JPH0519103B2 - - Google Patents

Description

[Detailed description of the invention]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates generally to analytical chemistry, and more particularly to analytical elements for analyzing predetermined specific components in fluids, and more particularly to quantitative analytical elements for analyzing lipase in biological fluid samples. Conventionally, many methods for analyzing analyte components in fluid samples have been developed, but these can be broadly divided into two types: reaction systems in solution and reaction systems in a solid phase. Analytical reactions in solution systems (hereinafter abbreviated as wet chemistry) are widely known, from analytical operations called manual methods that do not use any machinery at all, to automatic quantitative analyzers that have been widely used in clinical laboratories in recent years. There is. Among these, automatic quantitative analyzers are particularly useful for blood analysis. For example, US Patent No.
The analyzer based on continuous flow analysis described in No. 2797149 is representative of this. These involve mixing the fluid sample, diluent, and analytical reagents, transporting them into an analytical device, and performing analytical reactions and quantitative measurements. however,
Such continuous analysis devices are complex and expensive, require skilled operating techniques, and require repeated cleaning operations after each analysis operation, which requires a great deal of time and effort. They have the disadvantage that they are wasteful and these waste liquids inevitably pollute the environment. On the other hand, analytical methods using solid-phase analytical reactions (hereinafter abbreviated as dry chemistry) are also widely used. For example, US Pat. No. 3,050,373 or
As described in No. 3061523, etc., it is made by impregnating a water-absorbing carrier such as filter paper with a reagent solution and drying it. These are generally performed by dropping or immersing a fluid sample onto an analytical test strip or simply onto a test strip, and measuring the color change or density change in the test strip with the naked eye or with a reflectance densitometer. ,
It determines the concentration level of a particular component in a fluid sample. These sample pieces are useful because they are easy to handle and provide immediate results, but due to their construction, they remain in the realm of semi-quantitative or qualitative analysis. In contrast to the conventional analytical methods described above, a blood analysis element described in Japanese Patent Publication No. 53-21677 has been proposed as one that uses dry chemistry with a simple operation method and has high fixability. . This proposal consists of at least one reagent layer located on one side on a light-transparent liquid-impermeable support, containing at least one reagent that reacts with components in the fluid sample, and consisting of a hydrophilic colloid. , located on the opposite side of the reagent layer from the support, at least one non-container layer located on the reaction side of the support and allowing the components in the fluid sample to permeate to the reagent layer. The present invention relates to a blood analysis element having a fibrous porous media layer. However, the above-mentioned blood analysis element combined with a reagent layer consisting of a hydrophilic colloid such as gelatin is difficult to penetrate into the polymer matrix formed by the hydrophilic colloid, which is impermeable to flea having a high molecular weight such as lipase. Therefore, the analytical element proposed above has a fatal drawback in that quantitative analysis is not possible. SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide an analytical element that can quantitatively analyze lipase, which is high molecular weight flea white matter, present in a fluid sample and which has improved the above-mentioned drawbacks. The object according to the invention is to provide a light-transparent, liquid-impermeable support, a sensing layer located on one side of the support, and a sensing layer located on the opposite side of the support, An analytical element having at least one spreading layer for supplying the fluid sample to the sensing layer, the sensing layer comprising a hydrophobic and substantially non-swellable core to the fluid sample and a hydrophilic layer. This can be achieved by an analytical element for lipase analysis, which is composed of polymer particle units having a core-shell multilayer structure consisting of shells, and which contains a compound represented by the following general formula in the core detection layer. can. general formula In the formula, M represents an alkali metal, and n is 0 to 30
indicates an integer. That is, the present invention provides a detection layer or a development layer of an analytical element having a detection layer and a development layer on a support. An improved analytical element is characterized in that it contains a compound represented by This is a proposal. The present invention will be explained in more detail below. First, the analytical element of the present invention basically has a structure in which a detection layer and a developing layer are sequentially provided on a support. The detection layer according to the present invention is disclosed in Japanese Patent Application No.
The polymer particle bonding layer described in No. 155788 is used. The sensing layer consisting of this polymer particle bonding layer is
It is suitable for accommodating substances with large molecular weights such as enzymes within the layer, so that said enzymes can be detected within the layer. The thickness of the sensing layer in the present invention is about 80 microns to 10 microns, preferably about 60 microns to about 20 microns. Further, the developing layer according to the present invention is a layer having the following three functions as described in Japanese Patent Publication No. 53-21677. (1) uniformly distribute a fixed volume of fluid sample within the sensing layer at a fixed volume per unit area; (2) remove substances or factors that inhibit the analytical reaction in the fluid sample; and (3) perform spectrophotometric analysis. During this process, a background effect is performed which reflects the measuring light transmitted through the support. Therefore, the expansion layer according to the present invention can perform all of these three functions, but it is also possible to separate the three functions as appropriate and use a separate layer for each function. Furthermore, it is also possible to use a combination of a layer having two of the three functions and a layer having the remaining functions. For example, a spread layer of a non-fibrous porous medium called brush polymer consisting of titanium dioxide and cellulose diacetate is described in the above-mentioned publication, JP-A-56-
No. 24576, Patent Application No. 56-13203 and No. 56-65446
Examples include the fiber structure spread layer described in No. 1, etc. In particular, this fiber structure spreading layer is useful as a material capable of rapidly transporting blood cell portions, and can be used as a spreading layer effective in spreading and transporting macromolecules according to the present invention. The thickness of the spreading layer is determined by the porosity of the spreading layer and the amount of fluid sample dropped, but is preferably about 400 microns to about 80 microns, more preferably about 350 microns to about 100 microns. .
For example, when a sample of about 10 microliters is dropped using the fiber structure spreading layer, the thickness of the layer is preferably about 120 microns to about 220 microns. Next, the compound represented by the general formula to be contained in the detection layer is prepared according to the following reaction formula:
Azo dye compounds are formed by the action of lipase. This formed azo dye compound has a characteristic absorption at 475 nm, and in the present invention, lipase activity is detected by measuring the absorption of the dye in a solid-phase analytical element. . As is clear from the above, the compound according to the present invention is a type of dye precursor that is hydrolyzed by lipase, and the number of n in the general formula is preferably 5 to 20, more preferably 8 to 16. is an integer. When the compound represented by the above general formula is contained in the detection layer, the above-mentioned patent application
It is necessary to use the polymer particle binding layer described in No. 155788 as the sensing layer. In order to incorporate the compound according to the present invention into the detection layer, any conventional dispersion method such as an oil protect dispersion method or a direct dispersion method can be used. Alternatively, it may be contained in a uniformly dissolved state. Furthermore, when the compound according to the present invention is incorporated into the detection layer, it may be mordanted in advance with a compound having a quaternary ammonium base by ionic bonding,
It is also possible to stabilize and then disperse. This quaternary ammonium salt may have a low molecular weight or a high molecular weight. 1 of the above low molecular weight quaternary ammonium salt compounds
Examples include, for example, N,N,N-tributyl-N
-Octadecylammonium bromide, N,N
-dimethyl-N-benzyl-N-octadecylammonium chloride, N,N-dimethyl-N-
Examples include benzyl-N-cetylammonium chloride. In addition, one of the polymer quaternary ammonium salts
Examples include JP-A-50-80132 and JP-A-50-61228.
No. 51-73440, No. 54-74430, No. 55-
The polymers described in each specification of No. 22766 can be usefully used. In particular, JP-A-51-73440, JP-A No. 55-
The polymeric quaternary ammonium salts described in No. 22766 are useful in the form of polymeric latexes. The dye formed by the reaction does not diffuse into the sensing layer, and therefore undesired migration of the resulting dye is greatly reduced. The liquid-impermeable, light-transparent support according to the present invention (hereinafter referred to as the support according to the present invention) can be of any type as long as it is liquid-impermeable and light-transmissive, but for example, Various polymeric materials such as cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene and glasses are suitable for this purpose. The thickness of the support in this case is arbitrary, but typically is about 50 to 250 microns.
Further, one side surface of the support according to the present invention on the observation side can be arbitrarily processed depending on the purpose. When the sensing layer according to the present invention is provided on a support, it may be directly coated, but in some cases, a light-transmitting undercoat layer may be used to bond the sensing layer and the support. It is preferable to increase the quality. In addition to the compound represented by the general formula according to the present invention, the detection layer and the development layer may contain additional substances used in analytical reactions, such as buffers, preservatives, surfactants, etc. I can do it. Compounds useful as the above-mentioned surfactants are effective regardless of whether they are ionic (anionic or cationic) or nonionic, but nonionic surfactants are preferably effective. Examples of these nonionic surfactants include 2,5-di-t-butylphenoxypolyethylene glycol, p-octylphenoxypolyglycidyl ether, and p-isononylphenoxypolyethylene glycol. Examples include polyalkylene glycol derivatives of alkyl-substituted phenols and polyalkylene glycol esters of higher fatty acids. These surfactants have the effect of regulating the rate of penetration of the fluid sample into the sensing layer and at the same time suppressing the occurrence of undesirable "chromatographic phenomena."
Furthermore, the surfactant also has the effect of reducing various undesirable effects caused by flea white matter contained in a biological fluid sample. This surfactant can be used in a wide range of dosages, but
It can be used in an amount of from 0.005% by weight, preferably from 6% to 0.05% by weight. An aqueous liquid can be used as the liquid carrier for dispersing the compound according to the present invention in the sensing layer. Representative liquid carriers other than water include water-miscible organic solvents, aqueous mixtures of water and water-miscible organic solvents, suitable water-immiscible organic solvents, and the like. Water-miscible organic solvents include
Examples include lower alcohols (alcohols in which the alkyl group has 1 to 4 carbon atoms), acetone, and tetrahydrofuran. Additionally, water-immiscible solvents include
Examples include lower alkyl esters (ethyl acetate, etc.), halogenated hydrocarbons (chloroform, methyl chloride, carbon tetrachloride, etc.). Methods for coating the detection layer and development layer according to the present invention, or other constituent layers provided as necessary, on the support include, for example, dip coating, air knife coating, curtain coating, or the US patent The coating can be applied by a variety of coating methods, such as the extrusion coating method using a hopper as described in US Pat. They can also be applied simultaneously using this method. The analytical elements of the invention can take any one of a variety of different configurations. In addition, the detection layer according to the present invention, the functional layer of each layer, the reagent-containing layer, the reflective layer undercoat layer described in U.S. Pat. No. 3,992,158, the radiation blocking layer described in U.S. Pat. barrier layer, the registration layer described in No. 4144306,
The migration prevention layer described in No. 4160093, the scintillation layer described in No. 4127499,
The cleaning layer described in JP-A No. 55-90859 and the destructible pot-like member described in U.S. Pat. No. 4,110,079 may be arbitrarily combined to construct an analytical element suitable for the purpose of the present invention. can. The production of each of the constituent layers as described above and the incorporation of each of these layers into the analytical element of the present invention can be carried out by the same or similar methods as described in the above-mentioned publications. The analytical element of the present invention can be used for the analysis of whole blood, serum, and plasma without any disadvantage.
Other body fluids such as urine, lymph, and spinal fluid can also be used without any disadvantage. Particularly when whole blood is used, a radiation blocking layer or other reflective layer may be provided if desired to avoid interference of the detection radiation by blood cells. The amount of fluid sample applied to the analytical element of the present invention can be determined arbitrarily, but is preferably about
50μ to 5μ, more preferably about 20μ to
It is 5μ. In the present invention, the activity of lipase in a fluid sample can be detected by reflection spectrophotometry using an initial velocity method. That is, a certain amount of a fluid sample is dropped onto the spreading layer of the analytical element of the present invention, and the compound represented by the general formula contained in the detection layer is hydrolyzed by lipase in the fluid sample,
The activity of lipase can be determined by observing the absorption at 475 nm of the azo dye compound formed from the support side of the analytical element and examining the rate of increase in concentration over a certain period of time. EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the embodiments of the present invention are not limited thereto. Example 1 A sensing layer and a fiber structure developing layer having the following compositions were sequentially coated on a transparent subbed polyethylene terephthalate support having a thickness of 180 μm. (1) Sensing layer Copoly (styrene-glycidyl methacrylate) particles with a particle size of approximately 5μ encapsulating deionized gelatin in approximately 4μ particles 34.5g/m ² 1.3g/m ² Disodium phosphate 1.08g/m ² Sodium chloride 0.54g/m ² Octyl phenoxy polyethoxy ether
1.08g/m ² (Dry film thickness approx. 55μ) (2) Fiber structure spread layer powder filter paper (C) 91.0g/m ² (More than 300 mesh manufactured by Toyo Roshi Co., Ltd.) 91.0g/m ² Copoly(styrene-glycidyl) (methacrylate) (weight ratio 90:10) 13.9g/m ² octyl phenoxy polyethoxy ether
3.15g/m ² (dry film thickness approximately 160μ) Samples containing 0, 25, 50, 100, 250, and 500 units of lipase were added to each 10μ fiber structure spread layer on the analytical element thus obtained. dripped. The analytical element was incubated at 47° C., and the reflection density at 475 nm was continuously measured for 5 minutes starting 1 minute after dropping, and the slope of the increase in concentration was examined. The measurement results are shown in Table-1.

[Table] As is clear from the results, lipase can be measured by using the analytical element of the present invention. Comparative Example Beads-poly(styrene-covinylbenzyl chloride-co-methacrylic acid (75:
20:2) diameter of approximately 100 μm and adhesive poly(n-
A structure similar to that of Example 5 was prepared using butyl acrylate-costyrene-co-2-acrylamido-2-methylpropanesulfonic acid (76:19:5), and a sensing layer was used to prepare an analytical element. The substrate and buffer for lipase are Example-1
was added to the sensing layer as used in . 10 μl drops of each fluid sample containing approximately 0, 50, 100, and 500 lipase units were added to the analytical element thus obtained. The subsequent operations were performed in the same manner as in Example-1. The results are shown below (Table 2).

[Table] In organic polymer beads without a hydrophilic shell as in the present invention, it is difficult for lipase to enter the spaces between the particles, resulting in extremely poor color development efficiency and poor sensitivity.

Claims (1)

[Scope of Claims] 1. A light-transmissive, liquid-impermeable support, a sensing layer located on one side of the support, and a sensing layer located on the opposite side of the support for receiving a fluid sample. An analytical element having at least one spreading layer feeding the sensing layer, the sensing layer comprising a core that is hydrophobic and substantially non-swellable to the fluid sample and a hydrophilic outer shell. An analytical element for lipase analysis, characterized in that it is composed of polymer particle units having a shell multilayer structure, and the detection layer contains a compound represented by the following general formula. general formula (In the formula, M represents an alkali metal, and n is 0 to
Indicates an integer of 30. )