WO2008086809A1

WO2008086809A1 - A microfluidic device and a kit for performing a test

Info

Publication number: WO2008086809A1
Application number: PCT/DK2008/050002
Authority: WO
Inventors: Ole Kring; Jacques Jonsmann; Niels Kristian Bau-Madsen
Original assignee: Scandinavian Micro Biodevices Aps
Priority date: 2007-01-18
Filing date: 2008-01-04
Publication date: 2008-07-24

Abstract

The invention relates to a microfluidic device comprising a flow path with a first and a second opposite solid flow path surfaces (1) and a liquid inlet for said flow path. In the microfluid device at least one solid side wall free section (2) of the flow path (1) is provided by the first and the second opposite solid flow path surfaces in at least said one solid side wall free section (2) and a pair of borderlines (3, 31) defines the edges of the side wall free section (2) of said flow path (1). The pair of borderlines is placed on the first surface of a first and a second opposite surfaces to define the flow path surfaces of said first and second opposite surfaces. The pair of borderlines (3, 31) is defined by an abrupt change of the first and/or the second surface, e.g. in the form of an abrupt change of surface tension and/or an abrupt change of distance between the first and second surfaces beyond and adjacent to the borderlines (3, 31). For example the borderlines (3, 31) may be arranged with a sharp edge with an angle between a flow path surface in the side wall free flow path section and the respective surfaces beyond and, adjacent to the borderlines which is less than 135 degrees, such as about 90 degrees. The flow path comprises an immobilized capture reagent (7) downstream from or in said solid side wall free flow path section (2), said immobilized capture reagent (7) preferably being immobilized onto at least one of said first and second opposite solid flow path surfaces.

Description

A MICROFLUIDIC DEVICE AND A KIT FOR PERFORMING A TEST

TECHNICAL FIELD

The invention relates to a microfluidic device for use in immuno assays such as a sandwich assay. The invention also relates to a kit and a method for performing a sandwich test, in particular for detecting the presence the amount of a target component, such as an antibody.

BACKGROUND ART

Immunoassays, such as sandwich immunoassays are widely used for the performance of standard test such as for the detection and/or monitoring of antigenic substances in body fluids.

Sandwich immuno assays are based on a highly specific binding reaction between a target component, e.g. an antigen and two different reagents (often called probes), e.g. antibodies or antigens and/or antigen receptors that bind the same target component. One of the reagents is marked and the other one of the reagents is immobilized to a substrate. In most non competitive Immunoassays the liquid sample to be tested is first reacted with the immobilized reagent, the non-bound part of the sample is washed away and the marked reagent is brought into contact with the substrate to react with possibly captured target components. Thereafter the substrate is washed again and the possibly captured marked reagents can be detected and quantified via the marker. This method requires several washing steps and a relatively large amount of sample and therefore the method is not suitable for use in a totally or partly capillary driven microfluidic device.

WO 90/15327 discloses a method which does not require any washing and quenching step. This method comprises that the fluid test sample is admixed with a solution of labelled capture reagent against the target antigen followed by contacting the reaction admixture with an immobilized capture reagent. After a brief incubation, the reaction that has occurred is determined by visualization or measurement, preferably by detection of labelled capture reagent, as an indication of the presence of the target antigen in the fluid sample. For example, an antigen present in the fluid test sample will bind to the labelled antibody capture reagent against the target antigen when the body fluid test sample is first admixed with the labelled antibody, and then upon contacting the admixture with the bound antibody capture reagent, the antigen also binds to available sites on such bound antibody to form an antibody-antigen-antibody "sandwich."

WO 90/15327 also discloses a "dip-stick" type device having affixed to a portion of the surface thereof a solid polymeric carrier member with a capture reagent against the target antigen strongly bound to a portion of its surface and at least one container in which a target antigen containing sample and a labelled capture reagent against the target antigen may be admixed and the dip-stick device may be inserted to contact the admixture of the target antigen containing sample and the labelled capture reagent to the target antigen. The presence of labelled capture reagent on the "dip-stick" device is indicative of the presence of the antigen in the sample.

Such dip stick devices have for instance been used by non-technical persons in the home for private determinations of medical conditions such as pregnancy and ovulation.

Immunoassays are also used for the detection of environmental contaminants. More recently, immunoassays have been used by nontechnical persons in the home for private determinations of medical conditions such as pregnancy and ovulation.

US 2006/0216195 discloses a device and a process for testing a sample liquid wherein the sample liquid flows laminarly by capillary in a channel to completely filling a reaction area which has a soluble and/or reacting reagent and defines a reaction volume of the sample liquid. The reaction volume is temporarily stopped in the reaction area for dissolving or reacting the reagent in the defined reaction volume of the sample liquid. After the temporarily stop, the sample flows together with the reaction volume into a test area which is formed by the channel downstream of the reaction area. The sample liquid flows with an essentially straight liquid front. The channel is provided with delay structures in the form of projections or elevations (microstructures).

US 6663833 discloses a flow device with a continuous liquid flow channel having a proximal and a distal end, with a detection membrane in fluid communication with the distal end of the flow channel. Interspersed between the assay buffer and detection membrane, and continuous with the liquid flow channel, are a sample delivery means, one or more reservoirs containing the reagents necessary for conducting the assay, and, optionally, mixing or incubation reservoirs for combining the sample and reagents. The geometry of the liquid flow channel regulates the flow rate of the liquids through the channel, thereby controlling incubation, mixing and reaction time. The preferred detection membrane is an immunochromatographic test strip containing immobilized reagents. The detection of labelled reagent in a particular area of the detection membrane reflects the presence or relative amount of analyte in the sample. Detection may be achieved visually. One or more liquid flow channels may be contained within a single housing for simultaneous, consecutive, or comparative sample analysis.

SUMMARY OF INVENTION

The object of the present invention is to provide a micro fluidic device for use in immunoassays which device requires a minimum of sample to perform a test.

A further object of the invention is to provide a micro fluidic device for use in immunoassays which device is reliable and accurate, is simple and inexpensive to produce and easy to use.

These and other objectives are fulfilled with the invention and/or its embodiments as they are defined in the claims and disclosed in the following. The microfluidic device of the invention comprises a flow path with a first and a second opposite solid flow path surfaces and a liquid inlet for the flow path. At least a section of the flow path is a solid side wall free section which means that the edge of the flow path is not defined by a physically solid wall in that section. The solid side wall free section of the flow path is provided by the first and the second opposite solid flow path surfaces and a pair of borderlines defining the edges of the side wall free section of the flow path. The pair of borderlines is placed on the first surface of a first and a second opposite surfaces to define the flow path surfaces of the first and a second opposite surfaces. In other words the first and a second opposite solid flow path surfaces are parts of the first and a second opposite surfaces defined by the pair(s) of borderlines.

The borderlines are provided by an abrupt change of the first and/or the second surface.

It is especially preferred that the solid side wall free section of the flow path is provided from two essentially plane and parallel surfaces, free of protrusions/depressions other than what is naturally occurring in the used material (i.e. on a microscopic level). In one embodiment the solid side wall free section of the flow path is provided from two essentially plane and parallel surfaces, wherein both of the surfaces are plane on a macroscopic level. Any depressions/protrusions may result in undesired reduction of flow with the temporally trapping of non-reacted reagent, which again will require an increased amount of sample and/or require increased washing to remove non-reacted reagent from a depressions/protrusions comprising solid side wall free section of the flow path.

The flow path surfaces comprises downstream from or in said solid side wall free flow path section an immobilized capture reagent.

Due to the small dimensions of a flow path in a microfluidic device, the

Reynolds number (Re) is usually much less than 100, often less than 1.0. In this Reynolds number regime, flow is completely laminar and no turbulence occurs. The transition to turbulent flow generally occurs in the range of Reynolds number 2000 or higher. According to the invention laminar flow provides a means by which components can be transported in a relatively predictable manner through micro channels. However the laminar flow also means that the flow of the liquid has a flow velocity which is highest in the central part of the cross section of the flow path and which closest to the physical walls of as well as closest to any depressions/protrusions in the flow path approaches zero. Usually the height of the flow path i.e. the distance between the first and a second opposite solid flow path surfaces is of capillary dimension, whereas the width of the flow path may be much larger. This means that molecules in the liquid has a much higher tendency to stop flowing or to flow very slowly closest to solid side walls of such a flow path. By providing at least a section of the flow path as a solid side wall free section the flow in this section is unaffected of a physically side wall and the flow profile in a cross-section of the flow path in said solid side wall free section will be essentially equal in its entire width direction. This effect is even more pronounced when the flow path is essentially free of depressions/protrusions, which is accordingly preferred. This effect is used in the present invention to provide that reagents carrying a marker element and which is not captured by the immobilized capture reagent require less liquid to be removed sufficiently from the site of the immobilized capture reagent to avoid false signal.

Thanks to the invention, the amount of sample to be used may thus be reduced compared to the amount of sample employed using prior art microfluidic device for immunoassays.

The invention thus comprises a microfluidic device with a flow path and a liquid inlet for said flow path, at least a solid side wall free section of said flow path, wherein a flow of a liquid along the edge of said solid side wall free section is higher than what it would have been if the edge was solid, and wherein said flow path downstream from or in said solid side wall free section of said flow path comprises an immobilized capture reagent.

The invention also comprises a microfluidic device with a flow path with a flow path section comprising an immobilized capture reagent, wherein said flow path section is arranged so that the flow of a liquid through the flow path section has essentially same velocity along the edge of the flow path as in the center of the flow path.

In a preferred embodiment the flow path in the solid side wall free section of said flow path is essentially free of depressions/protrusions as well as in order to provide the same velocity along the edge of the flow path as in the center of the flow path.

It should be observed that the flow front of the liquid sample are highly influenced by the wetting properties (hydrophilic/hydrophobic character) of the surfaces of the flow path, whereas, when these surfaces are wetted, the flow velocity of the liquid sample is essentially unaffected by the initial wetting properties of the surfaces. To adjust the shape of the flow front is therefore a completely different task than to adjust the general flow velocity of the whole liquid sample. Consequently, in one embodiment the wetting properties of the first and/or second surfaces of the flow path is selected such that the flow front of the liquid sample is higher along the borderlines of the first and/or second surfaces than on the middle of first and/or second surfaces. For example the first and/or second surfaces of the flow path may be more hydrophilic along the borderlines of the first and/or second surfaces than on the middle of first and/or second surfaces. In this embodiment the hydrophilic surface areas along the borderlines will provide a pull in the liquid sample which will draw the liquid sample forward in a controlled fashion, where any risk of overflowing the borderline, to emerge from the flow path will be highly reduced.

The term 'upstream' means closer to the inlet. Thus 'upstream to the solid side wall free section' means between the inlet and the solid side wall free section. On the other hand 'downstream' means closer to the end of the flow path opposite the inlet. Often the end of the flow path opposite the inlet is provided by an end opening of the flow path. As it is known a flow path need to have an end opening through which the displaced gas (normally air) can escape to the environment or to a chamber e.g. an inflatable chamber. In the microfluidic device of the invention the flow path may comprise one two or several solid side wall free sections.

By arranging the flow path with solid side wall free section(s) in relating to the site of the immobilized capture reagent an optimal solution which requires minimum of sample can be provided.

In one embodiment the entire flow path is a solid side wall free section.

In one embodiment the entire flow path is free of protrusions/depressions from the first and the second surfaces.

In one embodiment the major part of the flow path upstream to the site of the immobilized capture reagent is a solid side wall free section. In one embodiment the site of the immobilized capture reagent is within a solid side wall free section of the flow path.

The length of the solid side wall free section along the flow path should be sufficient long to provide a flow velocity profile which is essentially equal along the width of the flow path. The minimum length of the solid side wall free section for obtaining the desired flow velocity profile over the width of the flow path in the solid side wall free section depends on several factors, including the surface tension of the liquid sample, and the surface tension of and the distance between the first and a second opposite solid flow path surfaces.

Preferably the length of the solid side wall free section along the flow path is at least 1 mm, such as at least 2 mm, such as at least 5 mm, such as at least 10 mm. In one embodiment the length of the solid side wall free section along the flow path is at least 15 mm. In one embodiment the length of the solid side wall free section along the flow path is at least 20 mm.

In section where the flow path is not solid side wall free section(s) the flow path may be in the form of section(s) of traditional channel type. In one embodiment the device comprises at least one channel section upstream to and/or downstream from the solid side wall free section. The first and second opposite solid flow path surfaces provides bottom and a top surfaces of the channel section. These first and a second opposite solid flow path surfaces may e.g. be provided with desired openings as it is known from prior art. The channel section additionally comprises one or more side surfaces.

The channel section may be as channel sections of prior art microfluidic devices e.g. as disclosed in US 6890093, US 4756884, US 6637463, US 2005/0000569, US 2004/020399, US 4618,476 US 5300779, US 6451264, PA 2004 01913 DK (US provisional 60/634,289), PA 2005 00057 DK (US provisional 60/642,987), PA 2005 00732 DK (US provisional 60/684,158) and PA 2005 01000 DK (US provisional 60/696,786).

In one embodiment the side surfaces of a channel section, comprise geometric side microstructures, preferably in the form of one or more of the structural shapes selected from the groups consisting of gaps, protrusions, and depressions, wherein the side microstructures preferably being of substantial smaller dimension than the height of the channel. Useful geometric side microstructures are e.g. as disclosed in PA 2004 01913 DK (US provisional 60/634,289) and PA 2005 00057 DK (US provisional 60/642,987).

In one embodiment the solid side wall free section is provided by the first and a second opposite solid flow path surfaces and only one pair of borderlines placed on the first surface of the first and a second opposite surfaces to define the flow path surfaces of said first and a second opposite surfaces. The first flow path surface in the solid side wall free section is thus constituted by the surface area of the first surface between the borderlines of the pair of borderlines. The second flow path surface in the solid side wall free section is for simplification calculated as the surface area opposite the first flow path surface in the solid side wall free section. In practice it may be a little broader or a little smaller dependant on the surface tension of respectively the second surface and the liquid sample.

In order to provide a highly reliably solid side wall free section with high control of the edge of the flow path, it in one embodiment desired that an additional pair of borderlines is placed on the second surface of the first and a second opposite surfaces to define the flow path surfaces of said first and a second opposite surfaces. The two pairs of borderlines on respectively the first and the second surfaces are preferably arranged opposite to each other.

In embodiment where the solid side wall free section is provided by the first and a second opposite solid flow path surfaces and by a pair of borderlines placed on both of the a first and a second opposite surfaces, the second flow path surface in the solid side wall free section is constituted by the surface area of the second surface between the borderlines of the pair of borderlines on this second surface.

The surface areas of the first and a second opposite surfaces which are beyond and adjacent to the borderlines along the flow path, in the solid side wall free section of said flow path is referred to as the external surface area of respectively the first and a second surfaces.

The two borderlines of a pair borderline may be parallel or they may not be parallel. The relation between the borderlines may vary along the length of the flow path, so as to vary the width or the shape of the flow path along its length. The borderlines may thus be straight lines or they may be curved. In one embodiment the borderlines is straight in the major part of their length. In one embodiment the borderlines of the pair of borderlines are parallel in the major part of their length, such as in 90 % of their length or more. Curved borderlines may e.g. be desired in sections of the flow path which should be designed with a chamber or an opening.

The respective borderlines are provided by an abrupt change of the surface comprising the borderlines. The abrupt change may preferably be selected from the group consisting of a change of surface tension, a stepwise displacement, and a combination thereof.

In a preferred embodiment the abrupt change is in the form of an abrupt change of surface tension.

In a preferred embodiment the abrupt change is in the form of an abrupt stepwise displacement and the first and the second surfaces of the wall free channel section is essentially free of protrusions/depressions seen on a macroscopic scale.

In one embodiment the respective borderlines are at least partly provided by an abrupt change of the surface tension of the surface comprising the borderlines. The abrupt change of the surface tension preferably is a change of at least 5 mN/m, such as at least 10 mN/m, such as between 15 and 60 mN/m.

The abrupt change of the surface tension is arranged so that the surface tension of the surface in question is higher within flow path part of the surface than it is in the external part of the surface.

In one embodiment the surface tension of at least the first flow path surface, in the solid side wall free section of the flow path is at least 60 mN/m, preferably at least 70 mN/m, more preferably at least 85 mN/m.

In one embodiment the major part, such as at least 60 %, such as at least 75 %, such as at least 95 % of the first flow path surface, has a surface tension of at least 60 mN/m, preferably at least 70 mN/m, more preferably at least 85 mN/m.

In one embodiment where the abrupt change is a change of surface tension, the surface tension of at least the external part of the first surface i.e. the part of the first surface beyond and adjacent to the borderlines along the flow path, in the solid side wall free section of said flow path is less than 80, preferably less than 73, such as less than 60, such as between 20 and 50 mN/m.

In one embodiment wherein the flow path further being provided by a pair of borderlines on the second surface, these borderlines on said second surface are totally or partly provided by an abrupt change of the surface tension which may be as disclosed for the first surface above.

In one embodiment of the microfluidic device of the invention the first and optionally the second surface comprises borderline external paths beyond and adjacent to the borderlines along the flow path. These borderline external paths have a surface tension which is less than the surface tension of the first and optionally second flow path surfaces. Such borderline external path provided by the first and optionally second external surfaces have respectively a surface tension which is preferably less than the surface tension of the respectively first and a second opposite solid flow path surfaces. Preferably the first and optionally second external surfaces have a surface tension which is less than 80, preferably less than 73, such as less than 60, such as between 20 and 50 mN/m.

The borderline external paths in the above embodiment may in principle have any width, such as a width of at least 25 μm, such as at least 100 μm, such as at least 1 mm. The wider the external paths are the less risk is there that a liquid sample will overflow the borderlines.

In one embodiment the abrupt change may preferably be provided by a stepwise displacement. The stepwise displacement should displace one of the first and the second opposite surfaces away from the other one of the first and the second opposite surfaces in the external part of the surface i.e. outside flow path surface of said surface. In principle the distance between the a first and a second opposite surfaces need not be larger in the external part than in the flow path part of the first and second surfaces, because the other one of the surfaces could be displaced equally to provide the same distance. In practice, however it is preferred that the distance between the first and a second opposite surfaces is larger in the external part than in the flow path part of the first and second surfaces.

In one embodiment the step wise change provides borderlines in the form of a pair of edges between the flow path part of at least one of the first and second surfaces and the adjacent external parts of the surface. The edges of the borderlines preferably have a relatively short corner radius, such as shorter than 1 mm, such as shorter than, 100 μm, such as shorter than 40 μm or even shorter. The corner radius means the radius of a circle with which the edge is concurrent. The shorter the corner radius, the sharper is the edge and the higher will the capillary forces that keep the flow from flowing beyond the borderline be. Furthermore the shorter the corner radius, the longer can the distance between the flow path part of the first and second surfaces be. A very sharp corner may be more difficult to produce than a less sharp edge, due to difficulties in withdrawing the produced element from the mold (e.g. using injecting molding). I practice the edges will always have a certain corner radius.

In one embodiment the borderlines are totally or partly provided by an abrupt change of the distance between the first and the second surfaces. The abrupt change thus provides at least one pair of borderlines provided by a stepwise displacement. Preferably the distance in the external part beyond the borderlines preferably increases with at least the length of the corner radios of the borderline edge, such as at least 40 μm, such as at least 200 μm, such as at least 1 mm, such as at least 4 mm compared to the distance of the first and a second opposite flow path surfaces.

In one embodiment both the first and the second surface comprises a pair of borderlines provided by a stepwise displacement of the external part of the respective surface away from the opposite surfaces.

The borderlines of the first surface and preferably also of the second surface may in one embodiment be arranged with a sharp edge, in other words the step of the stepwise displacement is very steep. Preferably the angle between the first flow path surface and the respective external surfaces is less than 135 degrees, such as between 45 and 125 degrees, such as about 90 degrees.

In general and independent the way the borderlines are established, it is in one embodiment desired that the major part, such as at least 60 %, such as at least 75 %, such as at least 95 % of the entire of one or both of the first and second flow path surfaces have a surface tension of at least 60 mN/m, preferably at least 70 mN/m, more preferably at least 85 mN/m. This surface tension is to provide for a good and preferably optimal wetting of the flow path surfaces which is a precondition for an optimal capillary flow.

One or both of the first and second flow path surfaces may comprise a pattern having a lower surface tension than the area of the respective flow path surfaces surrounding the pattern such as it is e.g. disclosed in PA 2005 00732 DK (US provisional 60/684,158). The flow path may additionally comprise one or more chambers with increased cross-sectional areas (e.g. increased width and or height). The chamber may be within or outside the solid side wall free section(s).

The dimension of the flow path may be as it is generally known and e.g. as disclosed in the prior art references referred to above, to provide a capillary flow. In one embodiment the distance between the first and the second flow path surfaces in the major part of the flow path, such as at least 60 %, such as at least 75 %, such as at least 95 % of the flow path is of capillary dimension, preferably in the range 1 μm - 1000 μm, such as 25 μm - 250 μm, such as 50 μm - 100 μm.

In one embodiment the distance between the first and the second flow path surfaces in the major part of the solid side wall free flow path section, such as at least 60 %, such as at least 75 %, such as at least 95 % of the solid side wall free section of said flow path being of capillary dimension, preferably in the range 1 μm - 1000 μm, such as 25 μm - 250 μm, such as 50 μm - 100 μm. The distance between first and the second flow path surfaces are also referred to as the height.

The width of the flow path is not important for the function of the device, but naturally it is important for the amount of sample necessary to use the microfluidic device. The width is defined as the distance between the pair of borderlines of the first surface, or if both the first and the second surfaces comprise a pair of borderlines the width is defined as the average of the distances between the pairs of borderlines of respectively the first and the second surface. If the width becomes very large in the solid side wall free section the effect of having such solid side wall free section may be diminished, it is thus preferred that the flow path at least in the solid side wall free section has a width which is less than 20 times, such as less than 10 times the distance between the first and the second flow path surfaces is said section.

In one embodiment the flow path at least in the solid side wall free section has a width which is at least 5 μm, such as between 10 μm, and 20 mm, such as between 200 μm and 4 mm.

In one embodiment the flow path in the solid side wall free section has a width which is less than 1 mm. Due to the solid side wall free section it is possibly to have a flow path with a small width while still have a very good flow along the flow path.

The immobilized capture reagent is immobilized in or downstream from the solid side wall free section of said flow path. Preferably the immobilized capture reagent is immobilized onto one or both of the first and a second opposite solid flow path surfaces. For practical reasons the immobilized capture reagent is most often immobilized onto one of the first and a second opposite solid flow path surfaces.

The place where the immobilized capture reagent is fixed along the path is also referred to as the site of the immobilized capture reagent. In one embodiment the immobilized capture reagent is immobilized onto a secondary solid substrate placed in the flow path, e.g. a membrane. This embodiment provides for a simple production as the secondary solid substrate can be prepared with the immobilized capture reagent, cut to desire size and arranged in the microfluidic device. Often a microfluidic device is produced from two element comprising respectively the first and the second surfaces, the surfaces are treated, desires elements, reagents and similar is placed or fixed and the elements are joint to each other.

Method of immobilizing capture reagents are well known and further information can e.g. be found in WO 90/15327.

The immobilized capture reagent may be arranged in any pattern e.g. a pattern selected from the group consisting of a square, a circle, a line, such as a line crossing the flow path. The immobilized capture reagent may e.g. be captured in a cavity arranged in a solid surface e.g. one of said first and second opposite solid flow path surfaces. In one embodiment the immobilized capture reagent being captured in an island shaped segment of at least one of said first and second opposite solid flow path surfaces. Further information about an island shaped segment and the formation hereof can be found in PA 2005 00732 DK (US provisional 60/684,158).

The immobilized capture reagent may in one embodiment be immobilized onto the solid surface downstream from said solid side wall free flow path section, i.e. in the section of the flow path between the solid side wall free section and the end of the flow path opposite the inlet.

In another embodiment the immobilized capture reagent is immobilized in said solid side wall free flow path section. This embodiment provides an optimal effect of the solid side wall free section.

The immobilized capture reagent may as example be selected from the group consisting of antibodies, antigens, nucleic acids, such double stranded, partly single stranded and single stranded DNA, RNA, LNA and PNA, polypeptides, enzymes, sugars, gold-particle and thiols. In one embodiment the flow path further comprises a non immobilized reagent, preferably in form of a marked non immobilized reagent.

A non immobilized reagent is a reagent which will not remain immobilized when contacted with water (representing a sample) or a sample. The non immobilized may be fixed to a surface within the flow path e.g. by applying it in dissolved state and allowing it to dry, or by using other methods which result in a fixation which is not permanent when the flow path is filled with sample.

The non immobilized reagent may e.g. be selected from the group consisting of antibodies, antigens, nucleic acids, such double stranded, partly single stranded and single stranded DNA, RNA, LNA and PNA, polypeptides, enzymes, sugars, gold-particle and thiols.

The non immobilized reagent may preferably comprise a marker, selected from the group consisting of such a color marker, a fluorescence marker, an enzyme, a metal particle and a magnetic particle.

In one embodiment the non immobilized reagent is a labeled antiimmunoglobulin such as a floorochrome-conjugated antiimmunoglobulin.

The place where the non immobilized reagent is arranged along the flow path prior to use of the microfluidic device is also referred to as the site of the non immobilized reagent.

The immobilized capture reagent and the non immobilized reagent may preferably be capable of binding to the same target component

In one embodiment the non immobilized reagent is placed in a distance from the immobilized capture reagent, such as at least 2 mm, such as at least 5 mm or longer. The non immobilized reagent is placed upstream to the immobilized capture reagent, so that a sample can first react with the non immobilized reagent, whereby it will be marked. The marked target component as well as non reacted non immobilized reagent will flow along the flow path and reach the immobilized capture reagent where the marked target component will be captured.

Preferably at least a part of the flow path between the site of the non immobilized reagent and the site of the immobilized capture reagent is a solid side wall free section, thereby non-reacted non immobilized reagent will be much faster to bring beyond the site of the immobilized capture reagent whereby false signal due to the non-reacted non immobilized reagent can more simple and by use of only little fluid be avoided.

In one preferred embodiment the major part and preferably all of the flow path between the site of the non immobilized reagent and the site of the immobilized capture reagent is a solid side wall free section, thereby non- reacted non immobilized reagent will be removed very fast using only little fluid.

In one embodiment even the non immobilized reagent is placed in the solid side wall free section of the flow path.

The non immobilized reagent may preferably be placed in a reaction chamber.

In one embodiment the flow path comprises a flow stop junction adjacent to the non immobilized reagent, such as between the non immobilized reagent and the immobilized capture reagent. This flow stop junction result in stopping or delaying the flow to allow the non immobilized reagent to react with target component in the sample. Further information about flow stop junctions can be found in the above cited prior art documents and in particular in PA 2005 01000 DK (US provisional 60/696786).

In one embodiment the target component being an antibody and the immobilized capture reagent being an antigen. The non immobilized reagent may in this example e.g. be a labeled antigen In one embodiment the target component being an antigen, and the immobilized capture reagent being an antibody. The non immobilized reagent may in this example e.g. be a labeled antibody

The invention also relates to a kit comprising a microfluidic device as described above e.g. in combination wit the desired reagents and/or the sample

The microfluidic device of the invention may be produced by production steps of the standard technology and as described above. The microfluidic device may for example be provided in a polymeric material using injection molding. The microfluidic device may be produced in two or more parts e.g. using injection molding, where after the parts are treated e.g. as disclosed in

PA 2005 00732 DK (US provisional 60/684,158) to provide the desired surface tension character, the reagents may be placed or immobilized and the microfluidic device may be assembled.

In use the sample may prior to injecting into the microfluidic device be reacted with a non immobilized reagent, if the microfluidic device in itself does not comprise the non immobilized reagent. If the non immobilized reagent is placed in the microfluidic device, the sample can be introduced into the microfluidic device directly. After a short reaction time the sample has passed the immobilized capture reagent and a little amount of sample free of the non immobilized reagent has passed the immobilized capture reagent as well, and the site of the immobilized capture reagent can be examined to observe if the target was in the sample or to calculate/count the amount of target components.

BRIEF DESCRIPTION OF DRAWINGS

Examples of embodiments of the invention will be described below with reference to the drawings: FIGs. 1a, 1 b, 1c and 1d show cross-sectional top views of four different variations of a microfluidic device according to the invention.

FIGs. 2a, 2b and 2c show cross-sectional cuts through a flow path of 3 different variations of a microfluidic device according to the invention.

FIG. 3 is a cross-sectional top view of a microfluidic device with a reaction chamber according to the invention.

FIG. 4 is a perspective view of a cross-sectional cut through a flow path of a microfluidic device according to the invention.

FIG. 1a shows a cross-sectional cut through a flow path 1 of an embodiment of a microfluidic device according to the invention. The sectional cut is made through the microfluidic device so that only one of the first and second opposite surfaces is visible. The flow path 1 comprises a solid side wall free section 2 with a pair of borderlines 3, 3' defining the edges of the solid side wall free section of the flow path 1. The flow path 1 further comprises a first and a second channel section 4, 5. The first channel section 4 is placed upstream to the solid side wall free section, and the second channel section 5 is placed downstream from the solid side wall free section. The channel sections 4, 5 comprise sidewalls 6 providing side surfaces 6' of the channel sections 4, 5. In the second channel section 5, the flow path 1 comprises an immobilized capture reagent 7 fixed to the surface in a pattern shaped as a line crossing the flow path 1. In the solid side wall free section the surfaces providing the flow path are essentially plane and free of depressiond/protrusions. In the solid side wall free section the flow path comprises a non-immobilized reagent 8, placed in an island shaped segment 9 formed by a hydrophobic pattern 9, which hydrophobic pattern has a lower surface tension than the surrounding flow path surface. Further information about this type of island shaped pattern can be found in PA 2005 00732 DK (US provisional 60/684,158). Reference is in particular made to FIG. 5 in that document. The flow direction is from the not shown inlet and as indicated with the arrow A. In use a sample is introduced into the inlet. The sample flow along the flow path as indicated with the arrow A, and reaches first the island shaped segment 9. The front part of the liquid sample passes into the central part of the island shaped segment 9, dissolve the non-immobilized reagent placed therein and possibly target components will react with the non-immobilized reagent. Simultaneously the hydrophobic pattern 9 will be wetted so that this hydrophobic pattern 9 does no longer affect the flow along the flow path 1. The sample continues its flow along the solid side wall free section. In this section the flow velocity profile is essentially equal over the width of the flow path and consequently essentially all of the front part of the sample which now comprises the non-immobilized reagent will flow with an equal velocity and reach the second the second channel section 5 almost simultaneously. In the second channel section 5 present target components will react with the immobilized capture reagent 7, whereby it will be captured. The remaining part of the front part of the sample will continue its flow along the flow path and sample free of the non-immobilized reagent will overflow the immobilized capture reagent. A slightly amount of non-immobilized reagent may be delayed immediately adjacent to the side walls 6' of the second channel section 5, but due to the solid side wall free section the amount of non-immobilized reagent delayed immediately adjacent to the side walls 6' will very fast be diluted to almost nothing. The non-immobilized reagent which reacted with a target and which is now captured via this target to the immobilized capture reagent can be measured using standard technology.

FIG. 1 b shows a cross-sectional cut through a flow path 11 of an embodiment of a microfluidic device which is slightly different from the microfluidic device shown in FIG. 1a. The flow path 11 comprises a solid side wall free section 12 with a pair of borderlines 13, 13' defining the edges of the solid side wall free section of the flow path 11. The flow path 11 further comprises a first and a second channel section 14, 15. The first channel section 14 is placed upstream to the solid side wall free section, and the second channel section 15 is placed downstream from the solid side wall free section. The channel sections 14, 15 comprise sidewalls 16 providing side surfaces 16' of the channel sections 14, 15. In solid side wall free section the flow path 11 comprises an immobilized capture reagent 17 fixed to the surface in a pattern shaped as a line crossing the flow path 11 upstream to but also in the solid side wall free section the flow path further comprises a non-immobilized reagent 18, placed in an island shaped segment 19.

The microfluidic device shown in FIG. 1 b is used as described for the microfluidic device of FIG. 1a with the difference that also the immobilized capture reagent 17 is placed in the solid side wall free section 12 which means that essentially all of the not reacted non-immobilized reagent will be carried away from the area of the flow path 11 with the immobilized capture reagent 17, and consequently the risk of false signal is extremely low.

FIG. 1c shows a cross-sectional cut through a flow path 21 of another embodiment of a microfluidic device which is slightly different from the microfluidic device shown in FIG. 1a. The flow path 21 comprises a solid side wall free section 22 with a pair of borderlines 23, 23' defining the edges of the solid side wall free section of the flow path 21. The flow path 21 further comprises a first and a second channel section 24, 25. The first channel section 24 is placed upstream to the solid side wall free section, and the second channel section 25 is placed downstream from the solid side wall free section. The channel sections 24, 25 comprise sidewalls 26 providing side surfaces 26' of the channel sections 24, 25. In the second channel section 25, the flow path 21 comprises an immobilized capture reagent 27 fixed to the surface in a pattern shaped as a line crossing the flow path 21.

In the first channel section 24 the flow path 21 comprises a non-immobilized reagent 28, placed in an island shaped segment 29

In use the microfluidic device of FIG. 1c will function almost as the microfluidic device of FIG. 1a with the difference that a small amount of non- immobilized reagent may be delayed immediately adjacent to the side walls 26' of both of the first and the second channel sections 24, 25. However, due to the solid side wall free section the amount of sample necessary to remove a sufficient part of the not reacted non-immobilized reagent from the area of the flow path 21 comprising the immobilized capture reagent 27 will be reduced compared to when using prior art methods.

FIG. 1d shows a cross-sectional cut through a flow path 31 of yet another embodiment of a microfluidic device which is slightly different from the microfluidic device shown in FIG. 1a. The flow path 31 comprises a solid side wall free section 32 with a pair of borderlines 33, 33' defining the edges of the solid side wall free section of the flow path 31. The flow path 31 further comprises a second channel section 34, placed upstream to the solid side wall free section 32. The channel section 34 sidewalls 36 providing side surfaces 36' of the channel section 34. In the solid side wall free section 32, the flow path 31 comprises an immobilized capture reagent 37. In the first channel section 34 the flow path 31 comprises a non-immobilized reagent 38, placed in an island shaped segment 39

In use the microfluidic device of FIG. 1c will function almost as the microfluidic device shown in FIG. 1 b with the difference that a small amount of non-immobilized reagent may be delayed immediately adjacent to the side walls 36' of the channel section 34. However, due to the solid side wall free section the amount of sample necessary to remove a sufficient part of the not reacted non-immobilized reagent from the area of the flow path 31 comprising the immobilized capture reagent 37 will be reduced compared to when using prior art methods.

FIG 2a shows a cross-sectional through a flow path 43 of a microfluidic device according to the invention wherein the borderlines 50 is provided by an abrupt change in the form of a stepwise displacement of the first surface 48, 48'. The external parts of the first surface 48' is displaced by the steps 45 from the first flow path surface 48. The distance between the first and a second opposite solid flow path surfaces 48, 49 is thereby larger in the external parts 46 than in the flow path part 43 of the first and second surfaces.

The borderline 50 has a geometrical radius of curvature (a corner radius) associated with it. The ability to act as a capillary stop is improved as this corner radius is decreased. Other factors governing the detailed shape of the fluid flow along the borderline are the distance between lid 42 and base 41 and the surface energies of the parts 41 and 42.

As it is indicated the microfluidic device may be made from two parts a first bottom part 41 and a second top part 42 which has been connected in the joints 44.

FIG. 2b shows a variation of the embodiment of FIG. 2a, wherein the only difference is that the step 45 is slightly angled which makes a production using injecting molding easier, since the first part 41 will be more simple to remove from the mold. The reference numbers used in FIG. 2b has the same meaning as in FIG 2a.

FIG. 2c shows a variation of the embodiment of FIG 2a, wherein the top part 42' differs from the top part 42 of FIG. 2a, in that the second surface 49, 49' of FIG. 2c also comprises a pair of borderlines 50' formed by the sharp edge provided by an abrupt change in the form of a stepwise displacement of the second surface 49, 49'. The remaining reference numbers used in FIG. 2c has the same meaning as in FIG 2a.

FIG. 3 is a cross-sectional top view of a microfluidic device with a flow path 53 comprising a reaction chamber 59. The figure shows only the first surface of the microfluidic device. The flow path is in the form of a solid side wall free section, wherein the pair of borderlines 51 , 51 ' is provided by an abrupt change of surface tension, wherein flow path surface 53 has a higher surface tension than the external paths 56. As shown the borderlines51 , 51 ' is curved to form the reaction chamber 59 of the flow path 53. In the reaction chamber 58 immobilized capture reagents is fixed in a square formed pattern 59. The arrow A indicates the flow direction.

FIG. 4 is a perspective view of a cross-sectional cut through a flow path 63 of a microfluidic device according to the invention. The microfluidic device is provided by a first bottom part 61 and a second top part 62 which has been connected in the joints 64. The flow path 63 is in the form of a solid side wall free section, wherein the pair of borderlines 67, 67' is provided by an abrupt change of surface tension, wherein flow path surface 63 has a higher surface tension than the external paths 66.

Claims

1. A microfluidic device comprising a flow path with a first and a second opposite solid flow path surfaces and a liquid inlet for said flow path, at least one solid side wall free section of said flow path being provided by the first and the second opposite solid flow path surfaces in at least said one solid side wall free section and a pair of borderlines defining the edges of the side wall free section of said flow path, said pair of borderlines being placed on the first surface of a first and a second opposite surfaces to define the flow path surfaces of said first and second opposite surfaces, wherein said pair of borderlines being defined by an abrupt change of the first and/or the second surface, said flow path comprises an immobilized capture reagent downstream from or in said solid side wall free flow path section, said immobilized capture reagent preferably being immobilized onto at least one of said first and second opposite solid flow path surfaces

2. A microfluidic device as claimed in claim 1 , wherein an additional pair of borderlines being placed on the second surface of the first and a second opposite surfaces to define the flow path surfaces of said first and a second opposite surfaces, said two pairs of borderlines on respectively the first and the second surfaces preferably being arranged opposite to each other.

3. A microfluidic device as claimed in any one of the claims 1 and 2, wherein said borderlines being totally or partly provided by an abrupt change of the surface tension of at least the first surface, the abrupt change of the surface tension preferably is a change of at least 5 mN/m, such as at least 10 mN/m, such as between 15 mN/m and 60 mN/m.

4. A microfluidic device as claimed in claim 3, wherein the surface tension of at least the first flow path surface, in the solid side wall free section of said flow path being at least 60 mN/m, preferably at least 70 mN/m, more preferably at least 85 mN/m.

5. A microfluidic device as claimed in claim 4, wherein the major part, such as at least 60 %, such as at least 75 %, such as at least 95 % of the first flow path surface, has a surface tension of at least 60 mN/m, preferably at least 70 mN/m, more preferably at least 85 mN/m.

6. A microfluidic device as claimed in any one of the claims 2-5, wherein the surface tension of at least the first surface beyond and adjacent to the borderlines along the flow path, in the solid side wall free section of said flow path is less than 80 mN/m, preferably less than 73 mN/m, such as less than 60 mN/m, such as between 20 mN/m and 50 mN/m.

7. A microfluidic device as claimed in any one of the preceding claims, wherein said flow path further being provided by a pair of borderlines on said second surface, said borderlines on said second surface totally or partly being provided by an abrupt change of the surface tension, the abrupt change of the surface tension preferably is a change of at least 5 mN/m, such as at least 10 mN/m, such as between 15 and 60 mN/m.

8. A microfluidic device as claimed in claim 7, wherein the surface tension of at the second flow path surface, in the solid side wall free section of said flow path being at least 60 mN/m, preferably at least 70 mN/m, more preferably at least 85 mN/m.

9. A microfluidic device as claimed in claim 8, wherein the major part, such as at least 60 %, such as at least 75 %, such as at least 95 % of the second flow path surface has a surface tension of at least 60 mN/m, preferably at least 70 mN/m, more preferably at least 85 mN/m.

10. A microfluidic device as claimed in any one of the claims 7- 9, wherein the surface tension of at the second surface beyond and adjacent to the borderlines along the flow path, in the solid side wall free section of said flow path is less than 80, preferably less than 73, such as less than 60, such as between 20 and 50 mN/m.

11. A microfluidic device as claimed in any one of the claims 6 and 10, wherein the first and optionally the second surface comprises borderline external paths beyond and adjacent to the borderlines along the flow path, said borderline external paths have a surface tension which is less than the surface tension of the first and optionally second flow path surfaces, said borderline external paths preferably have a surface tension which is less than 80, preferably less than 73, such as less than 60, such as between 20 and 50 mN/m.

12. A microfluidic device as claimed in claim 11 , wherein the borderline external paths has a width of at least 25 μm, such as at least 100 μm, such as at least 1 mm.

13. A microfluidic device as claimed in any one of the preceding claims, wherein the distance between the first and the second flow path surfaces in the major part of the solid side wall free flow path section , such as at least 60 %, such as at least 75 %, such as at least 95 % of the solid side wall free section of said flow path being of capillary dimension, preferably in the range 1 μm - 1000 μm, such as 25 μm - 250 μm, such as 50 μm - 100 μm.

14. A microfluidic device as claimed in any one of the preceding claims, wherein said borderlines totally or partly being provided by an abrupt change of the distance between the first and the second surfaces, the distance beyond the borderlines preferably increases with at least 40 μm, such as at least 50 μm, such as at least 200 μm, such as at least 1 mm.

15. A microfluidic device as claimed in any one of the preceding claims, wherein said borderlines totally or partly being provided by an abrupt stepwise change of the distance between the first and the second surfaces, which provides a pair of borderline edges, the borderline edges have a corner radius shorter than 1 mm, such as shorter than, 100 μm, such as shorter than 40 μm.

16. A microfluidic device as claimed in any one of the preceding claims wherein the borderlines of the first surface is arranged with a sharp edge, preferably the angle between the first flow path surface and the respective surfaces beyond and adjacent to the borderlines is less than 135 degrees, such as between 45 and 125 degrees, such as about 90 degrees.

17. A microfluidic device as claimed in any one of the preceding claims wherein the borderlines of the second surface is arranged with a sharp edge, preferably the angle between the second flow path surface and the respective surfaces beyond and adjacent to the borderlines is less than 135 degrees, such as between 45 and 125 degrees, such as about 90 degrees.

18. A microfluidic device as claimed in any one of the claims preceding claims, wherein the major part, such as at least 60 %, such as at least 75 %, such as at least 95 % of the entire of one or both of the first and second flow path surfaces have a surface tension of at least 60 mN/m, preferably at least 70 mN/m, more preferably at least 85 mN/m.

19. A microfluidic device as claimed in any one of the preceding claims, wherein one or both of the first and second flow path surfaces comprises a pattern having a lower surface tension than the area of the respective flow path surfaces surrounding the pattern.

20. A microfluidic device as claimed in any one of the preceding claims, wherein the flow path, at least in the solid side wall free section has a height, defined as the distance between the first and the second flow path surfaces, which is of capillary dimension, preferably in the range 1 μm - 1000 μm, such as 25 μm - 250 μm, such as 50 μm - 100 μm.

21. A microfluidic device as claimed in any one of the preceding claims, wherein the flow path at least in the solid side wall free section has a width, defined as the distance between the pair of borderlines of the first surface, or if both the first and the second surfaces comprises a pair of borderlines the with is defined as the average of the distances between the pairs of borderlines of respectively the first and the second surface, wherein the width is at least 5 μm, such as between 10 μm, and 20 mm, such as between 20 μm and 10 mm.

22. A microfluidic device as claimed in any one of the preceding claims, wherein the solid side wall free section has a length along the flow path of at least 1 mm, such as at least 5 mm, such as at least 10 mm.

23. A microfluidic device as claimed in any one of the preceding claims, wherein the device comprises at least one channel section upstream to and/or downstream from the solid side wall free section, said first and second opposite solid flow path surfaces optionally provided with desired openings, preferably constitutes respectively a bottom and a top surfaces of the channel section, more preferably the channel section additionally comprises one or more side surfaces.

24. A microfluidic device as claimed in claim 2 wherein the side surfaces of a channel section, comprise geometric side microstructures, preferably in the form of one or more of the structural shapes selected from the groups consisting of gaps, protrusions, and depressions, wherein the side microstructures preferably being of substantial smaller dimension than the height of the channel

25. A microfluidic device as claimed in any one of the preceding claims, said flow comprises one or more chambers constituted by an increased width and or height.

26. A microfluidic device as claimed in any one of the preceding claims wherein at least one of said first and second opposite solid flow path surfaces downstream from said solid side wall free flow path section comprises an immobilized capture reagent, said immobilized capture reagent preferably being arranged in a pattern, such as a pattern selected from the group consisting of a square, a circle, a line, such as a line crossing the flow path.

27. A microfluidic device as claimed in any one of the preceding claims wherein at least one of said first and second opposite solid flow path surfaces in said solid side wall free flow path section comprises an immobilized capture reagent, said immobilized capture reagent preferably being arranged in a pattern, such as a pattern selected from the group consisting of a square, a circle, a line, such as a line crossing the flow path.

28. A microfluidic device as claimed in any one of the preceding claims wherein the immobilized capture reagent being captured in a cavity arranged in at least one of said first and second opposite solid flow path surfaces.

29. A microfluidic device as claimed in any one of the preceding claims wherein the immobilized capture reagent being captured in an island shaped segment of at least one of said first and second opposite solid flow path surfaces.

30. A microfluidic device as claimed in any one of the preceding claims wherein the immobilized capture reagent being selected from the group consisting of antibodies, antigens, polypeptides, enzymes, nucleic acids, such double stranded, partly single stranded and single stranded DNA, RNA, LNA and PNA.

31. A microfluidic device as claimed in any one of the preceding claims wherein the flow path further comprises a non immobilized reagent.

32. A microfluidic device as claimed in claim 31 wherein the non immobilized reagent being selected from the group consisting of antibodies, antigens, enzymes, nucleic acids, such double stranded, partly single stranded and single stranded DNA, RNA, LNA and PNA.

33. A microfluidic device as claimed in claim any one of the claims 31 and 32 wherein the non immobilized reagent comprises a marker, such as a color marker, a fluorescence marker, a light reflective marker, an enzymatic marker, a radioactive marker and a chemiluminescent marker.

34. A microfluidic device as claimed in claim any one of the claims 31-33, wherein the non immobilized reagent being placed in a distance from the immobilized capture reagent, the non immobilized reagent being placed upstream to the immobilized capture reagent.

35. A microfluidic device as claimed in any one of the claims 31 - 34, wherein the non immobilized reagent being placed in a reaction chamber.

36. A microfluidic device as claimed in claim any one of the claims 31 - 35, wherein the flow path comprises a flow stop junction adjacent to the non immobilized reagent, such as between the non immobilized reagent and the immobilized capture reagent.

37. A microfluidic device as claimed in claim any one of the claims 31 - 36, wherein the non immobilized reagent being placed in the solid side wall free section of the flow path.

38. A microfluidic device as claimed in any one of the preceding claims in combination with a sample to be tested for a target component, wherein the target component being an antibody, the immobilized capture reagent being an antigen.

39. A microfluidic device as claimed in claim 38 wherein the non immobilized reagent being a labeled antiimmunoglobulin, such as a floorochrome-conjugated antiimmunoglobulin.

40. A microfluidic device as claimed in any one of the preceding claims in combination with a sample to be tested for a target component, wherein the target component being an antigen, the immobilized capture reagent being an antibody.

41. A microfluidic device as claimed in claim 40 wherein the non- immobilized reagent being a labeled antibody.

42. A microfluidic device as claimed in any one of the preceding claims wherein the solid side wall free section of the flow path is provided from two essentially plane and parallel surfaces, free of protrusions/depressions other than what is naturally occurring in the used material (i.e. on a microscopic level).

43. A microfluidic device as claimed in any one of the preceding claims wherein the solid side wall free section of the flow path is provided from the first and the second surfaces, said first and the second surfaces being essentially plane and parallel surfaces, wherein both of the surfaces are plane on a macroscopic level.

44. A microfluidic device as claimed in any one of the preceding claims wherein the first and the second surfaces along the entire length of the flow path solid side wall free section of the flow path being essentially free of protrusions/depressions.

45. A kit comprising a microfluidic device as claimed in any one of the preceding claims.

46. A kit comprising a microfluidic device as claimed in any one of the preceding claims 1-31 , 39 and 41 further comprising a reagent, preferably selected from the group consisting of antibodies, antigens, polypeptides, enzymes, nucleic acids, such double stranded, partly single stranded and single stranded DNA, RNA, LNA and PNA.

47. A microfluidic device comprising a flow path and a liquid inlet for said flow path, at least a solid side wall free section of said flow path, wherein a flow of a liquid along the edge of said solid side wall free section is higher than what it would have been if the edge was solid, and wherein said flow path downstream from or in said solid side wall free section of said flow path comprises an immobilized capture reagent, preferably said solid side wall free section of said flow path comprises essentially plane and parallel opposite surfaces.

48. A microfluidic device comprising a flow path with a flow path section comprising an immobilized capture reagent, wherein said flow path section is arranged so that the flow of a liquid through the flow pat section has essentially same velocity along the edge of the flow path as in the center of the flow path, said flow path section preferably comprises surfaces which are essentially free of protrusions or depressions..