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Biosensor

Inactive Publication Date: 2009-09-24
CCI HLDG INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009]Recently, the number of patients of hyperlipemia, which is a lifestyle-related disease, has been growing steadily, and thus it is a present state that development of a sensor, which is capable of measuring, quickly and in high accuracy, concentration of neutral fat contained in a sample like blood or the like, is strongly desired.
[0010]In these circumstances, it is an object of the present invention to provide a sensor, which is capable of measuring, quickly and in high accuracy, concentration of neutral fat from a sample like a biological sample or the like, without executing pretreatment of the sample.
[0011]The present inventors have found that, in a biosensor for measuring concentration of neutral fat, based on value of current flowing in the electrode system, having: an insulating substrate; an electrode system having a working electrode and a counter electrode, formed onto the insulating substrate; and a reaction layer formed at the upper part or the vicinity of the electrode system; by inclusion of a lipoprotein lipase, a glycerol dehydrogenase and an electron mediator in the reaction layer, concentration of glycerol in a sample can be measured rapidly and in high accuracy, and have thus completed the present invention.

Problems solved by technology

However, a triglyceride sensor described in the above JP-A-59-210356 measures fatty acids liberated by a glycerol ester hydrolase, using a pH sensitive, ion selective electric field effect transistor, and because response value is proportional to logarithm of sample concentration, measurement accuracy is insufficient.
And this sensor also has a problem that an apparatus becomes complicated.
Since the sensor requires supply of a measurement sample from its outside, it requires a large quantity of samples, thus makes measurement difficult using a small amount of a sample.
Further, a sensor described in the above JP-A-2001-343349, is configured by different layers for an enzyme and an electron mediator, which makes an apparatus extremely complicated, and requires use of two kinds of expensive enzymes, a glycerol kinase and a glycerophosphate oxidase, as well as a lipoprotein lipase.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

reference example 1

[0071]Into a 500-mL Sakaguchi flask, 400 mL of culture media composed of sorbitol (2% by weight), yeast extract (0.3% by weight), meat extract (0.3% by weight), corn steep liquor (0.3% by weight), polypeptone (1% by weight), urea (0.1% by weight), KH2PO4 (0.1% by weight), MgSO4.7H2O (0.02% by weight), CaCl2 (0.1% by weight), with a pH of 7.0, was transferred, by 100 mL per one flask, and autoclaved at 121° C. for 20 minutes.

[0072]As an inoculum, a strain of Gluconobacter Oxydans NBRC 3291, was inoculated by one platinum loop and cultured at 30° C. for 24 hours to make a seed culture media.

[0073]Then, 6.6 L of the culture media prepared with the same composition as in the above, was transferred to a 10-L jar fermenter, and autoclaved at 121° C. for 20 minutes, followed by gradually cooling and subsequently transferring of a 400 mL of the seed culture media, which was cultured under 750 rpm and an airflow amount of 7 L / minute, at 30° C. for 24 hours.

[0074]The culture media was subject...

example 1-1

[0075]As a sensor substrate formed with an electrode system, a commercial sensor electrode (manufactured by BVT Co., Ltd; ACl.W5.R1) was prepared. In this sensor substrate, the electrode system has a diameter of about 6 mm, and nearly circular shape. Furthermore, at the upper part of the electrode system prepared above, a PET sheet, with a thickness of 0.2 mm, and having a hole with a diameter of 6 mm, was glued together to form a spacer. Into the inside of the hole of the spacer on the electrode system, 10 μL of phosphate buffered saline mixed with GlyDH (20 U / mL) obtained in the above Reference Example 1, the lipoprotein lipase (manufactured by Amano Enzyme Inc., 10,000 U / mL), and 1-methoxy-5-methylphenazinium methylsulfate (manufactured by DOJINDO Laboratories, 10 mmol / L; hereinafter referred to as “m-PMS”) as an electron mediator, was dropped, and dried at room temperature, to form a reaction layer.

[0076]Onto this reaction layer, 10 μL of a sample liquid containing 50 mg / dL of t...

example 1-2

[0081]After dropping an aqueous solution of 0.5% (w / v) of sodium carboxymethyl cellulose (manufactured by Wako Pure Chemical Industries Ltd; hereafter may be referred to as “CMC”) onto the electrode system used in Example 1-1, and drying, a 0.5% CMC solution containing GlyDH (20 U / mL) obtained similarly as in Example 1-1, the lipoprotein lipase (10,000 u / mL) and m-PMS (10 mmol / L) was dropped, and dried to form a reaction layer.

[0082]Correlativity between response current value and triolein concentration was studied similarly as in Example 1-1, by using the biosensor. The result is shown in FIG. 5.

[0083]As shown in FIG. 5, although the resultant response current value was reduced a little by effect of CMC, higher correlativity between response current value and concentration of neutral fat was observed as compared with Example 1.

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Abstract

It is an object of the present invention to provide a sensor, which is capable of measuring, quickly and in high accuracy, concentration of neutral fat from a sample such as a biological sample or the like, without executing pretreatment of the sample. This object is attained by a biosensor for measuring concentration of neutral fat, based on value of current flowing in the electrode system, having: an insulating substrate; an electrode system having a working electrode and a counter electrode, formed onto the insulating substrate; and a reaction layer having a lipoprotein lipase, a glycerol dehydrogenase and an electron mediator, formed at the upper part or the vicinity of the electrode system.

Description

TECHNICAL FIELD[0001]The present invention relates to a biosensor, which is capable of determining, quickly and in high accuracy, quantity of neutral fat contained in a biological sample or the like.BACKGROUND ART[0002]In general, as a measurement method for neutral fat, there are (1) a method using chromotropic acid, (2) an acetylacetone method, (3) an enzyme method, and (4) a method using a nephelometer, however, any of these methods requires complicated analysis operations and long analysis time. Therefore, a biosensor has been developed, as a method for determining, simply and quickly, quantity of objective substances without execution of dilution or stirring of a sample liquid, in measurement of specific components contained in the sample.[0003]As such a biosensor for measurement of neutral fat, there is a triglyceride sensor characterized in that a glycerol ester hydrolase is immobilize onto a porous polymer membrane or a hydrophilic uniform polymer membrane covered onto a gat...

Claims

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Application Information

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IPC IPC(8): G01N27/26G01N27/327
CPCC12Q1/004C12Q1/61C12Q1/005C12N11/096G01N27/327G01N33/48G01N27/3272
Inventor MURASE, HIRONOBUKUWAHARA, MOTOAKIYAMADA, MASAYUKI
Owner CCI HLDG INC
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