JP3278109B2 - Standard solution for lipid determination - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a standard solution used for determining lipids, particularly free cholesterol.

[0002]

2. Description of the Related Art Lipids are classified into simple lipids, complex lipids and derived lipids, and are generally known to be insoluble in water, acids and alkalis. These lipids have been receiving attention in recent years as being involved in arteriosclerosis and ischemic heart disease when contained in blood. Conventionally, the following biosensors have been considered as a method for easily quantifying lipid contained in blood without performing dilution or centrifugation of a sample solution. In this biosensor, an electrode system including a working electrode and a counter electrode is formed on an electrically insulating substrate by a method such as screen printing, and a reaction reagent layer is formed on the electrode system. The electrode system may be formed using a noble metal such as platinum. In the case of a disposable sensor, silver is coated with carbon in consideration of cost and the like. The reaction reagent layer contains a hydrolase or oxidoreductase using a hydrophilic polymer, an electron acceptor and a lipid as substrates.

When a sample solution containing a substrate is dropped onto the enzyme reaction layer of the biosensor thus prepared, the enzyme reaction layer dissolves, the substrate reacts with the enzyme, the substrate is hydrolyzed, and then oxidized. Accordingly, the electron acceptor is reduced. After completion of the enzymatic reaction, the reduced electron acceptor is electrochemically oxidized, and the substrate concentration in the sample solution can be determined from the oxidation current value obtained at this time. In such a biosensor, various substrates can be quantified by using various oxidoreductases. In addition, the substrate concentration can be measured with a simple operation just by introducing the sample into the sensor chip inserted into the measuring instrument, and since it is a disposable type, it is effectively used as a diagnostic guideline in various medical institutions. .

[0004]

The standard solution for quantifying lipids using the above-described biosensor is prepared by solubilizing lipids in water by using an organic solvent such as isopropyl alcohol and a surfactant. Prepared. This standard solution was originally prepared for lipid determination by spectroscopic measurement. In the case of quantifying lipid by spectrophotometry, there is no problem because the influence of an organic solvent or a surfactant can be sufficiently reduced by diluting a standard solution with water.

[0005] However, when this standard solution is used for a biosensor, the reaction when the sample solution is introduced into the sensor depends on the type and concentration of the surfactant used and because of the organic solvent contained. The solubility of the reagent layer is reduced, and the surface of the carbon electrode formed on the substrate is eroded.
As a result, there has been a problem that the response current value decreases, or a current response called an electrode blank occurs even when a sample solution having a substrate concentration of 0 is introduced into the sensor. The present invention has been made in view of the above problems, and provides a lipid quantification standard solution that can increase the amount of lipid that can be solubilized without reducing the solubility of a reaction reagent layer and that does not erode the electrode surface. Aim.

[0006]

Means for Solving the Problems A standard solution for lipid determination according to the present invention is formed on an electrically insulating substrate and the insulating substrate.
An electrode system including a working electrode and a counter electrode and a reaction reagent layer
And introducing a reagent solution containing lipid into the reaction reagent layer.
The lipid is determined by measuring the response current value
Standard solution for lipid quantification used in biosensors
Therefore, it consists of a liquid in which lipids are dispersed in an aqueous solution of a surfactant.
In addition, a nonionic sugar ester-based surfactant is used as the surfactant. In addition,
The standard solution for lipid determination according to the present invention essentially contains an organic solvent.
It is characterized by not having.

[0007]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As described above, by using an aqueous solution of a surfactant as a dispersion medium of a lipid, erosion of a carbon electrode caused when a sample solution is supplied into a sensor can be suppressed. Therefore, the electrode blank value can be reduced, and the electrode blank can be suitably used as a standard solution for lipid quantification. In addition, by using a nonionic sugar ester surfactant as the surfactant, the amount of lipid that can be solubilized in an aqueous solution can be increased, so that a standard solution that can be used in a wide concentration range can be prepared. Examples of such nonionic sugar ester surfactants include sucrose fatty acid esters, octylthioglucoside, sucrose monolaurate, n-dodecyl-β-D-maltoside and decanoyl-N-methylglucamide. is there. As lipids, cholesterol,
Free cholesterol, phospholipids, glycolipids, fatty acids, steroids, neutral fats and the like can be used.

As the enzymes contained in the reaction layer, cholesterol oxidase, cholesterol dehydrogenase and the like can be suitably used. As the electron acceptor, dissolved oxygen in a sample solution, ferricyan ion, ferrocene, and derivatives thereof can be used. In the reaction layer, the effect of the sample solution on the electrodes is reduced,
In order to stably form the reaction layer on the electrode system, it is preferable to include a hydrophilic polymer. As such a hydrophilic polymer, carboxymethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, a water-soluble cellulose derivative, and the like can be used.

[0009]

The present invention will be described below in more detail with reference to specific examples. FIG. 1 is an exploded perspective view of a biosensor used for quantifying free cholesterol, from which a reaction layer has been removed, using a standard solution for quantifying lipid according to the present invention. Silver paste is printed by screen printing on an electrically insulating substrate 1 made of polyethylene terephthalate to form leads 2 and 3. Next, the working electrode 4 is formed by printing a conductive carbon paste containing a resin binder on the substrate 1. The working electrode 4 is connected to the lead 2
Is in contact with Further, an insulating paste is printed on the substrate 1 to form an insulating layer 6. The insulating layer 6
The outer peripheral portion of the working electrode 4 is covered, thereby keeping the area of the exposed portion of the working electrode 4 constant. Then, a conductive carbon paste containing a resin binder is printed on the substrate 1 so as to be in contact with the leads 3 to form a ring-shaped counter electrode 5. The biosensor is manufactured by bonding the insulating substrate 1, the cover 9 having the air holes 11, and the spacer 10 in a positional relationship shown by a dashed line in FIG. 1.

In such a biosensor, the slit 13 of the spacer 10 is provided between the substrate 1 and the cover 9.
A space that forms the sample liquid supply path is formed in the portion.
Then, the sample liquid is easily introduced into the reaction layer portion through the sample liquid supply path by a simple operation of bringing the sample liquid into contact with the distal end portion (sample liquid supply path opening) 12 of the slit 13 serving as the opening. You. Since the supply amount of the sample solution depends on the space volume generated by the cover and the spacer, it is not necessary to determine the amount in advance. Furthermore, evaporation of the sample liquid during measurement can be minimized, and highly accurate measurement can be performed. Here, when a transparent polymer material is used for the cover and the spacer, the state of the reaction layer and the state of introduction of the sample liquid can be easily observed from the outside.

Example 1 A mixed aqueous solution of cholesterol oxidase, potassium ferricyanide and carboxymethylcellulose was dropped on the electrode system of the insulating substrate 1 shown in FIG. 1 and dried to form a reaction layer. Next, a 20% (V / V) aqueous solution of sucrose fatty acid ester (DK ester S-L18A; manufactured by Daiichi Kogyo Seiyaku) was prepared. Free cholesterol was not dispersed in the aqueous solution because it was prepared to measure a blank value. 5 ml of this aqueous solution
Was dropped on the reaction layer, 5.5 seconds after supplying the sample solution, a voltage of +0.5 V was applied to the working electrode 4 with reference to the counter electrode 5, and the response current value (electrode Blank value) was measured. The result is shown in FIG.

Comparative Example 1 An electrode blank value was measured in the same manner as in Example 1 using water and a liquid containing 8% isopropanol in water (commercially available cholesterol standard solution) as a sample liquid. The result is shown in FIG. As shown in FIG. 2, when an aqueous solution of a sucrose fatty acid ester was used, the electrode blank value was about the same as that of water, and was about half that of a conventional commercially available standard solution.

Example 2 The aqueous solution of sucrose fatty acid ester prepared in Example 1 was added with a concentration of 0, 120, 240,
The cholesterol standard solution was prepared by dispersing the free cholesterol powder so as to be 360 and 480 mg / dl while heating. Then, the response current value was measured in the same manner as in Example 1. The result is shown in FIG. As shown in FIG. 3, as the cholesterol concentration increased, the response current value also increased, and the linearity was excellent.

[0014]

As described above, according to the present invention, it is possible to provide a standard solution suitable for a biosensor for lipid quantification.

[Brief description of the drawings]

FIG. 1 is an exploded perspective view of a biosensor used in one example of quantifying lipid using a standard solution for quantifying lipid according to the present invention.

FIG. 2 is a diagram showing electrode blank values in one example of the present invention and a comparative example.

FIG. 3 is a response characteristic diagram of a biosensor measured using a standard solution for lipid quantification prepared in another example of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Electrically insulating substrate 2, 3 Lead 4 Working electrode 5 Counter electrode 6 Insulating layer 9 Cover 10 Spacer 11 Air hole 12 Sample liquid supply channel opening 13 Slit

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/96 G01N 33/92 G01N 27/26

Claims (2)

(57) [Claims] 1. An electrically insulating substrate having a shape formed on the insulating substrate.
Electrode system and reaction including formed working and counter electrodes
It has a reagent layer and leads a reagent solution containing lipid to the reaction reagent layer.
By measuring the response current value obtained by
Target for lipid quantification used in biosensors for lipid quantification
A standard solution for lipid quantification , which is a semi-liquid , comprising a liquid in which a lipid is dispersed in an aqueous solution of a surfactant, wherein the surfactant is a nonionic sugar ester-based surfactant. 2. A process according to claim 1 , wherein the process does not contain an organic solvent.
The standard solution for lipid quantification according to claim 1.