WO2002031105A1

WO2002031105A1 - Method and instrument for collecting fluid in the oral cavity

Info

Publication number: WO2002031105A1
Application number: PCT/JP2000/007075
Authority: WO
Inventors: Toru Yokoyama; Naoki Shinozuka; Kenji Nakamura
Original assignee: Sapporo Immuno Diagnostic Laboratory
Priority date: 2000-10-12
Filing date: 2000-10-12
Publication date: 2002-04-18
Also published as: AU2000276848A1

Abstract

A method of collecting a fluid in the oral cavity for quantifying D-glucose in the fluid in the oral cavity, wherein bacteria in the oral cavity have been preliminarily killed by a bactericide or the fluid is collected together with the bacteria to thereby inhibit the digestion of D-glucose by the bacteria; and a convenient instrument for collecting and recovering a fluid in the oral cavity wherein the fluid in the oral cavity is sterilized and collected with a hydrophilic absorbent containing a bactericide and then the absorbent is pressed to thereby recover the fluid having been absorbed thereby. Thus, specimens can be quickly collected and recovered.

Description

Specification

FIELD OF THE INVENTION Field of the Invention

The present invention relates to the presence of oral secretions, such as salivary glands, submandibular glands, and sublingual glands, of saliva from each salivary gland, and exudates from the gingival sulcus, etc. The present invention relates to a method for collecting an oral secretion fluid that is subjected to a treatment for sterilizing bacteria, and a simple instrument capable of performing sterilization, collection and collection. Background of the Invention

Diabetes, an explosive increase worldwide, has been shown to be closely linked to everyday lifestyles as an environmental factor. It is also being clarified that not only environmental factors but also genetic factors contribute significantly. For example, in the treatment of diabetes, strict blood glucose control by insulin has shown clinical significance such as suppression of complications, and self-monitoring of blood glucose (SMBG) is frequently used in treatment settings. . Here, the point-of-care testing (POCT) and POCT equipment typified by SMBG, which can be performed easily and quickly, are becoming established in recent years, and the following describes how to use POCT equipment for SMBG. In the above blood glucose measurement, a few microliters of whole blood obtained by puncturing a fingertip or the like is used as a test sample, and a test device consisting of a disposable biosensor chip and a sensor meter is used. After about 30 seconds, it is a system that can obtain the blood glucose level which is the test result (for example, WO00 / 07003). However, although the sample volume is as small as a few microliters and further volume reduction is being promoted, it is still an invasive or invasive measurement using a puncture needle, and the physical The pain and the danger of infectious diseases, especially, are inevitable No. For these reasons, there is an urgent need for noninvasive testing methods for glycemic patients who must perform frequent blood sampling for glycemic control. Under these circumstances, non-invasive measurement methods for blood glucose have been studied since the 1980s as measures against diabetes. Recently, a skin intercellular fluid, which has widened skin cell gaps by sonication and increased permeability to blood vessels, was suctioned, and the extracted D-glucose concentration was compared with blood glucose (Nature Medicine, Vol. 6, p. .347 (2000)) and comparison of blood glucose with D-glucose concentration in saliva (eg, Medical Electronics and Biotechnology, Vol. 38, ρ. 127 (2000)). Of the above two cases, the former requires ultrasonic irradiation, vacuum suction, extraction of intercellular fluid into salt solution, quantification of D-glucose, and complicated pretreatment. Regarding the latter, although the degradation of D-glucose by bacteria present in the oral cavity is known ('for example, Dental Microbiology, 5th Edition, Medical and Dental Medicine Publishing (1992)), sampling during and during collection It does not take into account bacterial effects later. In addition, a test using saliva as a specimen (Annals of the New York Academy of Sciences, Vol.694, p.216 (1993)) is based on a saliva collection device “OraSureJ (Epitope, Inc.)” (for example, US Patent No. 5,714,341). (1998)), saliva collection device r Omnisal (Saliva-Sampler) J (manufactured by Saliva Diagnostic Systems, Inc.) (US Patent No. 5,260,031 (1993)) and saliva collection device "Salivette" (manufactured by Sarstedt) (for example, US (Patent No. 4,774,962 (1988)) to conduct various screening tests such as HIV, hepatitis virus, syphilis, rubella, measles, therapeutic drugs such as theophylline and phenytoin, and drugs and drug monitoring tests such as nicotine and cocaine. (Journal of the Japan Society of Clinical Laboratory Automation, Vol.19, p.265 (1994)) ₀ The equipment described above performs the steps from collection to storage, and is made up of dedicated equipment for collection and storage tubing containing storage solution. Here, sodium azide is mixed in the above-mentioned preservation solution to prevent propagation of oral bacteria present in the collected sample (saliva collection device “Omnisal (Saliva · Sampler) J)”. There is no D-glucose quantification, no sterilization operation is performed during collection, and no testing is performed on the spot and it is not used as a POCT, so the specimens stored in storage containers are transported to an inspection center, etc. In recent years, the remarkable progress of biosensor technology has led to the development of various POCT devices, and the need for simple instruments that can be collected immediately after sample collection. As mentioned above, when collecting oral secretions, at least when quantifying D-dalcos in oral secretions, a treatment to sterilize oral bacteria beforehand Performing is not seen at all proposes the operation. Further, to the collection to a method and adopt Tonochi taken while sterilize oral bacteria nor suggestions instrument can also be performed. Summary of the Invention.

The present invention solves the above-mentioned problem by disinfecting oral bacteria with a disinfectant in advance when collecting oral secretions for the purpose of quantifying D-dalcos in oral secretions. It has been shown that it suppresses the decomposition of D-dalcos by bacteria.

In addition, the oral secretory fluid is sterilized and collected by a hydrophilic absorbent containing a bactericide, and the hydrophilic absorbent is pressed to collect the absorbed oral secretory fluid. It is intended to provide a collection method and a simple device for collecting and collecting oral secretion fluid using the above method, thereby realizing rapid collection and collection of a sample. Description of the invention According to the present invention, when quantifying D-glucose contained in the oral secretion fluid, bacteria which are a nutrient source of D-glucose, which are various and abundant in the oral cavity, are preliminarily sterilized with a bactericide. And a new method for collecting oral secretions. Furthermore, for POCT, it consists of only a hydrophilic absorber containing a bactericide, which has both the function of collecting oral secretory fluid at the same time as sterilization and the function of collecting oral secretory fluid after collection. Another object of the present invention is to provide a simple device comprising a combination of an absorber and a water-resistant support. Streptococcus mutans, a cariogenic bacterium that is present in dental plaque, is capable of fermentatively decomposing various monosaccharides and producing several types of organic acids, including lactic acid. It is known (eg, Dental Microbiology, 5th Edition, Medical and Dental Medicine Publishing (1992)), and it is difficult to quantify D-glucose in saliva, an oral secretion fluid. Among them, mouthwashes containing various disinfectants have been developed and sold as liquid toothpastes to prevent dental caries. For example, those containing cetylpyridinium chloride (for example, W091 / 18585 (1991), JP-A-10-251131 (1998)) (for example, US Pat.No. 5,948,390 (1999)), and containing triclosan (For example, JP-A-11-310522, JP-A-11-322553) and the effect of triclosan and triclosan-monophosphate on the growth of oral bacteria (Journal of Antimicrobial Chemotherapy, Vol. 45, p. 447 (2000 )) And so on, and various bactericidal effects are being obtained. In addition, in order to quantify D_glucose in saliva, there is a method in which sodium fluoride is added after saliva collection to suppress decomposition (Archs Oral Biology, Vol. 41, p. 141 (1996)). The measurement methods were high-speed ion exchange chromatography and pulse current measurement. The decrease in D-glucose after saliva collection was almost completely resolved by the addition of 0.1% sodium fluoride. However, considering high-speed centrifugation as a pretreatment, expensive measurement systems and complicated operating conditions, it is hard to say that it is for P0CT. Above all, the addition of sodium fluoride alone was not sufficient in our measurement system. As described above, the inclusion of a bactericide in the mouthwash is intended to prevent dental caries and periodontal disease, and a simple sampling method for the quantification of D-glucose in oral secretions has not yet been found. Therefore, in the present invention, as a method of collecting oral secretion fluid including a treatment of sterilizing oral bacteria for the purpose of quantifying D-glucose in oral secretion fluid, a method of collecting oral bacteria after disinfection using a bactericide is described. The present invention provides a method for collecting oral secretions using one or both of the methods, which are performed simultaneously with or after sterilization. In addition, in a structure in which only a hydrophilic absorber containing a bactericide or in combination with a water-resistant support, oral secretions are sterilized and collected by the hydrophilic absorber, and then the hydrophilic absorber is pressed. It also provides a method for collecting and collecting oral endocrine fluid for recovering absorbed oral secretion fluid, and a simple instrument for collection and recovery. By selecting a material that has the property of adsorbing contaminants in oral secretions, the hydrophilic absorber can be a collection and collection device with a separation function that can remove contaminants at the time of collection. By forming a multi-layer structure consisting of a filter layer and filtration layers of various pore sizes, the collection layer with a filtration function that allows the filtration of the oral secretory fluid components having a pore size equal to or less than the selected pore size when collected through the filtration layer. A collection device is also provided. Furthermore, the water-resistant support is made of a more flexible material in the form of a plate and a tube, so that when the oral secretory fluid is collected, the hydrophilic absorber placed above and inside the water-resistant support is used. The compression is easy due to its flexibility, and a device for collecting and collecting oral secretion fluid with a simple collection function is also provided. In addition, the sensor chip and sensor meter, which are POCT devices, are based on the reaction principle and fabrication technology that we have already invented (JP-A-2000-35413, WO00 / 04378, PCT / JP99 / 01392, PCT / JPOO / 05788, PCT / JPOO / 05789, PCT / JP00 / 05790), and the sensor chip consists of at least a working electrode and a counter electrode formed using a conductive material. The layered structure on which the reaction reagent is immobilized is arranged on the upper surface of the electrode reaction part. In addition, the sensor mechanism is composed of an electrochemical detection device and the like. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the time course of the D-glucose abundance in human whole saliva in Example 1 of the present invention.

FIG. 2 shows the time course of the D-glucose abundance in human whole saliva with respect to various sterilized components in Example 2.

FIG. 3 shows the time course of the D-glucose abundance in human whole saliva by the bactericidal component (cetylpyridinium chloride (CPC)) in Example 3.

FIG. 4 shows a simple device for collecting and collecting oral secretion fluid (1) in Example 4, FIG. 5 shows a simple device for collecting and collecting oral secretion fluid in Example 5 (2), and FIG. This is a simple device (3) for collecting and collecting oral secretion fluid provided with a filtration function in Example 6. The reference numerals in the above figures are explained as follows.

1 is a water-resistant support; 2 is a hydrophilic absorber; 3 is an oral secretion; 4 is an absorption layer; 5 is a filtration layer. Description of preferred embodiments

The bactericide used in the present invention can be used in the oral cavity without affecting the human body, sterilizes oral bacteria that degrade D-glucose, and does not inhibit D-glucose quantification. If so, there is no particular limitation. For example, CPC for quaternary ammonium salt-based germicides, ethanol for alcohol-based germicides, ozone water for peroxide-based germicides, triclosan and iodine-based germicides for phenol-based germicides. If this is the case, a povidone or the like may be used. Therefore, it can also be used. The hydrophilic absorber, which collectively refers to the absorption layer, the adsorption layer, and the filtration layer, can be used in the oral cavity without affecting the human body, can contain a bactericide, and is compatible with the collected oral secretions. There is no particular limitation as long as there is no unspecific unintended adsorption or contamination of oral secretions, and it is a material that absorbs oral secretions and is hydrophilic. For example, fibers such as cotton, glass fiber, silica fiber, and cellulose fiber, and carboxymethyl cellulose, getyl aminoethyl cellulose, cellulose acetate, cellulose mixed ester, nylon, nitrocellulose, polyethersulfone, and polyester; Polyethylene, polyvinyl chloride, polypropionate, polytetrafluoroethylene, polyvinylidene fluoride, polyvinylidene difluoride, polypropylene and the like can be used. The absorption layer is not particularly limited as long as rapid absorption of oral secretions is realized.

The adsorbent layer should be made of a material that has positive or negative charge or a material that has ionic and covalent bonding capabilities, so long as it can realize a separation function that can remove contaminants by adsorption. There is no limitation. The filtration layer is not particularly limited as long as it realizes a filtration function capable of preparatively determining the molecular size of the recovered sample by selecting various pore sizes. The water-resistant support can be used in the oral cavity without affecting the human body, and it can be applied with a bactericide, and non-specific unintended adsorption or oral secretion with the collected oral secretory fluid There is no particular limitation as long as it has no mechanical contamination, has flexibility and water resistance, has good workability, and has sufficient mechanical strength to prevent breakage during use. For example, tubes and films made of silicon, polyester, polyethylene, polyethylene terephthalate, polystyrene, polypropylene, etc. are inexpensive, and can be used because of their good adhesion to the hydrophilic absorber and good workability. The substance for adhesion between the water-resistant support and the hydrophilic absorber can be used in the oral cavity without affecting the human body, and does not adsorb to the oral secretory fluid or contaminate the oral secretory fluid. There is no particular limitation as long as the substance is suitable.

The adhesion between the water-resistant support and the hydrophilic absorber is not always required. Example

Hereinafter, examples of the present invention will be specifically described, but the present invention is not limited to these examples. Example 1 Change of D-glucose abundance in human whole saliva with time

FIG. 1 shows the change over time in the D-glucose abundance in human whole saliva in this example.

Immediately after collection, D-glucose was added to whole human saliva to a concentration of 5 mg ZdL. Subsequent changes over time were determined based on the D-glucose concentration immediately after the addition. As a result, it decreased to about 60% of the initial value after 12 minutes. Example 2 Time-dependent change of D-glucose abundance in human whole saliva for various sterilized components

FIG. 2 shows the change over time of the D-Dalcos abundance ratio in human whole saliva with respect to various sterilizing components in the present example. In the figure, a commercially available oral washing solution containing a bactericidal component, etc. was added to the same human whole saliva so as to have a volume of 1/20, and the time-dependent change based on the D-glucose concentration immediately after the addition was measured. I asked.

As a result, a clear difference was observed in the D-glucose abundance depending on the presence or absence of the sterilizing component.

No D-glucose was found in the added commercially available mouthwash. Also, the effect and content of each bactericidal component are not uniform. Therefore, each D-glucose abundance after 3 hours is a reference value. . Example 3 Time course of D-glucose abundance in whole saliva of humans by bactericidal component (CPC)

FIG. 3 shows the time course of the D-dalcos abundance in whole saliva of humans by the sterilizing component (CPC) in this example.

Immediately after collection, CPC (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the whole saliva of humans at a concentration of 0.02%, and D-glucose was added at a concentration of 5 mg / dL. did. Subsequent changes over time were determined based on the D-glucose concentration immediately after the addition.

The black circles in the figure indicate the results containing CPC, and the white circles in the figure indicate the results in FIG. 1 containing no fungicide.

Due to the inclusion of CPC, the concentration was reduced to about 93% of the initial value after 12 minutes. A dramatic improvement in the bactericidal action was confirmed by the inclusion of the bactericide CPC. Example 4 Simple device for collecting and collecting oral secretions (1)

FIG. 4 shows an example of a simple device for collecting and collecting oral secretions in this example.

Hydrophilic substrate 1 made of polypropylene (manufactured by Oji Yuka Synthetic Paper Co., Ltd.) impregnated with a CPC solution and dried to absorb hydrophilicity made of cellulose (manufactured by Nippon Pall Co., Ltd.)

+ Body 2 was placed with an adhesive. By contacting the oral secretory fluid 3 with the hydrophilic absorber 2, good absorbability was exhibited, sterilized and absorbed, and the oral secretory fluid 3 was rapidly collected.

Thereafter, the hydrophilic absorber 2 was pressed by bending the water-resistant support 1, so that the sterilized and collected oral secretions 3 could be collected from the end surface of the water-resistant support 1.

In addition, the stick shape of this simple device makes it easy to place it on the target site.It is easy to place saliva from the parotid gland beside the upper molars, and sublingual and submandibular gland saliva below the tongue. Sterilization and collection of spores were carried out easily.

In particular, in the sample collected after collection, the viscous substance considered to be mucin was trapped by the hydrophilic absorber 2 and became serous. In addition, some of the food residue is captured by the hydrophilic absorber 2. It was confirmed that they would be caught.

It is also possible to use only the hydrophilic absorber 2 containing a bactericide without using the water-resistant support 1. Example 5 Simple device for collecting and collecting oral secretions (2)

FIG. 5 shows an example of a simple device for collecting and collecting oral secretions in this example.

A hydrophilic absorber 2 made of cellulose (Nippon Pall Co., Ltd.) impregnated with a CPC solution in a water-resistant support 1 that is a silicon tube (inner diameter 2 mm x outer diameter 3 mm) is placed inside the tube. did.

The oral secretion fluid 3 sterilized and collected from one end of the tube of the water-resistant support 1 presses the tube itself, thereby pressing the hydrophilic absorbent body 2 inside, and the oral secretion fluid from the other end or both ends of the tube. 3 can now be collected.

In addition, by selecting a material having an adsorbing ability for the hydrophilic absorber 2, for example, various protein concentrations can be applied to the adsorbing layer made of nylon (manufactured by Nippon Pall Co., Ltd.) having an adsorbing ability for proteins by covalent bonding. When the oral secretion fluid 3 added and prepared in Example 1 was sterilized, collected, and collected, the protein concentration was lower than before collection, protein binding was recognized by the adsorption layer, and the separation function was provided. Was.

The use of the water-resistant support 1 in the form of a flexible tube facilitated handling in the oral cavity. In addition, when the hydrophilic absorber 2 is disposed in the tube, a treatment such as adhesion is not required. Further, by making the hydrophilic absorber 2 into a floss shape, the efficiency of production can be improved by impregnation with a germicide, arrangement in a tube, and the like. Example 6 Simple instrument for collecting and collecting oral secretion fluid provided with a filtering function (3) FIG. 6 shows an example of a simple device for collecting and collecting oral secretion fluid having a filtering function in this example. Things.

A two-layered hydrophilic absorber consisting of an absorbent layer 4 made of cellulose (manufactured by Nippon Pall Co., Ltd.) and a filtration layer 5 made of polyether sulfone (manufactured by Nippon Pall Co., Ltd.) is placed on the water-resistant support 1. And the intraoral portion where the residue of the food used in Example 1 can be seen Lactic acid 3 was sterilized and collected from one end of the tube, and collected from the other end of the tube. In the above-described embodiment, the specific shape of the simple device for collecting and collecting oral secretions is illustrated, but the shape and arrangement of other components are not limited.

Claims

The scope of the claims

1. A method for collecting oral secretion fluid, which includes a treatment for killing oral bacteria, for analyzing the components of oral secretion fluid.

2. The collection method according to claim 1, wherein the oral secretion liquid is collected after sterilizing the oral bacteria using a bactericide, or is collected simultaneously with the sterilization, or both.

3. The collection according to claim 1 or 2, wherein the oral secretion is a liquid from a specific site secreted in the oral cavity, such as saliva derived from each salivary gland, an exudate from the gingival sulcus, or a mixed liquid of each site. Method.

4-The collection method according to any one of claims 1 to 3, wherein the component analysis is a D-glucose determination.

5. The method according to any one of claims 1 to 4, wherein the oral bacteria are bacteria that degrade D-glucose.

6. The germicidal treatment includes one or more germicidal germs such as quaternary ammonium salt germicides, alcohol germicides, peroxide germicides, phenolic germicides, iodine germicides and enzymes. The collection method according to any one of claims 1 to 5, wherein an agent is used.

7. The method according to claim 6, wherein the quaternary ammonium salt fungicide is cetylpyridinium chloride.

8. In a structure in which only the hydrophilic absorber containing the bactericide or a structure combined with a water-resistant support, the oral secretion fluid is sterilized and collected by the hydrophilic absorber, and then the hydrophilic absorber is compressed. And recirculates the absorbed oral secretions. The collection method according to any one of claims 1 to 7, which collects.

9. A device for collecting oral secretions consisting of a hydrophilic absorber containing a bactericide and a water-resistant support.

10. The hydrophilic absorber is provided with a separation function capable of removing the contaminants during the collection of the secretion fluid by selecting a material having a property of adsorbing contaminants in the oral secretion fluid. 9. The sampling instrument according to 9.

11. The hydrophilic absorber has a multilayer structure including at least an absorbent layer and a filter layer of various pore sizes, and when the oral secretory liquid is collected after passing through the filter layer, the pore size is equal to or less than the pore size selected for the filter layer. 10. The collection device according to claim 9 or 10, further comprising a filtration function capable of separating said oral endocrine fluid component.

12. The water-resistant support is made of a material having more flexibility as a plate and a tube. When the oral secretory fluid is collected, the water-resistant support is disposed above and inside the water-resistant support. The sampling device according to any one of claims 9 to 11, wherein compression of the hydrophilic absorber is facilitated by its flexibility, and the collection device has a simple collection function.