GB2095404A - Method for supplying liquid samples to an analysis element - Google Patents

Abstract

In a method of supplying a liquid sample to a dry type analysis element having a porous spreading layer as the outermost layer, a very small amount of liquid sample can be very uniformly supplied and spread on the porous spreading layer, even though the liquid sample is highly viscous, by impregnating a liquid-carrier such as an applicator having a cotton ball at the top thereof with the sample liquid and bringing the top of the liquid-carrier into contact with the porous spreading layer.

Description

SPECIFICATION Method for supplying liquid samples to an analysis element by a dry process This invention relates to a method for supplying a liquid sample to an analysis element by a dry process.

In recent years, analysis elements using a dry process have been widely employed for quantitative analysis of liquid samples, e.g., body fluids (blood, serum, urine, saliva, etc.), industrial waste water, reaction liquids, fermentation liquids, etc.

That is, as such analysis elements by dry processes, not only paper filter type materials soaked with color-forming reagents such as pH test papers, etc. which have been known previously, but also filter paper type analysis sheets or multilayer analysis sheets for analyzing liquid samples such as blood, urine, etc. are known.

Multilayer analysis elements are known in, for example, the fields of chemical analysis, immunoassay, etc., and the details are described in, for example, Japanese Patent Application (OPI) 53888/74 (the term "OPI" as used herein refers to an unexamined published Japanese patent application), U.S. Patents 3,992,158,4,110,079, and 4,132,528, Japanese Patent Applications (OPI) 137192/75 (corresponding to U.S.

Patent 3,983,005)40191/76 (corresponding to U.S. Patent 4,042,335)3488/77 (Reissue 30,267), 131786/77 corresponding to U.S. Patent 4,050,898), 131089/78 (corresponding to U.S. Patent 4,144,306) ,29700/79 (corresponding to U.S. Patent 4,166,093), 34298/79,90859/80 (corresponding to U.S. Patent 4,258,001), 124499/80 (corresponding to U.S. Patent 4,269,938) and 164356/80 (corresponding to U.S. Patent 4,292,272); Japanese Patent Applications 124514/79,120599/80, 140532/80 and 144849/80; Japanese Utility Model Application 120229/80 and Ciin,caIChem,str vol.24, pages 1335 to 1350(1978).

Such a multilayer-type analysis sheet is generally of type that a reagent layer containing reagents necessary for the analysis, a reaction layer for performing the reaction necessary for the analysis, a spreading layer for spreading well a liquid sample on the analysis sheet, etc., are laminated on a support, and when conducting a chemical analysis using such a multilayer analysis sheet, the analysis is performed by the two fundamental operations, i.e., the operation of placing a small amount of a liquid sample onto the sheet and the measurement operation of measuring an optical density such as the change in color or change in optical density after performing a necessary treatment.Among these multilayer analysis sheets, a multilayer analysis element of integral type by dry process having a spreading layer as the uppermost layer is particularly excellent compared to other known analysis elements by dry process in the point that a measurement result having a highly quantitative analyzing property is obtained even if the amount of a liquid sample supplied to the analysis element is not strictly constant caused by the uniform spreading action based on the spreading layer.

The uniform spreading action in a multilayer analysis element of integral type means the action that an amount of a supplied liquid sample is proportional to a spread area of the liquid sample at the surface of the analysis element. In other words, the amount of a liquid test sample reaching per unit area of the reaction layer containing the reaction system for detecting a species to be detected in the test sample is always most constant, thereby, even if the amount of the liquid sample supplied onto the analysis element is changed in a certain range, the concentration of the species to be detected contained in the test sample is obtained as a constant value without being influenced by the change thereof.As a result of combining the action in the analysis element, the degree of quantitative analysis of the analysis element for a dry process (hereafter often referred to simply as "dry analysis element") is greatly improved.

The present invention relates to a particularly effective and advantageous method for supplying a sample to such a multilayer analysis element of integral type.

For supplying a liquid sample to an analysis element for dry process, other than the multilayer analysis element of integral type, having a filter paper as the carrier as represented by a pH test paper, there are generally a method of dipping the analysis element in a large amount of a liquid sample and a method of contacting the maximum liquid amount (saturation liquid amount) capable of being contained in the analysis element with the analysis element by an optional non-quantitative manner, for examply, by bringing an analysis element piece into contact with a small amount of a liquid sample drop collected on a glass rod or a plate (e.g., a plastic piece or a spoon for experimental use).In a so-called clinical analysis using blood or urine as a sample to be detected, a method has been practiced for a long period of time wherein the liquid sample absorbed in cotton wool, gauze, an applicator, a piece of filter paper, etc., is supplied to the analysis element for dry process but the purpose of supplying the liquid sample in the known method is not in a quantitative supply of the sample because of the property of the analysis element and is only in the supply of the foregoing saturation amount of the liquid sample onto the analysis element, i.e., is only in supplying empirically the liquid sample in the amount in a certain range.

On the other hand, in a dry analysis element for a quantitative analysis or assay, the amount of a liquid sample usually used is about 1 to about 50 Fl and by the above described means, it is difficult to supply an almost definite amount of a liquid sample to the analysis element.

Therefore, when a quantitative supply is required to a certain extent, a measuring pipet, a capillary pipet, various kinds of micropipets, and a microsyringe are used. Specific sample dispensers capable of forming specifically a constant amount of a liqud drop are described in Japanese Patent Application (OPI) 99066/76 (corresponding to U.S. Patent 3,977,568) and U.S. Patent 4,041,995.

However, in these liquid supplying methods, there are such problems that the capillary pipet or micropipet itself used in these method is expensive and also an operation for determining a liquid sample is required.

Furthermore, in these methods, it takes a long time to spread a liquid sample on an analysis element since the liquid sample is supplied to the analysis element as a liquid drop, thereby a concentric chromatographic phenomenon (ringing phenomenon) frequently occurs as well as in the case of supplying a highly viscous liquid sample such as whole blood, problems are likely to occur in the quantitative analyzing properties and reducibility of the measurement of a species to be determined in that the liquid sample non-uniforming spreads or does not spread.

A primary object of this invention is, therefore, to provide a method of supplying a liquid sample which can eliminate the foregoing problems.

That is, an object of this invention is to provide a method of supplying a liquid sample which enables the measurement of a species to be detected quantitatively and with a good reproducibility.

Another object of this invention is to provide a method of supplying a liquid sample by a simple operation (for example, without any need of metering operation) using inexpensive equipments.

A still other object of this invention is to provide a method for sampling a highly viscous liquid such as whole blood, highly viscous hemolytic blood, an aqueous solution having less fluidity, a highly viscous emulsion, etc. which are liable to give a serious error if known assaying methods as described above are relied upon, in a simpler and less inexpensive way.

It has now been discovered that the foregoing objects of this invention can effectively be attained by the following method. That is, the above mentioned objects of this invention can be attained by the method of supplying a small amount of a liquid sample onto a dry analysis element which comprises including, in an analyzing operation using a dry analysis element having a porous layer as the outermost layer, a step of retaining a small amount of a liquid sample containing a material to be detected in a liquid carrier and then supplying the liquid sample in the state that at least a part of the liquid carrier is brought into contact with the porous layer of the dry analysis element.

The term "liquid carrier" as used herein refers to a carrier having a function capable of easily absorbing a liquid and retaining it for a certain period of time sufficient for transferring the absorbed liquid onto the porous layer of the dry analysis element; in other words, a function capable of carrying a liquid from one to another The term "carrying" is thus used in such a sense, in the specification.

In the following description reference will be made to the drawings in which Figure lisa schematic sectional view showing an embodiment of the liquid carrier used in this invention; Figure 2 is a schematic sectional view showing an embodiment of the dry multilayer analysis sheet used in this invention; Figure 3 is a plane view showing the porous layer of an analysis sheet to which a liquid sample was supplied; Figure 4 is a schematic sectional view showing another embodiment of the liquid carrier used in this invention; Figure 5 is a plane view showing other embodiment of the porous layer of an analysis sheet to which a liquid sample was supplied; Figure 6 is a schematic sectional view showing still another embodiment of the liquid carrier used in this invention;; Figure 7 is a plane view showing still another embodiment of the porous layer of an analysis sheet to which a liquid sample was supplied; Figure 8 is a schematic sectional view showing a further embodiment of the liquid carrier used in this invention; and; Figure 9 is a plane view showing a further embodiment of the porous layer of an analysis element to which a liquid sample was supplied.

This invention is based on the discovery that by supplying a liquid sample onto the surface of a dry analysis element by the method of this invention, for example, a method of supplying a small amount of a liquid sample to the analysis element by contacting a carrier containing or having absorbed or retained therein the liquid sample with the analysis element or a method of transferring the liquid sample from the carrier onto the analysis element, the liquid sample can be uniformly supplied onto the analysis element and an analytical result having very high accuracy is obtained.

Thus, according to the method of this invention, a highly viscous liquid sample such as whole blood, the uniform spreading and supplying of which is difficult by conventional methods can be effectively supplied and thus the degree of quantitative analysis and the accuracy of the analysis method using a dry analysis element can be greatly improved.

An embodiment of the invention will now be described with reference to Figures 1 - 3 of the accompanying drawings. A small amount of a liquid sample containing a material or species to be detected is retained in a liquid carrier composed of rodlike small piece 1 and a liquid carrying material 2 shown in Figure 1. Then, the liquid carrier containing the liquid sample is brought into contact with the porous layer 3 of a multilayer analysis sheet composed of a support 6 having formed thereon, in succession, a reagent layer 5, a light-blocking or radiation-blocking layer 4 and a porous layer 3, thereby the liquid sample is supplied to the porous layer 3. The multilayer analysis sheet containing the liquid sample thus supplied is shown in Figure 3.

Thereafter, after performing a necessary treatment, the quantitative assay of the material or species is performed by measuring the change in color, the change in optical density, etc.

The liquid sample which can be analyzed by the dry analysis element used in the method of this invention is generally an aqueous solution, an aqueous dispersion, or a water-containing liquid, but, as the case may be, the method of this invention may be applied for analyzing a specific omponent in natural oil such as crude oil, etc., or non-aqueous solutions or extracts formed in various kinds of industrial steps. Furthermore, a highly viscous liquid such as, for example, whole blood, a highly viscous hemolitic blood, an aqueous solution or emulsion having less fluidity can be preferably analyzed in this invention.

For the liquid carrier used in this invention, any materials which can absorb or carry therein a liquid sample containing a species to be detected can be effectively used.

As the material (liquid-carrying material) used for the carrier, there are swellable materials, porous materials, spongy materials, water absorbing materials, etc. Practical examples of these materials are cotton, sponge, cloth, gauze, paper (e.g., ordinary filter paper, filter paper for chromatography, synthetic paper, etc.), a porous material composed of a natural or synthetic resin, a glass wool, gelatin, polyacrylamide, agarose, etc.

These materials may be used as the liquid carrier as they are or the liquid carrier may be prepared by attaching such a material to a wood piece or a plastic piece. For example, a liquid carrier prepared by attaching cotton balls, sponge, a cloth such as gauge, etc., a paper such as a filter paper or a small piece of a spongy porous material to a small rod-like piece or a plastic sheet strip is preferably used.

There is no particular restriction about the shape of the liquid carrier used in this invention but in this invention, it is required to bring a part of the liquid carrier with the dry analysis element in at least a plane form. Therefore, it is preferred that the portion of the carrier to be brought into contact with the analysis element may form a curved surface having less uneveness or a plane surface; it may at least have a form, flexibility and quality capable of being easily deformed by a small force.

Practical examples of such liquid carriers are illustrated in Figure 4, Figure 6 and Figure 8.

That is, Figure 4 is a schematic sectional view of an embodiment of the liquid carrier of this invention composed of a rod piece 9 and a liquid-carrying material 10 attached thereto, Figure 6 is a schematic sectional view of another embodiment of a liquid carrier composed of a rod-like piece 14 and a liquid-carrying material 15 attached thereto, and Figure 8 is a schematic sectional view of still another embodiment of a liquid carrier composed of a rod-like piece 19 and a liquid-carrying material 20 attached thereto. The states of the dry analysis elements after contacting the analysis elements with the liquid carriers containing or retaining a liquid sample as shown in Figure 5, Figure 7 and Figure 9, respectively.Also, the contact faces or areas of the dry analysis elements with the liquid carriers are shown by numerals 12, 17 and 22, respectively, in these figures and the spread portions of the liquid sample are shown by numerals 13, 18 and 23, respectively, in these figures.

As the practical shapes of such a liquid carriers, there may be employed various shaped such as a plane form as a paper, etc., a rod form and a spherical form. Practical examples are a so-called applicator composed of a thin rod having a cotton ball attached to the point, a brush, a writing brush, a stick, etc. As an applicator, there are an applicator comosed of a metallic pin having cotton wound around the screw-shaped top portion of the pin and a disposable applicator composed of a small wood or paper core having cotton wound around the top portion of it. The disposable applicator is particularly preferred in the case that the dry analysis element is used for clinical tests. The top of such an applicator is usually of a spheroidal form or a spindle form.Such an applicator may frequently be used as it is but it is sometimes advantageous to use the applicator, the top of which is cut so as to suitably adjust its contact surface to be brought into contact with the dry analysis element.

In the case of using the liquid carrier in this invention for analyzing an aqueous liquid sample, it is preferred to previously subject the liquid carrier to an hydrophilic treatment in various ways so that the permeability of the carrier for the liquid sample is improved, so that sampling and supplying of a liquid sample composed of a water-containing solution can be more effectively performed.

Means for rendering the surface of the liquid carrier hydrophilic, include a method of washing the carrier with water; a method of impregnating the carrier with a surface active agent such as an anionic surface active agent, a cationic surface active agent and a nonionic surface active agent; a method of chemically treating the surface of the carrier with an acid, an alkali, etc.; and a method of impregnating the carrier with a hydrophilic or hygroscopic compound such as glycerol, polyethylene glycol, etc. Also, a physicochemical treatment such as a glow discharge treatment, a corona discharge treatment, a flame treatment, a ultraviolet irradiation treatment, a heat treatment, etc., can also be effectively used for rendering the carrier hydrophilic.

In selecting these materials and treatment methods, it is particularly important that the concentration of a species to be detected in a liquid sample does not change during the carrying operation of the liquid sample by the liquid carrier. Thus, it is more preferred to use a liquid-carrying material having porous structure than the use of an adsorptive orswellable material which is liable to change the composition of the liquid sample carried thereby.

In the method of this invention, the optimum operation condition for bringing the liquid carrier into contact with the porous top layer of a dry analysis element depends upon the shape of the liquid carrier, the viscosity of the liquid sample, the state of the liquid sample and the kind of the dry analysis element used.

For example, in the case of a dry analysis element having a spreading layer composed of a membrane filter, a paper, or a fibrous material or cloth, it is preferred for obtaining better measurement results to establish such a state that a spread circular portion of a liquid sample having a diameter of 2 to 50 mm, preferably about 5 to 20 mm is obtained by the contact operation of the liquid carrier with the spreading layer for about 0.1 to about 10 secs., preferably about 0.5 to about 5 secs.

This invention will now be more fully explained with reference to the following examples.

Example 1 A transparent polyethylene terephthalate (PET) film of 185 ym thick having coated thereon a gelatin subbing layer was coated with a reagent layer for determining the concentration of glucose in blood having the following composition at a dry thickness of about 15 p followed by drying.

Glucose oxidase 55,000 U Peroxidase 25,000 U 1 ,7-Dihydroxynaphthalene 59 4-Aminoantipyrine 59 Gelatin 200 g Nonion HS 210 (polyoxyethylene nonylphenyl ether, a surface active agent, made by Nippon Oils and Fats Co., Ltd.) 29 On the layer thus formed was uniformly coated a coating composition consisting of diacetyl cellulose, triacetyl cellulose, titanium oxide, acetone, methylene chloride and water, which was then dried to provide a multilayer analysis element for assaying glucose, having an isotropically porous spreading layer.

Using the multilayer analysis element for assaying glucose thus prepared, a sample was spotted thereto by means of a micropipetto attempt to form a color. When a serum was used as a liquid sample, good spreading and uniform color formation were observed but when whole blood was used as the liquid sample, the blood formed a liquid drop on the spreading layer and thus neither spread nor colored. In some cases, the whole blood spread and colored irregularly but the colored state was very inferior and hence unsuitable for the analysis.

Then, the method of this invention was performed. That is, a cotton applicator (made by Cherry Seihin Hompo K.K.) having the cut top of about 5 mm diameter was impregnated with bovine serum and the serum was spotted on the spreading layer of the foregoing analysis element by bringing the applicator into contact with the spreading layer, thereby a good and uniform circular colored portion was obtained. Then, when the bovine whole blood was spotted in place of the bovine serum by the same manner as above, the diameter of the spread circular portion of the blood was reduced about 2/3 of the case using the serum but a good and uniform circular colored portion was obtained.

Example 2 A transparent polyethylene terephthalate film of 185 cm thick having coated thereon a gelatin subbing layer was coated with a reagent layer for assaying the glucose content in blood having the following composition at a dry thickness of about 15 m followed by drying.

Glucose oxidase 55,000 IU Peroxidase 25,000 IU 1,7-Dihydroxynaphthalene 5g 4-Aminoantipyrine 59 Nonion HS 210 (polyóxyethylene nonylphenyl ether, a surface active agent made by Nippon Oils and Fats Co., Ltd.) 29 Gelatin 200 g On the layer was formed a light-shielding layer composed of 8 parts by weight of titanium dioxide powders dispersed in one part by weight of gelatin in a dry thickness of about 15 calm followed by drying.

An adhesive layer composed of gelatin containing 0.2% Nonion HS 210 was then formed on the light-shielding layer in a dry thickness of 5 Fm and dried.

The coated film thus prepared was uniformly wet with an aqueous solution of a 0.2% nonionic surface active agent, Nonion HS 210 and then immediately closely laminated with a spreading layer composed of a microfilter (FM 120, made by Fuji Photo Film Co., Ltd.) and dried to provide a multilayer analysis sheet for assaying glucose. Furthermore, the analysis sheet was cut into a square sheet of 15 mm x 15 mm area and the square sheet was mounted in a plastic mount of 24 mm x 28 mm area having an opening or aperture of a 10 mm diameter at the center to provide a multilayer analysis slide for assaying glucose.

On the other hand, a liquid stick was prepared by cutting the top portion of a cotton applicator (made by Cherry Seihin Hompo K.K.) having cotton wound around the top portion of a thin wood rod at a diameter of about 6 mm. The foregoing stick was impregnated with a mixture of bovine whole blood, heparin and NaF, and then brought into contact with the spreading layer of the foregoing multilayer analysis slide to gently spot the blood onto the layer. When the whole blood uniformly spread to form a circular spread portion of about 10 mm diamter, the spotting was stopped and after incubating for 6 minutes at 37"C, an optical density of the dye colored depending upon the amount of glucose in the blood was measured by reflection using light of 500 nm through the PET film support side. The reflection optical density was 0.387.

The spotting, incubation and photometry were repeated 20 times by the same procedures as above. When the repeating reproducibility was determined on each reflection optical density obtained, the mean density value was 0.383 and the standard deviation was 0.0084, and the coefficient of variation (CV) was 2.2%, which showed good reproducibility.

Small amounts of glucose powders were also added to bovine whole blood to prepare four kinds of blood samples having different glucose contents. After separating blood plasma from each of the samples, the glucose content in each blood sample was measured by an oxygen electrode method using USI 205 (made by Yellow Spring Instruments Co., Ltd.). Then each of these whole blood samples was spotted on the multilayer analysis slide for assaying glucose by the same manner as described above and after incubating for 6 minutes at 37 C, the reflection optical density was measured. The results obtained are shown in the following table.

TABLE Assay Value Reflection Test No. (mg/dl) Optical Density 1 78 0.384 2 137 0.528 3 221 0.731 4 347 1.020 5 411 1.148 As is clear from the above results, the method of this invention is effective for the determination of glucose in blood.

The determination of glucose was performed in a conventional manner.

In this case the blood sample was spotted on the spreading layer of the same multilayer analysis slide for assaying glucose using a 10 microliter micropipet and after incubating by the same procedure as above, the optical density was measured. In this case, the spreading of the blood was very uneven and color formation was irregular. When the measurement was repeated 20 times, the mean density value (x) was 0.318, the standard deviation was 0.0308, and the coefficient of variation was 9.7%, which showed inferior reproducibility.

Whole blood samples having glucose added thereto in various concentrations were also subjected to analysis. Results were very variable and the quantitative correlation was very inferior to that of the method of this invention.

Example 3 A multilayer analysis slide for assaying glucose was prepared by laminating a cotton cloth (cotton broadcloth #80, made by Nisshin Spinning Co., Ltd.) on the coated film for assaying glucose, prepared in a manner similar to Example 2.

A cotton applicator not having a cut top was impregnated with bovine whole blood as used in Example 2 and brought into contact with the spreading layer of the analysis slide to spot the blood sample onto the analysis slide and after incubation for 6 minutes at 37"C, the optical density was measured. The repeating reproducibility was good and the coefficient of variation was 2.6%.

Example 4 In place of the applicator used in Example 4, a liquid-carrying stick prepared by winding gauze around the end point of a wood rod of 100 mm long and a 3 mm diameter at a diameter of 3 mm and after impregnating the carrying stick with bovine blood, the stick was brought into contact with the spreading layer of the multilayer analysis slide for assaying glucose as used in Example 2 to spot the blood onto the layer and the optical density was measured as in Example 2. When the repeating reproducibility test as in Example 2 was also performed, the coefficient of variation was 3.1%.

Example 5 A liquid-carrying stick was prepared by winding a stripe of a filter paper for use of chromatography (#533, may be Toyo Filter Paper Co., Ltd.) having a width of 3 mm around the end point of a wood rod as in Example 3 and using the liquid-carrying stick, bovine whole blood was spotted onto the spreading layer of the multilayer analysis slide for assaying glucose as in Example 3 and colored. A good and uniformly colored portion was obtained.

Example 6 An applicator-like material having the end point length about 5 mm was prepared by winding a highly hygroscopic tissue (Kimwipe, tradename) around the end point of a wood stick as in Example 4 using a cotton yarn. When bovine whole blood was spotted using the applicator-like material and the color formation test was performed as in Example 3, a good and uniform coloring was observed.

Example 7 A liquid-carrying stick was prepared by adhering a thick filter paper piece (5 mm x 5 mm) for qualitative analysis to a plastic stripe of 0.5 mm thick, 5 mm wide and 100 mm long using a double-faced adhesive tape.

When bovine whole blood was spotted using the liquid-carrying stick and the color formation test was performed as in Example 3, a good and uniform coloring was observed.

Claims (7)

1. A method of supplying a small amount of a liquid sample onto an analysis element of dry type which comprises including, in an analyzing operation using an analysis element having a porous layer as the outermost layer, a step of retaining a small amount of a liquid sample containing a species to be detected in a liquid carrier and then supplying the liquid sample to said porous layer so that at least a part of said liquid carrier is brought into contact with said porous layer of said analysis element of dry type.

2. A method as claimed in claim 1 in which said liquid carrier is composed of a small rod-like piece or strip and a liquid-carrying material supported at the top portion thereof.

3. A method as claimed in claim 2 in which the liquid-carrying material is cotton, sponge, cloth, gauze, a filter paper a glass wool or a natural or synthetic porous resin.

4. A method as claimed in claim 2 in which the liquid-carrying material is, gelatin, polyacrylic amide or agarose.

5. A method as claimed in claim 1 in which said analysis element of dry type comprises a support having at least a reagent layer, a light-shielding layer and a porous spreading layer.

6. A method as claimed in claim 1 substantially as herein described with reference to any one of the accompanying drawings.

7. A method as claimed in claim 1 substantially as herein described with reference to any one of the Examples.