MXPA98005608A - Proof of displacement on a membrane by - Google Patents

Abstract

Under non-equilibrium conditions, displacement tests are carried out by flowing a sample of liquid through a membrane (12) having binding elements with binding sites saturated with a marked form of the analyte. Under unbalanced conditions, the analyte in the sample displaces the labeled form of the analyte from the membrane. Then the marked displaced form of the analyte can be detected

Description

PROOF OF DISPLACEMENT IN A POROSA MEMBRANE BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION The present invention relates generally to tests and more specifically to displacement type tests. 2. DESCRIPTION OF THE BACKGROUND TECHNIQUE The patent of E.U.A. No. 5,183,740, incorporated in its entirety herein for all purposes, describes a flow immunoprotection system for conducting displacement immunoprotes. In a displacement test, unlike a competitive test, the antibody is exposed to labeled analyte before analyte exposure. The analyte is in contact with the antibody and labeled analyte, bound an insufficient amount of time to establish equilibrium. Since no time is needed to establish balance, displacement tests are faster than competitive tests. However, a displacement test usually provides a smaller signal than a competitive test. In a displacement test, available antibody binding sites are saturated or nearly saturated with labeled analyte before adding the unlabeled analyte. Since equilibrium has not been achieved (with labeled analyte and unlabeled analyte continuously uniting, releasing and competing with each other for re-binding to the available binding sites in the antibody in a stable state), most of the analytes labeled in a The displacement test remains attached to the antibody and unable to provide a signal. The relatively small signal provided by the displacement test places an additional value on ensuring the consistency of the test conditions. The globule-containing columns described in USP 5, 183,740 for displacement tests should be stored carefully, prepared and loaded to ensure chemical and physical consistency (ie, porosity, avoid channeling) of test to test. The need for this careful preparation and testing increases the manpower, experience and costs necessary to carry out accurate displacement tests. Additionally, problems associated with the use of columns containing globules limit the lower detection limit for displacement tests. In actual studies in U S Drug Testing, I nc. (Ranch Cucamonga, California), better results were obtained for a displacement test by using high, thin columns of beads coated with an antibody and antigen labeled with short, broad columns. In addition, the efficiency with which the labeled antigen was dissociated from the antibody in the presence of unlabeled antigen was greater when flow velocities were reduced and the antigen had more time to interact with the immobilized complex (Wemhoff et al., J. Immunol Methods, 223-230, 1992). Both sets of experiments suggested that immobilization of the antibody and labeled antigen on a porous membrane would not provide an adequate matrix for the displacement test since this geometry would not allow sufficient time, under flowing conditions, for the antigen to efficiently interact with the antigen. complex to displace quantities that can be detected of the labeled antigen. The patent of E. U.A. No. 5, 369, 007, to David A. Kidwell discloses a displacement test wherein the samples pass through a membrane having an antibody immobilized therein. The immobilized antibody binding sites bind to a enzyme-labeled analyte. The analyte in the sample displaces the labeled analyte, causing the labeled analyte and the rest of the sample to pass in a superabsorbent layer. The superabsorbent layer contains a substrate for the enzymatic label and any necessary indicator. However, the Kidwell patent teaches the need for a flow rate of about 0.02 ml / min and interaction times of about 1 to 5 minutes to ensure an interaction that can be detected between the analyte and the antibody. In many situations, even faster results are desired. Additionally, the Kidwell micro-test card does not can be reused.

BRIEF DESCRIPTION OF THE INVENTION Accordingly, an object of this invention is to carry out bio-tests capable of detecting minute quantities of an analyte in less than one minute. Another object of the present invention is to rapidly perform bioassays in a format that allows re-use of the matrix that selectively binds the analyte. # These and other objects of the invention are achieved by flowing A sample passing through a nonabsorbent membrane that has a binding element covalently attached thereto to form binding sites for the analyte is rapidly collected. The available binding sites are essentially saturated with a marked form of the analyte. The binding or specific sites are blocked for prevent non-specific binding. Additionally, the sample flows # passing the membrane at a rate greater than that necessary to achieve equilibrium between the cleavage of labeled analyte from the binding sites and the binding of analyte (labeled or unlabeled) to them. The processed sample is then analyzed for the presence of any labeled antigen that the unlabeled analyte has displaced from its binding site. This analysis can be qualitative or quantitative.

BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention will be obtained soon by reference to the following description of the modalities Preferred and drawings wherein similar numbers in different figures represent - the same structures or elements, wherein: Figure 1 schematically illustrates a device in accordance with the present invention. Figure 2 illustrates schematically an alternative embodiment of a device in accordance with the present invention. Figure 3 schematically illustrates another alternative embodiment of a device in accordance with the present invention. Figure 4 is a graph of data from a membrane test, in accordance with the present invention, wherein the # membrane was prepared by the incubation method of test tube. Figure 5 is a graph of data from a membrane test, according to the present invention, wherein the membrane was prepared by saturating the immobilized antibody with labeled analyte in the column instead of in a test tube. Figure 6 is a graph of data from a single membrane test, in accordance with the present invention, prepared by saturating the antibody directly on the column. Figure 7 is a flow chart that schematically uses a form of a compliance test ^? the method of the present invention. Figures 8a, 8b and 8c show the results obtained from the test performed in accordance with the flow chart method of Figure 7.

DESCRIPTION OF THE PREFERRED MODALITIES The membranes useful in the present invention are typically non-absorbent materials (with respect to aqueous materials). The nonabsorbent membrane helps provide a fast flow rate of flow. Additionally, the use of a non-absorbent membrane allows the membrane, once used, to be rinsed early from the sample and reused. If it has happened displacement, reloading with labeled analyte is an option. Typically, the membranes useful in the present invention have thicknesses, exposed surface areas, and porosities of from about 0.1 second to about 30 seconds, and typically from about 1 second to about 15 seconds. seconds, between a sample that is suspected of containing the analyte and the membrane that has an analyte labeled with the analyte in it. Generally, the pore sizes in the membrane are around 0.2-1.1 microns, and are typically about 0.45 microns. Of course, other pore sizes can be used to achieve the desired interaction time. Also, the thickness and surface area of the membrane can be adjusted to provide the «Desired interaction time. Any nonabsorbent membrane of appropriate pore size and site density can be used to immobilize elements of binding for the analyte. For example, the membrane can be a polyamide (e.g., Nylon ™ membranes such as Immunodyne ABC ™ (a Nylon ™ 6,6 membrane made by Pall Biosupport, Port Washington, New York)) or a fluoride • polyvinylidine, such as Immobilon ™ or Durapore ™ membranes made by Millipore, Bedford, Massachusetts. Other suitable membranes include, but are not limited to, cellulose, nitrocellulose, silica fiber, aluminum oxide, and polyvinyl chloride. You can immobilize binding elements in the selected membrane in any way that ensures availability in the immobilized binding element of at least one binding site to selectively bind the labeled analyte and target analyte in an aqueous medium. Various methods for attaching membrane attachment elements are well known and will therefore not be specifically described herein. The joint element can immobilized throughout the thickness of the membrane or only on one or both surfaces thereof. The binding element can be any substance that can be immobilized in the membrane and that specifically binds the target analyte and its labeled analogue. The elements of union include, but are not limited to, lectins, antibodies, antibiotics, and binding proteins other than antibodies and antibiotics. • Once the binding elements in the membrane are immobilized, their binding sites available to selectively go with the analyte will usually be saturated in essence with an analogous analyte labeling (denoted herein as a "labeled analyte"). The saturation of the available binding sites with the labeled analyte improves the sensitivity by ensuring that the maximum number of analyte molecules will displace labeled analytes, rather than directly attaching to unoccupied binding sites. The membrane can be oriented in any way with respect to the sample flow that allows the sample to flow past the complex of binding element and labeled analyte in the membrane over the desired interaction time. For example, the sample can flow through and essentially normal to the plane of the membrane. Alternatively, the membrane can be configured as a dip stick and the sample allowed to flow laterally through the membrane, for example by capillary action. In another alternative, the membrane support may be a hollow fiber configured so that the sample flows over the hollow core before moving to through the membrane. In any embodiment of the present invention, the flow of the sample through the membrane can be passive (ie, gravitational or capillary flow) or active (flow that results fully or partially from the action of a bo m). flow rate, manual pressure or vacuum). 25 Any useful mark can be used in tests for the analyte to mark the analyte. Fluorophores are particularly useful brands. Suitable fluorophores include, but are not limited to Flourescein, Cadaverine, Texas Red ™ (Molecular Probes, Eugene, OR) and Cyanine 5 ™ (BDS, Pennsylvania). If used, the fluorophore mark is typically one that can be detected on the scale of visible to near infrared. Once the sample completes its interaction with the membrane that has the analyte marked by binding element • immobilized in it, the sample processed (for example, the The liquid leaving a sample column or the portion of the sample that has passed through and beyond the marked portion of a test strip) is then analyzed to determine the concentration of displaced labeled analyte. The detection means for this analysis include a reader to inform the user that a threshold amount of the mark has been detected in P the sample. When the label is fluorescent, the detection means also includes a light source to excite the analytes labeled by fluorophore. The detection system can use various methods of optical measurement, including but not limited to a spectrophotometer, infrared spectrometer, fluorimeter, optical biosensor or eye. The present invention is useful in the detection, in aqueous media, of any analyte that specifically binds the binding element. The invention can be used, for example, to detect the The presence of analytes in body fluids (blood, semen, saliva, urine, etc.), water, pharmaceutical preparations, environmental samples, aerosols, food and beverages. If the suspected sample contains the analyte is originally in a viscous, liquid, solid, gaseous state, the sample is preferably dissolves more in water before being exposed to the membrane. Multiple binding elements can be immobilized for multiple analytes in a single membrane. The membranes containing the same or different connection element can be arranged in stacks. Where multiple joining elements are used for For multiple analytes, different labels can be used on the labeled analytes to distinguish which analyte is present. Figure 1 schematically shows a device 10 according to the present invention wherein the membrane is normal to the sample flow. The membrane 12, with connecting elements bound covalently or otherwise immobilized thereto and available binding sites saturated with a labeled analyte of the analyte, is placed on the column 15. An aqueous sample entering the top of column 14 flows through the membrane 12. The analyte in the sample interacts with membrane 12 and displaces the labeled analyte from the membrane 12. The labeled analyte, if it does not remove another labeled analyte or unlabeled analyte from the membrane, is attached to the liquid leaving the column 14. The liquid leaving the aqueous sample from the column 14 then enter line 16, which carries the liquid that comes out to detector 18 to detect the The presence of the labeled analyte in the liquid leaving the column 14. Figure 2 shows an alternative embodiment of the present invention, wherein the membrane is also normal to the sample flow. The porous membrane 102, with or without covalently bound binding elements immobilized thereto and available binding sites saturated with a labeled analyte analyte, is placed on the column 104 having an open tip. To prevent the flow of the sample between the outer edge of the membrane # 102 and the inner wall of the column 104, the membrane extends typically completely over the width of the column 104. The open tip of the column 104 is inserted into the upper part of the container 106 (typically through a septum (not shown)), which holds a sample that is suspected to contain the analyte The suction means can apply a vacuum to pull the sample from the container 106 through membrane 102 in column 104. It --- ^ f can detect any mark in the column by detection means external to the column. To facilitate this external detection, column 104 is preferably transparent to, or includes a window placed adequately transparent to, the energy used for detection. Although Figure 2 shows the suction means as a plunger and column 104 since the syringe accommodates the plunger, other vacuum arrangements are possible. For example, Figure 3 shows a design similar to that used by Vacuutainers ™. The evacuated tube 204 has porous membrane 205, with binding elements covalently attached or otherwise immobilized thereto and sites of * saturable union saturated with a analyte labeled analyte in the same. To prevent sample flow between the outer edge of the porous membrane 205 and the inner wall of the evacuated tube 204, the membrane typically extends completely over the width of the evacuated tube 204. The open end of the evacuated tube 204 is sealed by a cap 206 which has a rim 208 extending over the rim of the open end of the tube 204. The card 21 0 * extends from the cover 206 opposite the hollow needle 212, which also extends from the cap 206. The needle 21 2 extends near the septum 214 when the tube 204 is placed, with light pressure only, within the flange 208. The septum 21 4 maintains the vacuum in the tube portion 216 204. Although the septum 214 is essentially impermeable to liquid gas, it is pierced by the needle 212 once. that the tube 204 is completely inserted into the flange 208. When piercing the septum 214, the vacuum within the portion 216 withdraws liquid from the sample container 218 through the tip 210, into the hollow needle 212, through of membrane brane 205 and in portion 21 6. Any mark can be detected within portion 216 as with other modalities of the invention. To ensure that the needle 212 does not pierce the membrane 205, the gap between the bottom of the septum 214 and the bottom of the membrane 205 should be greater than the height of the needle 212. This method of the invention Ensure that the flow over membrane 205 is consistent from sample to sample.

After describing the invention, the following examples are presented to illustrate specific applications of the invention including the best mode now known to carry out the invention. These specific examples should not limit the scope of the invention described in this application.

EXAM PLOS EXAM PLO 1. DETECTION OF TNT To prepare the membranes, monoclonal antibody 1 1 B3 (mouse 1 gG 1) with specificity for TNT (trinitrotoluene) was immobilized on the membrane of I mmunodyne ™ ABC with a pore size of 0.45 μm. Antibody 1 1 B3, 100μl of a solution of 2 nmol / ml in phosphate-buffered saline (PBS), was fixed to the membrane by placing the solution in a test tube, with subsequent addition of the membrane, or by pipetting the antibody into the column that already contained the membrane. Either in a columan or a test tube, the membranes were incubated with the antibody for four hours at room temperature atmosphere. After incubation, the antibody solution was removed. The membranes exposed to the antibody in a test tube were placed on a column. Any unreacted binding site in the membrane was blocked with the addition of 100μl of 1M Tris for approximately 30 minutes. To reduce non-specific binding, the membranes were drained and washed three times with PBS containing 0.01% Triton X-100® detergent. The labeled analyte was prepared by fixing the fluorophore CY5® (BDS, Pennsylvania) to trinitrobenzyl cadaverine (CY5-TN B). For saturating the antibody binding site with the labeled antigen, a solution of CY5-TNB (4 nmoles in 50μl PBS) was added to each column, and the columns were placed on an oscillating bed overnight. The columns were connected to the fluorimeter and, they were washed # briefly. Samples were introduced at a flow rate of 1 m L / min. Analyte injections were made in triplicate with concentrations ranging between 18.75 ng / mL and 1200 ng / mL. Figure 2 illustrates data obtained for a membrane test prepared with the test tube insertion method. A fluorescence signal was obtained at all analyte concentrations that were proportional to the amount of analyte added to the column. • Figure 3 represents data from a membrane test prepared by saturating the immobilized antibody with labeled analyte in the column instead of in a test tube. Once again, an increase in signal intensity was observed with concentration of analyte increasing. However, an altiplano was observed between an analyte concentration of 700 ng / mL and 1200 ng / mL where a negligible increase in signal intensity was observed despite an increase of two doubling in analyte concentration suggesting that there is less analyte labeled on the membrane available for displacement, compared to membrane f prepared in the test tube. Both figures 3 and 4 show reproducible results, with minimum standard error as indicated by the error bars. The test times were fast with the exact time being simply a function of the flow rate (1 m L / min in this case) and the length of the tube between the analyte introduction site and the flow cell of the fluorimeter. For these experiments, signals were generated in less than 1 minute from the time of # sample introduction. 10 EXAMPLE 2. DETECTION OF RDX Similar experiments were carried out by which a monoclonal antibody with specificity was immobilized for the explosive, cyclonite (RDX) in the membrane. The procedure for F immobilization was identical to that used for the anti-TNT antibody. However, 100μl of 0.5% casein was used instead of Tris in order to block the remaining binding sites in the membrane. Figure 4 represents data from a single membrane test prepared at saturate the antibody directly in the column. A linear relationship is observed between the signal intensity and the analyte concentration. The lower limit of detection for this test is at 5 ng / ml which corresponds to part levels by billion (ppb).

F II. DISPLACEMENT STUDIES WITH IMMERSION BAT The main purpose of these experiments was to design a qualitative membrane-based immuno test for the detection of an objective analyte in solution. The tests depend on the displacement test to function on the Immunodyne membranes with the fluid flowing through the membranes laterally rather than perpendicular to the membrane as described above. Transported by capillary action, the fluid conducts the analyte in the sample to the immobilized antibody-labeled analyte complex and transports the labeled analyte displaced further onto the membrane strip. The immersion stick displacement test not only depends on the ability of the target analyte to displace the labeled analyte of the immobilized antibody, but also on many other factors such as the speed of the capillary action of the phase F mobile and transport speeds of analyte and analyte marked through the membrane. Figure 5 provides a schematic of the experimental protocol. First, in step (a), strips 100 of a membrane of ABC I mmunodyne® 1 10 were cut which were 30x5 mm or 50x10 mm. A monoclonal antibody specific for TNT (1 1 B3) in concentrations ranging from 2 to 10 nmol / ml was placed in 5 μL drops in the membrane strips and allowed to immobilize for thirty minutes. In step (b), the strips 100 were then soaked, using test tube 12, in a Tris solution for about one leaflet to block any other covalent binding site. A membrane strip wash 100 was followed which consisted of three consecutive exposures to PBS containing 0.01% Triton X-100 to wash off any excess TNT antibody (step (c)). After a final wash with PBS, the labeled analyte of CY5-TN B, in excess of five to thirty times the molar amount of antibody, was applied in 6.5 μL drops in the antibody and incubated overnight (step (d) )). In step (e), after # washed 100 strips in PBS containing 2.5% ethanol, and 1% Tween 20 ™ for ten minutes in order to remove the labeled analyte bound non-specifically. In step (f), before drying, strips 100 were placed in a solution of trehalose dihydrate at 100mM in phosphate buffer for ten minutes. Finally, in step (g) the strips were dried at room temperature. The displacement test 15 (step (h)) was carried out by immersing the end of the membrane strip 100 in solution of TNT 1 14, 16, 18 or 120 (from the concentration specified in Figure 5, step (h)), and allowing capillary action to bring the target analyte to the antibody / analyte complex labeled for displacement. A laser was used of 650 nm (not shown) connected to a fluorescence detector to observe any displaced labeled analyte (Cy5-TNB) in membrane strip 100. In the first experiment, TNT antibody was placed at a concentration of 2 nmol / min. in the center of a membrane strip rectangular 3 x 0.5 cm and saturated by five times of CY5-TN B in excess. The strip was then immersed in a sample solution that # contains 300 ng / ml of TNT. Figure 6a represents this strip when the submerged end is held under the first laser (left side). The highest altiplano indicates the fluorescence of CY5-TNB bound to the immobilized antibody. One shoulder is evident to the right of the highest altiplano, indicating displacement of the labeled analyte of the antibody. Figure 6b shows this same strip optically interrogated in the inverted direction where the submerged end is on the right. Another membrane strip, which also has 2 nmoles / ml immobilized anti-TNT antibody was treated identically and exposed to the same solution of 300 ng / ml TNT. After placing it under laser with the submerged end to the right, the data shown in Figure 6c was obtained. These experiments were carried out by manually moving the strip of membrane on the laser path. "Time" on the x axis refers to the time of scrutiny and has no relation to the test time. Obviously, many modifications and variations of the present invention are possible in view of the above teachings. By Therefore, it should be understood that, within the scope of the appended claims, the invention may be practiced differently than as specifically described herein.

Claims (9)

1 .- A test method to detect an objective analyte, comprising the steps of: 5 providing a porous membrane that has binding elements immobilized therein, each of said joining elements having at least one binding site capable of specifically binding said target analyte; exposing said binding sites to a labeled analogue of the target analyte to form complexes of membrane immobilized binding elements and labeled analytes; flowing an aqueous sample, which is suspected to contain the target analyte, passing said membrane having said complexes therein, at a flow rate which allows the target analyte to displace the labeled analyte from the complexes under non-equilibrium conditions, said velocity of flow that also provides a time of interaction between said analyte and said membrane of about 0.1 second to about 30 seconds; detect the displaced labeled analyte, the amount of said displaced labeled analyte being proportional to the concentration of said target analyte in said sample.

2. The method according to claim 1, wherein the binding element is an antibody.

3. The method according to claim 1, wherein said labeled analog is fluorescently labeled.

4. - The method according to claim 1, wherein said interaction time is not greater than about 15 seconds.

5. The method according to claim 1, wherein said membrane is oriented normal to the direction of the sample flow, such that said sample flows through said membrane.

6. The method according to claim 1, wherein said membrane is a dip stick and wherein said sample flows laterally through said immersion stick.

7. The method according to claim 1, wherein said membrane is not absorbent.

8. The method according to claim 7, wherein said membrane is selected from the group consisting of cellulose, nitrocellulose, silica fiber, aluminum oxide, and polyvinyl chloride.

9. A device for testing an aqueous sample suspected of containing an objective analyte, comprising: a porous membrane having binding elements 20 immobilized therein, each of said joining elements having at least one site of binding capable of specifically binding said target analyte, essentially all of said binding sites on said membrane being occupied by a labeled analog of the target analyte to form complexes of binding elements immobilized on membrane and labeled analytes; means of flow to flow an aqueous sample, which is suspected to contain the target analyte, said membrane having said complexes therein, at a flow rate that allows the target analyte to displace the labeled analyte from the complexes. under conditions without equilibrium to form a processed sample, said flow velocity also providing an interaction time between said analyte and said membrane of about 0. 1 second at about 30 seconds; and EP detection means to detect the presence of said analogue labeled in said processed sample. 1. The device according to claim 9, wherein said labeled analog is fluorescently labeled. The device according to claim 10, wherein said detection means further includes a light source for exciting any fluorescently labeled analog t in said processed sample. 12. The device according to claim 9, wherein said binding element is an antibody. 13. The device according to claim 1, wherein said detection means are further adapted to quantitatively determine the amount of said analogue labeled in said processed sample. 14. The device according to claim 1, wherein said detection means also comprise a n 25 spectrophotometer, infrared spectrometer, fluorimeter or an optical biosensor. 5. The device according to claim 9, wherein said membrane has pores and defines a plane that is normal to the flow direction of the sample, so that said flow means flow said sample through said samples. pores of said membrane. The device according to claim 9, wherein said membrane has pores and defines an immersion stick and said flow means cause said sample to flow laterally through said membrane. 17. The device according to claim 1, wherein said flow means comprise a conduit through which said sample flows by the action of gravity or by manual force. 8. The device according to claim 16, wherein said membrane has pores and said flow means comprise pores in said membrane through which said sample flows by capillary action. 9. The device according to claim 9, wherein said membrane is not absorbent. 20. The device according to claim 9, wherein said flow means are adapted to provide an interaction time between said analyte and said membrane of no more than about 15 seconds. 21. A device for testing an aqueous sample suspected of containing an objective analyte, comprising: a container, said container including an open end for receiving a sample of liquid suspected of containing an analyte, a closed end, a porous membrane that 5 extends completely over the width of said container, and a deposit between said membrane and said closed end, said membrane having attachment elements immobilized therein, each of said joining elements having at least one attachment site capable of to specifically bind to said target analyte, Essentially all of said binding sites in said membrane being occupied by a labeled analog of the target analyte to form complexes of membrane-immobilized binding elements and labeled analytes; means of flow to flow a sample of liquid through Said open end, through said membrane, and in said reservoir at a flow velocity that allows the target analyte to displace the labeled analyte from the complexes under unbalanced conditions, said flow rate providing an interaction time between said analyte and said membrane of about 0.1 20 seconds to about 30 seconds; detection means for detecting the presence of said labeled analogue in said deposit. 22. A device for the immunoassay of an aqueous sample suspected of containing an objective analyte, which 25 comprises: a container, said container including an open end and a closed end; a cap inside said open end of said container can be adjusted in a first position wherein the end 5 closed from said container is at a first distance from said lid and a second position and at a second position wherein the closed end of said container is at a second distance from said lid, said second distance being less than said first distance; A tip for receiving said aqueous sample extending outward from said lid; a hollow needle extending from said cap in a direction opposite said tip; a septum that extends over the width of said container, Said septum placed between said closed end of said container and an end of said needle remote from said cover when said container is seated in said first position, said septum being essentially impermeable to fluid; a porous membrane placed between said septum and said The closed end of said container, said membrane extending over the width of said container, and a reservoir between said membrane and said closed end, said membrane having joint elements immobilized therein, each said joint elements having at least one binding site capable of 25 specifically binding said target analyte, essentially all of said binding sites in said membrane being occupied by a labeled analogue of the target analyte to form complexes of membrane-immobilized binding elements and labeled analytes; a deposit evacuated between said membrane and said closed end 5 of said container; detection means for detecting the presence of said labeled analogue in said deposit; said hollow needle placed to perforate said septum, but not said membrane, when said container is in said second position. 23. The device according to claim 22, further comprising a sample holding means having sample of liquid therein, wherein said perforation of said membrane releases liquid sample from the sample holding means 15 to flow through. of said open end, through said membrane, and in said reservoir.