JPH058384B2 - - 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). In other words, blood cells are unable to penetrate the filtration layer and remain on the surface of the filtration layer, resulting in so-called surface filtration.
It is separated from liquid components such as serum and plasma. 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 No. 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 function 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 limited to 50% or less of the absorption amount of the layer. Practical blood cell/serum (plasma) separation was achieved for the first time by providing a hydrophobic barrier layer. 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 filtration layer that cannot be avoided in solid-liquid separation based on surface filtration, or unsatisfactory solid-liquid separation performance. This is an attempt to solve problems with conventional methods. 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 non-fibrous microporous liquid sample development layer (hereinafter referred to as "deployment layer") is formed by closely integrating a layer for storing solids made of this fibrous material (hereinafter referred to as "volume filtration layer"). It has been found that when used in combination, complete solid-liquid separation is achieved almost instantaneously. The present invention is an analysis method for separating solids from a liquid sample containing solids, which is composed of a volume filtration layer made of a fibrous material and a spreading layer (non-fibrous porous spreading layer) made of a non-fibrous microporous medium. Regarding the element, the liquid retention capacity of the expansion layer is greater than that of the volumetric filtration layer, and the fibrous material of the volumetric filtration layer anchors to the pore inner wall of the expansion layer at the interface between the two layers, making them tightly integrated ( It is characterized by being constructed by integral molding. To integrate the above-mentioned spread layer and volume filtration layer by anchoring, for example, a dispersion of the fibrous material of the volume filtration layer is placed on the spread layer prepared in advance, and this dispersion is filtered through the spread layer. This can be achieved by using a method of performing a filtration operation using the filter as a material. 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 achieving a 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 about 0.1 to 5 μm, and about 100
Those having a length of ~4000 μm are advantageous for the purpose of the present invention, and the desired fibrous material can be obtained by classifying in a conventional manner, for example, using a sieve of about 10 to 200 meshes (Tyler standard). be able to. These fibrous materials are prepared to have a smaller liquid-retaining power than that of the fibrous material 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, the pore diameter of the non-fibrous material, etc. Once the material of one of the layers is determined,
By selecting the other material according to the above requirements, both layers can be configured functionally. For example, if a membrane filter, which is a non-fibrous porous material with relatively strong liquid holding power and excellent liquid spreading properties, is selected as the spreading layer, the volumetric filtration layer has a coarse density and is bulky. It can be easily obtained by paper-forming a slurry in which fibers with properties (specifically, thick or long fibers) are dispersed into the membrane filter described above. That is, it is convenient to first select the material for the spreading layer, and then to integrally mold the volumetric filtration layer, which is bulkier than the spreading layer, over the spreading layer by selecting the material and the paper making method. In that case, the points to be especially noted are:
The aim is to coexist relatively short fibers that can be anchored into the pores of the non-fibrous porous spread layer member. Regarding the method of adjusting the liquid retention capacity of the volumetric filtration layer, in terms of material selection, a denser layer can be formed by using finer fibrous porous media, and in terms of papermaking methods, it is possible to form a denser layer by using a finer fibrous porous medium. If paper is made using a slurry with a higher liquid specific gravity, a denser layer will be obtained. Furthermore, performing so-called calendering, which involves applying pressure to shape the paper after papermaking, is an effective method for producing a dense layer. For a volumetric filtration layer with a smaller liquid retention capacity, the material and method may be selected in the opposite direction, and the layer may be integrally molded with the spreading layer. In the present invention, the non-fibrous porous medium constituting the spreading layer is commonly used in the art for so-called metering or "metering" for uniformly spreading a liquid sample.
Known non-fibrous porous materials can be cited as functional materials. Typical examples of such non-fibrous porous materials include brush polymers obtained by known methods from polymers such as polycarbonate, polyamide, cellulose ester, and the like. Brush polymers are commercially available as ``Fuji Microfilter'' (registered trademark, manufactured by Fuji Photo Film), ``Millipore'' (trade name, manufactured by Millipore Corporation), and ``Metricel'' (trade name, manufactured by Gellman Instruments). Appropriate is what you have. Non-fibrous porous media are described in detail in JP-A-49-53888, US Pat. No. 3,992,158, and the like. 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. However, as a rough guide, it is convenient to set the porosity to about 85% or more (preferably 95% or more). The analytical element of the present invention is characterized in that it is constructed by closely integrating a volume filtration layer and a spreading layer made of the above-mentioned materials. 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 in which, at the interface between the volume filtration layer and the spread layer, the fibers of the fibrous material that achieves the volume filtration layer stick to the inner walls of the pores of the spread layer, forming an anchoring structure. Although this three-dimensional anchoring structure is merely physical rather than chemical, it is sufficiently strong that it cannot be separated into 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 volume filtration layer into pieces and adjust its length by sieving as necessary. When short fibers are removed by sieving, it is necessary to add a certain amount of short fibers for anchoring. The short fibers mentioned here are used for anchoring, so their length or thickness varies depending on the pore size of the non-fibrous porous medium, but they are usually fibers that pass through a sieve of about 150 meshes (Tyler standard). , using such short fibers. (b) The fibrous material obtained in (a) is dispersed in a liquid dispersion medium (for example, water or a mixture of water and a water-compatible organic solvent) to prepare a slurry (paper stock liquid).
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.). 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 of preparing the slurry; for example, there are various methods such as using ordinary mixing equipment such as a magnetic stirrer, stirring spring, homogenizer, or ball mill, or methods commonly used in slurry production such as a heater. Any method can be used. (2) On the other hand, the non-fibrous porous material for the spreading layer is selected from the above-mentioned materials depending on the desired liquid retention capacity. (3) A spreading layer with a volumetric filtration layer can be easily prepared by filtering and paper-making the slurry prepared in (1) on the non-fibrous porous material selected in (2).
That is, a non-fibrous porous material is placed on a suitable filter (e.g. long rope, round rope, paper making rope, filter filter) in place of the filter material, and the slurry is suction filtered to remove the dispersion medium. . During this papermaking process, some of the fibers in the slurry become stuck in pores at the interface of the spreading layer, forming a so-called anchoring structure and forming a strong interfacial bonding state. (4) Next, the thickness of this paper body is defined using a member having a certain clearance. For example, various methods can be used, such as sandwiching the paper body between two sheets and compressing it to a certain thickness, or passing the paper 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 the thickness of the paper body is determined in this manner, 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 volume 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 volume filtration layer, solid-liquid separation is performed without clogging. At this time, the liquid retention power of the non-fibrous porous spread layer, which is closely integrated with the volume filtration layer at the interface, is linked to the volume filtration phenomenon so that the liquid sample moves smoothly between the layers. It is believed that solid-liquid separation 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 expansion 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 microfilter spreading layer with volumetric 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 square pieces. Place the cut filter paper in an agate mortar and beat it while moistened with water. 800 ml of water was added and dispersed using an Ace homogenizer model AM-11 (manufactured by Nippon Seiki Seisakusho Co., Ltd.). The dispersion was strained using Tyler standard sieve mesh No. 12 to remove clumps of fibers to obtain a glass fiber dispersion. The glass fiber solid content in the dispersion is 23mg/ml
It was a dispersion liquid. The solid content is a microfilter with a diameter of 47 mm and a pore size of 0.22 μm (manufactured by Fuji Photo Film Co., Ltd.).
After filtering 10 ml of the dispersion using a Millipore filter equipped with a filter, the filter was dried and weighed, and the weight of the filter was calculated by subtracting the weight of the filter that had been weighed in advance. Preparation of spreading layer with volume filtration layer: Microfilter FM-80 (Fuji Photo Film Co., Ltd.) with a pore size of 0.8 μm was set in a Millipore filter (for 47 mm diameter), and 10 ml of the glass fiber dispersion prepared with The mixture with 100 ml of water was transferred to a filter and filtered paper was made. Next, the microfilter was sandwiched between two Teflon-coated glass plates having a clearance of 350 μm, and excess water was squeezed out to make the thickness constant. Next, it was frozen on dry ice, the glass plate was removed, and then freeze-dried. In this way, a spreading layer with a volume filtration layer [Element A] was obtained. Evaluation of blood (whole blood) development performance: In order to investigate the effect of the volumetric filtration layer prepared in step 1, fresh blood containing an anticoagulant was dripped onto the device A and the microfilter (FM-80) alone. After exactly 5 minutes had elapsed, the diameter of the circular portion developed on the microfilter portion was measured. for each
Measurements were carried out on 10 specimens, and the average value of the diameter and its standard deviation σ were calculated and shown in Table 1 (unit: mm).

[Table] Note that with the microfilter alone, only a portion of the blood was aspirated even after 5 minutes, and most of it remained on the surface. Moreover, blood cells were spread all over the area. 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. In addition, in a device obtained by preparing a volumetric plasma layer separately and simply pressing it onto a microfilter, the transition from the filtration layer to the microfilter was irregular, and the variation was so great that it was unusable. 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 non-fibrous porous expansion layer having a larger liquid retention capacity than the volume filtration layer, the fibrous material of the volume filtration layer expands at the interface between them. An analytical element for a solid-containing liquid sample, characterized in that it is integrally molded by anchoring to the inner wall of a pore of a layer. 2. The analytical element for a solid-containing liquid sample according to claim 1, wherein the non-fibrous porous spreading layer is made of a brushed polymer. 3. The solid content-containing liquid according to claim 1, wherein the volumetric filtration layer has a density in the range of 0.02 to 0.1 g/cm ³ , and the spreading layer has a density in the range of 0.1 to 2.0 g/cm ³ Analytical element for samples. 4. In the integral molding, a dispersion of the fibrous material of the volumetric filtration layer is placed on the non-fibrous porous spread layer prepared in advance, and a filtration operation is performed with this dispersion using the spread layer as a filtering material. An analytical element for a solid content-containing liquid sample according to claim 1, which is manufactured by. 5 The thickness of the fibrous material of the volumetric filtration layer is 0.1~
The analytical element for solid content-containing liquid samples according to claim 1, wherein the length is 1.0 μm and the length is in the range of 10 to 4000 μm. 6. 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. 7. The analytical element for a solid content-containing liquid sample according to claim 1, wherein the fibrous material of the volumetric filtration layer is glass fiber.