WO2019135526A1 - Bande de mesure de substance biologique - Google Patents

Bande de mesure de substance biologique Download PDF

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Publication number
WO2019135526A1
WO2019135526A1 PCT/KR2018/016433 KR2018016433W WO2019135526A1 WO 2019135526 A1 WO2019135526 A1 WO 2019135526A1 KR 2018016433 W KR2018016433 W KR 2018016433W WO 2019135526 A1 WO2019135526 A1 WO 2019135526A1
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WO
WIPO (PCT)
Prior art keywords
matrix
biomaterial
strip
sample
blood
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Ceased
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PCT/KR2018/016433
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English (en)
Korean (ko)
Inventor
이주원
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1Drop Inc
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1Drop Inc
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Publication of WO2019135526A1 publication Critical patent/WO2019135526A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Definitions

  • the present invention relates to biomaterial measurement, and more particularly, to a measurement strip for measuring a substance produced in the body using body fluids.
  • immunoassays In the field of medical diagnosis, immunoassays, chemical colorimetric assays, electrochemical assays are used to detect or quantify specific substances contained in biological samples such as blood, serum, urine, And so on.
  • biological samples such as blood, serum, urine, And so on.
  • analytical techniques are used in large-scale equipment such as automated analyzers used in clinical centers in large hospitals or in test strips and cartridges used in platform for point-of-care test (POCT).
  • POCT point-of-care test
  • the POCT device measures a specific substance with the same reaction principle as a large-sized device, but since it measures by using a strip or a cartridge, it is easy to measure and manage, Although it is less reliable than large equipment, it is widely used in medical diagnostics.
  • Platforms used in these field inspection devices use dry reagents that react with specific materials and are classified into strips and cartridges depending on the type of measurement platform.
  • the strip type the method of separating erythrocytes from the blood using glass fiber or porous membrane and measuring the change of color according to the concentration.
  • the cartridge type the separated serum (plasma) (Blood plasma) is measured according to the concentration of a specific substance.
  • the present invention relates to a strip-type platform used in a field-of-view inspection (POCT) apparatus, and is a porous film used for making strips. It is a chemical colorimetric assay in which red blood cells of blood are removed, As well as a technique using the same.
  • POCT field-of-view inspection
  • a technique for separating red blood cells from blood to measure specific substances in the blood and a technique for measuring by measuring the specific reaction of specific substances present in separated plasma of red blood cells are used.
  • a method of removing red blood cells by stacking a plurality of pads (two or more sheets) made of glass fiber or the like is often used. This method is effective in separating red blood cells, There was pain and difficulty in collecting during the procedure.
  • the present invention is a strip type platform used in a field inspection apparatus. It aims at improving the accuracy of measurement and reproducibility of measurement even when a small amount of sample is used.
  • the present invention provides a means that can be used to acquire information about a sample using a light source and a camera of a portable terminal.
  • the strip for biomaterial measurement of the present invention comprises a copolymer selected from the group consisting of polysulfone, polyethersulfone, and polyarylsulfone, A porous matrix comprising a ball; An enzyme, a dye, and a hydrophilic polymer material reacting with the biomaterial are included in the matrix;
  • the matrix is characterized in that the biomaterial contained in the sample which is in contact with the sample containing the biomaterial and diffused into the lower layer of the matrix is measured from the color development of the matrix in the upper layer.
  • the thickness of the matrix is 100 to 240 ⁇ .
  • the mean size of the micropores is 0.2 to 0.6 mu m.
  • the micropores of the matrix gradually decrease in average size in the region from the upper layer to the 1 ⁇ 2 to 2 ⁇ 3 of the thickness.
  • the micropores of the matrix gradually decrease in size from the upper layer to the lower layer of the matrix.
  • the micropores of the matrix have an average size in the region from 1/3 to 2/3 of the matrix thickness larger than in the remaining regions.
  • enzymatic reaction of the biomaterial and color development by the dye occur throughout the thickness of the matrix.
  • the hydrophilic polymer material is polyethylene glycol.
  • the polyethylene glycol has an average molecular weight in the range of 200 to 8,000.
  • the dye reacts with a substance produced from the reaction of the biomaterial or biomaterial in the sample.
  • the dyes are selected from the group consisting of Methyl Orange, Chlorophenol red / Cresol red / Phenol red, BTB / Bromophenol Blue, Bromocresol Green BCG, Bromocresol Purple BAP, AAP + MADB, AAP + MAOS, At least one selected from the group consisting of Methylene Blue, Indigo Carmine, WST-4, WST-8, WST-9, DA67, New Trinder's Reagents and Tetrazolium Salts.
  • the color of the matrix can be photographed by the camera of the portable terminal.
  • the present invention it is possible to provide a strip capable of increasing the measurement efficiency, accuracy and reproducibility in the field inspection apparatus. Accordingly, the present invention makes it possible to more easily and quickly acquire information on specific substances in blood, and information such as disease information and health information therefrom. Further, when the strip of the present invention is used for measurement in a mobile terminal, utilization can be expanded.
  • FIG. 1 is a scanning electron microscope (SEM) photograph of a section of the strip of the present invention as an embodiment.
  • 2A to 2C are SEM (scanning electron microscope) photographs of cross sections of the respective porous matrices shown in Table 1.
  • Fig. 3 shows the results of measurement on the day of manufacture for the strips prepared in Examples and Comparative Examples 1 and 2.
  • Fig. 4 shows the results of measurements on the strips prepared in Examples and Comparative Examples 1 and 2 on the seventh day after the production.
  • the present invention relates to a strip-shaped platform for use in an on-site inspection apparatus, and more particularly to a strip-type platform for use in a field inspection apparatus comprising a copolymer selected from the group consisting of polysulfone, polyethersulfone, polyarylsulfone, And a porous matrix containing the micropores.
  • the inside of the matrix includes a hydrophilic polymer material, an enzyme reacting with the biomaterial, or a dye reacting with the biomaterial or a material generated from the reaction of the biomaterial.
  • the strip measures the biomaterial contained in the sample that is diffused into the lower layer of the matrix by contacting the sample containing the biomaterial in the upper layer of the matrix from the color development of the matrix.
  • the biomaterial is contained in a body fluid (e. G., Blood, saliva, urine, sweat, etc.) and is capable of acquiring information about the disease or health from measurements on it.
  • a body fluid e. G., Blood, saliva, urine, sweat, etc.
  • the present invention relates to a method for measuring blood glucose, cholesterol, cancer marker, hepatic level, lung level, triglyceride, potassium, uric acid, calcium receptor protein (Troponon ) Information on at least one of the calcium-containing protein, CRP, NT-proBNP, and creatinine kinase.
  • the thickness of the porous matrix in the strip of the present invention is preferably 100 to 240 ⁇ m. This thickness makes it possible to use less samples containing biomaterials. That is, when a sample thicker than 240 ⁇ m is used, the amount of sample for measurement must be increased. Therefore, in an embodiment, when a sample is blood, a large amount of blood must be collected, which may cause pain to the user. Therefore, the present invention proposes a thickness range as described above, thereby enabling accurate and reproducible measurement while using a small amount of sample.
  • micropores distributed within the matrix of the strip of the present invention have an average size of 0.2 to 0.6 um.
  • FIG. 1 is a cross-sectional view of a strip according to an embodiment of the present invention
  • FIGS. 2 (a) to 2 (c) are cross-sectional views of respective porous matrices manufactured in the embodiment described below. Each of them is arranged vertically in the thickness section, the upper surface of which is in contact with the sample, and the sample is diffused from top to bottom.
  • micropores distributed within the matrix of the present invention vary in size depending on the thickness or area of the matrix. That is, there are regions where micropores having a small size are densely distributed, and regions having micropores having a large size are distributed. In some areas, the size of the micropores is so small that almost no micropores seem to be distributed.
  • the micro pores distributed in the matrix of the present invention have a shape in which the average size gradually decreases in the region from the upper layer of the matrix to the half to 2/3 of the thickness. This aspect may continue until it reaches the bottom of the matrix.
  • the average size in the 1/3 to 2/3 region of the matrix thickness may be larger in the remaining region.
  • micropores may be selected to increase the accuracy and reliability of the measurement depending on the sample used in the strip and the biomaterial to be measured.
  • micropores may be used to separate impurities in a sample, in one embodiment, plasma and blood cells by filtering out red blood cells when the sample is blood. In this process, And when the ball is required, the size of the fine holes distributed in the upper layer of the matrix is greatly adjusted.
  • the separation process may occur simultaneously with the reaction or color reaction of the biomaterial contained in the plasma. That is, separation and color development can occur simultaneously in the same area, not in the separated areas. This is due to the fact that enzymes and dyes are included throughout the matrix in the manufacturing process, which will be described later.
  • the separation process occurs mainly in an upper layer corresponding to a half to two-thirds of the thickness of the matrix after the sample contacts the upper layer of the matrix.
  • the strap of the present invention is formed so as to include a hydrophilic polymer material inside the matrix.
  • the hydrophilic polymer material assists the adsorption of the sample on the strip and makes the separation process effective.
  • the hydrophilic polymer material is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the accuracy and reliability of the measured value of the strip are improved compared to the case of using other hydrophilic polymer materials. This is believed to be due not only to the hydrophilicity of PEG, but also to the fact that it has a particularly favorable hint of its structural separation, for example in the case of blood, the adsorption of blood cells.
  • the PEG is a means for enabling accurate and reproducible measurement of biomaterials even if the strip of the present invention has a thin thickness of 240 ⁇ or less. Accordingly, the strap of the present invention can be measured even by using a small amount of sample.
  • the polyethylene glycol (PEG) has an average molecular weight in the range of 200 to 8,000. It is confirmed that one having such a molecular weight range exhibits an excellent effect for removing blood cells in the sample blood as an example.
  • Plasma serum
  • a biomaterial that is, enzyme or dye
  • the enzyme contained in the strip matrix of the present invention may be one which reacts with blood substances such as blood glucose, cholesterol, triglyceride, uric acid, total protein, albumin and the like in the blood as an embodiment. Further, ≪ / RTI >
  • when measuring blood glucose it may include an enzyme required for the following reaction.
  • the dye contained in the matrix of the present invention reacts with a substance generated from the reaction of a biomaterial or a biomaterial as in the above formula.
  • These dyes include Methyl Orange, Chlorophenol red / Cresol red / Phenol red, Bromothymol Blue / Bromophenol Blue, Bromocresol Green (BCG), Bromocresol Purple (BCP), AAP + MADB, AAP + , Indigo Carmine, WST-4, WST-8, WST-9, DA67, New Trinder's Reagents and Tetrazolium Salts.
  • the strip of the present invention can be applied to Korean Patent Application No. 10-2016-0174253 as an embodiment. That is, it can be used as a dye pad in the above patent. In this case, the color on the second surface of the strip of the present invention can be photographed by the camera of the portable terminal. Accordingly, the user can acquire information on the biomaterial in the sample using a portable terminal such as a conventional smart phone without using a separate measuring device.
  • Sample No. 1 4 kg of a polyether sulfone resin as a polymer resin, 25 kg of dimethylformamide as a solvent, 15 kg of p-toluenesulfonic acid as a coagulation catalyst and 6 kg of polyvinylpyrrolidone as a swelling agent were dissolved in a dissolution tank at a concentration of 65 ⁇ Lt; 0 > C. After the solution was filled with nitrogen, it was decompressed for 12 hours or more in a vacuum state to sufficiently remove bubbles in the solution and transferred to the casting surface. A polyester film having a width of 2 m was used as a support layer.
  • the solution was passed through a casting surface having a distance of 140 ⁇ m between the casting knife and the surface of the polyester film at a width of 0.75 m, and then immersed in a coagulation bath consisting of water at 5 ° C. After confirming that the solution solidified sufficiently in the coagulation bath, it was moved to a cleaning bath at 65 ° C.
  • the polyether sulfone film and the polyester film as the support layer were removed after about one minute after being received in the washing tank and further washed in water for 20 minutes. After the washing, the water on the surface of the excess porous Madrix was removed with an air knife and dried in a dryer to prepare a polyethersulfone porous matrix membrane.
  • Sample No. 2 A porous matrix membrane having an average size of different micropores was prepared by controlling only the contents of 5 kg of the polyether sulfone resin as the polymer resin and 5 kg of the polyvinylpyrrolidone as the swelling agent.
  • Sample No. 3 In the above method, only the content of 6 kg of polyetetesulfonic resin as a polymer resin and 4 kg of polyvinylpyrrolidone as a swelling agent were controlled to produce porous matrix membranes having different average micropore sizes.
  • Figs. 2A to 2C show photographs of cross sections of the matrix. Fig.
  • Sample No. 1 in Table 1 was used as the porous matrix used in the manufacture of the strip.
  • the reagent to be treated in the porous matrix was dissolved in 10 ml of 20 mM MES buffer by quantitatively measuring 16.9 kU of glucose oxidase, 13.3 kU of Horseradish peroxide, 131.8 mg of MAOS, 152.4 mg of AAP and 20% of PEG 6000 (average molecular weight 6000). After the reagents were completely dissolved, 20 mM MES buffer solution was added to a final volume of 15 ml. The porous matrix was placed in the prepared reagent, and when completely absorbed, the strip was taken out and dried in an oven at 40 ° C for 30 minutes.
  • Strips were prepared in the same manner except that 20% PEG 6000 (average molecular weight 6000) was omitted from the strip manufacturing method of the above example.
  • a strip was prepared in the same manner except that 1% of PVP 20000 (average molecular weight 20,000) was added instead of adding 20% of PEG 6000 (average molecular weight 6000) in the strip manufacturing method of the above example.
  • Sample blood was prepared at a glucose concentration of 50 mg / dL using a reference instrument (Cobas ® 8000).
  • a commercially available glucose (Sigma Aldrich) reagent was added to the blood of the sample to prepare a solution having concentrations of 100, 150, 200, 250, 300 and 350 mg / dL, respectively.
  • the sample blood was injected into the strips prepared in Examples and Comparative Examples 1 and 2, three times each, in an amount of 10 ul, and the average value was written.
  • sample blood was prepared by the above method and the same experiment was carried out.
  • Fig. Fig. 3 shows the results measured on the first day of the strip production
  • Fig. 4 shows the results measured on the seventh day after the production.
  • the strip of Comparative Example 1 showed a large difference from the strip of Example and Comparative Example 2 on the day of manufacture.
  • the straps of Examples and Comparative Example 2 did not show a large difference, but showed a difference on the seventh day after the production. That is, the measurement performance of the strap of the present invention was maintained even after the lapse of time, but the measurement performance of the strap of Comparative Example 2 decreased with time.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
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Abstract

La présente invention concerne une bande de mesure de substance biologique, en tant que plate-forme de type bande destinée à être utilisée dans un dispositif d'examen sur site. La bande est une matrice poreuse comprenant des micropores, et un copolymère choisi dans le groupe constitué de polysulfone, de polyéthersulfone et de polyarylsulfone, afin d'augmenter l'efficacité de séparation des cellules sanguines contenues dans un échantillon et d'améliorer la précision et la reproductibilité de mesures avec une petite quantité de sang, la matrice comprenant une enzyme réagissant avec la substance biologique, un colorant et un matériau polymère hydrophile, et la matrice étant mise en contact avec l'échantillon contenant la substance biologique dans la couche supérieure pour mesurer la substance biologique contenue dans l'échantillon, se diffusant dans la couche inférieure de la matrice, à partir du développement de couleurs de la matrice.
PCT/KR2018/016433 2018-01-05 2018-12-21 Bande de mesure de substance biologique Ceased WO2019135526A1 (fr)

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KR10-2018-0001815 2018-01-05
KR20180001815 2018-01-05

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WO2019135526A1 true WO2019135526A1 (fr) 2019-07-11

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3995826A4 (fr) * 2019-07-03 2023-07-19 1Drop Inc. Bande de dosage de biomatériau
US12169193B2 (en) 2019-10-25 2024-12-17 1Drop Inc. Sample-collecting device capable of quantitative sampling

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20030078613A (ko) * 2002-03-28 2003-10-08 후지 샤신 필름 가부시기가이샤 혈액 검사 유닛
KR100490185B1 (ko) * 1996-04-04 2005-08-31 라이프스캔, 인코포레이티드 혈중글루코스함량측정용시약시험스트립
JP2009244014A (ja) * 2008-03-31 2009-10-22 Cci Corp 中性脂肪測定用バイオセンサ
KR20130142768A (ko) * 2012-06-20 2013-12-30 (주)미코바이오메드 센서 스트립
KR20170133238A (ko) * 2016-05-25 2017-12-05 삼성전자주식회사 시료에 대한 정보를 획득하기 위한 스트립 모듈 및 이를 이용한 휴대 단말의 제어 방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100490185B1 (ko) * 1996-04-04 2005-08-31 라이프스캔, 인코포레이티드 혈중글루코스함량측정용시약시험스트립
KR20030078613A (ko) * 2002-03-28 2003-10-08 후지 샤신 필름 가부시기가이샤 혈액 검사 유닛
JP2009244014A (ja) * 2008-03-31 2009-10-22 Cci Corp 中性脂肪測定用バイオセンサ
KR20130142768A (ko) * 2012-06-20 2013-12-30 (주)미코바이오메드 센서 스트립
KR20170133238A (ko) * 2016-05-25 2017-12-05 삼성전자주식회사 시료에 대한 정보를 획득하기 위한 스트립 모듈 및 이를 이용한 휴대 단말의 제어 방법

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3995826A4 (fr) * 2019-07-03 2023-07-19 1Drop Inc. Bande de dosage de biomatériau
US12169193B2 (en) 2019-10-25 2024-12-17 1Drop Inc. Sample-collecting device capable of quantitative sampling

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