EP0882230A1

EP0882230A1 - Chemiluminescent assay methods and devices for detecting target analytes

Info

Publication number: EP0882230A1
Application number: EP96940503A
Authority: EP
Inventors: Mary Ann Childs; Joseph A. Mcclintock; Gregory K. Shipman; William P. Trainor; Erick Gray; David Bernstein; David W. Laub; Lyle K. Kimms; Craig Chung
Original assignee: Universal Health Watch Inc
Current assignee: Universal Health Watch Inc
Priority date: 1995-11-17
Filing date: 1996-11-18
Publication date: 1998-12-09
Also published as: AU7736796A; JP2000500568A; EP0882230A4; WO1997019353A1

Abstract

A sampling-test device and a method for its use provide rapid and easy detection of analytes. The device and method utilize chemiluminescence for sensitive detection and should find applications in such areas as the detection of bacteria on surfaces. The test device comprises a sampling portion and a reagent portion. The sampling portion is an adsorbent that collects analytes from a test sample such as a surface or volume of a liquid. The reagent portion comprises an adsorbent material that holds one or more chemiluminescent components such as luciferase enzyme and cofactors in a dry state. The device optionally comprises a movable shield to protect the sampling portion from cross contamination. In a preferred embodiment the sampling portion is swabbed over a contaminated surface. A bacteriolytic solution is then added to the adsorbent and releases ATP from sampled bacteria found there. The ATP diffuses into the reagent portion of the device. As it diffuses, luciferase and other cofactors present in the reagent portion react with it. Light is produced by chemiluminescence in response to bacteria originally present in the sample. Light can be detected, for example, electronically by insertion of the device into a mating light detector box, or chemically, for example, by an optional light detection portion comprised of a film such as an instant photographic film.

Description

CHEMILUMINESCENT ASSAY METHODS AND DEVICES FOR DETECTING TARGET ANALYTES

Field of the Invention

The present invention relates generally to chemilummescent assay methods and devices for detecting target analytes. In a preferred embodiment, the present invention relates to chemilummescent assay methods and devices for detecting bacteria on contaminated surfaces.

Background of the Invention In th"^> course of food processing, food becomes contaminated with bacteria and can spoil. If food is contaminated with pathogenic bacteria or its toxic products and then is ingested without proper cooking, human food poisoning can occur. The ability to reduce bacterial contamination of food is of major importance improving food safety.

Standard culture plate methods for monitoring surfaces for bacterial contamination require a sterile sample collection device (generally a swab or sponge) and suitable culture media which, after inoculation, must be incubated at an appropriate controlled temperature for a minimum of several hours or days.

Unfortunately prior art methods for detecting bacterial contamination are too cumbersome and time consuming for immediate use by untrained workers. In particular, rapid bacteria tests are needed in slaughterhouses and food handling establishments. In these locations one must rapidly determine whether additional cleaning methods are required or whether proper safety procedures have been followed. Bacteria measurements would be a useful component of a "hazards and critical control points program" (HCCP) to monitor and control bacterial contamination. Unfortunately this is often not possible because present methods require several hours or even days by trained laboratory technicians .

One strategy to overcome these shortcomings has been the use of chemiluminescence detection methods to increase sensitivity (and thus decrease t me) of the analytical method One such chemiluminescence method measures adenosme triphosphate (ATP) to indirectly measure bacteria. ATP detection is a reliable means to detect bacteria because all bacteria contain some ATP Chemical bond energy from ATP is utilized the chemilummescent reaction that occurs m the tails of the firefly Photinus . The mechanism of this chemiluminescence r. - ϋ j I 11U U l- i W i~ .J- .J- ^ jiU J- t ^ ^ ^ _\ j_ C-. -j ^- s_- (-J. , i i . , C- . Cl -L ,

1979 Anal. Biochem. 95 194-198) The components of this reaction can be isolated free of ATP and subsequently used to detect ATP m other sources by a reaction that begins with formation of an enzyme bound luciferyl- adenylate complex and free inorganic pyrophosphate and ends with a rapid reaction of this complex with molecular oxygen to produce light, C0₂ and A P.

The traditional method for measuring light has been by photon counting with a photomultiplier tube Photographic film has also been used For example, U.S Patent 4,396,579 describes use of photographic film to monitor chemilummescent reactions. This patent discloses a light detection device inside a closure which admits a cannula whereby fluid is introduced into a compartment A drawback of the described device is that it is complex and requires the addition of fluid in the dark

Luciferm-luciferase reactions of the firefly have been used for detecting a threshold level of microorganisms as described in U S Patents 4,385,113 and 5,366,867 These reported methods, however, suffer a number of deficiencies Lyophilized luciferase-luc fenn reagent is unstable at room temperature during long term storage and is unstable after liquid reconstitution over short time intervals Additionally, after reconstitution, solutions of this reagent display significant emission of light the absence of ATP This background decreases detection sensitivity and persists for several minutes to an hour.

The reagent instability problem was partly addressed by drying luciferin-luciferase reagents separately onto plastic surfaces. However, this solution to the problem requires the transfer of microorganisms from a collection device to a plastic surface . This transfer can occur by adding a solution to the collection device followed by transfer of the solution to a plastic surface that co ta s luc rerase j. c j_ πn e gents. j.n_- c e e in complexity means that the user of the device must be trained in its use.

Unfortunately, this solution to the instability problem lowers sensitivity of the detection method. This solution creates a new problem of incomplete transfer of ATP from the collection device to a separate plastic surface that contains the luciferase-luciferin reagent. Furthermore, this solution introduces a new variable of time between the transfer and the light emission measurement.

Adding reagent at timed intervals causes a further problem because the light emission kinetics become shorter as the light intensity decreases.

The twin problems of timing and reagent instability also plague other chemiluminescence chemistries that have been developed to detect target analytes. For example, U.S. Patent 4,396,579 (Luminescence detection device, Schroeder and Vogelhut) describes a complicated and expensive automated machine that was designed to add chemilummescent reagent at fixed time intervals in order to overcome the problem of light emission kinetics. This machine was unusually complex because of the timing problem and because of the instability of reagents used with it . Thus, there is a need for an assay which utilizes the high sensitivity and speed of chemiluminescence detection but which does not include the above mentioned problems of complexity, timing, reagent instability and high background light emission

Conventional instruments for measuring chemiluminescence, including lummometers and fluorometers, however, are not particularly suited for such an assay device that has a fiat geometry To this end, there is a need for a portable interface readily nterfaceable with a photomultiplier or other known light detector to provide a simple, efficient light intensity reading j_ cύ tne s r --ι g t pe uioCiuScd i. the j_ i_>L copendmg U.S patent application SN. 08/577107, filed December ^2, 1995 entitled SAMPLING-ASSAY INTERFACE SYSTEM AND METHOD, describes a system that fulfills this need, the disclosure of which is incorporated herein by reference

Specifically, the first copendmg application describes a sampling-device holder interface system and a method for performing an assay for a target analyte from a sampling device of the type disclosed in the first copendmg application. The sampling system includes a sampling-device holder interface and a quantifier for converting the output signal to quantifiable data indicative of the amount of the target analytes Specifically, the interface comprises a sampling-device holder and a light detector - means for converting light generated from the sampling device to an output signal corresponding to the amount or intensity of the light generated such as a photomultiplier or photodetector

The interface holds a sampling device, which comprises a container and a sampling strip inside the container. The sampling strip has a sampling portion for introducing a sample, a reading portion containing a reagent for producing a chemilummescent reaction with the target analytes, and a transfer portion connecting the sampling and reading portions foi transferring the sample from the sampling portion to the reading portion The container has an opening to permit introduction of samples to the sampling portion It also has a light transmissive portion, such as a window or opening, visibly exposing the reading portion.

The holder includes a housing and a tray. The housing has at least first and second walls forming a cavity therebetween. One of the first and second walls has an opening or light transmissive window. The tray is received in the cavity and movable between opened and closed positions. The tray has a compartment adapted to seat and support the sampling assay device. The first opening s n c j- y witn the ci π ^" υ txυh iicn ne; tray is in the closed position to enable observation of the reading portion through the first opening. The light detector is connected to the housing, registry with the first opening. The tray has a second opening (or light transmissive window) extending through the compartment, which opening is registry with the reading portion of the seated sampling device. The second opening is registry with the first opening to enable observation of the reading portion through both the first and second openings when the tray is m the closed position.

A second copending application, SN. 08/580096, filed December 22, 1995, SAMPLING -ASSAY DEVICE, INTERFACE SYSTEM, AND METHOD, describes a system similar to that of the second copending application, but has a light source opposite the light detector for illuminating a luminescent fluorescent or phosphorescent agent bound to the target analyte, the disclosure of which is also incorporated herein by reference. The sampling device in that copending application uses a binding agent immobilized to the reading zone to capture the luminescent agent bound to the target analyte

The sampling device disclosed m the first and second copending applications provides a unique means for allowing light detection using known light detection devices. Because the sampling device, however, is, rather flexible, thin, and flat, it can be challenging to remove the same from the tray compartment. The sampling device needs to be removed by prying out with a fingernail or some sharp instrument . One can also turn the interface upside down and drop the sampling device. But in any event, it would be desirable to ease the sampling device removal from the tray, in addition, there is a need to protect the exposed sampling portion from cross contamination. For instance, if the interface or holder is shaken or otherwise turned sideways or upside down, it is possible for the exposed sampling portion to contact the underside cf the housing upper wall, which contact could possibly introduce other samples that made contact therewith. The present invention fulfills both of these needs .

Summary of the Invention Accordingly, it is an object of the invention to eliminate or reduce the complexity of manually measuring and timing reagent additions required for the detection of target analytes.

A second object of the invention is to prevent cross- contamination of samples.

A third object of the invention is to provide enhanced stability for chemiluminescent reagent (s) used in detection of target analytes.

A further object of the invention is to incorporate a light detection means into an assay device and to allow rapid measurement of target analytes at a sample site.

In accomplishing these and other objectives, one aspect of this invention provides a device for conducting a chemiluminescent assay. The device comprises a container, and sampling and reagent portions that are physically associated with the container and with each other. The reagent portion is made from absorbent material into which one or more chemiluminescent reagents have been dried. The device has a light-permeable portion that permits light generated by a chemiluminescent reaction within the container to exit the container. The invention also provides a sampling device for performing an assay for a target analyte with a shield to prevent cross-contamination. According to the present invention, the sampling device has a container and a sampling strip inside the container. The sampling strip has a sampling portion for receiving a sample, a reading portion for holding the sample with a compound that can emit light, and a transfer portion connecting the sampling and reading portions for permitting transfer of the cample from the campling portion to the reading portion. The container has means to permit introduction of the sample to the sampling portion and a light transmissive portion exposing the reading portion. A shield extends from the container adjacent the exposed sampling portion. The shield has a portion extending beyond one end of the container and is movable to and from the sampling portion and can be wrapped around the one end.

Specifically, the container comprises a first layer and a second layer sandwiching the sampling strip. The shield is attached to or integral with the first layer and has means for -permitting the extending portion to wrap around the one end of the container. The wrap around means is preferably a preformed fold or crease, or even a perforation. A tab or handle is attached to or formed integrally with the first layer. Alternatively, the tab can also be attached to or integral with the shield.

The sample introducing means preferably is a first opening formed through the first layer and aligned with the sampling portion. Similarly, the light transmissive layer is preferably a second opening through the second layer and aligned with the reading portion. The second layer preferably also includes a light transmissive member to cover at least the second opening.

The device of the present invention eliminates or reduces much of the complexity associated with prior art assay methods and, as a result, decreases the cost and training requirements for detecting target analytes.

According to one embodiment, the sampling strip is composed of an adsorbent material . In another embodiment, the sampling strip includes a poly-carbonate membrane that is light transmissive.

The sampling device can further include a sample collecting member, which preferably is adsorbent, in contact with the sampling portion inside the container a d l gjie witu t e fix-A o eni . The toαia c collecting member receives the sample and transfers the sample to the sampling portion.

According to one embodiment, the compound is a reagent, preferably an enzyme in a dried state, contained within the reading portion. When the reagent is mixed with the analyte, it produces chemiluminescent light. More preferably, the reagent is luciferase-luciferin in a dried state .

According to another embodiment, the compound, which preferably is a luminescent labeling agent

(phosphorescent or fluorescent) with a binding agent for tagging the analyte, is contained within at least a portion of the transfer portion. The labeling agent glows when it is exposed to light. Preferably, the labeling agent is chelated europium or europium compound. The reading portion contains an immobilized binding agent, such as an antigen, specific to the analyte, for capturing the labeled analyte within the reading portion. In this embodiment, the sampling strip further includes a collecting portion contiguous with the reading portion. This collection portion is designed to absorb any excess liquid containing the labeling compound not coupled to the binding agent. Accordingly, as the analyte immobilized in the reading portion carries the labeling agent that glows when exposed to light, the amount of light produced after exposure to light correlates to the amount of analyte present in the sample. In operation, as the sample contained in liquid travels across the transfer portion, the target analyte will pick up the labeling agent. Other organisms mixed with the labeling compound and the excess labeling compound, however, are not specific to the binding agent. Thus, they will not be captured in the reading portion, but rather will flow through. The target analyte, however, since it is specific to the binding agent, will be captured and remain in the reading portion.

The sampling device according to the present invention is pα 'cic ±άi'iy aαapted ov se wxtπ a sampling-device holding interface, which has a housing and a tray for seating the sampling device. The housing has a cavity for accepting the tray with the sampling device. The cavity is light-light tight when the tray is closed. The housing is connected to a light detector or the like to measure the amount of light generated by the sample. The housing can have also a light source for triggering reaction of the luminescent labelling agent.

In a preferred embodiment, the device of this invention includes a film such as self-developing photographic film. This feature facilitates convenient readout and monitoring.

In accordance with another aspect of this invention, there is p^rovided a convenient method for performing an assay for a target analyte. In this method, the sampling portion of the above-mentioned device is made to contact a sample, for example, by wiping the sampling portion over a surface suspected of containing the target analyte. A carrier liquid is then added to the sampling area, which liquid transports target analyte into the reagent portion. The carrier liquid also re-wets chemiluminescence reaction components located in the reagent portion, and thus allows a chemiluminescence reaction to begin. The instability problem suffered by prior art methods is overcome by providing the chemiluminescent reagent in a dried state within the reagent portion and/or sampling portion. In another preferred embodiment the method is used to rapidly detect bacteria on surfaces such as countertops and equipment used meat or food production In this method, high sensitivity is achieved by incorporating components of the luciferase chemiluminescence reaction m a dried form into the reagent portion A bacteπolytic agent ( e . g . , a detergent) m the carrier liquid lyses bacteria that have been collected the sampling portion ATP liberated by

^{. . . ι} -- Lii ii reaction to produce light. The advantage of rapid and sensitive detection of bacteria can be realized, for example, through sensitive light detection with a photomultiplier or high speed film that is physically part of the device container.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled the art from this detailed description Brief Description of the Drawings

Figure 1 is a top perspective exploded view of a device m accordance with this invention.

Figure 2 is a top perspective exploded view of another device accordance with this invention depicting the positioning of a fibrous adsorbent sample portion .

Figure 3 is a top perspective exploded view of another device in accordance with this invention comprising a carrier liquid and a photographic film detection means.

Figure 4 is an exploded view of a sampling device accordmg to one embodiment of the present invention. Figure 5 is an exploded view of a sampling device accordmg to another embodiment of the present invention

Figure 6 is a schematic side view of the present sampling device showing the manner which the shield operates . 5 Detailed Description of the Preferred Embodiments

The present inventors discovered that, for a chemiluminescence system, the luciferase enzyme and necessary cofactors could be dried onto a porous adsorbent material and subsequently reconstituted m an

_- -; u b α^ _ ^ _- i -- LL. l j. α cin-O ϋliiLi αnαl i- c o . ■->. x \— u v e J- , u uu present inventors discovered that such reagents could be combined into a convenient device that included a sampling portion and a reagent portion.

In its most basic form for the detection of analytes

15 from surfaces and liquids or other areas where analytes can be found, the process of this invention comprises three steps- 1) contacting the surface, liquid, or other area suspected of containing the analyte by swabbing with the sample portion; 2) applying a carrier liquid to the

20 sample portion to wash target analyte from the sample portion to the reagent portion and, at the same time reconstitute dried reagent, and 3) detecting lignt produced by chemiluminescence m response to the presence of target analyte a sample It is noted that step 2

25 (application of a carrier liquid) may not be needed if the target analyte to be tested is m a liquid.

A wide range of target analytes can be detected by the invention In fact, the sampling portion of the device can collect virtually any type of target analyte

30 not only from physical contact with a solid but also from a fluid applied externally.

A target analyte as used herein, is a molecule such as a protein, cell metabolite or microorganism such as a prokaryotic cell, virus, microplasma or free living

35 eukaryotic cell The target analyte can be for example, introduced to the sampling portion of the device by application of a fluid by means of, for example, an eye dropper or other dispenser, or by brief immersion of the device m a fluid or stream of fluid. Alternatively the target analyte can be introduced by physical contact such as by swabbing a suspected contaminated surface with the device . In one preferred embodiment of the invention, suspect surfaces such as countertops, kitchen utensils and slaughterhouse machinery are tested for bacterial contamination by swabbing the suspect contaminated surface with the sampling portion of the device. nit. a pxi g portion xt> Oi an aasi--.ru xt material. The adsorbent material may be fibrous, such as glass fiber, cotton, dacron, or paper, and it may be porous, such as porous polyethylene or sintered glass

The sampling portion is in proximity to or physical contact with a reagent portion such that fluid applied to the sample receiving portion will enter the reagent portion.

The reagent portion also is comprised of an adsorbent material. This material may be fibrous, such as glass fiber, cotton, dacron, or paper and the like, and it may be porous, such as porous polyethylene or sintered glass and the like. The porous material is generally flat and can be m various shapes, including rectangular, or narrow in the middle and wider at each end. The shape provides for diffusion and mixing of reagents and, advantagecαsly, allows a maximum exposure of surface area to the light detection means. Those skilled m the art will recognize many useful materials, such as those used in chromatographic-type assays currently available. The reagent portion often will be only part of a porous adsorbent strips shown m the Figures . The reagent portion can contain one or more reagents for the chemilum scent reaction, generally m dried form. Additionally, it can contain other reagents useful for the assay including, for example, the detergent or other bacteπolytic reagent necessary to extract ATP from bacteria. The reagents can be mixed together the reaction portion, or placed sequentially so that the diffusing fluid contacts the reagents sequentially.

The chemiluminescent reaction may occur within the reagent portion or may occur downstream of the reagent portion (where the chromatically moving reagents will be) , once the reaction has become sufficient to generate detectable light. The location on the adsorbent material for detecting the reaction will depend upon such factors as the type and amounts of reagents, type of adsorbent iiLdL ^' tl , ei-L. A s Lisi tLui lutαLiuii S LL Lilt; ddt>ui.bc:ιiL for detecting the reaction will be easily determined.

A portion of the device is at least partially transparent to the light emitted by the chemiluminescent reaction. This can be achieved, for example, by using transparent plastic for the device although other means, such as windows or sonic welded transparent portions are suitable. Particularly suitable is the use of a transparent covering over a surface of the reagent portion of a strip so that light can be detected at a location distant from a sample application area.

A wide variety of chemiluminescent chemistries can be used in the devices and methods of this invention. Acceptable chemiluminescence chemistries include, among others, the reaction of hydrogen peroxide with horseradisn peroxidase labelled antibodies and luminol , enhanced horseradish peroxidase, reactions that include the use of diacylhydrazides, acridinium salts, dioxitanes, and bioluminescent reactions involving cofactors such as reduced nicotine adenine dinucleotide in the case of marine bacteria.

A particularly preferred chemiluminescent chemistry is the firefly ATP assay which utilizes luciferase and at least one cofactor to generate light from ATP that is present in a sample. At least one chemiluminescent reaction reagent is present in the reagent portion adsorbent material in a dry state. During preparation of the reagent portion adsorbent material a reagent may be conveniently applied as a wet water solution and dried during manufacture or it may be applied a dry form, such as a powder or suspension in a organic solvent or slurry. Other methods are known in the art and the preferred one is determined by characteristics of the reaction components.

Acceptable carrier liquids include, among others, buffer, buffer with detergent, water, blood and urine Buffer solutions of TRIS, HEPES buffers at pH 7 0 to 9.0, and most preferably HEPES buffer at 7 8 with EDTA arc preferred EDTΛ is a preferred ingredient because ΛT? degrading enzymes require divalent metal cations for activity and EDTA chelates these.

In an optional embodiment, detergent is present in the sample or reagent portion, or elsewhere the porous adsorbent material that contains the reagent portion The detergent dissolves fluid that is added to the device and it serve to open cells and liberate cell components

An optional further use of the carrier liquid is to release one or more substances from microorganisms the sample portion In this embodiment the carrier liquid lyses any collected bacteria m the sampling portion and releases ATP from the bacteria into the solution for transport into the reagent portion Detergent can be included the carrier liquid for this purpose.

In an advantageous embodiment the carrier liquid is present a reservoir that is present in the assay device For example, the reservoir can be positioned on the device adjacent to a flexible area (e.g., hinge, or integral hinge) The flexible area permits the reservoir to be positioned proximate the sampling portion In this way, after the sampling portion contacts the target analyte, the portion of the device containing the reservoir can be contorted or folded over so that the reservoir is positioned proximate (e g , directly on top of) the sampling portion The reservoir then can be broken (e.g. by finger pressure) so that its contents are released directly on the sample portion In this embodiment, therefore, there is no need for additional, separate containers for reagents since all reagents are present in the test device.

Several suitable detergents or combination of detergents are known to those skilled in the art and include, nonionic detergents such as Triton X-100. Nonidet P40, n-Undecyl Beta-D glucopyranoside, Zwitteriomc detergents such as n-hexadecyl-N, N-dimethyl- 3-ammonιo-l- propanesulfonate, and cationic detergents

chloride, cetyldimethyl-ethylammomum bromide, dod e c y 1 t r l me t hy 1 - a mmo n l um bromide , and cetyltπmethylammonium bromide. The concentration of detergent solution varies for each type of detergent and can range from 0.1% to 6%, and preferably from 0.5% to 2.0%.

The container itself should be constructed of liquid impermeable material, such as a plastic. The container can be made, for example, by molding a single piece of plastic into a shape that can house the adsorbent material of the sampling and reagent portions. Alternatively, the container can be constructed from multiple elements that are sealed to provide a liquid impermeable seal. The container optionally may have an opening to enhance the diffusive or chromatographic flow of liquid along the adsorbent material therein Alternatively, an air pocket within the container can be provided for such purpose .

The step of detecting chemilum escent light emitted from the container can be accomplished by a number of means known to those skilled m the art. Chemilum escent light can be detected electronically by, for example, a photomultiplier, photo diode, photo fet or charge coupled device. The most preferred electronic light detector is a photomultiplier because of its sensitivity.

Chemilummescent light also can be detected chemically, for example by the use of a film. Particularly preferred is high speed photographic film such as Polaroid #612 which has a speed equivalent to ASA 20,000. Those knowledgeable in the areas of films will appreciate many other types of suitable films. 5 The intensity of light emitted from the chemiluminescent reaction obeys an inverse square relationship to distance following Lambert's Law. Therefore, if a light detector is used, detection sensitivity is optimized by placing the detector as close x _/ U ϋ -, -_J _- X <-- cA __- ι L iX L. -'m Oiic Ii -' -J - i iu j- α - C rtπci c the reaction occurs.

For light detection with electronic readout methods, the device is may be inserted into a complementary fitting dark chamber wherein a light detector means is

15 proximal to the test area and distal to the sampling area of the device. This latter means overcomes the complexity limitation of some of the prior art in that no timed or measured reagent addition in the dark is required. This lowers the cost and improves the

20 convenience of the detection device and method.

Referring now to the Figures, several embodiments of this invention are provided.

Figure 1 depicts a basic form of the sample-test device 102 in which a sample portion and reagent portion

25 are combined as one part. The first element 104 comprises an impervious material such as a plastic film having an opening 106. The second element 107 is an adsorbent material that has dried reagent in it . An area distal from the sampling portion 108 is a detection area

30 110. Light is preferably detected from detection area 110 although, if desired, light can also be detected from other parts of the device. During operation, the sampling portion is contacted with an area suspected of containing the target analyte. Then, a carrier liquid

35 (if necessary) is added to the sampling portion and carries with it any analyte into the second element, and mixes the analyte with the chemiluminescent reagent within the second element 107. The reagent diffuses into the detection area 110. For example, dried luciferase reagent can be present throughout the second element 107 between sampling portion region 108 and detection area 110. 5 The third element 112 is comprised of an impervious material such as plastic film and is light permeable at least region 114 directly below the detection area 110. The light permeable region 114 can be positioned close to a light detector. The adsorbent material of the

-* -_ _, ~ _r. -1 _ " _ ___ι -_, "i π *7 . ~ — ^ ^. . _nu . i _ _ ,_ , , _. _ _, , ! -.. _. .' -ι _ _ _- -

_ι_ u L x v_-i[iLjι _ __ U , x ->u44_4W . _.IlCu __/<_ -, γv t_- .--± _ i- - L i - x cJl - X o

104 and 112 which are sealed together, for example, by heat, adhesive, or physical means that retain the sides of the two elements clamped together.

Figure 2 depicts another device in accordance with

15 this invention 202 that comprises separate sampling portion and reagent portion adsorbent materials. The first element 204 comprises a liquid impervious material such as a plastic film having an opening 206. The sampling portion 208 is comprised of fibrous adsorbent

20 material. The sampling portion 208 is positioned directly under opening 206 between the top impervious layer 204 and one end 210 of a porous absorbent material 211. Reagent portion 212 is wider at each end and narrow m its middle (although many other configurations are

25 possible) . Element 214 comprises a liquid impervious material which is light permeable, at least in the area where chemilummescent light will be emitted from 211. Although not shown in the figure, one or more holes may be present m elements 204 and 214 near end 216 m order

30 to facilitate the passage of fluid away from sampling portion 208.

Figure 3 depicts another device in accordance with this invention that is useful for detecting the presence of bacteria. The device comprises separate sampling

35 portion and reagent portion adsorbent materials as shown Figure 2, but additionally comprises a carrier liquid (located n reservoir 308) and instant photographic film light detection means. The first element 310 comprises a liquid impervious material such as a plastic film having an opening 304 and a reservoir 308 with a surface 312 attached at flexible area (e.g. , hinge) 310. Sampling portion 306 is comprised of fibrous adsorbent material, and is positioned directly under opening 304 between the top impervious layer 310 and one end 314 of porous absorbent material 316. Reagent portion 318 is wider at each end and narrow m its middle (although many other ^JT_ ___ ,-_----^, _c ^,_^.-_^, _αrc possible) . Another clement 320 comprises a liquid impervious material which is at least partially light permeable. Although not shown m the figure, one or more holes may be present in element 310 and 320 in order to facilitate the passage of fluid away from sampling portion 306.

The device of Figure 3 also comprises negative film layer 322, compressing bar 324, developing gel container 326, and positive print film 328 with attached tab 330. These components allow detection of chemilummescent light by instant photographic means.

In using the assembled device, finger pressure is placed on backing 302 in an area behind opening 304 through which exposed adsorbent of sample portion 306 is used, for example, to wipe the surface of a test area. After wiping, the portion of the device comprising carrier liquid reservoir 308 is folded over at flexible area 310. Pressure is applied to reservoir surface 312 which causes the reservoir to break and release the carrier liquid from the reservoir and into sample portion 306.

The carrier liquid comprises a bacteπolytic agent that releases ATP from any bacteria present in the sampling portion 306. The ATP diffuses through sampling portion 306 and into absorbent material 316 at point 314. The solution diffuses through the body of the adsorbent strip where it rehydrates dried chemilummescent reagent. Any ATP present in the carrier liquid reacts with the rehydrated chemilummescent reagent present in portion 318 and light is emitted

Light from chemiluminescence passes through light- permeable element 320 and enters negative film layer 322. 5 After appropriate incubation time for exposure of the film, compressing bar 324 is moved across the device from the direction of point 314 to detection area 318, and compresses developing gel container 326. This forces developing gel from the developing gel container 326 x α t_ 1 _->--> ir. SJ -- x ι_ x v c_ print film 328 After an appropriate development time, tab 330 s pulled and the positive print film is viewed to determine the presence or absence of the target analyte (bacteria)

15 The sampling features of the invention are illustrated in Figures 4 through 6 For convenience, the same or equivalent elements m figures 4-6 of sampling the embodiments illustrated have been identified with the same reference numerals.

20 Figure 4 illustrates a first embodiment of a sampling assay device 400 accordmg to the present invention, which device is particularly adapted for use with the interface described m the second copendmg application. Figure 5 illustrates a second embodiment of a sampling

25 assay device 400 accordmg to the present invention, which device is particularly adapted for use with the interface described the third copending application The device 400, 400' includes a housing 410 for containing a sampling strip 440, 410' , which has a

30 sampling portion 442, 442' for receiving a sample, a reading portion 444, 444' for emitting light, and a transfer portion 446, 446' for transferring the sample to the reading portion The housing 410 is defined by a top layer 420 and a bottom layer 430, each having an opening

35 422, 432 m alignment respectively with the sampling portion 442, 442' and the reading portion 444, 444' .

Each of the top and bottom layers 420, 430 is preferably composed of a thin liquid impervious material, such as a plastic film. The sampling strip 440, 440' is sandwiched and retained between the top and bottom layers 420, 430, which can be sealmgly joined together, for example, by heat, adhesive, ultrasonic welds, or any physical means that retain layers together while containing the sampling strip therein. A single film sheet can also be folded to form the upper and lower layers. Alternatively, a housing with three pre-sealed sides can also be used. -j J.ic otto.Ti u/ci J rcj-t α y iiicluoes ct --jeal ng film 450 positioned underneath as shown Figures 4 and

5. This film 450 is preferably light transmissive

(clear) to transmit light emitting from the sampling portion. The film 450, however, could be structured so 5 that only the region directly below the bottom layer opening 432 is clear. Although Figures 4 and 5 show the film 450 positioned below the bottom layer, it can also be positioned above the bottom layer, below the sampling strip 440, 440' . The sealing film 450 can be omitted 0 altogether if the bottom layer, at least the portion below and m registry with the reading portion 444, 444' , is formed of a clear material to provide a window for the reading portion In this regard, the entire bottom layer could be made of a clear material if desired Even ust 5 the opening 432 could be covered with a light transmissive material, from either above or below the bottom layer 430. The sampling portion 440, 440' is positioned directly under the top-layer opening 422, sandwiched between the top layer 420 and the bottom layer 0 430.

In the embodiments shown, an optional sample collecting member 460 is sandwiched between the top layer 420 and the sampling portion 442, 442' , with the collecting member exposed to the top layer opening 422 5 The sampling portion is thus accessible through the envelope 410 The collecting member is preferably adsorbent, which is preferably composed of fibrous material, such as glass fiber, cotton, dacron, or paper,

SUBSTTI UTE SHEET (RULE 26) and it may be porous, such as porous polyethylene or sintered glass.

In the first embodiment, the sampling strip 440 is also composed of a similar adsorbent material, which may be fibrous, such as glass fiber, cotton, dacron, or paper and the like, and it may be porous, such as porous polyethylene or sintered glass and the like. An ordinary skilled artisan will recognize many useful materials, such as those used in chromatographic-type assays

In the second embodiment, the sampling strip 440' preferably is made of a similar material as on the first embodiment, particularly a material that permits lateral liquid flow, such as a bibulous, adsorbent, or hydrophilic membrane. At least the reading portion, however, is light transmissive so that it transmits light. A poly-carbonate membrane suits this requirement. The transfer portion 446' and the sampling portion 442' can be made of glass fiber for example. An ordinary skilled artisan will recognize many useful materials that permit lateral liquid flow and are light transmissive.

The sampling strip 440' also has a collecting portion 448 for absorbing excess liquid and sample components not immobilized in the reading portion 444' . In this regard, the collec ing portion preferably is made of an adsorbent or absorbent material such as paper, cellulose filter paper, etc. If the entire sampling strip is formed of the light transmissive material, such as a polycarbonate membrane, the collection portion 448 preferably includes an absorbent layer, such as cellulose paper to wick away excess liquid away from the reading portion.

The sampling and reading portions 442, 442' and 444, 444' are preferably wider than the transfer portion 446 connecting these wider portions, although many other configurations are possible. The sampling and reading portions can come in various shapes, including circular (as shown) , rectangular, or triangular. The shape can be maximized J or particular needs. In both embodiments, the sample collecting member 460, if used, is preferably m physical contact with the sampling portion 442, 442' to maximize liquid transfer In the first embodiment, the reading portion 444 contains one or more chemilummescent reagents, preferably a dried form to produce a chemilummescent reaction with the target analyte. Additionally, the reading portion can contain other reagents useful for the assay including, for example, the detergent or other agent for extracting ΛTP fro"¹ bacteria.

A wide variety of chemilum escent chemistries can be used with the present sampling device Acceptable chemiluminescence chemistries include, among others, the reaction of hydrogen peroxide with horseradish peroxidase labelled antibodies and lummol , enhanced horseradish peroxidase, reactions that include the use of diacylhydrazides, acridimum salts, dioxitanes, and biolum escent reactions involving cofactors, such as reduced nicotine adenme dmucleotide m the case of marine bacteria. A particularly preferred chemilummescent chemistry is the firefly ATP assay, which utilizes luciferase and at least one cofactor to generate light from ATP present m the sample

Reagents may be conveniently applied as a solution and then dried or they may be applied m a dry form, such as a powder or suspension m an organic solvent or slurry other methods are known m the art and the preferred one can be determined by characteristics of the reaction components desired In the second embodiment, the transfer portion 446' further has a labeling portion 447 adjacent or near by the reading portion This labeling portion 447 contains a luminescent (fluorescent or phosphorescent) labeling agent, such as chelated euiopium or europium compound (phosphorescent) or phycobiliprotems (fluorescent) for labeling the analyte

Specifically, the labeling portion 447 contains a conjugate (typically two molecules held together by one or more bonds) of a luminescent reporter molecule (agent) and a first binding agent that binds the target analyte. The luminescent agent can be either fluorescent such as, for example, fluorescem or a phycobiliprotem, or phospnorescent, such as a chelated europium or europium compounds. The "reporter molecule" for purposes of this invention can include larger associations of molecules and atoms such as liposomes filled with fluors or enzymes, and particles such as gold or polystyrene coated

The first binding agent binds to the target analyte m a solution. A preferred first binding agent is an antibody although other substances that can bind a particular antigen such as, for example, protein A (antibody antigen) , lectm (glycolipid or glycoprotem antigen) , streptavidin (antibody antigen) , avidm (biotin target analyte) , and hormone or trophic factor (cell surface receptor) .

The first binding agent and luminescent reporter molecule are conjugated by any of a number of techniques known to an ordinary skilled artisan. In practice, the conjugate formed from a first binding agent and a luminescent reporter molecule is placed into the labeling portion 447 by preparing a solution of the conjugate by spotting the solution onto labeling portion 447. The solution is then dried. Depending upon the composition of the labeling portion 447, however, it is preferred to include a carrier protein such as bovine serum album or milk in the solution to help prevent the conjugate from binding to the labeling portion 447. Detergent such as T EEN-20 or TRITON X-100 can also be included to prevent non-specific binding of the conjugate to the labeling portion

It is ⁱmportant to introduce detergent or other agent for releasing the target analyte or to releasing the labeling agent from the labeling portion before the labeling portion 447. Preferably, they are introduced with the carrier or buffer The labeling agent does not spontaneously react with the target analyte, but rather attaches thereto and glows when it is exposed to light. It thus needs to be triggered. This advantageously enables selective light 5 measurement to be taken at anytime between 2 to 60 minutes after sampling

As the sample m liquid, such as carrier liquid or buffer, travels toward the reading portion, it releases the labeling agent, which has a specific binding agent

-I 4- _, ,-, _ "I „ *- — , rp^' -. "l „. _Λ ,- ^~l _ ,__ ,-. _ _, _ —, *_ j_ -, α i iu v L l uuc l i iiy y t-li , however, will attach only to the target analyte. The carrier liquid now with the tagged or labeled target analyte (and any excess labeling agent not tagged with the target analyte) continues into the reading portion

15 The reading portion 444' contains a second binding agent, which could be same as the first, such as an antibody or antigen complementary to the target analyte to capture the labeled analyte within the reading portion. This time, however, the second binding agent is

20 bound to the base, the light transmissive member, εo that it does not flow out of the reading portion. Thus, the labeled analyte remains immobilized within the reading zone .

Specifically, the second binding agent functions to

25 bind to the target analyte and prevent or significantly slowing its movement. The second binding agent is preferably an antibody but any substance that can bind the target analyte, is suitable The primary difference between the second binding agent and the first binding

30 agent is that the second binding agent is immobilized to, for example, the polycarbonate member such that the target analyte that flows over or through the polycarbonate can react with the second binding agent to become immobilized there

35 Immobilizing the second binding agent can be achieved by a number of methods known to the ordinary skilled artisan For some membranes and support materials, non¬ specific absorption is sufficient For others, and foi polycarbonate membranes, the second binding agent can be indirectly immobilized through an intermediary material such as latex articles or deπvatized glass particles, which are commercially available. For example, an antibody can coat latex and deπvatized particles by non¬ specific absorption, followed by washing and blocking with a second protein in excess, such as bovme serum albumin The coated latex or glass beads can then be immobilized within the polycarbonate membrane by applying t iC α _- v_ OoT x c± S -- p __nS lθr_ t O t -ixβ u y Cα l nα membrane a solution form.

The collecting portion 448 absorbs excess liquid containing the labeling agent that has not been coupled to the second binding agent m the reading portion. Acceptable carrier liquids include, among others, a buffer solution or a buffer solution with detergent. Buffer solutions neighboring in the pH range of 5-10, and more preferably 6-8 (neighboring neutral pH) and compatible with the labeling agent can be used, such as TRIS, HEPES. Detergent, which is preferably present m the carrier liquid, dissolves in liquid that is added to the sampling device and serves to improve flow as surfactants at one concentration, and if necessary, to solubilize the cell wall or organism for release of antigen or the element to be detected. Several suitable detergents or combination of detergents are known to those skilled m the art and include, nonionic detergents such as TRITON X-100 and NONIDET P40 The concentration of detergent solution varies for each type of detergent and can range from 0 01% to 6%, and preferably from 0.5% to 1.0%

In both embodiments, carrier liquid or buffer is preferably introduced into the sampling portion and preferably includes an agent for facilitating the detection.

In the first embodiment, the agent releases ATP from any bacteria present the sampling portion Acceptable carrier liquids include, among others, a buffer solution or a buffer solution with detergent. Buffer solutions of TRIS, HEPES buffers at pH 7.0 to 9.0, and most preferably HEPES buffer at 7.8 with EDTA are preferred when used with firefly luciferase from Photinus pyral i s (first embodiment) . EDTA is a preferred ingredient because ATP degrading enzymes require divalent metal cations for activity and EDTA chelates these. The pH of the chemiluminescent reaction determines the ratio of the 562 nm peak to the 616 nm peak emission associated with

616 nm peak is at a maximum and there is no 562 nm peak. The emission spectra change with pH due to the protonation of the oxyluciferin molecule (the light emitter) . Oxyluciferin as a dianion emits a yellow-green light (562 nm) and as a monoanion it emits a red light (616 nm) .

In the first embodiment, detergent also can be present in the sampling or reading portion or included with the carrier, which detergent dissolves in liquid that is added to the sampling device and serves to open cells and liberate cell components. Several suitable detergents or combination of detergents are known to those skilled in the art and include, nonionic detergents such as Triton X-100, Nonidet P40, n-Undecyl Beta-D glucopyranoside, zwitterionic detergents such as n- hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate , and cationic detergents such as alkyltrimethylammonium bromides, benzalkonium chloride, cetyldimethyl- etiylammonium bromide, dodecyltrimethylammonium bromide and cetyltrimethylammonium bromide. The concentration of detergent solution varies for each type of detergent and can range from 0.1% to 6%, and preferably from 0.5% to 2.0%.

In a second embodiment, acceptable carrier liquids include, among others, a buffer solution or a buffer solution with detergent. Buffer solutions neighboring in the pH range of 5-10, and more preferably 6-8 (neighboring neutral pH) and compatible with the labeling agent can be used, such as TRIS, HEPES

In the second embodiment, detergent, which is preferably present the carrier liquid, dissolves m liquid that is added to the sampling device and serves to improve flow as surfactants at one concentration, and if necessary, to solubilize the cell wall or organism for release of antigen or the element to be detected. Several suitable detergents or combination of detergents

nonionic detergents such as Triton X-100 and Noni et P40 The concentration of detergent solution varies for each type of detergent and can range from 0 01% to 6%, and preferably from 0.5% to 1 0% Devices for detecting intensity of luminescent light

(including chemilummescent, fluorescent or phosphorescent) emitted is generally known It is preferable, however, to use the interface described in the first and second copendmg applications In this regard, a hield 470 is attached to or made integral with the top layer 420 The shield preferably extends beyond the proximal end 424 of the sampling device 400 and preferably has a preformed crease or fold 472 m line with the proximal end so that a portion 474 extending beyond the proximal end 424 can easily be tucked unαerneath the bottom layer 430 or the sealing f lm 450, as shown in Figure 6

Referring to Figure 6, the sampling device preferably comes packaged in a sealed envelope with the shield m the folded condition as shown solid This will maintain the shield 470 m place when seated m the tray, for example, even when the tray is opened The shield 470 also is preferably composed of a thin liquid impervious material, such as a plastic film and it can be attached A the top layer by any conventional means, such as heat (melding) , adhesive, ultrasonic welds, etc The attachment area 476 forms a hinge 477, which is defined by the attachment area demarcation line to allow the shield to move or pivot thereabout. If the hinge is made of a relatively rigid material, the hinge should be made so that it preferably does not have any positional retention. Sometimes, however, it may be useful to bias the shield toward the closed position A as shown in Figure 6, where the shield is substantially parallel to the housing 410. This may be achieved by playing desired creases or folds, even along the hinge 477.

As shown in Figures 4-6, a tab or handle 480 is

removal of the sampling device 400, 400' from a tray for example . The tab also can be formed of the same material as the shield. The tab can be attached to the shield 470 or even to the top layer 420. It is, however, preferable to attach the tab with the shield, more preferably to the attachment area 476 as shown. The tab and the shield are preferably attached together in a single step, for instance by ultrasonically welding these members to the top layer 420 before the sampling strip 440, 440' is sandwiched between the top and bottom layers.

To ease the access to the tab, the tab is preferably biased in the upright position, away from the upper layer or the shield as shown in Figures 4-6. This advantageously serves another purpose when inserted in the interface described in the first and second copending applications - the wall underneath the upper housing where the tray is inserted pushes the tab down and holds the sampling device in place. It thus can prevent the sampling device from jumping around in the interface. In operation, the sampling device 400, 400' preferably comes packaged in another sealed container or envelope (not shown) . Once the sampling device 400, 400' is taken out, sampling can begin. The shield 470 is moved back from the position A toward the distal end 426 of the sampling device, toward the position B shown in Figure 6 to expose the sample collecting member 460. The collecting member is then exposed to the target area, for instance, by contacting or swabbing the suspected surface, liquid, or other area suspected of containing the analyte with the member 460 Alternatively, the sample to be tested can be directly introduced to the sample collecting member 460. Then, carrier liquid is introduced to the member, if needed, to wet the target analyte and move any analyte present to the reading portion. If the target analyte to be tested is m a liquid form, then the carrier liquid may not be necessary but s preferred Finally, the degree of luminescence X x c- p i OuuC cu I n i c opOliS c uO c ue pj. c 5ch _-c u l Lαi e c analyte tne sample is detected, using for example the interface disclosed m the second and third copending applications, where the degree of light detected correlates to the amount of analyte present The target analyte can be introduced by physical contact such as by swabbing a suspected contaminated surface with the device or by introducing the sample m liquid form by, for example, an eye dropper or other dispenser, or by brief immersion of the device in the liquid to be tested.

If wiping or swabbing is carried out, then finger pressure is preferably placed on the bottom layer 430, behind the opening 422 to more fully expose the sample collecting member 460 to the sample. In the first embodiment, the applied carrier liquid diffuses through the sampling strip, where it, upon reaching the reading portion, rehydrates the dried chemilummescent reagent contained therein Any ATP present m the carrier liquid reacts with the rehydrated chemiluminescence reagent present m the reading portion to spontaneously emit light.

In the second embodiment, the applied carrier liquid carrying the sample diffuses through the transfer portion and passes through the labeling portion, which contains luminescent agent attached to a binding agent that is complementary with the target analyte As the carrier liquid passes therethrough, the liquid releases the labeling agent, which attaches only to the target analyte The labeled or tagged analyte, along with the excess released labeling agent, now moves into the reading portion 444' , which also contains an immobilized binding agent (which could be same as the binding agent m the labeling portion) that is complementary to the target analyte. Here, the binding agent captures the target analyte, which carries the labeling agent, while the non-complementary elements, including excess labeling agent, (excess) pass through the reading zone The

the reading portion 444' . Whereas the chemilummescent reaction in the first embodiment provides a spontaneous light emission upon mixing the chemilummescent reagent with the target analyte, the luminescent reaction m the second embodiment does not produce light until it is first triggered with light exposure The labeling agent associated with the captured target analyte glows or emits light when exposed to light The amount of analyte present can be calculated based on the amount of light exposed and collected thereafter Accordingly, the reading phase can advantageously take place at a later time, about between 2 to 60 minutes depending upon the target analyte Thereafter, luminescence light produced response to the presence of target analyte the sample can be triggered and read, as described m the third copendmg application

Once the sampling has been made, the shield 470 is placed over the sampling portion, with the extending portion 474 folded below the bottom layer 430 or the sealing film 450 if used, as shown by the position A m Figure 6 This prevents the sampling portion from accidentally receiving any additional sample or contamination. The sampling device 400 can then be positioned in the interface Because the shield is implemented, any accidental cross-contammation between samples within the interface is eliminated After the reading is completed, the sampling device can easily be removed by conveniently grabbing the tab and lifting the sampling device out of tray.

Given the disclosure of the present invention, one versed m the art would readily appreciate that there may be other embodiments and modifications well within the scope and spirit of the present invention. Accordingly, all expedient modifications readily attainable by one versed m the art from the present disclosure within the scope and spirit of the present invention are to be

The following example is presented by way of illustration and not by way of limitation.

EXAMPLE 1 A cellulose filter paper (Scientific Products Filter paper Grade #361) is cut into a strip 10mm x 35mm Luciferase-luciferm (Analytical Luminescence Systems) is reconstituted w th a solution of 5% alpha-D- Glucopyranosyl alpha-D-glucopyranoside m 0 05M Dithiothreitol . Fifteen microliters of the Luciferase- luciferm solution are pipetted at approximately 15mm from the distal end of the filter paper strip and dried m vacuo

An adhesive coated plastic is cut into a 12.5mm x 40mm strip. A 8.0mm diameter hole is punched out at the proximal end of the strip The perimeter of the hole is centrally located approximately 3 5mm from the proximal end of the strip.

An adnesive coated translucent plastic strip is cut into a 12.5mm x 40mm strip. With the adhesive side of the translucent plastic strip facing up, the filter paper strip is positioned wherein the edge of the distal end of the filter paper is 2.5mm from the distal end of the translucent plastic strip and the proximal portion of the filter paper is 2.5mm from the proximal end of the translucent strip. A plug of dacron fibers approximately 10mm m diameter is centered close to but not extending beyond the edge of the proximal portion of the filtei paper strip The adhesive coated plastic strip with adhesive side facing downward is positioned directly over the translucent strip and oriented so that the punched hole is directly over the dacron plug Pressure is applied to the device to seal the adhesive backings together.

An individual Polaroid film packet (#612 ISO 20,000) is cut down into a 12 5mm x 20mm rectangle and glued to the proximal undersurface of the device, becoming an integral part of the device forming a dark chamber

portion of the filter paper

The device is held between the fingers with dacron side facing the surface to be monitored for bacteria Finger pressure is applied on the back side of the device directly behind the sampling area, and the dacron sampling portion is used to wipe a defined area One hundred and fifty microliters of an extraction buffer that cont-iin 0 05M HEPES buffer pH , 8 and 0.5% Cetyldimethyl-ethylammonium bromide is added to the punched out hole containing the dacron sampling area The extracted ATP, if present, diffuses across the porous filter, the luciferase-lucifeπn reagent dissolves, and light is emitted as the solution flows across the distal portion of the filter paper A film is exposed to light emitted over a given period of time at the test area After 2 minutes, the film is developed and peeled back from the device to reveal a white spot if positive, or no white spot if negative

EXAMPLE 2 In this example, strips were made and used as described for Example 1 except that luciferase and lucifeπn were separately applied to the paper strip Three ug Firefly Luciferase dissolved 8 microliters of 0 05M HEPES buffer pH 7 8 , 0 025M dithiothreitol , 0 05% Triton X-100 as described above were applied and dried onto the distal ends of 10mm by 35mm cellulose filtei paper strips (Whatman #1) Three ug of sodium D luciferm (Sigma Chemical Company, St Louis, Mo.) in water were applied and dried onto the proximal ends of each strip. Various dilutions of fresh bacterial suspensions in broth were reacted with equal volumes of bacterial releasing agent (0.4% chlorohexidine 5 digluconate, 0.1% hexadecyltrimethylammonium bromide) . Each extract was absorbed by a dacron absorbant swab and transferred to the proximal portion of a cellulose filter paper strip that had been impregnated with luciferase and luciferin. Each test strip was then inserted into a i n -ι ,,_r~._ __^--._τ.-_^ - -.-- /π_ ; ... _^._{^ ^}^ ττj_ -,i x τ,ι--4- -,' - ._^ ~ ^--_ -. ,,,-, -; - τr .3 .

The amount of relative light units emitted were integrated for 10 seconds and background values were subtracted.

The data of Table 1 indicates that the detection 15 limit for Z . Coli was found to be well below 1000 cells.

Table 1

E. Coli Cells Relative Light Units

10'' 4844

10⁵ 956

20 10⁴ 342

10¹ 123

10² 33 0 0

The above examples are presented for purposes of 25 clarity of understanding. It will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

What Is Claimed Is:

1. A device for performing a chemiluminescent assay, comprising:

A a container constructed of material that is substantially impermeable to liquid;

B. a sampling portion physically associated with said container, said sampling portion comprising a first adsorbent material,

C. a reagent portion, said reagent portion within said container, said first and second adsorbent portions being in proximity to or in physical contact with each other, said second adsorbent material comprising at least one reagent that participates in a chemiluminescent reaction,

wherein said container comprises a light-permeable portion that permits at least a portion of light generated from a chemiluminescent reaction within said container to pass to the outside of said container, where said light can be detected.

2. A device according to claim 1, further comprising a detector located outside of said container for detecting light transmitted through said light-permeable portion.

3. A device according to claim 2, wherein said detector is a film.

4 A device according to claim 3, wherein said film is a self-developing photographic film.

5 A device according to claim 1, wherein said reagent is an enzyme.

6. A device according to claim 1, wherein said reagent is present in a dried state.

7 A device according to claim 6, wherein said reagent is luciferase.

8 A device according to claim 1, wherein said adsorbent materials in said sampling portion and said reagent portion are a single unit.

9. A device according to claim 1, further comprising a reservoir comprising a carrier liquid.

10. A device according to claim 9, wherein said carrier liquid comprises a bacteriolytic solution.

11. A device according to claim 9, further comprising a flexible area adjacent to a reservoir which permits the reservoir to fold or bend such that it can be positioned proximate to the sampling portion.

12. A method of performing an assay for a target aricixyte, comprisirig one steps of:

A. contacting a test sample suspected of containing the target analyte with a sampling portion of a device, said device comprising: i. a container constructed of material that is substantially impermeable to liquid; ii. a sampling portion physically associated with said container, said sampling portion comprising an adsorbent material; and xii. a reagent portion, said reagent portion comprising an adsorbent material located within said container, said adsorbent material containing at least one reagent that participates in a chemiluminescent reaction,

wherein said container comprises a light-permeable portion that permits at least a portion of light generated from a chemiluminescent reaction within said container to pass to the outside of said container, where said light can be detected;

B. contacting said sampling portion with at least one carrier liquid that transports target analyte from said sampling portion into said reagent portion; and

C. detecting light produced within said container.

13. A method according to claim 12, wherein, after

Step A, said sampling portion is contacted with a carrier liquid comprising a target analyte extraction reagent that extracts target analyte present in said sampling portion.

14 A method according to claim 13, wherein, said target analyte extraction reagent is present in said at least one carrier liquid.

15 A method according to claim 14, wherein, said t arget analyte is ATP .

16 A method according to claim 13, wherein said target analyte extractor reagent comprises a compound selected from the group consisting of cetyldimethyl-ethylammonium bromide, Triton X-100, Tween 20, Noindet P40 and n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.

17 A method according to claim 12, wherein, Step C comprises detecting said light using an electronic instrument.

18 A method according to claim 12, wherein, Step C comprises detecting said light using a film.

19. A method according to claim 18, wherein said film is a self-developing photographic film.

20 A method of performing an assay for bacteria, comprising the steps of:

A. wiping a test surface suspected of containing the bacteria with a sampling portion of a device, said device comprising:

i. a container constructed of material that is substantially impermeable to liquid, ii. a sampling portion physically associated with said container, said sampling portion comprising an adsorbent material,

in. a reagent portion, said reagent portion comprising an adsorbent material located within said container, said adsorbent material containing at least one reagent that participates in a chemiluminescent reaction, wherein said container comprises a light- permeable portion that permits at least a portion of light generated from a chemiluminescent reaction within said container to pass to the outside of said container, where said light can be detected;

B. contacting said sampling portion with a reagent that extracts ATP present in said sampling portion; and

detecting light produced within said container.

21. A method according to claim 20, wherein said reagent comprises a compound selected from the group consisting of cetyldimethyl-ethylammonium bromide, Triton X-100, Tween 20, Noindet P40 and n-hexadecyl-N,N-dimethyl-2 ammonio-1-propanesulfonate.

22. A method according to claim 20, wherein, Step C comprises detecting said light using an electronic instrument.

23. A method according to claim 22, wherein, Step C comprises detecting said light using a film.

24. A method according to claim 23, wherein said film is a self-developing photographic film.

25. A method according to claim 20, wherein said device further comprises:

A. a reservoir comprising a carrier liquid, said carrier liquid comprising a bacteriolytic solution that extracts ATP from bacteria, and B. a flexible area adjacent to a reservoir which permits the reservoir to fold or bend such that it can be positioned proximate to the sampling portion, and

wherein, Step B is carried out by bending the device at the flexible area so that the reservoir is positioned proximate to the sampling portion, and then breaking the reservon so as to release carrier liquid into the sampling portion.

26. A method according to claim 25, wherein, Step C comprises detecting said light using a film.

27. A method according to claim 26, wherein said film is a self-developing photographic film.

28. A sampling device for performing an assay for a target analyte, comprising:

a container;

a sampling strip having a sampling portion for receiving a sample, a reading portion for emitting light, and a transfer portion connecting the sampling and reading portions for permitting transfer of the sample from the sampling portion to the reading portion, the sampling strip contained inside the container, the sampling strip containing an agent that emits light, wherein the container has means to permit introduction of the sample to the sampling portion and a light transmissive portion exposing the reading portion; and a shield extending from the container adjacent the exposed sampling portion, wherein the shield has a portion extending beyond one end of the container and is movable to and from the sampling portion.

29. A sampling device according to claim 28, wherein the container comprises a first layer and a second layer sandwiching the sampling strip.

30. A sampling device according to claim 29, wherein the shield is attached to or integral with the first layer and has means for permitting the extending portion to wrap around the one end of the container.

31 A sampling device according to claim 30, wherein the means for permitting the extending portion to wrap around is a preformed fold, crease, or perforation.

32. A sampling device according to claim 29, further comprising a tab attached to or integral with the first layer.

33 A sampling device according to claim 32, wherein the tab is attached to or integral with the shield.

34. A sampling device according to claim 29, wherein the sample introducing means is a first opening formed through the first layer and aligned with the sampling portion.

35. A sampling device according to claim 29, wherein the light transmissive layer is a second opening though the second layer and aligned with the reading portion.

36. A sampling device according to claim 35, wherein the second layer includes a light transmissive member at least to cover the second opening.

37. A sampling device according to claim 28, wherein the sampling strip is composed of an adsorbent material.

38. A sampling device according to claim 34, further comprisinga sample collecting member incontact with the sampling portion inside the container and aligned with the first opening.

39. A sampling device according to claim 38, wherein the sample collecting member is composed of an adsorbent material.

40. A sampling device according to claim 37, wherein the agent is a chemiluminescent reagent contained within the reading portion.

41. A sampling device according to claim 40, wherein the reagent is luciferase-luciferin in a dried state.

42. A sampling device according to claim 29, wherein the sampling strip is made of a material permitting lateral flow of the sample in liquid, wherein at least the reading portion is light transmissive.

43. A sampling device according to claim 42, wherein the light transmissive material is a poly carbonate membrane.

44. A sampling of device according to claim 43, wherein the agent is contained within at least a portion of thetransfer portion.

45. A sampling device according to claim 44, wherein the agent is a luminescent labeling agent for labeling the analyte, wherein the labeling agent glows when exposed to light.

46. A sampling device according to claim 45, wherein the labeling agent is chelated europium or europium compound.

47. A sampling device according to claim 45, wherein the reading portion contains an immobilized binding agent for immobilizing the target analyte.

48. A sampling device according to claim 47, wherein the binding agent is an antigen complementary to the target analyte.

49. A sampling device according to claim 47, wherein the sampling strip includes a collecting portion contiguous with the reading portion for absorbing excess liquid containing the labeling agent not coupled to the binding agent.

50. A sampling device according to claim 49, wherein the collecting portion is absorbent or adsorbent.