WO1999056111A1

WO1999056111A1 - Electronic test device

Info

Publication number: WO1999056111A1
Application number: PCT/EP1999/002840
Authority: WO
Inventors: Thomas Augustinus Maria Beumer; Hermanus Johannes Maria Kreuwel; Petrus Franciscus Hendrikus Maria Verheijden
Original assignee: Akzo Nobel N.V.
Priority date: 1998-04-28
Filing date: 1999-04-22
Publication date: 1999-11-04
Also published as: AU3824099A

Abstract

The invention relates to a device for the detection or determination of the amount of an analyte in a test liquid. The assay device is equipped with a light source (11) and light detector (13), processing (14) and display means (15) for electronic reading of the assay results, while the actual assay is based on radial solid phase diffusion. In this way the necessity for an exact positioning of the optical components and the measuring area are eliminated.

Description

Electronic test device

The invention relates to a device for the detection of an analyte in a test liquid by means of a radial solid phase diffusion assay. The assay device is equipped with a light source and detector, processing and display means for electronic reading of the assay results and is especially suitable for home use or use by non-professional organisations.

Since the introduction of the first immunological pregnancy test in 1969 a great variety of test principles and formats have been used for detection of hCG. Initial tests were based on either haemagglutination, resulting in sensitive tests which required long incubation times, or latex agglutination, which provided quick but rather insensitive tests. In the following generation tests the ELISA principle was used which gave tests with a high sensitivity and relatively short incubation time, but which required quite a number of handlings. A great breakthrough was achieved when the ELISA principle was used in combination with porous materials, instead of the tubes and plates used sofar, in the so-called filter tests as described in EP 180 638 US.4,632,901. The porous materials enabled a close contact between the various reagents thereby reducing the reaction time dramatically. The drawback of these filter tests was that quite a number of handlings were required, which made them less suitable for use by unskilled people. This problem could be solved when porous test strips were used, which contained all or part of the reagents required, and which could be dipped into the test liquid. Although in part of these dipstick tests enzyme labels were used, a further simplification was achieved by using the so-called "direct" labels which did not require any additional reagents or equipment, such as enzymes or radioisotope labels. Well-known direct labels are the particulate labels as for example described in EP 291 194. The above mentioned dipstick tests, comprising an immobilized capture antibody and a freely mobile particle labelled antibody, proved to be highly suited for use by unskilled people. Upon dipping the test strip into the test liquid, a complex is formed between the labelled antibody and the analyte when present. Subsequently this complex is transported to the capture antibody whereby a sandwich is formed. When, for example, colloidal gold particles are used as a label, a magenta coloured spot is observed when the analyte to be determined, for example hCG, is present in the test liquid. This colour can be observed visually. In general no problems are observed with strongly positive or absolutely negative samples. However, confusion arises when samples are measured from very early pregnancies comprising low levels of hCG, i.e. corresponding to the lower limit of detection of the test. In those cases a faint colour will be observed, which will not always be interpreted unambiguously. Moreover there will be a difference in interpretation between individual users. By using for example an optical instrument for reading of the colour, the subjective interpretation of the test results is eliminated, thereby providing an unambiguous interpretation of the result.

US 5,580,794 describes an electronic assay device comprising a bibulous support with dried assay reagents, a light source and a light detector, as well as a data processor and a display unit, all encapsulated in a unitary housing. The test liquid is applied to the oblong bibulous support through an opening in the housing, This oblong bibulous support comprises various zones with reagents, such as a mobile labelled binding reagent for the analyte to be determined and an immobilized binding reagent for said analyte. The label is preferably a physically detectable label such as colloidal metal particles. Upon contact of the test liquid with the bibulous support said liquid is transported through the support by capillary action. The analyte to be detemined reacts with the mobile labelled binding reagent and the analyte/labelled binding reagent complex formed, as well as any unreacted reagent, is transported to a detection zone with the immobilized binding reagent for the analyte. The amount of physically detectable label present in the complex of immobilized binding reagent/analyte/labelled binding reagent formed in the detection zone, is measured using a light source and light detector.

This device suffers from several drawbacks, the most critical ones being

•the accuracy requirements concerning positioning of the coated measurement spot with respect to the porous strip,

•the accuracy requirements concerning mutual positioning of optical components and the coated measuring spot Both requirements effect manufacturability and costprice The object of the present invention is to overcome these disadvantages. These improvements have been achieved with an assay device for detection or determination of the amount of an analyte in a test liquid comprising - a bibulous support comprising a zone with an immobilized first reagent and a sample application spot;

- a light source for projecting light onto the immobilized first reagent;

- a light detector capable of producing an electrical signal, the light source and the light detector defining a measuring area; -electronic processing means for deriving an output signal from the electrical output signal of the light detector; at least one of the first reagent and a mobile, labelled second reagent being capable of reacting specifically with an analyte to be determined in the test liquid, the electronic processing means being capable of determining the electrical signal in a time-dependant manner and capable of processing the data obtained to yield a value corresponding to the analyte concentration; and

- means for presenting the output signal indicative of said value in a form which can be interpreted by a non-skilled person; wherein, the sample application spot and the measuring area are located within the perimeter of said zone containing an immobilized first reagent.

The test liquid is applied to the bibulous support, that has been pre-coated with a first immobilized reagent, and allowed to diffuse radially through said bibulous support. A mobile labelled second reagent, capable of reacting directly or indirectly with the analyte to be determined in the test liquid, is applied to said bibulous support coated with a first immobilized reagent before, together with, or after application of the test liquid, and allowed to diffuse radially. The amount of labelled second reagent bound is then measured kinetically.

Such a radial solid phase diffusion assay is also described in European Patent No. 207 152, wherein a test liquid comprising a labelled compound diffuses radially from a point of application on a bibulous support comprising immobilized first reagent. Generally, the size of a labelled spot is measured as an indication for the amount of analyte present. EP 207 152 does, however, not suggest or disclose an electronic measurement for an accurate indication of the amount of analyte, let alone a kinetic measurement.

The device of the present invention comprises a bibulous support comprising a material which transports the test liquid essentially by capillary forces. Advantageously, absorbent, porous or fibrous material is used, which is suitable for rapid uptake and transport of liquid. Suitable materials involve amorphous sponge-like structures or various porous synthetic materials such as polypropylene, polyethylene, polyvinylidene fluoride, ethylene/vinyl acetate copolymer, polyacrylonitrile and polytetrafluoroethylene In addition certain materials with an inherent hydrophobicity can also be pretreated with surface-active agents to such an extent that they are able to take up a test liquid and transport it by capillary forces. Preferably, multilayer materials are used and materials which are already generally applied in analytical test strips such as paper or paper-like materials. A most preferred material is nitrocellulose. Said bibulous support is coated with an immobilized first reagent which is capable of reacting directly or indirectly with the analyte to be determined. Said first reagent is preferably a member of a specific binding pair such as an antigen or antibody or their fragments, a DNA or RNA fragment, avidin or biotin, a hormone or hormone receptor, etc. The first reagent can be immobilized onto the bibulous support in a variety of ways. For example, the first reagent such as an antibody can be immobilized onto the bibulous support by adsorption or covalent binding. If the bibulous support consists, for example, of nitrocellulose, the antibodies can be coupled directly without a previous chemical treatment of the bibulous support. In order to prevent non-specific binding to the bibulous support the remaining binding sites can be blocked with, for example, treatment with hydrophilic synthetic polymers, such as polyvinylalcohol, or hydrophylic biopolymers, such as human and bovine serum albumin, ovalbumin, casein and the like. It is, however, also possible to add these compounds to the labelled second reagent. If the bibulous support consists of other materials such as paper, covalent coupling can be achieved with CNBr or carbonyldiimidazole. It is also possible to use printing processes, such as ink jet and screen printing techniques, for this purpose. In this case, the reagent may either merely be applied to the surface of the bibulous support or impregnated into the bibulous support. Either the entire surface of the bibulous support is coated with the immobilized first reagent or that part which is defined by the borderline of the recess of the absorbing element described below.

In a preferred embodiment of the present invention an absorbing element provided with a recess is placed on top of said bibulous support to enhance the diffusion rate and to speed up the reaction. The recess defines a borderline in contact with the bibulous support, which borderline is substantially continuous and substantially circular, and which defines a centre substantially coinciding with the sample application spot. The material of the absorber is not critical and can be any material that is capable of absorbing liquids for a sufficient volume and/or period of time, for example a paper-like material or a sponge-like material such as, for example, cotton wool or hydrophilic and hydrophilized synthetic polymer materials, such as polyethylene vinylacetate, as well as other polyesters, and polypropylene.

Advantageously the dimensions of the absorbing element are chosen in such a way that it is capable of maintaining a substantially constant flux through the bibulous support.

In order to obtain a well-defined measuring area with a homogeneous distribution of the labelled component it is advantageous to apply the labelled component and/or the test liquid in such a way that a precise geometric origin of application is obtained and the sample application spot always has the same, preferably small diameter. This is achieved by using a cover sheet that includes a hole on top of the bibulous support. Liquid enters the bibulous support through the latter hole. The sample application spot is thereby well defined. The size of this hole has also an effect on the liquid mass flow into the bibulous support. In order to avoid that the test liquid is applied outside the sample application spot the cover sheet is prepared from a water-impermeable material, such as thermoplastic material, polystyrene, polypropylene or the like. A hole with well-defined dimensions can be prepared by treating the water- impermeable material with a laser beam, matched metal cutting, rotary die cutting, or other cutting techniques.

The reaction between the analyte to be determined, the mobile labelled second reagent and the first immobilized reagent is accompanied by a physically detectable change, for example a change in colour. Such a change can be detected with a light source and light detector positioned under the bibulous carrier.

Preferably the light source couples light into the bibulous support in a direction suitable to cause the substance of the light to enter the bibulous support, as described in our copending unpublished European patent application PCT EP 97/05984, the contents of which are hereby incorporated by reference. As described in said patent application a prolonged path for the light imparted in the carrier's interior for travelling to the detector is realised. Along that path in the interior of the carrier the light is scattered and diffused allowing for an increased interaction through absorption of the light by such area of the carrier where the analyte in the test liquid and the mobile labelled second reagent are bound to the first reagent immobilized to said carrier. By measuring the transmitted rather than the reflected light, the sensitivity and accuracy for measuring the occurrence and the extent of any such reaction is highly increased. The sensitivity of the test device for the light transmitted by the light source is improved by the application of blocking means to prevent the light to travel directly from the light source towards the detector.

Preferably, the blocking means are formed as a light shield, such as the shield of material which is not translucent.

In a preferred embodiment the centre of the measuring area is defined by the light source and light detector, and substantially corresponds to the sample application spot. In this way it is possible to start the actual measurements involving a reaction with the mobile labelled second reagent, the analyte to be determined and the first immobilized reagent as soon as the mobile labelled second reagent and/or test liquid are applied to the spot. When no analyte is present in the test liquid, the mobile labelled second reagent will not be bound to the bibulous support with the immobilized first reagent. Instead, the mobile labelled second reagent will diffuse radially to the circular borderline of the absorber and is ultimately taken up by the absorber. If however analyte is present, the test liquid comprising said analyte, the mobile labelled second reagent and the complex formed between analyte and labelled second reagent, will diffuse radially through the bibulous support with the immobilized first reagent, whereby the mobile labelled seccond reagent will eventually be bound to said immobilized first reagent via the analyte. The higher the concentration of the analyte in the test liquid, the more labelled second reagent will be bound per time unit onto a certain circular area on the bibulous support, resulting in a faster increment in light absorption. The distribution of the labelled second has substantially the shape of a 2-dimensional bell-curve. Although in principle the whole volume under this curve can be measured as an indication for the total amount of bound particles, it is advantageous to restrict the measuring area to the upper part, close to the axis of symmetry of said bell-curve; in this way the highest signal value is obtained. Furthermore, the positioning of the light source and detector, defining said measuring area, is less critical: a minor shift in the position of light source and detector will cause a decrease of the measuring area in one half of the curve, while at the same time the measuring area is increased similarly in the other half of said curve.

According to a preferred embodiment, the light detector is located substantially under the sample application spot, whereas the light source is positioned at a small distance from said sample application spot. This allows for a simple construction of the assay device, where the light source and detector are positioned in substantially the same plane substantially parallel to the bibulous support. Although it might be expected that the light source and the light detector are located optimally at a substantially equal distance from said sample application spot, surprisingly, it was found that more accurate signals are obtained when the detector is mounted under the sample application spot. In fact, the optical axis of symmetry of a LED-diode combination is not the same as the mechanical axis of symmetry.

Preferably the bibulous support is illuminated by a LED that is used without its normal housing with lens. Such a device is cheap and is easy to mount using state of the art SMD techniques. The wavelength should be selected to match with the absorption maximum of the applied labelled second reagent. In its preferred embodiement this label is a carbon sol particle. In that case, the particles behave like a neutral density filter with absorption rougly constant over the entire UV-VIS spectrum.

In that case, the wavelength can be selected on (a) economic arguments and (b) to fit with the detector's optimum spectral sensitivity. Today, GaAs-based materials show best performance round 950nm, and are the cheapest available. The detector's output current is led to the processing means, for example an ASIC. The current value is digitized using a known capacitor and a timer, in this way digitization accuracies better than 99% can be achieved at any current value The time series of these digitized values is then used for quantitative or qualitative interpretation of the concentration of the analyte to be determined. The ASIC forms the electronic interface between light source/light detector and the output device that the user experiences

As mentioned above the electrical current is measured as a function of the time. This current-to-time curve can ideally be described by a single exponential function, which is based on the following assumptions the amount of bound labelled second reagent, for example the number of colloidal carbon sol particles coated with second reagent, increases approximately linear with time and analyte concentration, furthermore the photometric reading behaves like an absorption measurement, following Lambert-Beer's law and thereby causing output currents to behave exponentially to the above number of bound particles The dynamics of such an exponential are fully described by its so-called time constant value τ, which is a direct measure for the speed at which photometric current decreases with increasing amount of bound second reagent labelled with colloidal carbon sol particles This time constant value τ can be approximated from only two data points using a first order Taylor series expansion These two data points are with best possible reliability obtained as described below It should be considered that any other method of reading two dinstinctly different data values will enable a similar mathematical means to estimating τ

The first point is obtained by reading the current value as short as possible after wetting of the bibulous support, which is characterized by a sharp decrease in the current value The current value immediately measured after said sharp decrease is defined as I₀ and the corresponding time as t=0 The second data point is obtained when the current level reaches a fixed fraction α of I₀ by recording the corresponding time value t' (as the time expired after t=0). The time constant value τ is then calculated from τ =t'/(l-α) This value can be used for both qualitative and quantitative measurements. For a qualitative measurement the τ value is calculated for a reference (cut-off) value and the τ value found for a test sample is subsequently compared with that of the cut-off value, which gives a positive/negative or yes/no answer Such an answer can easily be visualized on a display means for reading by, for example, non-skilled persons. The mobile labelled second reagent can either be included in the device or supplied separately. In the latter case the mobile labelled second reagent is mixed with the test liquid outside the device and the mixture is applied onto the sample application spot. Mixing of test liquid and label can also effectively be accomplished by using a porous element (static mixing means) which is placed on top of said bibulous support. Upon application of test liquid and mobile labelled second compound to said static mixing means, an adequate mixing between the test liquid comprising the analyte to be determined, and the mobile labelled second reagent takes place, while at the same time a pre-incubation between analyte and labelled second component is achieved. Said static mixing means is preferably made of porous sintered. material, and comprises most preferably polyethylene. The pore size typically is 5 to 70 μm.

Upon contact between said static means and the bibulous support the reaction mixture is transferred to the sample application spot of the bibulous support. Contact between said static means and said bibulous support can be enhanced by placing a transfer element on top of said bibulous support, which transfer element is capable of establishing a direct contact between said static means and said bibulous support. Such a transfer element comprises preferably a porous sheet, which is most preferably made of a non-woven material, for example glass fibre with a negligible flow resistance. Ideally, the pore size distribution of the transfer element has intermediate values between those of the bibulous support and static means respectively. Preferably the test liquid is filtered prior to application onto the bibulous support in order to avoid clogging by non-soluble materials that can be encountered in every urine specimen. Suitable filter materials are rigid porous media (e.g. plastics, sintered metals), plastic sheets (e.g. plastic porous sheets), membranes (e.g. polymeric membranes), woven fabrics (e.g. staple fibre yarns), non woven media (e.g. paper media such as cellulose, glass and polymeric non wovens such as melt blown and spun bounded materials). Defining the required filter specifications depend very much on the filtering characteristics of the bibulous support. Normally, particle diameters must exceed half the average pore diameter of the filter before it is certain whether they will be retained by the bibulous support.

In a preferred embodiment of the device the mobile labelled second reagent is incorporated into the device. Said reagent can be diffusibly immobilized onto the bibulous support, but most preferably said mobile labelled second component is incorporated in a separate porous element which is either placed directly onto the bibulous support or onto said static means, so that said porous element with the mobile labelled second compound is in direct or indirect fluid flow contact with the bibulous support.

In another embodiment of the device the mobile labelled second reagent is incorporated in the upper part of said porous element, while the lower part of said porous element serves as the static measn described above.

The porous element with the labelled second reagent should be capable of a rapid uptake of test liquid and an easy release of test liquid and labelled second reagent. Suitable materials for use in said porous element are sintered plastics, paper-like materials (for example glass, cellulose) and polymeric non-woven materials. In a most preferred embodiment sintered polyethylene beads are used. Ideally, the pore size distribution of the porous element has intermediate values between those of the sample collector and the static means. The mobile labelled second reagent is capable of reacting directly or indirectly with the analyte to be determined and is preferably a member of a specific binding pair such as an antigen or antibody or their fragments, a DNA or RNA fragment, avidin or biotin, a hormone or hormone receptor, etc. The mobile second reagent is preferably provided with a label. In principle all kinds of labels can be used, provided that this label is capable of absorbing and/or scattering light. Preferably a direct particulate label is used, which gives a direct visible test result without the need for additional reagents. Advantageously said direct particulate label comprises small coloured particles, such as gold sol particles, latex particles, dyestuff particles, liposomes or microcapsules including a dye, carbon- and selenium sol particles etc. These particles are as such insoluble in water, but resuspendible in solution. All these particulate labels are well known in the literature (see Clin. Chem. 27, 1157, 1981, EP 007 654, EP 032 270, EP 291 194, EP 154 749, EP 321 008). Carbon sol particles are particularly advantageous. The carbon sol particles preferably have a diameter of about 60 to 250 nm, most preferably 90 to 140 nm.

10 The preparation of carbon sol particles conjugated to an antibody or antibody fragment is o a described in European Patent No 689 673, which is incorporated herein by reference

These conjugates are preferably incorporated in a porous element by application of a dispersion of these conjugates in a buffer solution to said element, followed by a drying process, preferably a freeze drying process Suitable buffer solutions comprise non-ionogemc buffers such as CHES, Hepes, MES, CHAPS and preferably Tπs Advantageously these buffers comprise complexing agents such as EDTA, proteins and sugars A preferred protein is casem, while suitable sugars comprise dextran and sucrose Addition of a sugar to the buffer composition enables a controlled release of the conjugate, 1 e the mobile second reagent labelled with colloidal particles This controlled release is either based on a gradual dissolution or on an instantaneous dissolution In the latter case the dissolution time is much faster than the time needed to fill the porous carrier with sample fluid

The test liquid possibly containing the analyte to be determined can directly be introduced into the device, for example with a pipette, or indirectiv with a separate sample collector, the latter obviously being preferred In a preferred embodiment the sample collector is incorporated into the device In a most preferred embodiment said sample collector can temporaπlv be removed from the device for uptake of test liquid and then replaced into the device in such a way that contact is made with the porous element compπsing the mobile labelled second reagent The sample collector comprises a material which can readily absorb test liquid, but also easily releases this test liquid for example under mechanical pressure or capillary transfer It can thus be a spongelike mateπal such as, for example, cotton wool or hydrophilic and hydrophi zed synthetic polymer materials, such as polyethylene vinylacetate as well as other polyesters, and polypropylene To this material additional reagents can be added as, for example, buffering compounds to adjust the pH of the test liquid, or compounds able to eliminate possibly interfering substances present in the test liquid Another example of a sample collector compπses mateπal that as such is not able to absorb and release test liquid, but is able to do so merely by its shape or construction

The device preferably comprises a housing which is

11 advantageously produced from a material which is impermeable to moisture, such as thermoplastic material, polystyrene or the like. In a preferred embodiment this housing is equipped with an opening for introduction of the sample collector and a window for observation of the test result.

The present invention is further directed to the use of a device as described above for the detection or determination of the amount of an analyte, such as an antigen, hapten, antibody or antibody fragment, DNA- or RNA fragment, and in particular hCG, in a test liquid, such as urine, and a method wherein such a device is used.

Such a method comprises for example the following steps:

* pipetting a sample of test liquid and a certain volume of a labelled second reagent, for example a dispersion of colloidal particle labelled second reagent in a container; * after mixing, applying said mixture onto the sample application spot of the bibulous support and allowing said mixture to spread radially from the sample application spot;

* projecting light from the light source onto the measuring zone and measuring the electrical signal from the light detector at two predefined points of time or measuring the points of time required to obtained two predefined values for the electrical signal;

* numerically processing the measurement data to compute a value representative for the presence or amount of the analyte to be determined. The above method is applicable to devices comprising at least a bibulous support with a sample application spot located within the zone coated with an immobilized first reagent, a light source and light detector defining a measuring area within said zone, processing and display means. These devices may further comprise an absorbing element and a cover sheet with a hole.

In case that said hole is provided with a tranfer element, the mixture of test liquid and labelled second reagent is applied to said transfer element.

The above described devices are preferably also provided with a static means, which is an element meant for mixing and pre-incubation of the test liquid and mobile labelled second reagent. Said static means is placed on top of said devices and the mixture of test liquid and mobile

12 labelled second reagent is then applied on top of said static means. Said mixture is then allowed to be transferred to the bibulous support by capillary forces, during which transfer incubation takes place between the analyte, when present, and the mobile labelled second reagent. The assay method is further identical to that described above.

In another embodiment of the present invention the mobile labelled second reagent is incorporated into the bibulous support. The method for detection of the presence or amount of the analyte to be determined is then carried out by applying the test liquid directly, or via the transfer element, onto the sample application spot on the porous support, followed by measuring and processing the values obtained from the light detector as described above.

In a preferred embodiment of the present invention the device further comprises a porous element comprising the mobile labelled second reagent. This element is placed on top of the bibulous support, provided with a coversheet with a hole and an absorbing element, and optionally with a transfer element. When the device also comprises a static means, said porous element is placed on top of said static means. Test liquid is then applied to said porous element by means of, for example, a pipette, or a separate sample collector which is preferably incorporated into the device. The method for detecting the presence or amount of the analyte to be determined with the above described device then comprises the following steps:

* applying the test liquid onto the porous element of the above device;

* allowing the test liquid to dissolve or re-disperse the mobile labelled second reagent present in said element;

* allowing the reaction mixture of test liquid and mobile labelled second reagent to be transferred to the bibulous support, while incubation takes place;

* projecting light from the light source onto the measuring zone and measuring the electrical signal from the light detector at two predefined points of time or measuring the points of time required to obtain two predefined values for the electrical signal; * processing the measurements to derive a value representative for the presence or amount of the analyte to be determined.

13 The present invention is also directed to a test kit for determination of the presence or amount of an analyte in a test liquid, which comprises a device as described above and, depending on the integration of the various reagents and components into said device, other reagents and components required

Exemplary embodiments of the invention are explained in detail hereinafter.

Figure 1 is a schematic cross-sectional view of an embodiment of the device described in the present invention, wherein the housing surrounding the various elements is omitted

Figure 2B is an embodiment of the presentation of the device described in the present invention, while Figure 2A shows the separate sample collector incorporated in said device

The device depicted in Figure 1 comprises a sample collector with a handle 1 and an absorbent tip 2 which fits into a housing, but which can temporarily be removed from said housing for collection of test liquid After collection of test liquid the sample collector is brought back into the housing whereby contact is made with a filtration means 3, which is placed on top of a porous element 4 comprising a labelled reagent for the analyte to be determined in the test liquid The lower part of said porous element 4 is in contact with the upper part of a static means 5, i e a porous element for mixing and pre-incubation of analyte and labelled reagent Said static means is in fluid flow contact with a bibulous support 9, comprising an immobilized reagent for the analyte, via a transfer element 7 The upper part of said bibulous support is in contact with the lower part of an absorber 6 with a recess, defining a circular borderline in contact with said bibulous support The area on the upper part of said bibulous support which is within said borderline, is covered by a water- impermeable sheet 8 Said sheet 8 comprises a hole 12 through which the reaction mixture in the static means 5, through transfer element 7, is applied onto the bibulous support 9 At the lower part of said bibulous support 9 a light source 1 1 and a light detector 13 are placed The output signal from the light detector is led into the processor 14, which is connected to a battery 10, for analysis of the test data Said processor is in contact with the display unit 15 on which the final test result can be read

14 The sample collector 1 presented in Figure 2A, comprises a handle 2 and an absorbent tip 3.

The complete device depicted in Figure 2B has a housing 2 comprising the handle 1 of the sample collector (as depicted in Figure

2A), which can be removed from said housing for uptake of test liquid, a part 3 suπounding the chemical- and opto-electronic unit, and a cap 6 which can also be removed from the housing to take out the battery (shown in Figure 1). The housing 1 further comprises a window 5 through which the test result 4 can be read.

15

Claims

1 Assay device for detection or determination of the amount of an analyte in a test liquid comprising - a bibulous support comprising a zone with an immobilized first reagent and a sample application spot,

- a light source for projecting light onto the immobilized first reagent,

- a light detector capable of producing an electrical signal, the light source and the light detector defining a measuring area, -electronic processing means for deriving an output signal from the electrical signal from the light detector, at least one of the first reagent and a mobile, labelled second reagent being capable of reacting specifically with an analyte to be determined in the test liquid, the electronic processing means being capable of determining the electrical signal in a time-dependant manner and capable of processing the data obtained to yield a value corresponding to the analyte concentration, and

- means for presenting the output signal indicative of said value in a form which can be interpreted by a human being, wherein, the sample application spot and the measuring area are located within the perimeter of said zone with an immobilized first reagent

2 Assay device according to claim 1, wherein the device comprises a strongly absorbing element provided with a recess, said recess defining a borderline in contact with the bibulous carrier, the borderline being substantially continuous and substantially circular, the borderline defining a centre substantially coinciding with the sample application spot

3 Assay device according to claim 2, wherein said absorbing element is capable of maintaining a substantially constant flux through the bibulous carrier 4 Assay device according to any of claims 1-3, wherein the size of the sample application spot is determined by a hole in a cover sheet provided on top of said bibulous support

5 Assay device according to claim 4, wherein said cover sheet comprises water-impermeable material 6 Assay device according any of claims 1-5, wherein the centre of the measuring area substantially corresponds to the sample application spot 7 Assay device according to any of claims 1-6, wherein the light detector

16 is located substantially under the sample application spot 8 Assay device according any of claims 1-7, wherein the device comprises static means for mixing and pre-incubation of test liquid with said labelled mobile second reagent 9 Assay device according to claim 8, wherein said static means is a porous element positioned on top of the bibulous carrier 10 Assay device according to any of claims 1-9, wherein the device comprises a transfer element capable of establishing a direct contact between the bibulous carrier and the static means 11 Assay device according to claim 10, wherein said transfer element is a porous sheet positioned on top of said hole in the cover sheet

12 Assay device according to claim 11, wherein said porous sheet comprises a non-woven material with a negligible flow resistance

13 Assay device according to any of claims 1-12, wherein the assay device comprises a mobile labelled second reagent being capable of reacting specifically with the analyte to be determined

14 Assay device according to any of claims 1-13, wherein the label is capable of absorbing or scattering light

15 Assay device according to claim 14, wherein the label is a colloidal particle

16 Assay device according to claim 15, wherein said colloidal particle is chosen from the group consisting of metal sol particles, non-metal sol particles and dyestuff sol particles

17 Assay device according to claim 16, wherein said non-metal sol particles are carbon sol particles

18 Assay device according to any of claims 1-17, wherein said assay device comprises a porous element containing the labelled second reagent, which element is capable of an easy direct or indirect release of said labelled second reagent to the bibulous support 19 Assay device according to claim 18, wherein said porous element comprises a reagent composition allowing a controlled release of said mobile, labelled second reagent

20 Assay device according to any of claims 1-19, wherein said assay device comprises a sample collector for uptake and release of test liquid, said sample collector comprising a handle and absorbing material, which absorbs the test liquid rapidly and is capable of an easy direct or indirect release of test liquid to the bibulous support

17 21 Assay device according to any of claims 1-20, wherein the assay device comprises an element for filtration of the test liquid.

22 Assay device according to any of claims 1-21, additionally comprising a housing 23 Use of an assay device according to any of claims 1-22 for detection of an analyte in a test liquid

24 Method for detection or determination of the amount of an analyte in a test liquid, comprising the steps of a) mixing the test liquid with the labelled second reagent b) supplying said mixture directly or indirectly onto said sample application spot of the device according to any of claims 1-12 c) allowing said mixture to spread radially from the sample application spot of the bibulous support d) while projecting light from the light source onto the measuring zone, measuring the electrical signal from the light detector in a time-dependent manner e) processing the measurements to derive a value representative for the concentration of the analyte f) presenting the output signal indicative of said value in a form which can be interpreted by a human being

25 Method for detection or determination of the amount of an analyte in a test liquid, comprising the steps of a) applying the test liquid and the labelled second reagent to the static means of the device according to claim 8 or 9 b) allowing the test liquid and the labelled second reagent to be transferred to the sample application spot on the bibulous support c) allowing said mixture to spread radially from the sample application spot of the bibulous support d) while projecting light from the light source onto the measuring zone. measuring the electrical signal from the light detector in a time-dependent manner e) processing the measurements to derive a value representative for the concentration of the analyte f) presenting the output signal indicative of said value in a form which can be interpreted by a human being

26 Method for detection or determination of the amount of an analyte in a test liquid, comprising the steps of

18 a) applying the test liquid to the sample application spot of the device according to claim 13 b) allowing the test liquid and the second labelled reagent to spread radially from the sample application spot of the bibulous support c) while projecting light from the light source onto the measuring zone, measuring the electrical signal from the light detector in a time-dependent manner d) processing the measurements to derive a value representative for the concentration of the analyte e) presenting the output signal indicative of said value in a form which can be interpreted by a human being.

27. Method for detection or determination of the amount of an analyte in a test liquid, comprising the steps of: a) contacting the test liquid with a device according to claim 18 or 19; b) allowing the test liquid and the mobile labelled second reagent to be transferred to the sample application spot on the bibulous support; c) allowing said mixture to spread radially from the sample application spot of the bibulous support d) while projecting light from the light source onto the measuring zone, measuring the electrical signal from the light detector in a time-dependent manner e) processing the measurements to derive a value representative for the concentration of the analyte f) presenting the output signal indicative of said value in a form which can be interpreted by a human being.

28. Method for detection or determination of the amount of an analyte in a test liquid, comprising the steps of: a) collecting the test liquid with the sample collector of the device according to claim 20 b) contacting said sample collector with the porous element of said device c) allowing the test liquid and the labelled second reagent to be transferred to the sample application spot on the bibulous support d) allowing said mixture to spread radially from the sample application spot on the bibulous support d) while projecting light from the light source onto the measuring zone, measuring the electric signal from the light detector in a time-dependant manner.

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29. Method according to any of claims 24-28, wherein at least two subsequent readings of the electrical signal at either a preset time interval or at a predefined reading interval are used to calculate a single characteristic number that, when compared to a preset reference value, enables a determination of the presence of said analyte

30. Method according to any of claims 24-28, wherein at least two subsequent readings of the electrical signal at either a preset time interval or at a predefined reading interval are used to calculate a single characteristic number that enables a determination of the amount of said analyte

31. Test kit for detection or determination of the amount of an analyte in a test liquid comprising: a) a device according to any of the preceding claims b) other reagents and materials required

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