AU2011246529B2

AU2011246529B2 - Microfluidic system with sample pretreatment

Info

Publication number: AU2011246529B2
Application number: AU2011246529A
Authority: AU
Inventors: Franz Aberl; Sebastian Klaus; Torsten Stadthagen
Original assignee: Securetec Detektions Systeme AG
Current assignee: Securetec Detektions Systeme AG
Priority date: 2010-04-26
Filing date: 2011-04-26
Publication date: 2014-07-31
Anticipated expiration: 2031-04-26
Also published as: AU2011246529A1; EP2564196B8; WO2011134946A1; ES2559977T3; HRP20151436T1; PL2564196T3; EP2381258A1; EP2564196B1; EP2564196A1

Abstract

The present invention relates to a method for determining an analyte and to an apparatus which is suitable for this purpose.

Description

1 WO 2011/134946 Microfluidic system with sample pretreatment The present invention relates to a method for determining an analyte and to a device which is suitable for this purpose. Immunoassays are an important component of clinically relevant analysis methods, which are based on the formation of immune complexes from antigens and antibodies and are used to trace analytes in liquid samples, for example in the field of medicinal, environmental, food and agro analytics. Because of the biophysical and biochemical properties of antigen antibody binding, immunoassays generally provide high specificity and sensitivity with a comparatively simple and inexpensive apparatus, and thus have significant advantages over alternative detection methods. A further field of application of immunoassays, which is important in practice, is detecting drugs in bodily fluids or on surfaces which are contaminated with drugs. In tests of this type, a sample of the analyte is conventionally taken from a surface, using a suitable sampling element, the sampling element which has been wetted with the analyte is brought into contact with a test strip, and the analyte which has been extracted from the sampling element and transferred to the test strip is detected by means of an immunological detection reaction. An aspect which is of major importance especially for detecting drugs (for example amphetamines, methamphetamines, cannabis, cocaine, heroin) is the specificity, sensitivity and rapidity of the tests which are used. In this context, on the one hand there is a need for highly sensitive detection methods, so as to be able to trace the presence of drugs reliably and rapidly even with small sample volumes or when using complex sample material such as saliva. On the other hand, the test formats should also have a high specificity to the substance which is to be traced in each case, so as to exclude false-positive measurement results, and thus provide authoritative information as to which specific drug the tested substance is. EP 0 699 906 A2 discloses a rapid drug test which is commercially available as a surface, sweat and saliva test known as DrugWipe* (from Securetec Detektionssysteme AG). The 2 test makes use of a wiping element ("wiper") which consists substantially of plastics material, comprising a bonded-on non-woven, by means of which a sample of the analyte (for example from a surface or from a solution) is taken and subsequently transferred directly to an immunochromatographic test strip which is stored in a one-way housing ("lateral flow" technology). Chromatography is started by immersing the test strip in an aqueous solution (water or buffer, comprising various reagents), it being possible to read the result of the determination by eye or. by using a suitable measurement device. The analyte is traced in that antibodies which are bound to drug molecules (antigens) bind onto a test line, and, as a result of the gold labeling thereof, form a coloured line which can be optically detected in the read-out window. Before reaching the test line, antibodies which do not carry drug molecules are captured by means of haptens which are present on the test strip in an immobilised form. By comparison with all the other rapid drug tests on the market, DrugWipe* and Triage* (from Biosite) are the only two products which form a coloured line if a particular analyte is present in a sample, and thus have what is known as a positive display. All other commercial products have a negative display, in which the failure of a line to appear is evaluated as a positive detection of the respective analyte. A further major advantage of DrugWipe* is the low sample volume which is required for detecting drugs and other substances. Whilst a sample volume of approximately 1-20 pl is sufficient for DrugWipe*, other commercial tests require a sample volume of at least 100 pl and up to several millilitres. The rapid test OraLab* developed by Varian is used to detect drugs from saliva samples, and is also based on lateral flow technology. Whilst the specificity of the test is about 90-100 %, the sensitivity to amphetamines and opiates is only between 50 and 90 %, and for A 9 tetrahydrocannabinol and cocaine it is even well below 50 % in some cases (see DRUID study, http://www.druid-project.eu). Further, a major drawback of this test is that relatively large sample volumes are required, and these are often not available for acute drug users. According to the data from the DRUID study (http://www.druid-project.eu), the rapid test DrugCheck* 5000 from Drager, which is also based on lateral flow technology, is a viable test system for detecting drugs. However, this test format has the significant drawback that the results on the test strip can only be evaluated by means of a read-out device which has high acquisition costs and is only somewhat suitable for rough use outdoors in the field.

3 Therefore, significant problems occur with this test as regards cost-efficiency and ease of use. The Rapid STAT* test kit supplied by Mavand is a further commercially available rapid drug test in which a saliva sample diluted with buffer is incubated on an immunochromatographic test strip having labeled binding partners, and which according to the manufacturer's specifications makes it possible to trace A 9 -tetrahydrocannabinol down to a lower limit of 15 ng/ml. A significant drawback of this test is the extremely complex and cumbersome implementation thereof, which makes it unsuitable for use particularly by traffic police, and the comparatively low specificity of 80-90 % for A 9 -tetrahydrocannabinol (see DRUID study, http://www.druid-project.eu). According to a study by the University of Mainz, the rate of false-positive test results for A 9 -tetrahydrocannabinol is over 10 %, and the overall specificity is 84 % (published on the GTFCH 2009, Mosbach, http://gtfch.org/cms/images/stories/media/tk/tk76_2/abstractsposter.pdf). US 7,090,803 B1 and US 7,507,374 B2 (or US 2006/0292035 Al) each disclose devices for determining an analyte in a saliva sample, which comprise a housing for receiving a sampling element, a holding device for receiving at least one chromatographic test strip, a first chamber for storing a first reagent such as a buffer solution, a second chamber for storing a second, analyte-specific reagent, means for bringing the sample into contact with the first reagent, means for bringing the mixture of the sample and the first reagent into contact with the second reagent in the second chamber, and means for bringing the mixture of the sample, the first reagent and the second reagent into contact with the chromatographic test strip. Specifically, in this context a sampling element, which at one end comprises a sponge material comprising a sample of the analyte received therein, is introduced into the housing via a cylindrical recess in the housing, and the sample of the analyte is pressed out of the sponge material by the sampling element pressing into the recess. The sample thus passes through an opening located at the base of the recess, into the second chamber, in which it is mixed with the first reagent which is released from a deformable shell by being brought into contact with a sharp-edged object, and passes through an opening located at the base of the first chamber, into the second chamber, and is mixed with the second reagent. The

I

4 mixture is subsequently incubated for several minutes, and afterwards is brought into contact with the chromatographic test strip, which test strip makes it possible to determine the test result by eye. The devices and methods disclosed in US 7,090,803 B1 and US 7,507,374 B2 have the drawback that the sample of the analyte is merely pressed out of the sponge material mechanically, by the sampling element pressing into the cylindrical recess of the test device. In this way, on the one hand, only a small part of the sample originally taken passes into the second chamber in which the sample is incubated with the first and second reagent and the analyte binds to its analyte-specific binding partner, which ultimately reduces the sensitivity of the analyte determination. On the other hand, because of the absorptive action of the sponge material, large sample volumes are required, and these are generally not available in practice. US 2008/0166820 Al discloses a kit for determining an analyte in a bodily fluid, which comprises a sampling element for receiving a sample of the analyte, an ampulla having a buffer solution contained therein, a transfer element for withdrawing the solution from the ampulla and for transferring the solution into an analysis device, and an analysis device. The analysis device in turn comprises at least one reaction region which is configured for receiving the buffer solution and contains a binding partner which is specific to the analyte, a chromatographic test strip, and means for bringing the chromatographic test strip into contact with the buffer solution. To determine the analyte, the sampling element is introduced into the ampulla which is filled with buffer solution, the region of the sampling element which is soaked with a sample of the analyte being saturated with the buffer solution, and the analyte being eluted by the sampling element subsequently compressing into the buffer solution. After the sampling element is removed, part of the buffer solution containing the analyte is removed from the ampulla and transferred into the analysis device by means of the transfer element , in which analysis device the analyte is incubated for several minutes with the analyte-specific binding partner and subsequently brought into contact with the chromatographic test strip. The kit disclosed in US 2008/0166820 Al has the drawback that because of the large number of individual components it is cumbersome to use, and thus is not considered a viable test in terms of reliable application, for example as a rapid drug test in the field.

5 Further, because it is necessary to withdraw the buffer solution containing the analyte from the ampulla and to transfer it into a separate analysis device, there are risks of sample losses and of sample contamination, which can ultimately lead to a reduction in the sensitivity and specificity of the test result and severely limits the overall reliability of the test. EP 0 634 215 Al discloses a method for detecting analytes in a bodily fluid, which method makes it possible to determine the analytes on a plurality of test elements substantially simultaneously and substantially uniformly at different sampling points. In this context, to carry out the method, a device is used which comprises a sample deposit point for applying the sample, a plurality of separated sampling zones, which are each connected to the sample deposit point by a capillary transport path, and a plurality of test elements for individually determining analytes, a delay zone for slowing down the transport of the fluid from the sample deposit point to the sampling zone being provided on at least one of the transport paths. A drawback of the test format described in EP 0 634 215 Al is that the analyte which is located in the sample is not incubated with an analyte-specific binding partner in a region separate from the test element, since the delay zone, the presence of which is obligatory, does not contain an analyte-specific binding partner, and is merely intended to prevent the test from being flooded by the sample or ensure that the individual test elements are wetted with the sample simultaneously. This does not provide a basis for optimum kinetics between the analyte and the analyte-specific binding partner, and as a result sensitivity and specificity are severely limited, in particular when detecting poorly water-soluble (hydrophobic) analytes. EP 0 811 852 A2 discloses a method for detecting an analyte in a bodily fluid or on a contaminated surface. In this context, to carry out the method, a test kit is used which comprises a test strip consisting of one or more capillary-active chromatography-compatible materials, a sampling element separate from the test strip surface, and a pressing device for bringing the test strip surface and the sampling element into contact, it being possible for the sampling element to be moistened with an aqueous buffer, a detergent or/and an organic solvent, so as to receive the analyte better. The test disclosed in EP 0 811 842 A2 has the drawback that the sample containing the analyte is not pretreated in any way before being brought into contact with the test element, 6 and this :is of significance in particular when determining biological samples having a complex chemical composition (for example blood, saliva or sweat). Ignoring the fact that biological samples inherently fluctuate within a wide range depending on the individual, the constitution of the sample matrix in particular (for example viscosity, protein content etc.) has a major influence on the analyte detection, since the sensitivity of the detection method is highly dependent on the accessibility of the analytes to the analyte-specific binding partner. A further drawback of the test format disclosed in EP 0 811 842 A2 is also that the analyte which is located in the sample is not incubated with an analyte-specific binding partner in a region separate from the test element. In this way, optimum kinetics are not provided between the analyte and the analyte-specific binding partner, and as a result sensitivity and specificity are severely limited, in particular when tracing poorly water-soluble analytes. US 6,203,757 B1 discloses a method for detecting analytes in a bodily fluid, which provides simultaneous determination of a plurality of analytes by means of a single sample. To carry out the method, a test device is used which comprises at least two test strips which are arranged substantially mutually parallel, and a sample distributor element which is V-shaped or W-shaped in form and which makes fluid communication possible between the sample deposit point and the individual test strips. In one embodiment, the analyte may be a drug molecule which is detected for example by means of an enzymatic immunoassay. A drawback of the detection method disclosed in US 6,203,757 B1 is that the sample which is dripped onto the test device is transported along a fabric-like material before the analyte and the analyte-specific binding partner come into contact. Because fabric-like materials generally have very large surface areas, to which the analyte can bind non-specifically, in tests of this type the total amount of analyte present in the sample is often no longer available for a reaction with the analyte-specific binding partner. This is highly disadvantageous in particular when detecting poorly water-soluble analytes, and in some cases dramatically limits sensitivity. A further drawback of the test format disclosed in US 6,203,757 B1 is also that the analyte which is located in the sample is not incubated with an analyte-specific binding partner in a region separate from the test element. In this way, the sensitivity and specificity of the test are severely limited, in particular when detecting poorly water-soluble (hydrophobic) H1iparnIem-ovenNRPartblDCC\PAR'635322_I.do-23/o5/2I4 7 analytes, and there are therefore problems in particular when detecting analytes which are only present in low concentrations in the sample which is to be analysed. Based on the above-described test systems, the present invention seeks to provide a method for determining an analyte, in particular for determining drugs, in which method the drawbacks of the prior art are overcome at least in part. In particular, the method should have a high sensitivity and specificity to all the analytes which are to be tested, be simple to implement, and make rapid determination possible without the use of additional technical aids for reading the test results. Accordingly, the present invention provides a method for determining an analyte comprising the steps: (a) providing a microfluidic analysis device, comprising at least a first, a second and a third region, the first region being in fluid communication with the second region and the second region being in fluid communication with the third region, (b) receiving a sample containing the analyte from a sample surface by means of a sampling element, (c) introducing the sampling element into the first region of the microfluidic analysis device and eluting the analyte in the first region by means of an eluent, (d) transferring the eluate which was obtained in step (c) into the second region of the microfluidic analysis device, and bringing the eluate into contact with an analyte specific binding partner, which is stored in the second region, for a period of at least 3 seconds, the analyte binding to the analyte-specific binding partner, (e) transferring the mixture which was obtained in step (d) into the third region of the microfluidic analysis device, and bringing the mixture into contact with at least one test element, and (f) determining the presence or/and the amount of the analyte on the test element. Surprisingly, in the context of the present invention, it has been found that by means of the method according to the invention analytes can be detected in a simple and reproducible manner with a high sensitivity and specificity, without large sample amounts of the analyte or/and complex technical aids for reading the test results being required for this purpose. The first step of the method according to the invention requires the provision of a microfluidic analysis device which is suitable for determining the analyte and which comprises at least a 8 first, a second and a third region. The first region of the microfluidic analysis device is configured for introducing a sampling element by means of which a sample of the analyte was taken in advance, and for subsequently eluting the analyte from the sampling element with an eluent, whilst the second region contains a binding partner which is specific to the analyte and is in fluid communication with the first region. Finally, the third region comprises at least one test element for determining the presence or/and the amount of the analyte and is in fluid communication with the second region. The term "in fluid communication", as used in the context of the present application, means that the respective regions of the microfluidic analysis device are interconnected by microfluidic structures, such as microchannels, stages, branches or/and chambers, and thus make it possible to transfer or process fluids within the analysis device. Microfluidic structures, such as those mentioned above, can be manufactured by methods known to a person skilled in the art, such as chip milling or injection moulding, from suitable materials in particular from plastics material, according to the respective demands on the analysis device. In the next step of the method according to the invention, to determine the analyte, a sample containing the analyte is received by means of a suitable sampling element from a sample surface which is to be analysed (for example tongue, skin, other surface). In this context, as a sampling element in principle any element can be used, which is capable of receiving a sample of the analyte and releasing it virtually quantitatively upon subsequent contact with an eluent, that is to say at an amount 95 % by weight based on the total weight of the received sample. Sampling elements which are particularly suitable for the purposes of the present invention are disclosed for example in EP 1 608 268 Al and WO 2004/086979 Al, the disclosure of which is hereby incorporated by reference. The sampling element, or a region of the sampling element which is configured for receiving the sample, may in principle consist of any material which appears useful to a person skilled in the art for the purposes of the present invention, and which makes it possible both to accumulate the analyte on the sampling element and subsequently to release it upon bringing it into contact with an eluent. Thus, as well as the sampling elements disclosed in EP 1 608 268 Al and WO 2004/086979 Al, sampling elements may also be considered, which comprise absorbent materials, in particular fabrics, non-wovens or/and porous matrices (for example membranes and sponges). Suitable non-wovens are disclosed for 9 example in DE 38 02 366 Al and EP 0 699 906 A2, the disclosure of which is hereby explicitly incorporated by reference. So as to provide high sensitivity and specificity when determining the analyte, the surface of the sampling element may be chemically pretreated before being used for the first time; in this way, it is possible to improve the receipt of the analyte during sampling or/and to minimise adhesion of the analyte to the sampling element. Thus, the method according to the invention provides that the sampling element comprises a transfer reagent containing at least one protein, at least one carbohydrate, at least one sugar alcohol or/and at least one salt, in particular an inorganic salt. The transfer reagent which promotes the transfer of the analyte from the sample surface to the sampling element or/and the subsequent release of the analyte onto the eluent, in particular by blocking free binding sites on the sampling element or/and influencing the analyte properties, can for example be impregnated on the sampling element for this purpose. Techniques which may be used for applying the transfer reagent to the sampling element are generally known to a person skilled in the art. The term "carbohydrate", as used in the present application, refers to monosaccharides, oligosaccharides and polysaccharides of the general empirical formula CnH 2 nO,, which may each be of natural or synthetic origin. In the context of the invention, monosaccharides or oligosaccharides are preferably used, in particular naturally occurring tetroses, pentoses and hexoses, such as erythrose, threose, ribose, arabinose, lyxose, xylose, allose, altrose, galactose, glucose, gulose, dose, mannose, talose and fractose, which may each be present in the D form or in the L form, being used as monosaccharides. In particular naturally occurring disaccharides and trisaccharides, such as lactose, maltose, saccharose, trehalose, gentianose, kestose and raffinose, may be used as oligosaccharides. In a particularly preferred embodiment of the invention, the transfer reagent comprises a carbohydrate selected from the group consisting of glucose, lactose, maltose and saccharose. The term "sugar alcohol", as used in the present application, refers to monosaccharide sugar alcohols of the general empirical formula CnH 2 n, 2 O and disaccharide alcohols of the general empirical formula CnH 2 nOn 1 , which may in each case be of natural or synthetic origin. Preferred monosaccharide sugar alcohols include glycerol, erythritol, threitol, ribitol, 10 arabinitol, xylitol, allitol, altritol, galactitol, glucitol, iditol and mannitol, which may each be present in the D form or in the L form. In particular isomalt, lactitol and maltitol may be used as disaccharide sugar alcohols. In a particularly preferred embodiment of the invention, the transfer reagent contains a sugar alcohol selected from the group consisting of glucitol, glycerol, mannitol and xylitol. In the context of the method according to the invention, a transfer reagent is preferably used, which comprises (a) at least one protein selected from the group consisting of gelatine, ovalbumin and bovine serum albumin, (b) skimmed milk powder, (c) at least one carbohydrate selected from the group consisting of glucose, lactose, maltose and saccharose, (d) at least one sugar alcohol selected from the group consisting of glucitol, glycerol, mannitol and xylitol, or/and (e) at least one salt selected from the group consisting of calcium chloride, potassium chloride, magnesium chloride, sodium chloride and a borate. Particularly preferably, the transfer reagent which is used according to the invention comprises at least one protein selected from the group consisting of gelatine, ovalbumin and bovine serum albumin, or/and skimmed milk powder. The concentration of the at least one protein, at least one carbohydrate, at least one sugar alcohol or/and at least one salt in the above-described transfer reagent may be adapted by a person skilled in the art, according to the respective demands on the analyte, but is usually approximately 0.01 to approximately 15 % by weight, based on the total weight of the transfer reagent. If the transfer reagent comprises salts, they are usually added in concentrations of approximately 1 pM to approximately 1 M. In addition to the at least one protein, at least one carbohydrate, at least one sugar alcohol or/and at least one salt, the transfer reagent may optionally comprise further reagents which promote a transfer of the analyte from the surface which is to be analysed to the sampling element or/and the subsequent release of the analyte onto the eluent, such as a detergent or/and an organic solvent. Examples of detergents include, among others, cholamidopropane sulphonate, octyl glucoside, polidocanol, polyalkylene glycol ether (for example Brij*, Synperonic*) and polysorbates (for example Tween* 20, Tween* 80), which are conventionally used in concentrations of approximately 0.01 to approximately 5 % by weight based on the total weight of the transfer reagent. Examples of organic solvents include in particular dimethyl sulphoxide, ethanol, methanol, glycerine and mixtures thereof, which are added to the transfer reagent in a concentration of usually < 30 % by weight.

11 In a preferred variant, the method according to the invention provides the use of a sampling element which comprises a volume indicator. During sampling, the volume indicator displays to the user whether a sufficient, defined sample volume for determining the analyte has been taken. This is of decisive importance in particular when sampling fluids such as saliva since in general an optimum performance of the respective test system can only be provided if a defined sample volume is provided. A negative influence on the test system by the test subject, for example by the subject depositing too low a sample volume, can be prevented by the volume indicator, and as a result the sensitivity, specificity and overall reliability of the test system can ultimately be optimised. Particularly preferably, the volume indicator is a colour indicator which changes colour upon contact with a sufficient sample volume, for example upon contact with a sufficient volume of bodily fluid, and thus correlates with the sample volume required for determining the analyte. Any colour indicator which is known to a person skilled in the art and appears suitable for the purposes of the present invention may be used as a colour indicator, as long as it meets the above criteria and is also non-toxic. Examples of colour indicators of this type include in particular common pH colour indicators or plant dyes, which can be applied to the sampling element by vapour deposition, imprinting, spraying or/and soaking. In the next step of the method according to the invention, the sampling element which is wetted with the sample of the analyte is introduced into the first region of the microfluidic analysis device, which region is preferably in the form of a chamber (Fig. 1A). In this context, the sampling element and the first region of the microfluidic analysis device, which is configured for receiving or integrating the sampling element are configured in such a way that the first region is tightly sealed after the sampling element is introduced and no fluid communication can take place between the interior of the first region and the external environment. Once the sampling element has been received in the first region of the microfluidic analysis device, and it has been ensured that the first region is sealed off from the external environment, eluent is introduced into the first region of the microfluidic analysis device (Fig. 1B), the analyte being eluted from the sampling element and distributed preferably homogeneously in the eluent. Thus, in a preferred variant, the invention provides that after the introduction of eluent into the first region, a homogeneous mixture of sample or analyte 12 and eluent is provided, and this can be ensured for example by a microfluidic mixing path or another microfluidic mixing structure which is known to a person skilled in the art. This has the advantage that the sample is additionally pretreated before being brought into contact with a suitable test element, and as a result, the analyte is accessible in a simpler and more efficient manner to an analyte-specific binding partner, and fluctuations in particular in the determination of biological samples are minimised. Consequently, in this way the sensitivity of the method can be greatly increased. When a suitable combination of sampling element and eluent is used, it can be ensured by means of the above method sequence that the sample and the analyte which is contained therein are eluted from the sampling element substantially quantitatively, that is to say in an amount 2 95 % by weight based on the total weight of the received sample, the analyte is released substantially quantitatively from the sample matrix, and the analyte and eluent are mixed completely. Quantitative release of the sample from the sampling element and quantitative release of the analyte from the sampling matrix are of essential importance for the maximum sensitivity of diagnostic test systems since bodily fluids which contain large amounts of proteins, lipids and carbohydrates, which in turn can lead to undesirable consequences in immunochemical reactions, are often used as samples for detecting drugs for example. According to the invention, the eluent can be introduced into the first region of the microfluidic analysis device in any desired manner. Thus, in a variant of the invention, the eluent can be stored in the sampling element which is used according to the invention. For this purpose, the sampling element may for example comprise an ampulla which contains the eluent, and which is opened and releases the eluent when the sampling element is introduced into the first region of the microfluidic analysis device. Alternatively, the eluent can be stored in a fourth region of the microfluidic analysis device, which fourth region is in fluid communication with the first region and from which the eluent can be flushed out into the first region of the microfluidic analysis device, for example by way of an implementation step which is to be carried out separately by the user (Fig. 1 A and Fig. 1 B). In the context of the present invention, in principle any eluent which can release the analyte from the sampling element can be used as the eluent. However, in the method disclosed herein, buffer solutions are preferably used which may optionally contain further reagents, in particular at least one protein, at least one carbohydrate, at least one sugar alcohol, at least 13 one detergent or/and at least one organic solvent, in each case as disclosed above, in concentrations of usually approximately 0.05 to approximately 1.5 % by weight. In the context of the method according to the invention, a preferably aqueous eluent which comprises 3-[(3-cholamidopropyl)dimethyl-ammonio]-1 -propane sulphonate as a component is considered to be particularly preferred. By way of a suitable combination of the above reagents, synergy effects can be achieved as a function of the structure of the respective analyte, and as a result the elution of the analyte from the sampling element or the release of the analyte from the sample matrix can be improved, and in this way the sensitivity or/and specificity 'of the analyte determination can be increased. Subsequent to the elution of the analyte from the sampling element, in a further step of the method according to the invention the obtained eluate, that is to say the mixture of analyte containing sample and eluent, is transferred into the second region of the microfluidic analysis device by suitable means, in particular via a microfluidic structure such as a microchannel, and brought into contact with an analyte-specific binding partner, which is stored in the second region, for a defined period of time, the analyte binding to the analyte specific binding partner. According to the invention, the analyte is thus incubated with the analyte-specific binding partner in the absence of a test element which is used for detecting the analyte, and as a result both the sensitivity and the specificity of the detection method can be increased significantly, in particular when determining poorly water-soluble analytes. In this context, the transfer of the eluate from the first region into the second region of the microfluidid analysis device can be started for example by way of an implementation step which is to be carried out separately by the user, a defined volume of the eluate preferably being transported into the second region. The transfer of the eluate from the first region into the second region can additionally be used to achieve an even higher level of homogeneous mixing of the sample and the eluent. This may preferably be achieved by way of a microfluidic mixing path or another microfluidic structure which is known to a person skilled in the art. The second region of the microfluidic analysis device may in principle be configured in any form, as long as it can receive the eluate which is transferred from the first region and makes contact possible between the eluate and the analyte-specific binding partner which is stored in the second region. In a preferred variant of the invention, the second region of the microfluidic analysis device is in the form of a single chamber and comprises a single 14 analyte-specific binding partner. Alternatively, the second region may also comprise a plurality of chambers, for example 2 to 5 chambers, which are preferably arranged mutually parallel and may optionally contain different analyte-specific binding partners, if a plurality of analytes are to be determined simultaneously (Fig. 1C). The analyte-specific binding partner may be any chemical substance which binds to the analyte which is to be tested, but does not form bonds with other substances which may be present in the sample. Chemical substances which satisfy this requirement profile are generally known to a person skilled in the art, or can be produced in accordance with the demands on the respective analyte by means of routine experiments and by using known techniques. Preferably, the analyte-specific binding partner is an antibody or a functional antibody fragment, which antibody may optionally additionally comprise a binding site or a plurality of binding sites for a capture reagent, as described above. In this context, the term "functional antibody fragment", as used in the context of the present application, refers to an antibody fragment which can perform its intended function, the function of specifically binding the analyte. A "non-functional antibody fragment", by contrast, refers to an antibody fragment which does not form a bond or does not form a specific bond to the analyte, and thus does not perform its intended function, the function of specifically binding the analyte. To make it easier to detect the analyte, the analyte-specific binding partner may optionally comprise a detectable label, such as an enzyme label, dye label, fluorescence label, metal label or particle label. The use of analyte-specific binding partners which comprise an optically detectable label, in particular a metal label, has been found to be advantageous for the purposes of the present invention. Particularly preferably, the label is a gold label which has the advantage that the test result can be detected and evaluated optically by eye, directly by the user. Techniques with which above-described labels can be introduced into a molecule which is to be labeled are known to a person skilled in the art and are therefore not explained in greater detail. According to the invention, the second region may comprise the analyte-specific binding partner in any desired form, as long as a reaction with the analyte-specific binding partner can take place when the eluate enters the second region of the microfluidic analysis device. Preferably, however, the analyte-specific binding partner is stored in the second region of 15 the micr6fluidic analysis device in such a way that it is dissolved and homogeneously distributed in the solution when the eluate is introduced into the second region of the microfluidic analysis device. For this purpose, the microfluidic analysis device may contain the analyte-specific binding partner for example in a dried form, although in principle other storage forms which appear suitable to a person skilled in the art are also possible. After the eluate and the analyte-specific binding partner have been brought into contact and optionally. homogeneously mixed, an immunological reaction is usually started, an antigen antibody complex being formed from analyte molecules and an analyte-specific, optionally labeled binding partner. To maintain high sensitivity and specificity, the eluate containing the analyte is brought into contact with the analyte-specific binding partner for a period of at least 3 seconds, preferably of at least 5 seconds, during which the analyte binds to the analyte-specific binding partner. In this context, a reaction time of approximately 5 seconds to approximately 600 seconds, more preferably of approximately 60 seconds to approximately 300 seconds, most preferably of approximately 90 seconds to approximately 240 seconds, has been found to be advantageous. The reaction between the analyte and the analyte-specific binding partner may in principle take place in any desired manner, as long as sufficient contact between the analyte and the analyte-specific binding partner is ensured. For the purposes of the present invention, however, it has been found to be advantageous if the reaction between the analyte and the analyte-specific binding partner is carried out in the stationary phase, such that a sufficiently long contact time is available for forming a complex of the analyte and the analyte-specific binding partner. So as to promote the reaction between the analyte and the analyte-specific binding partner, the second region of the microfluidic analysis device may if required comprise further reagents. In this context, in particular reagents which promote the release of the analyte specific binding partner upon contact with the eluate, which promote complete mixing of the eluate and the analyte-specific binding partner, or which ensure an optimum reaction between the analyte and the analyte-specific binding partner are considered to be advantageous. Reagents which may be used for this purpose include for example proteins, carbohydrates, sugar alcohols, detergents or/and salts, as defined above.

16 In a further step of the method according to the invention, the reaction mixture which is obtained in the second region of the microfluidic analysis device, that is to say the mixture of the analyte-containing sample, the eluent and the analyte-specific binding partner, is transferred via suitable means, for example via a microchannel, into the third region of the microfluidic analysis device, where it is brought into contact with at least one test element on which the analyte can subsequently be determined (Fig. 1 D). The transfer of the reaction mixture from the second region into the third region can be started for example by way of an implementation step which is to be carried out separately by the user, for example by opening a microchannel, a defined volume of the mixture which is to be transported preferably being transferred into the third region of the microfluidic analysis device and, for example by using capillary effects, passing onto the at least one test element. In a preferred variant of the invention, the third region comprises more than one test element, for example 2 to 5 test elements, which in this case are preferably arranged mutually parallel. In principle, the test elements which are used according to the invention are of any physical form which is known to a person skilled in the art and which is suitable for determining the presence or/and the amount of an analyte in a sample. In this context, the test element is preferably configured in such a way that it generates an optically detectable signal, which makes a qualitative or/and quantitative determination of the analyte possible, if the analyte which is to be determined is present. Examples of test elements within the meaning of the present invention include, among others, test strips, test bands and test pads, to which the analyte can be applied for example in the form of an aqueous or non-aqueous solution. The test element is preferably a chromatographic test strip, which may, in a variant of the invention, be formed from a single chromatography-compatible, optionally strip-shaped material. Preferably, however, the chromatographic test strip comprises a plurality of capillary-active surfaces of the same or of different chromatographic materials, which are arranged overlapping on a support layer, and which are in fluid communication with one another and thus form a transport path along which a fluid, driven by capillary forces, can flow through all the regions of the test element. In this context, any known liquid-absorbing, porous or capillary-active material, such as cellulose and derivatives thereof, glass fibres, and non-wovens and fabrics of synthetic or natural materials, can be used as a chromatographic material. Chromatographic test strips which can be used in the context of the present invention are disclosed for example in EP 0 699 906 A2, the disclosure of which is hereby explicitly incorporated by reference.

17 To make it possible to determine an analyte, for example on the basis of a competitive test format or a sandwich test format, the test elements may comprise a plurality of zones which can be delimited from one another in space and which preferably perform a different function and, where required, are equipped with different reagents. In the context of the method according to the invention, the use of a test element is particularly preferred which comprises (a) a first zone which is configured for receiving a solvent, (b) a second zone which comprises a labeled control substance, (c) a third zone which is configured for the optical detection of the analyte, and (d) a fourth zone which is configured for receiving excess solvent. Preferably, the test element comprises an end zone which is configured for receiving solvent and usually comprises an absorbent material, such as fabric or/and non-woven. Once this zone has been wetted with solvent which in the context of the method according to the invention is the reaction mixture of the analyte-containing sample, the eluent and the analyte-specific binding partner, which mixture is received in the second region of the microfluidic analysis device, the solvent migrates through the various zones of the test element. In this context, the capillary action of the individual components of the test element can be exploited, said components being arranged or interconnected in such a way that an uninterrupted flow of solvent is ensured. The zone which is configured for the optical detection of the analyte usually comprises a plurality of defined portions in which different reagents can be immobilised. Therefore, this zone preferably comprises (a) a portion which is configured for binding unbound analyte specific binding partner, (b) a portion which is configured for binding the complex of the analyte and the analyte-specific binding partner, and (c) a portion in which a control signal is generated in a manner independent of the analyte. In this context, the zone which is configured for the optical detection of the analyte may be formed from one or more materials which appear to a person skilled in the art to be suitable for the purposes of the invention, such as membranes of Nylon*, nitrocellulose or polyvinylidene fluoride. The portion which is provided for binding unbound analyte-specific binding partner may for example comprise immobilised analyte analogues, in particular polyhaptens, which capture the analyte-specific, optionally labeled binding partner at a defined position on the test element capturee line) as a result of the formation of a complex, and thus prevent the 18 generation of false-positive results. The complex of the analyte and the analyte-specific binding partner is not usually immobilised on the capture line since the analyte blocks the binding sites on the analyte-specific binding partner, which are required for this purpose. The portion which is configured for binding the complex of the analyte and the analyte specific binding partner preferably comprises a binding partner which is specific to the analyte-specific binding partner and which causes immobilisation of the complex of the analyte and the analyte-specific binding partner at a predetermined position on the test element (test line) and thus makes optical determination of the analyte possible. The complex is generally immobilised by way of a free binding site on the analyte-specific binding partner, a positive detection of the analyte being accompanied by a colouration of the test line. So as to be able to read the signal on the test line clearly, and prevent confusion with the capture line, the capture line may optionally be covered in a suitable manner. The control portion preferably comprises a binding partner in an immobilised form, which is suitable for the control substance from the second zone of the test element, and as a result, the control substance is immobilised upon contact with the binding partner thereof and generates a detectable signal at a predetermined position on the test element (control line). The control line is formed independent of the presence of the analyte and acts as an indicator that the test element is working properly. Excess solvent, which leaves the zone of the test element which is configured for the optical detection of the analyte, can be received in a zone of the test element by means of a liquid-absorbing material, which zone is configured specifically for this purpose. The qualitative or/and quantitative determination of the analyte, which is carried out in the final step of the method according to the invention, can be carried out in any desired manner. For this purpose, in principle it is possible to use all of the methods known from the prior art for detecting immunological reactions, which generate a measurable signal which can be evaluated or read manually or by using suitable means. In the context of the present invention, optical detection methods, in particular photometric or fluorimetric detection methods, are preferably used. According to the invention, optical detection of the analytes by eye is particularly preferred.

19 In a preferred variant of the method according to the invention, the microfluidic analysis device comprises a further region in addition to the aforementioned regions, which comprises at least one timer function for temporally monitoring the processes or reactions taking place in the analysis device. The term "timer function", as used in the context of the present application, refers to a timing device which displays to the user, for example by means of an optical or/and acoustic signal, the end of a process taking place in the analysis device, in particular the end of a reaction taking place in the analysis device, and may optionally necessitate a further implementation step by the user, such as pressing a button on the analysis device to read the test result. Particularly preferably, by means of the aforementioned timer function the duration of the reaction between the analyte and the analyte-specific binding partner in the second region of the microfluidic analysis device [step (d)] or/and the duration of the analyte determination on the test element in the third region of the microfluidic analysis device [step (f)] is monitored, the microfluidic analysis device preferably providing a separate timer function for each of the two steps and thus preferably comprising two mutually independent timer functions. As regards step (d) of the method according to the invention, the timer function usually starts when the eluate, that is to say the mixture of the analyte-containing sample and the eluent, is introduced into the second region of the microfluidic analysis device. This first timer function displays to the user that the reaction between the analyte and the analyte-specific binding partner is complete, and thus the transfer of the mixture from the second to the third region of the microfluidic analysis device can be initialised. As regards step (f), the timer function usually starts with the transfer of the reaction mixture of the analyte-containing sample, the eluent and the analyte-specific binding partner into the third region of the microfluidic analysis device. This second timer function is set up in such a way that it correlates with the duration of the analyte determination on the test element, and displays to the user when he/she can read the test result. Thus, false test results as a result of the test element being read too soon, and unnecessary delays in evaluation as a result of excessively long waiting times can be prevented. A defined 'and constant timer function can, among other things, be represented by a chromatography which is carried out simultaneously with the reactions which take place in step (d) or/and in step (f). For this purpose, a lateral flow test strip may for example be used, 20 which consists of one or more capillary-active surfaces which are arranged side by side and which are in fluid communication and make continuous liquid transport possible. In a preferred variant, the test strip comprises at least one dye as an optical indicator, which can be applied to different positions on the test strip and, because the chromatography duration is dependent on the solvent, can be detected after a defined period of time, for example by a colour change or/and by its appearance in a read-out window. If the microfluidic analysis device comprises at least one timer function for temporally monitoring the reactions taking place in the analysis device, it preferably additionally comprises means for initiating or carrying out the timer function, such as at least one solvent which is suitable for chromatographic applications. These means for initiating or carrying out the timer function may be stored in one of the above-described regions of the microfluidic analysis device or else in a further, separate region, this second variant being preferred. Alternative configurations of the microfluidic analysis device or the individual regions thereof will be apparent to a person skilled in the art on the basis of his general technical knowledge in combination with the above explanations. The method according to the invention makes it possible to determine one or more analytes with high sensitivity and specificity. Thus, according to the invention it is preferable to determine analytes with a specificity of at least 95 % or/and a sensitivity of at least 90 %. More preferably, determination takes place with a specificity of at least 98 % or/and a sensitivity of at least 95 %, in such a way that, by the method disclosed herein, analytes can be detected down to a lower detection limit of approximately 1 ng/ml sample. The method according to the invention can be used for determining any biological or chemical substance which is immunologically detectable. Preferably, however, the method disclosed herein is used for tracing an analyte selected from the group consisting of amphetamines, methamphetamines, cannabinoids, in particular A 9 -tetrahydrocannabinol, opiates, in particular morphine, codeine or dihydrocodeine, opioids, in particular heroin, tropane alkaloids, in particular cocaine, or benzodiazepines, A 9 -tetrahydrocannabinol and cocaine being particularly preferred as analytes. The analyte may be from any desired source, such as the surface of an object which is wetted with the analyte or a bodily fluid, such as whole blood, plasma, serum, urine, saliva or sweat. Preferably, the presence or/and the amount of an analyte in a sample of whole blood, 21 urine or saliva are determined by the method disclosed herein. The sample amount required for carrying out the method is usually approximately 5 pl to approximately 500 pl, preferably approximately 10 pl to approximately 250 pl, and most preferably approximately 30 pl to approximately 150 pl. In a further aspect, the invention relates to a kit which is preferably used for carrying out the above-disclosed method and which comprises the following components: (a) a microfluidic analysis device for determining an analyte, comprising (i) a first region which is configured for introducing a sampling element with an analyte received thereon and for eluting the analyte from the sampling element, (ii) a second region which comprises a binding partner which is specific to the analyte and is in fluid communication with the first region, (iii) a third region which comprises at least one test element for determining the presence or/and the amount of the analyte and is in fluid communication with the second region, (iv) optionally a fourth region which comprises an eluent for eluting the analyte from the sampling element and is in fluid communication with the first region, (v). optionally a fifth region which comprises at least one timer function for temporally monitoring the reactions taking place in the analysis device, and (vi) optionally a housing, and (b) a sampling element which can be introduced into the microfluidic analysis device and which optionally comprises an eluent for eluting the analyte from the sampling element. As regards preferred configurations of the microfluidic analysis device, of the sampling element and of the test element which are contained in the above kit, reference is made to the statements made in connection with the description of the method according to the invention. The invention will be described in greater detail by way of the following drawings and examples. Description of the drawings 22 Fig. 1 is a drawing of an embodiment of a microfluidic analysis device for carrying out the method according to the present invention, comprising: (a) a first chamber for introducing a sampling element and for eluting analyte from the sampling element, (b) five second chambers which each contain an analyte-specific binding partner and are in fluid communication with the first chamber, (c) five test strips for determining the presence or/and amount of analyte, which are each in fluid communication with one of the second chambers, (d) a reservoir containing eluent, which is in fluid communication with the first chamber, and (e) two timers for temporally monitoring the reactions taking place in the analysis device. Fig. 1A shows a sampling element being introduced into the first chamber of the microfluidic analysis device. Fig. 1B shows analyte being eluted from the sampling element in the first chamber by means of the eluent contained in the reservoir. Fig. 1C shows the eluate being transferred into the second chambers and being brought into contact with the analyte-specific binding partner or partners. Fig. 1D shows the reaction mixture being transferred onto the test strips and the analyte(s) being determined. Fig. 2 is a cross-section of an embodiment of a test element for carrying out the method according to the present invention. The test element shown in the form of a test strip comprises: (a) a first zone which comprises a labeled control substance and optionally further reagents, (b) a second zone which is configured for the optical detection of the analyte and comprises a capture line, a test line and a control line, and (c) a third zone which is configured for receiving excess solvent. Fig. 3 shows the sensitivity of the determination of A 9 -THC as a function of the duration of the reaction between the analyte and the analyte-specific binding partner. Fig. 4 illustrates various test strips with a dye applied thereto, which make it possible to establish defined, constant periods of time.

23 Fig. 4A shows test strips comprising immobilised dye which changes colour after a defined period of time; Fig. 4B shows test strips comprising dye which appears in a read-out window after a defined period of time. Examples Example 1: Production of A 9 -THC immunogen 1.25 ml of a stock solution of A 9 -tetrahydrocannabinolic acid A (1-hydroxy-(-)-(6aR,10aR) 6,6,9-trimethyl-3-pentyl-6a,7,8,1 Oa-tetrahydrobenzo[c]chromene-2-carboxylic acid; from Lipomed) in N,N-dimethylformamide (concentration: 100.8 mg/ml) was diluted with 8.75 ml N,N-dimethylformamide to a final volume of 10 ml (final concentration: 12.6 mg/ml). 5 ml of this diluted solution was subsequently added, while stirring, to an aqueous solution of bovine serum albumin (BSA), which had previously been produced by dissolving 250 mg of bovine serum albumin (from Sigma Aldrich) in a mixture of 25 ml distilled water and 2.5 ml N,N-dimethylformamide. After adding 6.3 ml distilled water to the resulting reaction mixture, the following were added in succession: the remaining 5 ml of the aforementioned diluted solution of A 9 -tetrahydrocannabinolic acid A in N,N-dimethylformamide, a further 6 ml distilled water, and 0.2 ml of a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (from Sigma Aldrich) in N,N-dimethylformamide (concentration 500 mg/ml). The glass beaker with the solution contained therein was wrapped in aluminium foil and stirred for 24 h at 5 OC on a magnetic stirrer. Subsequently, the solution was transferred into a dialysis container and dialysed at 5 0C for a period of 4 days against a 25 % solution of N,N-dimethylformamide in water. The dialysate was tested by immunoelectrophoresis. Incubation with a goat anti-BSA antibody (from Genetex) and a precipitate band showed that the A 9 -THC immunogen was different from the band of the bovine serum albumin. Example 2: Obtaining antibodies against A 9 -THC 20 mice were immunised with the A 9 -THC immunogen obtained in example 1 in complete Freund's adjuvant (from Sigma Aldrich), the dose for the first and each subsequent 24 immunisation being 75 pg of immunogen per animal in each case. The immunisations took place over a period of 6 months, in each case at an interval of one month. The sera obtained from the immunised mice were subsequently analysed, in a microtitre plate assay, for the presence of antibodies against A 9 -THC. For this purpose, microtitre plates coated with streptavidin were initially incubated for 12 h at 4 0C with 10 ml of a solution of A 9 -tetrahydrocannabinol-[N'-biotinylaminocaproyl-(3,6-dioxa-8-aminooctyl)-amide in a phosphate-buffered saline solution, which had been produced in advance by reacting

A

9 -tetrahydrocannabinol succinimidyl ester and N-(biotinylaminocaproyl)-1,8-diamino-3,6 dioxaoctane (from Applichem). After washing with 20 ml of a 0.05 % solution of Tween 20 in phosphate-buffered saline solution, the microtitre plates were each incubated for 1 h at 20 0C with 10 ml of the sera which were to be analysed, and subsequently again washed with 20 ml of a 0.05 % solution of Tween 20 in phosphate-buffered saline solution. For the detection, incubation was carried out for 1 h at 20 0C with 10 ml of a solution of a conjugate of peroxidase and rabbit anti mouse lgG (from Dako), followed by washing with 20 ml of a 0.05 % solution of Tween 20 in phosphate-buffered saline solution and the addition of substrate. Sera having a high affinity for A 9 -THC were selected for obtaining antibodies against A 9 -THC. Example 3: Producing a gold-labeled antibody against A 9 -THC Gold sol, having a particle diameter of 20 nm as determined by photon correlation spectroscopy, was produced in accordance with the prior art (Frens, Nature (1973), 241, 20 22). The A 9 -THC-specific antibodies which were obtained in example 2 were labeled with the gold particles in accordance with known methods (Geoghegan et al., J. Immunol. Meth. (1980), 34, 11-31). Example 4: Influence of 3-[(3-cholamidopropyl)dimethylammonio-1 -propane sulphonate (CHAPS) in the eluent on sensitivity Pooled saliva (a saliva mix from five people) was adjusted to a A 9 -THC concentration of 50 ng/ml using a solution of A 9 -THC (from Cerilliant) diluted in methanol. Subsequently, 100 pl of this doped pooled saliva was received by a conventional commercial sampling element 25 (from CopanFlock Technologies), and the sampling element was introduced into the first chamber of a microfluidic analysis device as shown in Fig. 1. Subsequently, four differently concentrated solutions of 3-[(3-cholamidopropyl) dimethylammonio-1 -propane sulphonate (from Sigma Aldrich) in distilled water were added to the sampling element in the first chamber of the microfluidic analysis device for 20 seconds, the analyte-containing sample being eluted from the sampling element and distributed homogeneously in the eluent. The mixture of analyte-containing sample and eluent was subsequently transferred via a microfluidic capillary system into a second chamber of the microfluidic analysis device, which chamber contained a Ag-THC-specific antibody in accordance with example 3, and brought into contact with the analyte-specific binding partner in the stationary state for a period of 120 to 180 seconds. The geometry of the second chamber with a length of 3.2 cm, a width of 1 cm and a height of 2.1 mm was configured in such way that a large diffusion surface resulted between the eluate and the analyte-specific binding partner which was present on the base surface of the chamber in a dried form (dried-out solution of the A 9 -THC-specific antibody in 50 mM carbonate buffer, pH 6). Microscopic analyses showed that after the analyte-containing sample was incubated with the analyte-specific binding partner, there were no residues of the dried analyte-specific binding partner in the second chamber. After the completion of the stationary phase, the reaction mixture was transferred via a microfluidic capillary system onto a test strip and in this way the chromatography process was started. The complete test process lasted 5 to 10 minutes. The results of the four determinations are shown in Table 1: Table 1 CHAPS concentration in the eluent (% 0 % 0.01 % 0.1 % 1.0% by weight) Signal intensity for A9-THC 0 1 6 3 26 As can be seen from Table 1, elution with differently concentrated solutions of CHAPS in water leads to a different signal development on the test line of the chromatographic test strip. The scaling of the signal intensity has 9 levels, signals in the range of 0-3 being considered negative, signals in the range of 4-5 being considered weakly positive, and signals in the range of > 6 being considered positive. In detail, it is found that from a concentration of 0.01 % by weight of CHAPS in the eluent upwards, the sensitivity of the analyte determination increases. The best sensitivity is achieved with a concentration of 0.1 % by weight of CHAPS in the eluent. Example 5: Influence of thorough mixing of the analyte and the eluent on sensitivity Pooled saliva (a saliva mix from five people) was adjusted to a A 9 -THC concentration of 200 ng/ml using a solution of A 9 -THC (from Cerilliant) diluted in methanol. Subsequently, 300 pl of this doped pooled saliva was received by a conventional commercial sampling element (from CopanFlock Technologies), and the sampling element was introduced into the first chamber of a microfluidic analysis device as shown in Fig. 1. Subsequently, 900 pl of eluent (25 mM Tris with 0.5 % cholate) was added to the sampling element in the first chamber of the microfluidic analysis device for 40 seconds, the analyte containing sample being eluted from the sampling element. Subsequently, in the first chamber a homogeneous mixture of the analyte-containing sample and the eluent was formed by means of a microfluidic mixing structure; in a control test, the analyte was not homogenously distributed in the eluent. The mixture of analyte-containing sample and eluent was subsequently transferred into a second chamber which contained a A 9 -THC-specific antibody in accordance with example 3, and brought into contact with the analyte-specific binding partner in the stationary state for a period of 120 to 180 seconds. The geometry of the second chamber with a length of 3.2 cm, a width of 1 cm and a height of 2.1 mm was configured in such way that a large diffusion surface resulted between the eluate and the analyte-specific binding partner which was present on the base surface of the chamber in a dried form (dried-out solution of the A 9 -THC-specific antibody in 50 mM carbonate buffer, pH 6). Microscopic analyses showed that after the analyte-containing 27 sample was incubated with the analyte-specific binding partner, there were no residues of the dried analyte-specific binding partner in the second chamber. After the ,completion of the stationary phase, the reaction mixture was transferred via a microfluidic capillary system onto a test strip and in this way the chromatography process was started. The complete test process lasted 5 to 10 minutes. In this context, it was found that different mixing of the analyte-containing sample and the eluent in the first chamber of the microfluidic analysis device leads to a different strength of signal development on the test line of the chromatography test strip. The scaling of the signal intensity has 9 levels, signals in the range of 0-3 being considered negative, signals in the range of 4-5 being considered weakly positive, and signals in the range of > 6 being considered positive. The approach with the homogeneous mixture of the analyte-containing sample and the eluent led to a signal intensity of seven units, whereas the unmixed control approach resulted in a significantly weaker signal intensity of just two units. Example 6: Influence of the incubation time of the analyte and the analyte-specific binding partner on sensitivity. Pooled saliva (a saliva mix from five people) was adjusted to a A 9 -THC concentration of 50 ng/ml, using a solution of A 9 -THC (from Cerilliant) diluted in methanol. Subsequently, 100 pl of this doped pooled saliva was received by a conventional commercial sampling element (from CopanFlock Technologies), and the sampling element was introduced into the first chamber of a microfluidic analysis device as shown in Fig. 1. Subsequently, eluent (distilled water) was added to the sampling element in the first chamber of the microfluidic analysis device for 20 seconds, the analyte-containing sample being eluted from the sampling element and distributed homogeneously in the eluent. The mixture of analyte-containing sample and eluent was subsequently transferred via a microfluidic capillary system into a second chamber of the microfluidic analysis device, which chamber contained a A 9 -THC-specific antibody in accordance with example 3, and brought into contact with the analyte-specific binding partner in the stationary state for different periods of time (incubation times).

28 The geometry of the second chamber with a length of 3.2 cm, a width of 1 cm and a height of 2.1 mm was configured in such way that a large diffusion surface resulted between the eluate and the analyte-specific binding which was present on the base surface of the chamber in a dried form partner (dried-out solution of the A 9 -THC-specific antibody in 50 mM carbonate buffer, pH 6). Microscopic analyses showed that after the analyte-containing sample was incubated with the analyte-specific binding partner, there were no residues of the dried analyte-specific binding partner in the second chamber. After the completion of the stationary phase, the reaction mixture was transferred via a microfluidic capillary system onto a test strip and in this way the chromatography process was started. The complete test process lasted 5 to 10 minutes. The results of the four determinations, which may be obtained by eye or by means of an appropriate read-out device, are shown in Fig. 3. As can be seen from Fig. 3, different incubation times in the second chamber of the microfluidic analysis device lead to a different strength of signal development on the test line of the chromatographic test strip. The scaling of the signal intensity (y-axis) has 9 levels, signals in the range of 0-3 being considered negative, signals in the range of 4-5 being considered weakly positive, and signals in the range of > 6 being considered positive. The incubation time (x-axis) is given in minutes. It is found that the sensitivity can be greatly increased by setting an incubation time of 2 minutes, and an optimum is reached at approximately 3 minutes. Example 7: Providing defined, constant timer functions To provide a microfluidic analysis device having defined, constant timer functions, the pH indicator bromothymol blue (from Sigma) was dissolved in 1 mM citrate buffer, pH 5.0 (from Sigma) and adjusted to a concentration of 1 mg/ml. This solution was subsequently applied to a chromatographic test strip at a predetermined position. Chromatography was started by applying 20 mM tris buffer, pH 8.5 (from Sigma) to one end of the test strip. After a defined period had elapsed, the solvent reached the zone of the test strip labeled with bromothymol blue, the colour of the pH indicator changing from yellow to blue. The colour change displays to the user the end of a defined period which is dependent on the H.\palniew.oenm\NRPorib\DCCPAR63583221 dcc-23/5120 14 29 previous positioning of the dye solution on the test strip. Fig. 4A shows three test strips which define a period of 1 minute, 2 minutes and 3 minutes as a result of the different positioning of the dye solution. In a variant, methyl blue (from Sigma) was used as the dye and was added at 1:10 to distilled water. This solution was subsequently applied to a chromatographic test strip at a predetermined position. Chromatography was started by applying distilled water to one end of the test strip. After a particular time, the solvent reached the zone of the test strip which was labeled with methyl blue, the dye being transported along by the solvent and moved as far as a read-out window. The read-out window displays to the user the end of a defined period which is dependent on the positioning of the read-out window on the test strip. Fig. 4B shows a test strip of this type before the start of chromatography (1) and after a chromatography duration of approximately 8 minutes (2). Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), orto any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

1. Method for determining an analyte, comprising the steps: (a) providing a microfluidic analysis device, comprising at least a first, a second and a third region, the first region being in fluid communication with the second region and the second region being in fluid communication with the third region, (b) receiving a sample containing the analyte from a sample surface by means of a sampling element, (c) introducing the sampling element into the first region of the microfluidic analysis device and eluting the analyte in the first region by means of an eluent, (d) transferring the eluate which was obtained in step (c) into the second region of the microfluidic analysis device, and bringing the eluate into contact with an analyte specific binding partner, which is stored in the second region, for a period of at least 3 seconds, the analyte binding to the analyte-specific binding partner, (e) transferring the mixture which was obtained in step (d) into the third region of the microfluidic analysis device, and bringing the mixture into contact with at least one test element, and (f) determining the presence or/and the amount of the analyte on the test element.

2. Method according to claim 1, characterised in that a sampling element which comprises a volume indicator is used.

3. Method according to either claim 1 or claim 2, characterised in that the eluent is stored in the sampling element or/and in a fourth region of the microfluidic analysis device.

4. Method according to any one of claims 1 to 3, characterised in that an eluent is used which comprises: (a) an aqueous buffer solution and (b) optionally a protein, a protein mixture, a carbohydrate, a sugar alcohol, a detergent or/and an organic solvent.

5. Method according to any one of claims 1 to 4, characterised in that an antibody or a functional antibody fragment, which optionally comprises an optically detectable label, in particular a metal label, is used as the analyte-specific binding partner. reparflIcooewrNOt) NvW~ofLtArmacoonIU.vwomIt 31

6. Method according to any one of claims I to 5, characterised in that the analyte-specific binding partner is dissolved and homogeneously distributed in the solution when the eluate which was obtained in step (c) is introduced into the second region of the microfluidic analysis device.

7. Method according to any one of claims 1 to 6, characterised in that the reaction between the analyte and the analyte-specific binding partner in step (d) is carried out for a period of approximately 5 seconds to approximately 600 seconds.

8. Method according to claim 7, characterised in that the reaction between the analyte and the analyte-specific binding partner In step (d) is carried out for a period of approximately 90 seconds to approximately 240 seconds.

9. Method according to any one of claims 1 to 8, characterised in that the reaction between the analyze and the analyte-specific binding partner in step (d) is carried out in a stationary phase.

10. Method according to any one of claims I to 9, characterised in that a chromatographic test strip is used as the test element.

11. Method according to any one of claims 1 to 10, characterised in that the analyte is determined optically in step (f).

12, Method according to any one of claims 1 to 11, characterised in that the duration of step (d) or/and of step (f) is monitored by means of at least one timer function which is integrated into the microfluidic analysis device.

13. Method according to claim 12, characterised in that the duration of step (d) or/and of step (f) is monitored by two timer functions which are integrated into the microfluidic analysis device,

14, Method according to any one of claims 1 to 13, characterised in that an amphetamine, a methamphetamine, a cannabinold, in particular A 9 -tetrahydrocannabinol, an opiate, in particular morphine, codeine or dihydrocodeine, an opioid, a tropane alkaloid, or a benzodiazepine is used as the analyte.

15. Method according to claim 14, characterised in that the cannabinoid is A" tetrahydrocannabinol. H ipatYrwo etANRPo DbDCC AR436675 ,DOC- I %6O4 32

16. Method according to any one of claims 1 to 15, characterised in that a bodily fluid is used as the sample.

17. Method according to any one of claims 1 to 16, characterised in that a sample having a volume of approximately 5 pl to approximately 500 pl is used.

18. Method substantially as hereinbefore described, with reference to any one of the Examples and/or accompanying drawings.

19. Kit, when used for carrying out the method according to any one of claims I to 18, comprising: (a) a microfluidic analysis device for determining an analyte, comprising: (i) a first region which is configured for introducing a sampling element with an analyte received thereon and for eluting the analyte from the sampling element, (ii) a second region which comprises a binding partner which is specific to the analyte and is in fluid communication with the first region, (iii) a third region which comprises at least one test element for determining the presence or/and the amount of the analyte and is in fluid communication with the second region, (iv) optionally a fourth region which comprises an eluent for eluting the analyte from the sampling element and is in fluid communication with the first region, (v) optionally a fifth region which comprises at least one timer function for temporally monitoring the reactions taking place in the analysis device, and (vi) optionally a housing, and (b) a sampling element which can be received in the microfluidic analysis device and which optionally comprises an eluent for eluding the analyte from the sampling element.

20. Kit, substantially as hereinbefore described, with reference to any one of the Examples and/or accompanying drawings.