JPH058382B2 - - Google Patents

Description

[Detailed description of the invention]

Object of the Invention 1 Industrial Application Field The present invention relates to an analytical element effective for dry analysis of liquid samples containing solid content. 2. Prior Art Many analysis systems are already known for detecting and quantifying biochemically active components in sample liquids using so-called dry analysis elements configured in a layered (sheet-like) manner (U.S. Pat. No. 3,050,373, etc.). ). Generally used in these analysis methods, reactive components that cause a physical or chemical reaction upon contact with the target component (analyte) contained in the liquid sample are prepared in advance as analytical elements. The reaction between the analyte introduced into the analytical element and the reactive component proceeds in the biological reaction layer provided within the analytical element, and the reaction product or unreacted component is This is a method of quantifying analytes by measuring the amount of analytes spectroscopically, fluorescently, or using radioactive isotopes. Since the dry analysis method described above is relatively simple to operate, it is used for many purposes, such as immunological analysis using antigen-antibody reactions, and enzyme or substrate analysis using enzymatic reactions. There is. While methods using analytical elements generally have the advantage of being simple, various improvements have been made to the layer configuration of analytical elements in order to provide a wide latitude that can be adapted to the analysis of various liquid samples. Ta. In particular, when a liquid containing solids, such as whole blood, is used as an analysis sample, it has been proposed to provide a hemocyte filtration layer above the reagent layer to mainly filter red blood cells. A typical blood cell filtration layer is described in Japanese Patent Publication No. 53-21677, which filters blood cells through a material with appropriate porosity. It is therefore taught that the pore size of the filter layer should be set to 1-5 μm, which is smaller than the size of blood cells (7-30 μm). That is, blood cells are separated from liquid components such as serum and plasma by undergoing so-called surface filtration in which they cannot penetrate into the filtration layer and remain on the surface thereof. Blood cell separation using such surface filtration is simpler than the conventional method in which the whole blood sample is centrifuged in advance because the blood cell filtration layer is incorporated into the analytical element, but the filtration speed is not sufficiently fast. I can't say anything and it's easy to get confused. As a result, the liquid sample develops poorly, leading to a decrease in analytical sensitivity and impairing analytical accuracy. JP-A-57-53661 discloses a device for removing solids from blood and separating plasma and serum using a layer composed of specific glass fibers having an average diameter of 0.2 to 5 μm and a density of 0.1 to 0.5 g/cm ³ . Are listed. However, the blood cell separation ability of this separation device is also not satisfactory, and according to the example, in an analysis using a multilayer analytical element, the applied serum or plasma amount is measured to be less than 50% of the absorption amount of the layer. By providing a hydrophobic barrier layer, practical blood cells/
Serum (plasma) separation has been achieved. Moreover, the above separation device was proposed based on the recognition that serum or plasma passes through the glass fiber layer more quickly than blood cells, and there is no suggestion whatsoever regarding solid-liquid separation based on volumetric filtration, which is the characteristic concept of the present invention described later. There's nowhere to go. Problems to be Solved by the Invention The present invention solves problems such as the complexity of centrifuging blood in advance, clogging of the filter layer that is inevitable in solid-liquid separation based on surface filtration, and unsatisfactory solid-liquid separation. It attempts to solve problems in the law. Means for Solving the Constituent Problems of the Invention In the present invention, as described above, the phenomenon of efficiently utilizing the three-dimensional structure of the layer itself to store solids over its entire volume, without using surface filtration. In this specification, this phenomenon is referred to as "volume filtration")
The objective is to separate solids from liquid samples based on the following. As a result of repeated studies on various materials, the present inventors found that a fibrous material is effective in separating solids from a solids-containing liquid sample by volumetric filtration. In addition, a porous liquid sample spreading layer (hereinafter referred to as the ``deployment layer'') is formed by closely integrating a layer for storing solids made of this fibrous material (hereinafter referred to as the ``volume filtration layer'').
It has been found that when used in combination, complete solid-liquid separation can be achieved almost instantly. The present invention relates to an analytical element for separating solids from a liquid sample containing solids, which is composed of a volumetric filtration layer and a spreading layer made of a fibrous material, and in which the liquid holding capacity of the spreading layer is larger than that of the volumetric filtration layer. Both layers are characterized in that their respective fibrous materials are intertwined with each other at their interfaces and are tightly integrated (integral molding). The close integration of the above-mentioned expansion layer and volumetric filtration layer is
For example, a method (filtration method) in which a dispersion of the fibrous material of the volumetric filtration layer is placed on a pre-prepared spread layer and the spread layer is used as a filtering material (filtration method); This can be achieved by using a method such as a method (paper making method) in which a dispersion of the fibrous material and a dispersion of the fibrous material of the volumetric filtration layer are layered and a filtration operation is performed using a separately prepared filtration material. can. Fibrous materials that can be used for the volume filtration layer constituting the analytical element of the present invention include inorganic fibers such as glass fiber and asbestos, natural organic fibers such as cotton, hemp, pulp, and silk, viscose rayon, and copper. Semi-synthetic fibers and synthetic fibers such as ammonia rayon, cellulose acetate, partially formalized polyvinyl alcohol, polyethylene, polypropylene, polyvinyl chloride, polystyrene, and polyesters (polyethylene terephthalate, etc.) are typical. Among these, glass fiber is particularly preferred. These fibrous materials must, of course, be substantially non-reactive with the liquid sample or analyte. These fibrous materials that make up the volumetric filtration layer are approximately
It is desirable to have a density of 0.02 to 0.1 g/cm ³ .
In addition, these fibrous materials have a thickness of approximately 0.1 to 5 μm, approximately
Those having a length of 100 to 4000 μm are advantageous for the purpose of the present invention, and the desired fibrous material can be obtained by classifying it by a conventional method, for example, using a sieve of about 10 to 200 meshes (Tyler standard). Obtainable. These fibrous materials are adjusted to have a liquid retention capacity smaller than that of the fibrous materials constituting the spreading layer. The liquid holding power depends on the density of the voids in the layer (porosity),
It is determined by the clearance of the layer space, the thickness of the fibrous material, etc. Once the material for either layer is decided, the material for the other layer can be selected according to the above requirements to make both layers functional. It can be configured as follows. For example, if a filter paper made of relatively fine and short glass fibers (e.g. GC-50 manufactured by Toyo Roshi Co., Ltd.) is selected as the spreading layer, the volumetric filtration layer may be of the same quality, thicker or more densely formed. It can be easily obtained by forming a slurry in which long fibers are dispersed onto the above-mentioned filter paper.
Specifically, first, a material for the spreading layer is selected and a relatively dense spreading layer is formed. Next, depending on the material selection and paper making method, it is convenient to integrally mold a so-called bulky volumetric filtration layer, which has a lower density than the spread layer, over the spread layer. In terms of material selection, finer and shorter fibers can be used to form a denser spread layer, and in terms of papermaking methods, paper can be made by using a larger pressure difference and a slurry with a higher liquid specific gravity. If you do this, you will get a denser expanded layer. Furthermore, performing so-called calendering, which involves applying pressure to shape the paper after papermaking, is an effective method for producing a dense spread layer.
The volume filtration layer may be formed integrally with the expansion layer by selecting the material and method in the opposite direction. In the present invention, examples of the fibrous material constituting the spreading layer include fibrous materials commonly known in the art as having a so-called "measuring" or "metering" function for uniformly spreading a liquid sample. Typical such fibrous materials generally have a density of about 0.1 to 2.0 g/ ^cm3 , such as wood (cellulose) pulp, cotton, silk, and wool. Natural fibers; semi-synthetic fibers such as cellulose esters, viscose rayon; polyamides, polyesters, e.g.
Synthetic fibers such as polyethylene tereftate (polyethylene tereftate) and polyolefin; inorganic fibers such as glass fiber and asbestos; and fibers made by loosening filter paper.
These fibrous materials for the spreading layer may be subjected to a hydrophilic treatment using a conventional method. Details of the fibrous material related to the spread layer, its hydrophilic treatment, layer formation, etc. can be found in JP-A-55-
164356, 57-66359, etc. As mentioned above, the liquid retaining power varies depending on various factors, and porosity is one of them, so it is difficult to set a fixed range for the volumetric filtration layer and the spreading layer. However, as a rough guide, in the case of a volumetric filtration layer, it is approximately 85% or more (preferably 95%)
In the case of a spread layer, it is convenient to have a porosity in the range of about 50 to 90%. The analytical element of the present invention is characterized in that a volume filtration layer and a spreading layer made of the above-mentioned materials are closely integrated. In this specification, the term "adhesive integration" refers to the integrated type in the prior art (usually, by simply overlapping multiple layers and pressure molding, or by adhesive molding using a binder or similar substance). This refers to a state where, at the interface between the volume filtration layer and the spreading layer, the fibers of the fibrous material constituting both layers exhibit a random structure in which they are three-dimensionally intertwined with each other.
This three-dimensional random structure is not chemical but merely physical, but it is sufficiently strong.
It is not possible to separate the original two layers without destroying this interfacial state. The reaction element for solid-liquid separation of the present invention can be manufactured as follows. (1) (a) Loosen the fibrous material for the volumetric filtration layer into pieces and adjust its length by sieving, etc., if necessary. (b) A slurry (paper stock liquid) is prepared by dispersing the fibrous material obtained in (a) in a liquid dispersion medium (for example, water or a mixture of water and a water-compatible organic solvent).
At this time, a slurry having a desired specific gravity and viscosity can be obtained by appropriately selecting a dispersion medium or by adding a water-soluble solute (for example, sucrose, etc.). Selection of these dispersion media and additive substances is performed according to the known density gradient method. When preparing the slurry, various chemicals used in general papermaking operations such as dispersants, viscosity modifiers, and preservatives may be optionally added. There are no particular limitations on the method for preparing the slurry; for example, there are various methods such as methods using ordinary mixing equipment such as magnetic stirrers, stirring springs, homogenizers, and ball mills, and methods commonly used in slurry production such as beaters. Any method can be used. (2) On the other hand, in the same manner as in (1) (a), loosen the fibrous material for the spreading layer into pieces and adjust its length if necessary. In the state where the obtained fibrous material is loosened (1) (b)
Prepare a slurry in the same manner as above. (3) Confirm that the slurries obtained in (1) and (2) form an interface in which they do not mix with each other. In order to make the two slurries unmixable, it is most common to provide an appropriate difference in specific gravity between the two slurries, and for this purpose, a known density gradient method or the like can be applied. In addition, the state in which the two do not mix is due not only to the difference in specific gravity, but also to water-soluble polymers (so-called thickeners: for example, synthetic polymers,
This can also be achieved by adding a suitable viscosity difference between the two slurries by adding gum, polysaccharide, etc.). The above conditions are for when both are in a wet state, but if one is in a dry state, they will not mix by themselves, so it is not necessary to set the above conditions. (4) The two types of slurry prepared as above so that they do not mix with each other are placed in a container and paper is made. Specifically, a high specific gravity (and/or high viscosity) slurry forms the lower layer and a low specific gravity (and/or low viscosity) slurry forms the upper layer. suction through a membrane filter, etc.) to remove the dispersion medium. During this papermaking process, the fibers in the slurry become entangled with each other at the interface between the two layers, forming a three-dimensional random structure. (5) Next, the thickness of this layered body is defined using a member having a certain clearance. For example, various methods can be used, such as sandwiching the layered body between two sheets and compressing it to a certain thickness, or passing the layered body between rollers having a certain slit. The details are described in Japanese Patent Application No. 57-211382, an earlier application by the present inventors. After setting the thickness of the layered body in this manner, preferably within the range of 100 to 2000 μm, drying is performed without substantially changing the thickness. For such purposes, drying is preferably carried out at relatively low temperatures, with freeze-drying being a particularly preferred means. Drying may be performed before determining the thickness. Function The analytical element of the present invention is part of a multilayer analytical element having a structure in which a plurality of layers (e.g., a reaction layer, a reagent layer, a light shielding layer, etc.) are laminated, and the outermost layer on the side on which a liquid sample is deposited. It is used by providing it so that it becomes a volumetric filtration layer. When a liquid sample containing solids is deposited on the analytical element of the present invention, the solids are first stored in the volume filtration layer. Since storage is carried out as the liquid sample moves inside the volumetric filtration layer, solid-liquid separation is performed without clogging. At this time, the liquid retention power of the expansion layer, which is closely integrated with the volumetric filtration layer at the interface to facilitate the movement of the liquid sample between the layers, works in conjunction with the volumetric filtration phenomenon to achieve solid-liquid separation. It is believed that the process can be completed more effectively and in a shorter time. It goes without saying that the interfacial state greatly contributes to the uniform and rapid development of the liquid portion. A multilayer analytical element incorporating the element of the present invention is applied in actual analysis, for example, as follows.
A fixed amount of a solid-containing liquid sample containing an analyte is dropped onto the volumetric filtration layer of the element of the present invention located at the top layer of the multilayer analytical element. Solid-liquid separation is performed within the device, and only the liquid component moves to the reaction layer, reagent layer, etc., and a corresponding biological reaction occurs. As a result, a signal corresponding to the amount of analyte is generated. This generated signal is measured by a conventional method. The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto. Example 1 Preparation of a glass fiber filter paper spreading layer with a volume filtration layer Preparation of glass fiber dispersion: 2 g of glass fiber filter paper GA-100 (manufactured by Toyo Roshi Co., Ltd.)
was cut into 2 mm squares, 800 ml of water was added, and the mixture was dispersed using an Ace homogenizer model AM-11 (Nippon Seiki Seisakusho Co., Ltd.). Tyler standard sieve mesh No.12
The dispersion was strained using a sieve to remove clumps of fibers to obtain a glass fiber dispersion. The amount of glass fiber solids in the dispersion was determined by filtering 10 ml of the dispersion using a Millipore filter equipped with a membrane filter (microfilter, manufactured by Fuji Photo Film Co., Ltd.) with a 47 mm diameter and 0.22 μm pore size, and then drying the dispersion together with the filter. The weight was calculated by subtracting the weight of the filter weighed in advance, and the dispersion was found to be 22 mg/10 ml. Preparation of spreading layer with volumetric filtration layer: 55 mm diameter glass fiber filter paper GC-50 (Toyo Roshi Co., Ltd.)
was attached to a Millipore filter (for 47 mm diameter), and 15 ml of the slurry (solid content 33
A mixture of 100 ml of water and 100 ml of water was transferred to a filter and filtered paper was made. 500 μm from the thickness of GC-50 after filtration
The mixture was sandwiched between two Teflon-coated glass plates with a thick spacer in between, and after squeezing out excess water, it was frozen on dry ice, and the glass plates were removed before freeze-drying. In this way, a spreading layer with a volume filtration layer [element (A)] was obtained. Comparison of development performance of blood (whole blood): In order to investigate the effect of the filtration layer prepared in Arrived,
After exactly 5 minutes, the diameter of the circular portion developed on glass fiber filter paper (GC-50) was measured. Measurements were performed on 10 samples for each sample, and the average value ( ) and standard deviation (σ) of the diameter were calculated and shown in Table 1 (unit: mm).

[Table] In addition, when using glass fiber filter paper alone, blood clearly remained on the surface even after 5 minutes, and blood cells were spread over the entire surface. On the other hand, in the device (A) of the present invention, it was observed that the blood cells were accommodated in the volumetric filtration layer after about 1 minute, and only the plasma was rapidly expanded. Example 2 Preparation of a filter paper spreading layer with a volume filtration layer Preparation of a spreading layer with a volume filtration layer: A glass fiber dispersion (solid content 15 mg/10 ml) was obtained in the same manner as in Example 1-. Filter paper No. 5C (manufactured by Toyo Roshi Co., Ltd.) was poured with water onto a Millipore filter for 47 mm diameter, and a mixture of 15 ml of the above glass fiber dispersion (solid content 22.5 mg) and 50 ml of water was supplied. , filter paper was made. After filtration, the paper material is sandwiched between two glass flat plates with a 650 μm spacer in between to make the thickness constant, then frozen on dry ice, the glass plates are removed, and freeze-dried to form a volumetric filtration layer. A spreading layer (device B) was prepared. Evaluation of blood development performance: In order to examine the effect of the glass fiber volumetric filtration layer, the volumetric filtration layer and the volumetric filtration layer prepared as described above [element (B)] and the filter paper (No. 20,
30 μl of blood (containing anticoagulant) was dropped and its spreadability within the filter paper was examined. Ten samples were measured for each sample, and the direct average value ( ) and standard deviation (σ) of the developed area were calculated. The results are shown in Table 2.

[Table] As in Example 1-, blood remained on the surface of the filter paper alone, and blood cells were observed to be spread over the entire surface of the spread area. On the other hand, in the device (B) of the present invention, blood cells did not develop and plasma developed quickly and well. Effects of the Invention By incorporating the element of the present invention into a multilayer analytical element, it is possible to accurately and quickly analyze solid-containing liquid samples, which conventionally required special measures to be taken due to analytical errors. Summer. By using the element of the present invention, the solid content is stored in the volume filtration layer, so that clogging does not occur, and the spreading layer, which has a large liquid holding capacity, completes spreading of the liquid component in a very short time. The device of the present invention is particularly effective when using liquid samples such as whole blood, turbid urine samples, body fluids, and chyle serum. For example, when the device of the present invention is used to analyze whole blood with different solid content (whole blood with different hematocrit values), only the plasma portion is quantitatively sent to the developing layer and developed, regardless of the hematocrit value. Due to the metering effect of the layer, the area will be expanded according to the amount of plasma. In other words, the device of the present invention enables analysis using whole blood that is not influenced by hematocrit values and also allows analysis that is resistant to measurement errors.

Claims (1)

[Scope of Claims] 1. A volume filtration layer made of a fibrous material and a spread layer made of a fibrous material having a larger liquid retention capacity than the volume filtration layer, the fibrous materials of which are entangled with each other at their boundary surfaces. An analytical element for a liquid sample containing solids, characterized in that it is integrally molded with the elements. 2. The density of the volume filtration layer is in the range of 0.02 to 0.1 g/cm ³ , and the density of the expansion layer is 0.1 to 2.0 g/cm ³
An analytical element for solid content-containing liquid samples according to claim 1, which falls within the scope of claim 1. 3. The integral molding is manufactured by placing a dispersion of the fibrous material of the volumetric filtration layer on a previously prepared spread layer, and performing a filtration operation on this dispersion using the spread layer as a filtering material. Or manufactured by carrying out a filtration operation using a separately prepared filtration medium with the fibrous material dispersion of the spread layer and the fibrous material dispersion of the volumetric filtration layer layered. An analytical element for solid content-containing liquid samples according to claim 1. 4 The thickness of the fibrous material of the volume filtration layer and the spreading layer is 0.1 to 1.0 μm, and the length is 10 to 1.0 μm.
An analytical element for a solid content-containing liquid sample according to claim 1, which has a particle size in the range of 4000 μm. 5. The analytical element for a solid content-containing liquid sample according to claim 1, wherein the combined thickness of the volumetric filtration layer and the spreading layer is in the range of 100 to 2000 μm. 6. The analytical element for solid content-containing liquid samples according to claim 1, wherein the fibrous materials of the volume filtration layer and the spreading layer are both glass fibers.