IL96138A - Devices and methods for testing liquid samples - Google Patents

Description

DEVICES AND METHODS FOR TESTING LIQUID SAMPLES - ^nn Ο'ΟΑΠ η ·>Ί mo^i o^pnn The present invention relates to devices and methods for testing liquid samples.

More particularly the present invention relates to a device and method for subjecting at least one liquid sample to a detection test and for preserving the results thereof, as well as to devices and methods for subjecting at least one liquid sample to a plurality of discrete sequential reactions or for subjecting each of a plurality of liquid samples to a plurality of discrete sequential reactions.

Specifically, the present invention provides a new type of multilayer dry reagent carrier enabling the carrying out of detection tests and the observation and preservation of test results in an advantageous manner heretofore not achieved with state of art dry reagent chemistry carriers.

As described by Dr. Bert Walter in Analytical Chemistry, Vol. 55, No. 4. April 1983, pp. 498-514, providing a reagent in a dry format for rapid use is by no means new - a familiar example is litmus paper, which dates back to the 19th century. By introducing litmus, a colored extract from several lichens, into a paper matrix, the inventor provided a dry reagent chemistry for testing the akalinity of a solution.

The first major impact dry format chemistries had on clinical testing was the appearance in the 1950s of Ames Clinistix urine reagent strips for testing urinary glucose. By comparing the color developed on the reagent strip with a color chart provided on the product label, the user got a rapid qualitative glucose analysis that otherwise would require a laboratory and skilled personnel. Clinistix reagent .strips provided the groundwork for other reagent strips developed by Ames and other manufacturers for testing urine constituents. The appearance of Ames Dextrostix reagent strips in the 1960s for the semiquantitative analysis of blood sugar propagated the development of dry format chemistries for testing blood constituents. With the later introduction of instrumentation, the era of quantitative clinical analysis for glucose with reagent strips began.

In the 1970s, more sophisticated dry chemistries emerged for quantitative analysis of blood constituents. Whereas many of the clinical test formats available share the common trait of requiring reconstitution of reagents prior to use, either manually or automatically, the emerging reagents have a totally new format. In all cases, a complete chemistry is miniaturized into a dispensable dry format. No prior reconstitution of reagent is required, and many manipulations are replaced simply by applying the sample. An analysis is complete in 1-7 min.

The development of dry reagent chemistries is the cumulation of several technologies. These technologies provided crucial knowledge on how to prepare quantitative chemistries in dry media such as thin films, paper matrices and other synthetic porous materials. They also provided knowledge on how to cast thin films of desired porosity with high precision, how to make paper matrices and other porous matrices reproducibly with well-defined characteristics, and how to laminate various materials to each other. The photographic industry has made substantial contributions in advancing the technology of dry reagent chemistries. With the appearance of color photography, the industry had deveoped a technology that is based on conducting quantitative chemistries in discrete films arranged in multiple layers. This eventually resulted in instant photography. An instant color print may have as many as 15 layers of film, each with a specific chemical or physical function to perform in developing the photographic image. This very technology was used to develop several dry reagent chemistry formats for clinical testing. The coating and plastic industry provided a variety of techniques for precision casting of thin films, and it has developed lamination techniques frequenty required for bonding various materials together. , It also developed a variety of inert materials used in constructing dry reagent chemistries. The paper and fabric industries have developed techniques for making fibrous matrices with reproducble parameters to fit a variety of unique needs. Some of the parameters that can be controlled easily include matrix thickness, fiber density and solvent absorbency, hich are Important in generating dry reagent chemistries.

Contributions from these industries led to the evolution of dry regent chemistries that were adapted to meet many of the needs in the clinical laboratories.

Dry reagent chemistries have been described for a variety of blood analytes. These Include serum metabolites, enzymes, and serum electrolytes as well as therapeutic drugs. Many of these chemistries are available on the market and provide a unique approach to conducting a quantitative analysis of serum analytes. Each dry reagent chemistry provides an integrated assay for a specific analyte that requires only the application of the serum sample.

Thus, e.g., dry reagent chemistries are presently available for nearly all the commonly tested blood metabolites. These include glucose, blood urea nitrogen, uric acid, cholesterol, triglycerides, creatinine, bilirubin, ammonia, and calcium. Analysis of many of these metabolites by conventional means . requires several steps that are done either manually or by automation. The integration of several steps into a single-step analysis is exemplified by the dry reagent chemistries developed for whole-blood glucose analysis.

Two such carriers developed by Boehringer Mannheim Corporation (BMC) and Ames Division, Miles Laboratories, Inc., are described hereinafter to illustrate the present state of the art. Glucose is detected by a glucose oxidase-peroxidase procedure. In both cases, approximately 50-fL of whole blood is applied to the surface of the carrier (approximately 0.5 cm x 1 cm), where plasma containing glucose is separated from red blood cells by virtue of the selective permeability of the carrier matrix. After an allotted reaction time (usually 1-3 min), the red blood cells are removed by washing or wiping, and the color developed is analyzed and translated to blood glucose concentrations. Both carriers consist of a support material that also incorporates a reflective zone. Both devices depend on a film layer to selectively exclude red blood cells from the reagent zone and quantitatively meter a sample volume. In one case, the reagent zone is part of the film matrix ; in the other, the reagent consists of a paper matrix with a membrane. Both devices have reduced the blood glucose analysis from a laborious effort of removing red blood cells, either by centri fugation or by precipitation prior to analysis, to one simple step.

Separation steps or immobilized catalytic centers also can be integrated into carriers at any reaction step of an analysis. An example of a .dry reagent chemistry employing a separation step to isolate a product is the multilayer film chemistry (Ektachem slide) developed by Eastman Kodak for blood urea analysis. The carrier consists of a transparent support material, a reflective layer, and two reagent layers (film matrix) separated by a semipermeable membrane. A 10 v L sample of undiluted serum is applied to the reflective layer, which also acts as a spreading layer, to meter a uniform reaction volume. As the sample enters the first reagent, layer, the urea is converted to ammonia and CC^ by the enzyme urease. The semipermeable membrane acts as a barrier to hydroxyl ions and allows the diffusion of the ammonia into the second reagent layer where it deprotonates a pH indicator. The reaction is over in 7 min. The color developed is monitored by a reflectance meter from below the carrier, and the results are expressed as concentration of blood urea. The dimensions of the dry reagent carrier are 2.8 x 2.4 cm, with an application region of less than 0.8 cm . The thickness of the carrier is less than lOO^m.

Among the carriers incorporating catalytic centers is the dry reagent strip of blood urea analysis developed for the Seralyzer system by Ames. The carrier consists of a support that incorpo ates a reflective zone and a paper matrix reagent layer that contains uniformly distributed cation exchange centers. Upon application, the sample is distributed into the reagent zone via capillary action; this solubiHzes the reagents The blood urea reacts with o-phthalaldehyde to produce 1 , 3-dihydroxyiso1ndoine (DM). The cation exchange centers catalyze the coupling of DHI to 3-hydroxy-l , 2, 3, 4-tetrahydroben- zo(h)quinidine (HTBQ) to form a chromogen. The rate of color development, monitored by reflectance from above the carrier, is then converted into blood urea concentration units. The analysis requires 30 uL of solution after a threefold dilution of a serum sample with water (10 ut of undiluted serum is required per assay). The dimensions of the carrier are 0.5 x 1.0 cm, with a thickness of less than 0.5 mm.

As with serum metabolites, sophisticated dry reagent chemistries also are available for the analysis of serum enzymes. Dry reagent chemistries have been described for the analyis of creatin kinase, lactate dehydrogenase, aspartate transaminase, alanine transaminase, a-amylase, Y-glutamyltransferase, and akaline phosphatase. Some of these carriers are available on the market.

As will be realized from the above descriptions, the common features of most dry reagent chemistry carriers monitored by diffuse reflectance methods are a support material, a reflective zone, and a reagent zone. In some carriers these three features are distinctly defined; in others, the reflective zone may coalesce with the reagent zone or the support zone, or both. The support material usually consists of a thin, rigid plastic or a plasticlike material that may be transparent or reflective. The support material serves as a building base for the reagent chemistry carrier. The major function of the reflective zone is to reflect to a detecting system any light not absorbed by the chemistry of the carrier. The reflective zone is usually constructed with pigments such as TiC^ or BaS04 or reflective materials such as metal foils. Where paper constitutes the reagent zone, the paper matrix itself acts as a reflective layer.

It is important to note that in all of the said prior art dry reagent carriers, liquid samples pass spontaneously from one layer to another via porous, permeable or semipermeable films or membranes and cannot be purposely retained on the film or membrane for any measured length of time.

Furthermore, multi-layer reagent carriers are not designed as devices for the simultaneous testing of a plurality of samples.

Finally, the results of the tests are recorded by the testing personnel either in the form of a written description or with the aid of instruments, e.g., a reflectometer, but not by means of the device itself.

With this state of the art in mind, it is an object of the present invention to provide a simple means for screening large numbers of samples in a short time without the aid of any instrumentation and for retaining a permanent record of the results without any further handling and without the need for metering devices.

It is a further object of the invention to provide means wherein transference of a sample from first reaction zone to another zone of reaction and or test result preservation is manually controllable to assure reaction completion in each zone.

Thus, according to the present invention there is now provided a device for subjecting at least one liquid sample to a detection test and for preserving the results thereof comprising a first, transparent non-absorbent upper layer having at least one soluble reagent deposited thereon in an initial reaction zone and an absorbent lower layer for the absorption of a reaction mixture and the preservation of reaction test results, an openable channel being provided in said first layer to enable the sequential passage of said sample from said initial reaction zone to said absorbent lower layer.

In a preferred embodiment of the present invention there is provided a device for subjecting at least one liquid sample to a plurality of discrete sequential reactions comprising a first transparent, non-absorbent upper layer having at least one soluble reagent deposited thereon in an initial reaction zone and an absorbent lower layer having at least one reagent incorporated therein in a final reaction zone, an openable channel being provided in said first layer to enable the sequential passage of said sample from said initial reaction zone to said final reaction zone.

In another preferred embodiment of the present invention there is provided a device for subjecting each of a plurality of liquid samples to a plurality of discrete sequential reactions comprising a first, transparent, non-absorbent upper layer having at least one soluble reagent deposited thereon in a plurality of distinct initial reaction zones, an absorbent lower layer having at least one reagent incorporated therein in a plurality of final reaction zones, a plurality of openable channels being provided in said first layer to enable the sequential passage of said samples from said initial reaction zones to said final reaction zones.

In said embodiment said first layer may be provided with a plurality of perforations at predetermined intervals, each perforation being positioned to channel a liquid sample from an initial reaction zone to an associated final reaction zone.

In yet another preferred embodiment of the present invention there 1s provided a device for subjecting at least one liquid, sample to a plurality of discrete sequential reactions, comprising a first, transparent, non-absorbent upper layer having a predetermined sequence of discretely deposited soluble reagents deposited thereon in a plurality of distinct adjacent reaction zones, each of said reagents serving for one step in a sequential series of reactions, and an absorbent lower layer, an openable channel being provided in said first layer to enable the sequential passage of said sample from said upper layer to said absorbent lower layer after passage through said reaction zones.

Using the devices of the present invention there is provided a method for subjecting at least one liquid sample to a detection test and for preserving the results thereof, comprising applying a liquid sample to a first, transparent, non-absorbent upper layer having at least one soluble reagent deposited thereon in an initial reaction zone, to combine said sample with said reagent, and then causing a resulting reaction mixture to pass through a channel provided in said first layer to an absorbent lower layer to effect the absorption of a reaction mixture and the preservation of reaction test results in said absorbent layer.

The invention also provides a method for subjecting each of a plurality of samples to a plurality of discrete sequential reactions comprising applying a plurality of liquid samples which are to be individually and separately tested to a first, transparent, non-absorbent upper layer having at least one soluble reagent deposited thereon in a plurality of distinct initial reaction zones, wherein each of said samples is placed in a separate initial reaction zone to form a separate reaction mixture, and then causing each of said separate reaction mixtures to pass through a separate channel provided in said first layer and associated with each initial reaction zone, to an associated final reaction zone provided in an absorbent lower layer, said absorbent lower layer having at least one further reagent incorporated therein in each of said plurality of associated final reaction zones.

The invention also provides a method for subjecting at least one liquid sample to a plurality of discrete sequential reactions comprising applying a liquid sample to a first, transparent, non- absorbent, upper layer having a predetermined sequence of discretely deposited soluble reagents, deposited thereon in a plurality of distinct adjacent reaction zones, each of said reactants serving for one step 1n a sequential series of reactions, combining said sample with said reagents in predetermined sequence and at predetermined time intervals and then causing the resulting reaction mixture to pass through a channel provided in said first layer to an absorbent lower layer for the absorption of a reaction mixture and the preservation of reaction test results.

Also provided is a method for subjecting at least one liquid sample to a plurality of discrete sequential reactions comprising applying a liquid sample to a first, transparent, non-absorbent upper layer having a predetermined sequence of discretely deposited soluble reagents, deposited thereon in a plurality of distinct adjacent reaction zones, each of said reactants serving for one step in a sequential series of reactions, combining said sample with said reagents in predetermined sequence and at predetermined time intervals and then causing the resulting reaction mixture to pass through a channel provided in said first layer to a final reaction zone of an absorbent lower layer having at least one further reagent incorporated therein.

Preferably said detection tests include a color reaction the results of which are observable on said absorbent layer and which can then be photocopied therefrom for record purposes.

Alternatively, said detection test can include a color reaction, the results of which are observable on said absorbent layer, which absorbent layer is sandwiched between top and bottom non-absorbent, transparent layers which layers protect the inner absorbent layer and allow the same to be handled and itself stored for record purposes.

As will be realized, one of the unique features of the present Invention is the provision of a first transparent, non-absorbent, non-permeable layer upon which one or more controlled reaction tests are carried out and through which a reaction mixture can pass to a reaction test result preservation layer and/or to a final reaction zone only via an openable channel.

Another unique feature of the present invention is its dual functionality as a reagent site and a test result recording device which can be safely handled and filed for future reference.

The invention will now be described in connection with certain preferred embodiments with reference to the following illustrative figure so that it may be more fully understood.

With specific reference now to the figure in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

Referring now to the Figure in detail there is seen a first transparent, non-absorbing plastic sheet 2 (e.g. polyethylene) of convenient size (e.g. size of this page).

Beneath said top sheet is found a filter paper sheet 4 (e.g. Whatman No. 3) not exceeding the size of the plastic sheet which filter paper sheet or absorbent sheet 4 is immobilized (e.g. by stapling or joining the edges) between said top plastic sheet and a bottom plastic sheet 6 or by inserting said sheet 4 into a tightly fitting plastic envelope (not shown).

The device is provided with perforations 8 at desired intervals (e.g. 12 - 18 mm apart) with a standard gauge needle (e.g. 24 g syringe needle). Each perforation serves as a "channel" for one liquid test sample to reach the "absorbent".

The area surrounding each channel 8 (e.g. 10 - 15 mm diameter) is an initial test area 10. A typical device may thus provide over 150 test areas. Each area can be convenienty identified on a grid drawn on the plastic, or preferably, printed on the "absorbent" as grid lines 11, 13 illustrated in the Figure.

The face of the top layer 2 may be coated with the appropriate FSageht preparation. Preferably, a reagen pellet 12 is deposited on or near each "channel. This is done by allowing a drop of the appropriate reagent solution to dry in situ. All such pellets must adhere to the screen and retain activity under normal handling conditions.

Where appropriate, two or more kinds of pellets 12,14,16 may be placed in each "test area" to allow sequential treatment of a sample. The pellets can be color coded by having each reagent solution include a distinctive dye which will not interfere with the reaction.

Where appropriate, the "absorbent" sheet is impregnated with a reagent or an indicator solution 18, or with a plurality of concentrically deposited reagents 20, 22, 24 for effecting further sequential reactions and dried. The impregnated "absorbent'1 must retain activity under normal storage conditions.

The device and method for the present invention are particularly advantageous for tests which consist of several discrete, sequential reactions. In conventional tests of such "multi-step" nature, each step is initiated and terminated by an operation such as addition of measured amounts of reagents. In the present method the correct amounts of all the reagents required are present in the test area and each takes place in situ.

Where desirable, the following variations of the device can be introduced: 1. Since the top sheet is transparent, two or more such sheets may be used, with different reagents placed on each. Alternati ely, an additional upper transparent layer can be added on top of the device, which layer contains no reactive materials so that the device can be more readily handled without any precautions.

Furthermore, each test area can then be sealed off by fusing its edges after the sample has been introduced (e.g using a heated metal grid). This is important for trapping gas evolved, e.g. when testing for the presence of catalase in the sample.

The standard absorbent sheet can be replaced with several strips, each impregnated with different reagents when a multi-purpose device is desired. For example: a single device may then serve for the determination of pH, nitrite and hemoglobin in each of 100 urine samples screened.

The flow of the sample liquid can be controlled by the diameter or shape of the perforation or the number of channels per test area. When desirable, the device may be prepared without channels, and perforated by the user when actually used. (The kit may then include the necessary accessories). Alternatively, preformed channels may be sealed (e.g. with self-adhesive labels) until used.

Multi-purpose devices can be constructed with a single absorbent but varying reagents. For example, if a specimen is found to contain R -lactamase, it is often important to determine the specificity of the enzyme. The appropriate screen will be as in Example 2, but the pellets will represent a series of potential substrates, rather than benzylpenicillin alone.

In accordance with the present invention: a) Individual tests can utilize small devices with single test a,reas. b) For follow-up, multi-test area devices (e.g. with test areas for consecutive morning urine tests) can be used at home and brought for doctor's evaluation. c) Devices can serve for recording and preservation of results of conventional tests (e.g. run in part or entirely in test tubes and transferred with pipettes or a swab to a screen containing an appropriate absorbent).

While the invention will now be described in connection with certain preferred embodiments in the following examples so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims. Thus, the following examples which include preferred embodiments will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention.

Procedure: General Description Materials (for 100 tests) 1. 1 device as claimed; 2. 100 disposable tips (for a 20 - 50 vl pipette) or disposable capillary pipettes or equivalent; 3. 100 toothpicks or equivalent.

Steps: 1. Place a sample (e.g. 50 yL) of each specimen (e.g. urine) on pellet in test area. 2. Press each channel with toothpick (a round spot will immediately form under the plastic). 3. Wipe screen clean (e.g. with tissue paper) and read results or file for later reference.

COMMENTS: Step 1. A drop of sample solution will retain its near-spherical shape on the plastic face of the device until guided through a channel (see below). This allows the pellet to dissolve or the pretreatment to go to completion as required by the test.

Step 2. The spot provides the diagnostic information required of the particular test. It may be color, size or other properties (See Examples below).

Step 3. Typically, the appearance of the spots will not change substantially after drying and the diagnostic information on the device will not be distorted thereafter. This device can be examined at convenience and even filed to provide a direct and lasting record.

If samples are suspected of containing pathogenic material, the device should be wiped gently with a mild disinfectant before further handling. If, for similar reasons, filing of the device itself is not desirable, a copy (e.g. a photocopy) of the results seen on the absorbent layer through the transparent layer may be filed instead.

EXAMPLE 1 TEST: Bovine mastitis as evidenced by elevated somatic cell count of milk.

PRINCIPLE OF TEST: Rapid disruption of the cells by incubation of milk with a protease-detergent preparation leads to release of DNA which interferes with flow and absorption of the milk through the channel.

KIT: Standard polyethylene screen with channels covered by pellets consisting of dyed cell-disrupting preparation comprising protease combined with a compatible detergent. Absorbent: untreated Whatman No. 3 filter paper sheet.

PROCEDURE: 1. Place 50 yL samples of milk specimens on pellets. 2. Within 1 - 5 minutes, use toothpick to stir pellet into milk sample and press channel for about 5 seconds. 3. Wipe face of screen and file for examination and reference at any convenient time.

NOTE: Normal milk will form a blue, full-sized (ca. 10 mm dia.) spot under the plastic. Reduced spot size indicates mastitis and extent of reduction provides basis for estimating somatic cell count.

EXAMPLE 2 TEST: Indication for treatment of bovine mastitis.

PRINCIPLES OF TEST: Benzylpenicillin is the drug of choice, in terms of price and efficiency, for treating susceptible infections. Oectection of mastitis (see Example 1) is here followed by a test for 6-lactamase, a commonly encountered enzyme which inactivates benzylpenicillin and several related antibiotics. If positive, such antibiotics must not be used for treatment.

KIT: Standard polyethyene screen with" pellets consisting of 10 mg of procaine-pen1c1ll1n. Absorbent: - Whatman No. 3 filter paper impregnated with starch iodine (see Comments below).

PROCEDURE: 1. Place 50 yL samples of the same specimen in two adjacent test ' areas (A, B). Use toothpick to mix sample A with pellet. 2. After 20 minutes, mix sample B with pellet and use toothpicks to spread both milk drops over respective channels and press for 5 seconds to allow absorption. 3. Wipe" screen, observe and store.

COMMENTS: 1. The product of the β-lactamase reaction is penicilloic acid which removes iodine from the starch starch-iodine complex. Hence, if A goes white before B, the infection is associated with ^-lactamase producing bacteria and should not be treated with standard penicillins (i.e., benzylpenicillin, ampicillin phenoxymethylpenicillin, etc.). 2. The difference between A and B, if any, will increase with temperature (up to 42°C) with time allowed before absorption and with time after absorption. 3. Susceptibility of other β-lactam antibiotics to any 6 -lactamase detected can be similarly tested with the pellet of penicillin replaced by other β-lactams.

EXAMPLE 3 AIM: Detection of antibodies to bacterial cell surface antigens in serum samples.

FACE: Polystyrene sheet.

PELLET: Glutaraldehyde - immobilized bacteria X, blocked with BSA. ABSORBENT: Filter paper (Whatman 3MM) impregnated with a starch- iodine solution as described in example 2.

PROCEDURE: 1. Place aliquots (50 1) of serial dilutions of test serum on pellets. 2. Allow 20 min. at 22°-28°, wash and blot.* 3. Place on each pellet 50 L of the immunoglobulin detecting reagent (T 4) described in Example 2. 4. Wash and blot.* 5. Place on each pellet 50 L of the substrate solution as described in Example 2 and allow to stand 10 min. at room temperature. 6. Press so as to allow absorption of the reactants through the channel .

*A11 washes consist of placing 4x50 L of PBST (phosphate-buffer-saline containing 0.05% Tween 20) and blotting each aliquot of the wash solution with filter paper.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that tHe present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being 'made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (17)

WHAT IS CLAIMED IS:

1. A device for subjecting at least one liquid sample to a detection test and for preserving the results thereof comprising a first, transparent, non-absorbent upper layer having at least one soluble reagent deposited thereon 1n an initial reaction zone and an absorbent lower layer for the absorption of a reaction mixture and the preservation of reaction test results, an openable channel being provided in said first layer to enable the sequential passage of said sample from said Initial reaction zone to said absorbent lower layer.

2. A device according to claim 1 for subjecting at least one liquid sample to a plurality of discrete sequential reactions comprising a first transparent non-absorbent upper layer having at least one soluble reagent deposited thereon in an initial reaction zone and an absorbent lower layer having at least one reagent incorporated therein in a final reaction zone, an openable channel being provided in said first layer to enable the sequential passage of said sample from said initial reaction zone to said final reaction zone.

3. A device according to claim 2 wherein said absorbent layer has incorporated a sequence of reagents in said final reaction zone.

4. A device according to claim 2 for subjecting each of a plurality of liquid samples to a plurality of discrete sequential reactions comprising a first transparent non-absorbent upper layer having at least one soluble reagent deposited thereon in a plurality of distinct initial reaction zones, an absorbent lower layer having at least one reagent incorporated therein in a plurality of final reaction zones, a 1 - 25 - plurality of openable channels being provided in said first layer to enable the sequential passage of said samples from said initial reaction" zones to said final reaction zones.

5. A device according to claim 1 further comprising a bottom non-absorbent layer.

6. A device according to claim 5 wherein said bottom layer is transparent.

7. A device according to claim 4 wherein said first layer is provided with a plurality of perforations at predetermined intervals, each perforation being positioned to channel a liquid sample from an Initial reaction zone to a final reaction zone.

8. A device according to claim 1 for subjecting at least one liquid sample to a plurality of discrete sequential reactions, comprising a first, transparent, non-absorbent upper layer having a predetermined sequence of discretely deposited soluble reagents deposited thereon in a plurality of distinct adjacent reaction zones, each of said reagents serveing for one step in a sequential series of reactions, and an absorbent lower layer, an openable channel being provided in said first layer to enable the sequential passage of said sample from said upper layer to said absorbent lower layer after passage through said reaction zones.

9. A method for subjecting at least one liquid sample to a detection test and for preserving the results thereof, comprising applying a liquid sample to a first, transparent, non-absorbent upper layer having at least one soluble reagent deposited thereon in an initial reaction zone, to combine said sample with said . reagent, and then causing a resulting reaction mixture to pass through a channel provided 1n said first layer to an absorbent lower layer to effect the absorption of a reaction mixture and the preservation of reaction test results 1n said absorbent layer.

10. A method according to claim 9, for subjecting at least one liquid sample to a plurality of discrete sequential reactions comprising applying a liquid sample to a first transparent, non-absorbent, upper layer of a multi-layer reagent carrier, said upper layer having at least one soluble reagent deposited thereon in an initial reaction zone, to combine said sample with said reagent and then causing said sample to pass through a channel provided in said first layer to a final reaction zone of an absorbent lower layer having at least one further reagent Incorporated therein to combine said sample with said further reagent.

11. A method according to claim 10 wherein reaction results in said final zone are observed through said first upper transparent layer.

12. A method according to claim 10 wherein reaction results in said final zone are observed through a bottom transparent non-absorbent layer.

13. A method according to claim 10 for subjecting each of a plurality of samples to a pluraity of discrete sequential reactions comprising applying a plurality of liquid samples which are to be individually and separately tested to a first, transparent, non-absorbent upper layer having at least one soluble reagent deposited thereon in a plurality of distinct initial reaction zones, wherein each of said samples 1s placed in a separate initial reaction zone to form a separate reaction mixture, and then causing each of said separate reaction mixtures to pass through a separate channel provided 1n said first layer and associated with each initial reaction zone, to an associated final reaction zone provided in an absorbent lower layer, said absorbent lower layer having at least one further reagent incorporated therein in each of said plurality of associated final reaction zones.

14. A method according to claim 9 for subjecting at least one liquid sample to a plurality of discrete sequential reactions comprising applying a liquid sample to a first, transparent, non-absorbent, upper layer having a predetermined sequence of discretely deposited soluble reagents, deposited thereon in a plurality of distinct adjacent reaction zones, each of said reactants serving for one step in a sequential series of reactions, combining said sample with said reagents 1n predetermined sequence and at predetermined time intervals and then causing the resulting reaction mixture to pass through a channel provided 1n said first layer to an absorbent lower layer for the absorption of a reaction mixture and the preservation of reaction test results.

15. A method according to claim 10 for subjecting at least one liquid sample to a plurality of discrete sequential reactions comprising applying a liquid sample to a first, transparent, non-absorbent upper layer having a predetermined sequence of discretely depositee! soluble reagents, deposited thereon in a plurality of distinct adjacent reaction zones, each of said reactants serving for one step 1n a sequential series of reactions, combining said sample with said reagents 1n predetermined sequence and at predetermined time Intervals and" then causing the resulting reaction mixture to pass through a channel provided in said first layer to a final reaction zone of an absorbent lower layer having at least one further reagent incorporated therein.

16. A method according to claim 9 wherein said detection test Includes a color reaction the results of which are observable on said absorbent layer.

17. A method according to claim 9 wherein said detection test Includes a color reaction the results of which are observable on said absorbent layer and photocopied therefrom for record purposes. FOR THE APPLICANT WOLFF, BREGMAN AND GOLLER