coli lysis step, (c) Separation and removal of Escherichia coli by dehydration with a water-absorbent sheet, and fixing of DNA to membrane filter, (d) Membrane filter with Escherichia coli fixed Peeling process.

FIG. 4 is a membrane for DNA hybridization obtained by the process of FIG. 3;

FIG. 5 shows an example of a dot hybridization substrate using the porous membrane of the present invention. (A) A hybridization membrane of the present invention provided with an impermeable portion, and (b) a schematic diagram for explaining a state of blotting using a conventional hybridization substrate.

FIG. 6 shows electrodes and wirings for a blood glucose level measurement substrate formed on the porous membrane of the present invention.

FIG. 7 is a blood glucose measurement substrate intermediate in which an ink containing a ferrocene-modified glucose oxidase is further printed on an electrode. (A) Plan view, (b) Sectional drawing.

FIG. 8 is a biosensor substrate for measuring blood sugar level.

FIG. 9 is an immunochromatographic substrate for a myocardial injury test.

[Explanation of symbols]

1: Membrane filter, 1 ': Permeable part of membrane filter, Permeability part of membrane filter 2: Permeability part of membrane filter, 3: Molded body with hole for filtration holder (also well), 4, 5: Silicone adhesive Sheet: 6: Membrane filter for adsorbing DNA, 7: Waterproof sheet, 8: Adhesive, 9: Holder for filtration (well), 10: Cover sheet, 11: LB medium containing E. coli, 12: E. coli, 13: DNA in Escherichia coli, 13 ': DNA released and separated from Escherichia coli, 14: Escherichia coli residue, 15: Membrane for DNA hybridization, 16: Electrode part printed with gold paste ink, 17: Wiring part printed with gold paste ink , 18: Electrode section formed with ferrocene-modified glucose oxidase, 19, 20: Support, 21 Detection region antibody was applied, 22: water-absorbent structure, 23: colloidal gold-labeled antibody impregnated region, 24: sample donor region. 25: Hybridization substrate 26: Blotting solution fixed on a conventional hybridization substrate

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI B01D 71/50 B01D 71/50 71/56 71/56 G01N 33/543 525 G01N 33/543 525C

Claims (14)

[Claims] 1. A porous membrane having a porous region defined by providing an impermeable portion in a membrane filter. 2. Each of the partitioned porous regions has a diameter of 1 to 50.
The porous membrane according to claim 1, which has a circular shape of mm. 3. Each of the partitioned porous regions has a side of 1 to 50.
The porous membrane according to claim 1, which is a rectangle or a square of mm. 4. The porous film according to claim 1, wherein the porous film is a laminated film of one or more selected from the group consisting of nitrocellulose, regenerated cellulose, polyvinylidene fluoride (PVDF), nylon, polycarbonate, and cellulose acetate. 4. The porous membrane according to any one of 3. 5. A substrate for Southern, Northern or Western blotting hybridization using the porous membrane according to claim 1. 6. A substrate for immunoassay using the porous membrane according to claim 1. 7. A sample holding portion for a biosensor using the porous membrane according to claim 1. 8. A cell holding portion of a cell culture plate using the porous membrane according to claim 1. 9. A cell culture substrate using the porous membrane according to claim 1. 10. A substrate for immunochromatographic assay using the porous membrane according to claim 1. 11. The non-permeable portion according to claim 1, wherein thermocompression bonding is performed at a temperature equal to or higher than the melting point of the polymer resin constituting the membrane filter to melt and close the pores of the membrane filter. A method for producing a porous membrane. 12. Applying ultrasonic waves in a state where a jig is in contact with a portion of the membrane filter to be densified and heating the portion of the membrane filter in contact with the jig, thereby melting the membrane filter of the portion; The method for producing a porous membrane according to any one of claims 1 to 3, wherein a hole in the portion is closed to form an impermeable portion. 13. A dispenser for dissolving a solvent capable of dissolving a polymer resin constituting a membrane filter.
The method for producing a porous membrane according to claim 1, wherein the non-permeable portion is formed by welding and closing the pores of the membrane filter by bringing the membrane filter into contact with a specific region of the membrane filter by screen printing, roll coating, inkjet or the like. 14. The porous membrane according to claim 1, wherein a specific portion of the membrane filter is closed by impregnating with a coating solution containing a solid substance such as a resin or a filler to form an impermeable portion in the membrane filter. Production method.