KR20040063358A - Fluorescence bioscence for the measurement of total cholesteron in serum and whole blood - Google Patents

Abstract

PURPOSE: A method for measuring total cholesterol in serum and whole blood using fluorescence biosensor is provided, thereby rapidly and easily measuring the total cholesterol in serum and whole blood by directly contacting the sample with a reaction region of enzymes, and easily and cheaply installing the fluorescence biosensor in hospitals due to its small size. CONSTITUTION: The method for measuring total cholesterol in serum and whole blood using fluorescence biosensor comprises the steps of: adhering nitrocellulose membrane(2) to the surface of a plastic plate(1) with adhesive; spotting a mixed solution of AEC(3-amino-9-ethyl carbazole) solution, CO solution and HRP solution on the nitrocellulose membrane(2) in a line; attaching an absorption pad(4) to a blood corpuscle separating pad(3) treated with 1% BSA and 0.1% Tween 20 and cutting them in a certain size; covering the pads with a plastic upper and lower plates(1)(6), and introducing a sample(5) and 0.1M citric acid buffer solution(pH5.0) into a sample inlet(7); and measuring the intensity of fluorescence using a fluorescence detector after 5 minutes of reaction, wherein the pads are pretreated with cholesterol esterase or surfactant to destroy membrane protein such as lipoprotein during the sample flows.

Description

Fluorescence bioscence for the measurement of total cholesteron in serum and whole blood}

In general, plasma lipid proteins exist in the form of spherical particles, the surface of which is composed of phosphate and cholesterol and membrane proteins called apolipoproteins, inside of which are cholesterol esters and Consisting of triglyceryl molecules (see AL Lehninger, principles of biochemistry, second Edition NY, 1993, page 482). Cholesterol is a lipophilic monohydric alcohol, fat, bile, blood, brain tissue, milk, egg yolk, nerve fiber myelin, liver. Present in the kidneys, adrenal glands. Most cholesterol in the body is synthesized in the liver and some is absorbed in food. It is a precursor to bile acids and is important for the synthesis of steroid hormones. Cholesterol synthesized in the liver forms a complex with lipoproteins, proteins, fatty acids, and triglycerols and is transported through the blood to where it is needed in the body, when the content of lipoproteins is high (low density lipoprotein choresterol, LDL-C) In some cases, it is called high density lipoprotein choresterol (HDL-C). Normally, less than 200 mg / ml but hypercholesterolemia is above 240 mg / ml. Ingestion of foods high in saturated fatty acids and cholesterol increases the concentration of LDL-C in the blood, which leads to the deposition of cholesterol in the blood vessel walls, resulting in atherosclerosis (Ref .: PS Bachorik, et al., Clin. Chem. 1995, Vol. 41, page 1414). Atherosclerosis is caused by the accumulation of cholesterol in cells caused by the proliferation of leukocytes, fibrous tissues, or muscle cells. Monocytes gather under the epithelial tissues of the arteries to accumulate and receive cholesterol from LDL in the blood during the differentiation of macrophages, resulting in distortion of the vascular epithelial layer. These factors stimulate the proliferation of vascular epithelial cells by these factors, thereby reducing the diameter of blood vessels. This process continues, forming artheroscerotic plaques that block the blood vessels in the arteries, disrupting blood flow. Atherosclerosis, especially in the coronary arteries of the heart, can be fatal, such as myocardial infarction. Atherosclerosis interferes with blood flow, resulting in high blood pressure. Therefore, the higher the blood LDL cholesterol level, the higher the chance of hypertension. Increasing total cholesterol is also an indication of an increased risk of coronary artery disease. Total cholesterol above 180 mg / dL increases the likelihood of coronary artery disease, and at 220 mg / dL, the probability doubles. With a 2% drop in cholesterol at 200 mg / dL, the risk of coronary artery disease is reduced by 1%.

The conventional method for measuring cholesterol is as follows. By using the LIEBERMANN-BURCHARD reaction, cholesterol is gradually dehydrated under the presence of acetyl acid and sulfuric acid, and oxidized to finally sulfonate to produce a green compound. This reaction can be used to determine total cholesterol (S. H. Kim et al., Bull. Korean Chem. Soc. 2000, Vol. 21, 389). However, this method is limited to serum measurement, and the experiment is performed by separating LDL and HDL from other lipoproteins using ultra centrifugation, which is cumbersome to operate. At present, the method of measuring HDL which is used for clinical examination is precipitation method. A method of measuring cholesterol in a supernatant containing only HDL by aggregating lipoproteins other than HDL by centrifugation by adding a precipitant to the specimen (Ref .: SA Lottenberg et al., Atherosclerosis, 1996, Vol. 127 , 1115). This method is simpler than ultracentrifugation or electrophoresis, but involves a relatively large amount of sample, which involves the segregation of sediment, is largely error-prone and difficult to automate the entire process. In addition, biochemical reactions using enzymes take up the majority, and the reaction can be expressed as follows.

CE:

Cholesteryl ester + H ₂ O ---> Cholesterol + Fatty acid

CO:

Cholesterol + O ₂ ----> Cholesten-4-one + H ₂ O ₂

HRP:

H ₂ O ₂ + Chromogen (colorless) ----> Oxidized chromogen (color) + 2H ₂ O

Depending on the type of chromogen used, the wavelength and sensitivity required for measurement are determined. The method of absorbance at 405-415 nm using 3,5-diacetyl-1,4-dihydrolutidine as a colorant, phenol and 4-aminoantipyrine Method to measure absorbance at 515 nm by reacting the method, method to measure absorbance at 600 nm by reacting MBTH (3-methyl-2-benzothiazolinone hydrazone) with dimethylaniline, ABTS (2,2-azino-di- [3- ethylbenzthiazolinesulphonic acid] and the method for measuring the absorbance at 420-436 nm, the method for measuring the chemiluminescence generated by the reaction with luminol (US 5,597,703) and the like are known.

In addition, a method for fractionating and quantifying cholesterol in HDL using an enzymatic reaction has also been studied. For example, a method (Japanese Patent Laid-Open No. 63-126498) is known in which an enzyme reaction is carried out under conditions in which bile salts and nonionic surfactants are present. In this method, the initial enzyme reaction is proportional to the cholesterol concentration in the LDL and the next reaction rate is proportional to the cholesterol concentration in the HDL. It is not easy to control the reaction time by fractionation.

Most of the automated devices for cholesterol measurement developed so far are mostly using chemiluminescence generated by reacting with luminol. Other examples of measurement methods in the market are as follows.

TECO Diagnostics (http: // www. Teco diag.com) has developed a device to quantify total cholesterol in serum using 4-aminoantipyrine. CARESIDE (http:. // www careside .com) were also developed automated equipment for the determination of total cholesterol using the Leuco dye. Many patents have also been registered for dyes used in this process (US 4,251,222, US 4,340,392, US 5,004,685, US 5,024,935, US 5,047,318, US 5,366,864, US 5,589,347). These methods are rapid measurement methods using commercially available expensive equipment, simple semi-quantitative kits that can be easily measured, and fluorescence kits have not been published.

In the present invention, AEC (3-amino-9-ethyl carbazole) was used as a colorant. 20 mg of AEC is dissolved in 5 mL of DMF and diluted 10-fold with distilled water (about 0.4 mg / mL). In proportion to the amount of H ₂ O ₂ produced by cholesterol oxidase, the AEC participates in the reaction, and the colored material generated in the reaction remains on the ship in the form of a polymer, which can be measured using a portable and convenient fluorescent device. It was. The reaction formula at this time is represented by the formula shown in FIG.

In the experimental technique of the present invention, in order to solve the problem of semi-quantification of the conventional commercial cholesterol measurement kit, quantification is possible by using a portable fluorescence reader manufactured by itself. By forming a polymer at, as the progress of the reaction that was difficult to solve conventionally, the busy colored lines moved together to solve the problem of difficult quantification. In addition, by using a sample pad capable of separating whole blood, not only serum but also whole blood can be measured.

Figure 1 schematically shows the configuration of the free cholesterol measuring apparatus of the present invention.

Figure 2 schematically shows the configuration of the total cholesterol measuring device of the present invention.

Figure 3 illustrates the color development principle of the present invention.

4 is a graph showing the intensity of fluorescence according to the concentration of cholesterol measured by the present invention.

FIG. 5 confirms linearity by converting the intensity of fluorescence according to FIG. 4 into an area ratio.

6 is an image of the membrane actually developed by the present invention with a scanner and an image pickup device.

Figure 7 is a graph showing the measured value of cholesterol of 2mg / ml concentration by the present invention.

Figure 8 is a graph showing the measured value of cholesterol of 4mg / ml concentration by the present invention.

Looking at the present invention is as follows.

Since the reaction site of the enzyme is in direct contact with the sample sample, the sensitivity can be increased by reducing the loss due to adsorption and diffusion during the movement of other samples.

CO and HRP enzymes are distributed along a line with the chromophores participating in the reaction, thus increasing the reaction efficiency.

Cholesterol esterase was dispensed into the sample pad, so that it was immediately reacted with the sample to allow conversion to alcohol type cholesterol.

Pre-treated sample pads were attached and the type and concentration of surfactants were adjusted to allow the determination of free and total cholesterol. In other words, when quantifying free cholesterol, pretreatment is performed. Since the sample pad (FIG. 1) is not used, free cholesterol can be measured, and when the total cholesterol needs to be measured, as shown in FIG. The use of an active agent-treated sample pad made it possible to measure total cholesterol.

The surfactant used may be polyoxyethylene alkylene phenyl ether, polyoxyethylene alkylene tribenzyl phenyl ether, triton X-100, NP-40 and the like, and sodium cholate. The concentration can be 0.001-5%, but 1% is fine.

Quantitative measurement was possible by using a sensitive portable fluorescent reader.

The following preparation examples are presented as a means for implementing the present invention, the present invention is not limited by the preparation examples.

Preparation Example 1

Nitrocellulose manmbrene (2 in FIG. 1) is attached to a plastic plate (1 in FIG. 1) to which an adhesive is attached.

Dispense a mixed solution of 50 mL of 4 mg / ml AEC solution (solvent: DMF), 5 mL / ml CO aqueous solution, and 5 unit / ml HRP aqueous solution by wire.

As shown in FIG. 1, 1% BSA, 0.1% Tween 20 treated blood cell separation pad (3 in FIG. 1) and an absorption pad (4 in FIG. 1) were attached and then cut to a size of (60 × 4.1 mm).

Cover the upper and lower plates of plastics (1 and 6 in Figure 5) and add 40 mL of Sample 5 (Roche calibrator) and 0.1 M citric acid buffer (pH 5.0) to the sample inlet (7 in Figure 1).

After the reaction proceeds for 5 minutes, it is infused into a fluorescence reader to read the fluorescence intensity from the fluorescence window (8 in FIG. 1).

As a result of the experiment, as shown in FIG. 4, the fluorescence intensity of the acid increased with the increase of the concentration of cholesterol. At this time, the correlation coefficient was 0.997, indicating that the linear relationship was well established.

Preparation Example 2

Nitrocellulose manmbrene (2 in FIG. 2) is attached to a plastic plate (1 in FIG. 2) to which an adhesive is attached.

50 mL of a 4 mg / ml AEC solution (solvent: DMF), a mixture of 5 mL / ml CO aqueous solution, and 5 unit / ml HRP aqueous solution are dispensed linearly.

As shown in FIG. 2, a sample pad (9 in FIG. 2), a blood cell separation pad (3 in FIG. 2) and an absorption pad (4 in FIG. 2) were treated with 1% BSA, 0.1% Tween 20 and 1% NP-40. Then cut (60 x 4.1 mm).

Cover the upper and lower plates of plastic (5 and 6 in Figure 2) and add 40 mL of Sample 5 (Roche calibrator) and 0.1 M citric acid buffer (pH 5.0) to the sample inlet (7 in Figure 2).

After the reaction proceeds for 5 minutes, it is infused into a fluorescence reader to read the fluorescence intensity from the fluorescence window (8 in FIG. 2).

Experimental results also showed a good linear relationship with the increase in the concentration of cholesterol as in FIG.

Example 1

Images of membranes in which color signals were generated for the strips tested according to Preparation Example 1 were captured using a scanner (HP, Model 3300C) and a program built into a computer. The captured image is shown in FIG. 6, and a relatively uniform line-shaped band was identified. The color development of the colored portion also increased the amount of cholesterol (1 mg / ml, 2 mg / ml, 4 mg / ml) was found to increase.

Example 2

7 and 8 show the results of capturing the image of the membrane in which the color signal was generated with respect to the strip tested according to Preparation Example 1 using a CCD under development in the company. In the case of Figure 7 is 2 mg / ml and Figure 8 is 4 mg / ml. The color development of the colored part also increased with the increase of cholesterol.

The present invention can be used for diagnosis and tracking of diseases by using a pre-prepared strip to accurately quantify the presence and amount of cholesterol in both whole blood or serum by simple manipulation, and the results can be viewed quickly. There is an advantage. In addition, by using a much smaller device than the existing large serum analyzer, the burden of installation and use in small and medium-sized hospitals and clinics can be greatly reduced.

Claims (6)

Method of quantification by chromophore generated by using biochemical enzymatic reaction in detection line linearly dispensed on membrane The method of quantifying the extent of the color reaction in claim 1 using an image analysis such as a fluorescence reader or a charge imaging device (CCD) Apparatus and method for separating serum using a sample pad in claim 1 and reacting the separated serum by moving in a vertical direction Analytical method using AEC as a coloring material to quantify the amount of hydrogen peroxide produced in the biochemical enzyme reaction of claim 1 The method of claim 3, wherein the sample pad is subjected to cholesterol esterase treatment to enable a pre-reaction, and the sample pad is subjected to a surfactant treatment to destroy membrane proteins such as lipoproteins while the sample is flowing. The analytical method and apparatus of the present invention is not limited to cholesterol, but hydrogen peroxide is generated in an enzyme reaction such as glucose, ALT, AST, etc.