KR20180063646A - A strip for simultaneously measuring total-hemoglobin and glucose-6-phosphate dehydrogenase, a preparing method thereof, and a use of the same - Google Patents

A strip for simultaneously measuring total-hemoglobin and glucose-6-phosphate dehydrogenase, a preparing method thereof, and a use of the same Download PDF

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KR20180063646A
KR20180063646A KR1020160163662A KR20160163662A KR20180063646A KR 20180063646 A KR20180063646 A KR 20180063646A KR 1020160163662 A KR1020160163662 A KR 1020160163662A KR 20160163662 A KR20160163662 A KR 20160163662A KR 20180063646 A KR20180063646 A KR 20180063646A
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membrane
glucose
strip
hemoglobin
phosphate dehydrogenase
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KR101974645B1 (en
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이효근
원유덕
송병학
김병수
이세인
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에스디 바이오센서 주식회사
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Priority to PCT/KR2016/014426 priority patent/WO2018101525A1/en
Priority to BR112019010868-8A priority patent/BR112019010868B1/en
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • 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
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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    • G01N33/545—Synthetic resin
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
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Abstract

The present invention relates to a porous film; A solution-treated membrane containing glucose-6-phosphate, immobilized on top of the porous film; And simultaneous measurement of total-hemoglobin and glucose-6-phosphate dehydrogenase, comprising a mesh attached to the membrane with a structure that includes all of the membrane and a portion of the membrane protruding from the membrane at a size equal to or greater than the membrane. And a method for manufacturing the strip and its application. The strips of the present invention simultaneously and simultaneously measure whole-hemoglobin and glucose-6-phosphate dehydrogenase on the same strip, providing readings through accurate and accurate quantification, thereby providing an advantage of quickly and accurately measuring the concentration of G6PD corrected to total hemoglobin concentration .

Description

FIELD OF THE INVENTION [0001] The present invention relates to a strip for simultaneous measurement of total-hemoglobin and glucose-6-phosphate dehydrogenase, a method for producing the same, }

The present invention relates to a porous film comprising a solution-treated film containing glucose-6-phosphate immobilized on the top of the porous film, and a film-forming solution containing the whole of the film on the top of the film, Hemoglobin and a glucose-6-phosphate dehydrogenase simultaneous measurement strip including a mesh attached with a structure in which a part of the membrane protrudes from the membrane in a large size, a method for producing the same, and an application thereof.

Glucose-6-phosphate dehydrogenase (hereinafter referred to as 'G6PD') deficiency is a worldwide disease with a prevalence of 4.9%. It is estimated to have about 400 million patients and is the most common enzyme deficiency. G6PD deficiency is an X chromosomal genetic disorder and a variety of biochemical enzyme abnormalities and clinical symptoms arise due to genetic defects caused by mutations in the G6PD gene. The most common symptoms are neonatal jaundice and acute hemolytic anemia. Most acute hemolytic anemia is caused by external factors. In 1956, it was reported that G6PD activity was decreased in hemolytic patients after taking the malaria drug Primaquine. It has been found that most G6PD deficiency patients are asymptomatic and have a high likelihood of causing neonatal hemolysis or causing hemolytic anemia due to certain medications or some infections.

Most G6PD deficiency patients are often asymptomatic over a lifetime, often without knowing whether they are patients. Acute hemolysis is caused by oxidative stress on red blood cells when ingested with certain drugs, infections or beans. Oxidative stress oxidizes glutathione in red blood cells (Reduced glutathione, GSH -> Oxidative glutathione, GSSG). G6PD deficient red blood cells have a limitation in reducing glutathione, so that the remaining GSH is rapidly depleted, Irreversible damage to the red blood cells is destroyed.

The point-of-care test (POCT) is a direct examination from the patient's place. It is the formerly called examination of the patient side, the examination room, the bed examination, the auxiliary examination performed outside the hospital do.

In other words, the clinical pathology test is not carried out in the central laboratory of the medical institution but is performed by the patient himself or herself, at home, at the workplace, etc., or by the doctor, nurse or clinician in the ward, emergency room, surgical field or intensive care unit.

Particularly noteworthy is that the sample can be used directly at an individual clinic rather than sent to an inspection center. The main items of the field test are blood glucose analysis performed by the patient, the caregiver and the healthcare personnel, the blood gas analysis performed by the healthcare personnel, the cardiac enzyme test, the general blood test (CBC) There are routine chemistry tests, urine tests, immunoenzymes such as HBsAg / Ab, electrolyte tests, some microbiological tests, occult blood tests, lactic acid tests and so on.

The tests that can be performed by the field test are almost all items that can be inspected by the central laboratory today. In the past, field inspections which have been questioned about the reliability of inspections and which had to be verified as the inspectors of central inspections have been developed rapidly, so that the problem of reliability has been solved.

By inventing a POC product that enables field testing of G6PD through such a technology, it is demanding that it can be inspected at any time and place when necessary.

[Prior Patent Literature]

Korean Patent Publication No. 10-2014-0072023

The present invention has been made in view of the above needs, and it is an object of the present invention to provide a product capable of on-site inspection of G6PD.

It is an object of the present invention to provide a novel on-site inspection method of G6PD.

In order to attain the above object, the present invention provides a method for producing a porous membrane, which comprises mixing a solution prepared by mixing a solution containing glucose-6-phosphate with a surfactant and a polymer in a membrane, drying the membrane, , A mesh for uniformly spreading a blood sample on the top of the membrane, the membrane having a size equal to or greater than the membrane, a portion of the membrane protruding from the membrane, The present invention provides a method for producing a strip for simultaneous measurement of all-hemoglobin and glucose-6-phosphate dehydrogenase.

In one embodiment of the invention, the solution is selected from the group consisting of? -Nicotin amide adenine diclcreotide (NADP), tetrazolium, and 5-bromo-4-chloro-3- It is preferred, but not limited, to include one or more selected materials.

In another embodiment of the present invention, the membrane is preferably, but not limited to, selected from Poly sulfone, Poly ether sulfone, Nylon, and Nitro Cellulose .

In another embodiment of the present invention, the porous film is preferably made of polyethylene phthalate, but is not limited thereto.

In another embodiment of the present invention, the mesh is preferably made of a hydrophilic polyester material, but is not limited thereto.

The present invention also relates to a method for preparing a porous membrane, which comprises: a membrane pretreated with a solution containing glucose-6-phosphate immobilized on top of the porous membrane; Hemoglobin and glucose-6-phosphate dehydrogenase, comprising a mesh attached with a structure having the same size as the membrane or larger than the membrane and partially protruding from the membrane, Provide a strip for use.

The present invention also relates to a method for fabricating a strip, comprising: applying a sample to a mesh portion of the strip of the present invention; And

6-phosphate and glucose-6-phosphate dehydrogenase react with each other at the membrane site to cause a color reaction, thereby exhibiting a specific color, and coloring the resultant color to be quantified through light emission of the measuring instrument and light reception Hemoglobin and glucose-6-phosphate dehydrogenase from a sample containing the enzyme.

In the above, the 'quantification' principle is that hemoglobin (Hb), when the sample is mixed with Lysis Buffer before the G6PD color reaction, changes into HbO2 state by binding with oxygen, and the emission and the light immediately after addition to the sample film are quantified And the% Ref value of the difference between G6PD and 300Sec based on the% Ref value at the time of hemoglobin measurement is quantified based on the difference.

In one embodiment of the present invention, the sample is preferably whole blood, but is not limited thereto.

Hereinafter, the present invention will be described.

The present invention is based on the observation that the strip measurement structure is changed to G6PD Enzyme Activity reaction at the lower reaction membrane by flow through the sample, and then the Dye reaction by the oxidation-reduction reaction occurs to purple, The present invention relates to an analytical method that can be measured through a measuring instrument.

Internationally, it is important to develop products that can measure simultaneously with total hemoglobin, not just G6PD. For example, Carestart G6PD electrochemicals can not measure total hemoglobin simultaneously, Is a measure of U / dL only. These existing G6PD measurement products currently have the disadvantage that they can not quantify the G6PD IU / g Hb, which is an important unit measure for G6PD deficiency testing internationally.

The present invention can quantify IU / g Hb which is most important for G6PD deficiency measurement at present and does not provide an accurate result according to the total-hemoglobin concentration. In addition, it has developed a product that can measure anytime and anywhere advantage of POCT.

The strip of the present invention simultaneously measures both Total-Hemoglobin and Glucose-6-phosphate dehydrogenase within 5 minutes, eliminating any interference due to the total hemoglobin concentration by providing an easy and simple procedure and accurate quantification of the readings. The present invention provides advantages that include simple and straightforward procedures through quick and accurate quantification.

Figure 1 is a precision table. Total-hemoglobin and G6PD measurements were carried out using the strip of the present invention and G6PD measurement was performed and the results were shown to be sufficient to quantify. After preparing the experiment as in Example-1, the coefficient of variation CV% and the standard deviation STDEV As a result of measuring the reproducibility performance of a quantitative product commonly used in a measurement system, the result of measurement as shown in FIG. 1 is that of a normal (6 U / g Hb) or more and an intermediate & deficiency (less than 6 U / g Hb) Good results.
2 is a graph of linearity. The G6PD concentration was measured by the present invention as shown in Table 2. The instrument was named Hitachi Automatic Chemical Analyzer. The G6PD deficiency concentration and the normal concentration sample were measured in 9 steps As a result of measurement experiment made by making it Contrived.
Figure 3 shows the appearance of the device of the invention.
FIG. 4 shows a graph of the parameter measurement algorithm, and G6PD of the present invention shows a difference rate of change of 1 to 300 seconds according to the concentration as shown in the graph. The hemoglobin (Hb) When Hb is added to the HbO2 state, the light emission and the light emission after the addition to the sample film are quantified by the data. G6PD is the% Ref of the difference between 300Sec based on the% Ref value at the time of hemoglobin measurement Values are quantified based on differences.
5 is a comparison chart of the present invention and the Symmetric comparison Precision performance. Glucose-6-phosphate 5 mM, β-Nicotinamide adenine dinucleotide 5 mM (NADP) and Tetrazolium 0.5 mM BCIP 0.4 mM (Or BCPIP) were mixed with the appropriate TX-100 and polyvinylpyrrolidone in a Tris 50 mM pH 8.0 solution for G6PD measurement The prepared solution was treated with symmetric membrane, Nylon membrane and asymmetric membrane, poly ether sulfone membrane, and dried. This test was repeated 5 times using a sample of normal G6PD concentration and the performance of the asymmetric membrane was excellent as shown in FIG.
FIG. 6 is a diagram illustrating the measurement structure of the present invention in comparison with a conventional product. FIG. 6 is a diagram illustrating the structure of a product of the lateral flow method, which is a conventional G6PD RDT technology, and the strip structure of the present invention. It has the advantages of improved productivity and excellent performance.
FIG. 7 is a performance comparison chart according to the strip structure. As a result of the performance comparison, the flow through measurement method according to the present invention has a better performance than the conventional G6PD RDT method. It is confirmed that this is a big improvement in Precision performance which is important for quantitative measurement. The G6PD solution was treated on the membrane and dried. Later flow method, which is a conventional method for comparative evaluation, was made to measure side by side with a pad made of polyester material and a pad made of glass fiber. Membrane was made by attaching a mesh so that the entire pad was covered on the dried pad. The strips of these two structures were subjected to five repeated precision tests using a sample of normal G6PD concentration and the measurement performance of the flow through structure was excellent as shown in FIG.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

Example  1: Production and measurement principle of the strip of the present invention

For the G6PD assay, 5 mM Tris-6-phosphate, 5 mM N-acetylglucosamine adenine dinucleotide (NADP) and Tetrazolium 0.5 mM BCIP 0.4 mM (Or BCPIP) were added to a solution of Tris 50 mM pH 8.0 with appropriate detergent and polymer Polyvinylpyrrolidone) is mixed and dried in a membrane made of Poly sulfone, Poly ether sulfone, Nylon, Nitro Cellulose, etc.

After the sample is cut into the appropriate size (5mm), the sample is fixed on the cross-linked adhesive porous film of PET, and the mesh (15mm), which is a material for spreading the blood sample, is fixed on the top. Cut the immobilized film card into the appropriate size (5mm) and place it on the device, which is made of poly carbonate or ABS material, and prepare it for use.

Lysis buffer preparation for G6PD was performed by combining 0.5% of TX-100, polymer, and protein in a Tris 50 mM pH 8.0 solution.

The G6PD dedicated measuring instrument is able to measure Total-Hemoglobin & G6PD by changing the% reflectance on the principle of emitting and receiving 525nm wavelength. After inserting the prepared device strip into the measuring device, the whole blood specimen is diluted 21X in the lysis buffer. After the dilution is completed, if the sample is spotted on the strip, the measurement starts when the blood is detected and the measurement time is 5 minutes. When the measurement is completed, the displayed values can be checked at the same time for two values of Total Hemoglobin g / dL and G6PD U / g Hb.

When blood is detected in the device strip, Glucose-6-phosphate reacts with the G6PD enzyme and the NADP reagent is reduced to NADPH. The NADPH-reduced reagent is again oxidized to NAPD. This redox reaction causes the NADPH Appears as a purple or blue formazan color with reduced reduction of the tetrazolium salt. The NADP and NADPH redox reactions are more activated by the higher concentration of G6PD in the blood, and the resulting color is quantitated by measuring the light emission and receiving light.

The precision and linearity performance test for evaluating the G6PD measuring apparatus manufactured according to the above embodiment was carried out, and the results are shown in FIG. Through this technology, patients with G6PD deficiency, a global hereditary disease, developed a technique to use the easily measurable G6PD level as a quantified product, such as Primaquine, which is a treatment for malaria, before the drug prescription, so that the damage caused by acute hemolysis .

Example 2: Bonn  Invention measuring instrument

The outer appearance of the measuring device of the present invention is made in a compact size which can be held by one hand, and the measurement operating temperature is dynamic from 18C to 40C. As a result, it is used as a product suitable for measurement in various places, not limited to the laboratory. After the measurement is completed, the units for the displayed items are quantified by displaying the values of Total-Hemoglobin (g / dL) and G6PD (U / g Hb).

Experimental Example  One. G6PD  & Total Hemoglobin simultaneous measurement method (5 ~ 30 sec Total- Hemoglobin measurement , 300 seconds G6PD measurement )

Glucose-6-phosphate 5 mM, β-Nicotinamide adenine dinucleotide 5 mM (NADP) and Tetrazolium 0.5 mM BCIP 0.4 mM (Or BCPIP) were mixed with the appropriate TX-100 and polyvinylpyrrolidone in a Tris 50 mM pH 8.0 solution for G6PD measurement The prepared solution was treated with symmetric membrane, Nylon membrane and asymmetric membrane, poly ether sulfone membrane, and dried. G6PD is divided into normal and defective samples according to the concentration and then the measurement is progressed. By applying the linear function and the quadratic function to the difference of the reflectance (% Ref), the numerical value is changed to the actual concentration. The measurement principle is hemoglobin (Hb) When G6PD is mixed with Lysis Buffer, Hb is converted to HbO2 state by mixing with Lysis Buffer, and the light emission and light reception after addition to the sample film are quantified by the data. G6PD is the% Ref value at the time of hemoglobin measurement The% Ref value of the difference between 300Sec as a criterion is quantified based on the difference

Hemoglobin is converted to HBO2 by binding to oxygen when the sample is mixed with Lysis Buffer before the G6PD color reaction, and the light emission and the light received immediately after addition to the sample film are quantified by the data. G6PD is the% Ref value at the time of hemoglobin measurement , The% Ref value of the difference between the changes of 300 seconds is quantified based on the difference

Experimental Example 2: Bonn  Comparison of Asymmetric Structure Membranes and Porous Symmetric Membranes of the Invention

G6PD is a composition for measuring G6PD, which is composed of 5 mM of glucose-6-phosphate, 5 mM of β-nicotinamide adenine dinucleotide, and a solution of Tris 50 mM pH 8.0 in order to compare the performance of the membrane with asymmetric polymer structure to the porous symmetric membrane (NADP), Tetrazolium 0.5 mM BCIP 0.4 mM (Or BCPIP) were mixed with the appropriate detergent and polymer. The symmetric membrane was a membrane of Nitro Cellulose, the Asymmetric membrane used in the present invention was a membrane of poly sulfone And then dried at 40 ° C for 20 minutes. Measurements were carried out at room temperature.

The coefficient of variation CV% and the standard deviation STDEV are the units that represent the reproducibility of the quantitative products commonly used in quantitative measurement systems. The measured results are shown as Normal (6U / g Hb), Intermediate & Deficiency (6U / g Hb). Therefore, it shows excellent reproducibility and accuracy enough to quantify against the conventional RDT G6PD.

In addition, the G6PD solution was treated on the membrane and dried. In the lateral flow through method for comparative evaluation, a polyester sample pad and a glass fiber moisture absorbing pad were stacked on each other, Membrane was made by attaching a mesh so that the entire pad was covered on the dried pad. The strips of these two structures were subjected to five repeated precision tests using a sample of normal G6PD concentration and the measurement performance of the flow through structure was shown as shown in FIG.

Claims (11)

A solution prepared by mixing a solution containing glucose-6-phosphate with a surfactant and a polymer is dispensed onto a membrane and dried, and the membrane is cut and fixed on a single-sided adhesive porous film, and a blood sample is uniformly spread Hemoglobin and glucose-6 < RTI ID = 0.0 > (I) < / RTI > comprising a step of immobilizing and attaching a mesh to a structure comprising a portion of the membrane, - A method for preparing a strip for simultaneous measurement of phosphoric acid dehydrogenase. 3. The method of claim 1, wherein the solution comprises one or more compounds selected from the group consisting of [beta] -nicotinamide adenine diclcreotide (NADP), tetrazolium, and 5-bromo-4- Wherein the method further comprises the steps of: (a) contacting the substrate with a glucose-6-phosphate dehydrogenase enzyme.  The method of claim 1, wherein the membrane is selected from the group consisting of Poly sulfone, Poly ether sulfone, Nylon, and Nitro Cellulose. A method for producing strips for simultaneous measurement of 6-phosphate dehydrogenase. The method according to claim 1, wherein the porous film is made of polyethylene phthalate. 2. The method according to claim 1, wherein the porous film is made of polyethylene phthalate. The method according to claim 1, wherein the mesh is a polyester material having hydrophilic properties. 2. The method according to claim 1, wherein the mesh is a hydrophilic polyester material. Porous film;
A membrane pretreated with a solution containing glucose-6-phosphate immobilized on the top of the porous film;
A mesh attached to the top of the membrane such that a portion of the membrane protrudes from the membrane at a size greater than or equal to the size of the membrane;
And a strip for simultaneous measurement of total-hemoglobin and glucose-6-phosphate dehydrogenase. 7. The method of claim 6, wherein the membrane is selected from the group consisting of Poly sulfone, Poly ether sulfone, Nylon, and Nitro Cellulose. Strip for simultaneous measurement of 6-phosphate dehydrogenase.  The strip according to claim 6, wherein the mesh is a hydrophilic polyester. The strip for simultaneous measurement of all-hemoglobin and glucose-6-phosphate dehydrogenase according to claim 6, wherein the porous film is made of polyethylene phthalate. Applying a sample to the mesh portion of the strip of claim 6;
6-phosphate and glucose-6-phosphate dehydrogenase react with each other at the membrane site to cause a color reaction, thereby exhibiting a specific color, and coloring the resultant color to be quantified through light emission of the measuring instrument and light reception doing
A method for simultaneous measurement of total-hemoglobin and glucose-6-phosphate dehydrogenase from a sample in the same strip. 11. The method of claim 10, wherein the sample is whole blood.
KR1020160163662A 2016-12-02 2016-12-02 A strip for simultaneously measuring total-hemoglobin and glucose-6-phosphate dehydrogenase, a preparing method thereof, and a use of the same KR101974645B1 (en)

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Application Number Priority Date Filing Date Title
KR1020160163662A KR101974645B1 (en) 2016-12-02 2016-12-02 A strip for simultaneously measuring total-hemoglobin and glucose-6-phosphate dehydrogenase, a preparing method thereof, and a use of the same
PCT/KR2016/014426 WO2018101525A1 (en) 2016-12-02 2016-12-09 Strip for simultaneously measuring total-hemoglobin and glucose-6-phosphate dehydrogenase, manufacturing method therefor, and application thereof
BR112019010868-8A BR112019010868B1 (en) 2016-12-02 2016-12-09 STRIP FOR SIMULTANEOUS AND QUANTITATIVE MEASUREMENT OF TOTAL HEMOGLOBIN AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE BY THE COLORIMETRIC METHOD, AND METHOD FOR QUANTITATIVE MEASUREMENT OF TOTAL HEMOGLOBIN AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE SIMULTANEOUSLY ON THE SAME STRIP BY THE COLORIMETRY METHOD CO OF A SAMPLE

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KR20000017299A (en) * 1998-08-13 2000-03-25 리젠펠드 제임스 Visual blood glucose test strip
US6200773B1 (en) * 1998-09-28 2001-03-13 Lifescan, Inc. Diagnostics based on tetrazolium compounds
KR20100130902A (en) * 2009-06-04 2010-12-14 주식회사 인포피아 Test strip improved spreadability of plasma or serum
KR20120039774A (en) * 2010-10-08 2012-04-26 주식회사 메디센서 Diagnostic apparatus and diagnostic method using the same
KR20140072023A (en) * 2011-07-22 2014-06-12 액세스 바이오 인코포레이티드 A single-pad strip for an improved lateral flow assay and a test device using the same

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US6420128B1 (en) * 2000-09-12 2002-07-16 Lifescan, Inc. Test strips for detecting the presence of a reduced cofactor in a sample and method for using the same
JP4566983B2 (en) * 2003-02-24 2010-10-20 バイナックス インコーポレイティッド Lateral flow in dry chemistry-reconstituted chromatographic enzyme-driven assay
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Publication number Priority date Publication date Assignee Title
KR20000017299A (en) * 1998-08-13 2000-03-25 리젠펠드 제임스 Visual blood glucose test strip
US6200773B1 (en) * 1998-09-28 2001-03-13 Lifescan, Inc. Diagnostics based on tetrazolium compounds
KR20100130902A (en) * 2009-06-04 2010-12-14 주식회사 인포피아 Test strip improved spreadability of plasma or serum
KR20120039774A (en) * 2010-10-08 2012-04-26 주식회사 메디센서 Diagnostic apparatus and diagnostic method using the same
KR20140072023A (en) * 2011-07-22 2014-06-12 액세스 바이오 인코포레이티드 A single-pad strip for an improved lateral flow assay and a test device using the same

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