WO2009068027A1

WO2009068027A1 - Separation and detection device

Info

Publication number: WO2009068027A1
Application number: PCT/DK2007/000519
Authority: WO
Inventors: Peter Warthoe; Per Berden; Søren MENTZEL1; Klaus Rune Andersen; Jens M1Kkelsen; Jacob Hoist Madsen
Original assignee: Atonomics A/S
Priority date: 2007-11-26
Filing date: 2007-11-26
Publication date: 2009-06-04

Abstract

The present invention relates to a method and a device for separating a suspension into a liquid phase and a retentate phase. The device comprises a separation chamber comprising an application zone and a hydrophilic filter material. The separation chamber is connected to a first capillary channel, where the connecting junction between the separation chamber and the first capillary channel comprise a physical barrier preventing flow of residue retentate from a lower part of the chamber into the first capillary channel.. The invention further relates to a device and a method for quantitative detecting of the presence or absence of a target analyte in a liquid sample, the device comprising a reaction chamber in the form of a capillary channel having a volume of less than 200μl.

Description

Title: Separation and detection device

Technical Field

The present invention relates to a device for separating a suspension into a liquid phase and a retentate phase and to the use thereof

The invention further relates to a method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the suspended matter, and a liquid phase substantially free of suspended matter The suspension might be blood, the liquid phase plasma/serum and the retentate blood cells.

The present invention further relates to a device for quantitative detecting the presence or absence of a target analyte in a liquid sample, and to uses thereof

The invention further relates to a method for quantitative detecting the presence or absence of a target analyte in a sample consisting of less than 200 μl

The invention further relates to a kit of parts comprising the device according to the in- vention and magnetic particles.

The invention further relates to an apparatus comprising the devices according to the invention

Background

Many diagnostics are carried out in the clinical field utilizing blood as a sample. Although some of these techniques can be carried out on whole blood, it is necessary in many instances to utilize serum or plasma as the sample in order to obtain an accurate reading. For example, red blood cells (erythrocytes) scatter and absorb light and could adversely affect a measurement of either reflected or transmitted light of a diagnostic test relying on either of these measurement techniques.

Traditionally, plasma and serum have been separated from whole blood by centrifuging either before (for plasma) or after (for serum) clotting. However, centrifugation is time consuming and requires equipment that is not generally available outside the clinical laboratory, Accordingly, field testing of numerous blood substances that require serum or plasma is difficult.

A number of techniques have been devised to avoid this problem The techniques gen- erally utilize a filtering device capable of separating red blood cells from plasma. Numerous materials have been used in the past to form filters. Paper, non-woven fabric, sheet-like filter material composed of powders or fibers such as man-made fibers or glass fibers, and membrane filters having suitable pore sizes have been proposed.

However, these prior art techniques have proven to be unsuitable for use in applications which, because of space and volume restraints, can only utilize a small filter in a device in which a single drop of blood is separated and the plasma is transported through the device solely by means of capillary action. Thus, most prior art devices for separation suffers from dealing with sufficient to separate undiluted whole-blood by use of capillary and/or hydrostatic pressure without the use of an external force. Accordingly, further refinement in blood separation techniques is desirable.

Accordingly one object of the present invention was to develop a device and a method capable to separate undiluted whole-blood into a plasma/serum phase and a blood cell phase in a short time, where the plasma/serum phase is substantially free of blood cell contamination, and wherein the blood sample comprises less than 200 μl_.

Another object of the invention was to develop a device and a method capable to separate undiluted whole-blood into a plasma/serum phase and a blood cell phase in short time, where the separation is driven without the use of an external force, and wherein the blood sample comprises less than 200 μL,

Over the years, numerous simplified test systems have been designed to rapidly detect the presence of a target analyte of interest in biological, environmental and industrial fluids. In one of their simplest forms, these assay systems and devices usually involve the combination of a test reagent which is reacting with the target analyte to give a visual response and an absorbent paper or membrane through which the test reagents flow.

The contact may be accomplished in a variety of ways. Most commonly, an aqueous sample is allowed to traverse a porous or absorbent member, such as porous polyeth- ylene or polypropylene or membranes by capillarity through the portion of the porous or absorbent member containing the test reagents, In other cases, the test reagents are pre-mixed outside the test device and then added to the absorbent member of the device to ultimately generate a signal.

Many commercially available devices and assay systems also involve a wash step in which the immune absorbing zone is washed free of non specifically bound signal generator so that the presence or amount of target analyte in the sample can be determined by examining the porous member for a signal at the appropriate zone.

In addition to the limitations of the assay devices and systems of the prior art, including the limitations of using absorbent membranes as carriers for sample and reagents, assay devices generally involve numerous steps, including critical pipetting steps which must be performed by relatively skilled users in laboratory settings. Accordingly, there is a need for one step assay devices and systems, which, in addition to controlling the flow of reagents in the device, control the timing of the flow of reagents at specific chambers in the device. In addition, there is a need for assay devices which do not require critical pipetting steps and are performing in a full quantitative way,

Today most target analyte are measured using large equipment (immune analyzers) located at central laboratories One of the major reasons for this is that no small handheld instrument exist today that can fulfil the critical parameters for a highly sensitive, reproducible and quantitative immune as well as DNA assay,

Accordingly, an object of the present invention was to develop a handheld device and a method capable of reliably and efficiently detecting the presence or absence of target analytes in small samples.

One major concern when quantitatively detecting presence or absence of analytes in small samples is the elimination or reduction of background signal, which disturbs the reliability and reproducibility of detecting small amounts of analyte

Accordingly another object of the present invention was to develop a device and a method for quantitatively detecting the presence or absence of a target analyte in a small liquid sample, wherein the background unspecific signal is reduced or eliminated Disclosure of the Invention

An object of the invention was to develop a device and a method capable to separate a suspension into a liquid phase and a retentate phase in a short time, where the liquid phase is substantially free of retentate contamination.

A further object was to develop a device and a method capable to separate a suspension into a liquid phase and a retentate phase in a short time where the separation is driven without the use of an external force.

Another object of the present invention was to develop a device and a method for quantitatively detecting the presence or absence of a target analyte in a small liquid sample, wherein the background uπspecific signal is reduced or eliminated

During the developments leading to the present invention the inventors found several ways to improve the devices and methods in this respect. In summary the inventors found that the use of a) corona treated surfaces channels in the capillary channels, and b) microcapillary channels in the capillary channels, and c) a physical barrier between the capillary channel and the separation chamber all enhanced the performance of the separation significantly. Further the inventors found that separation of detection steps and the separation steps in the quantitative detection cycle increased the sensitivity significantly, primarily by lowering the background noise.

Accordingly, in one embodiment the invention relates to a device for separating a sus- pension comprising 200 μl or less into a liquid phase and a retentate phase, the device comprises a separation chamber (2) comprising an application zone (1) and a hydro- philic filter material (17), said separation chamber being connected to a first capillary channel (3), where the connecting junction between the separation chamber and the first capillary channel comprise a physical barrier (10) preventing flow of residue reten- tate from a lower part of the chamber into the first capillary channel

In a preferred aspect the sample to be analysed preferably has a volume of less than

200μl. In an even more preferred aspect the sample to be analysed has a volume of less than 150μl, even more preferred less than 100μl, even more preferred less than 90μl, such as less than 80μl, less than 70μl or even less than 60μl. In an even more preferred aspect the sample to be analysed has a volume of less than 50μl, even more preferred less than 45μl, even more preferred less than 40μl.

In a preferred aspect the first part of the capillary channel has a volume of less than 100μl. In an even more preferred aspect the capillary channel has a volume of less than 90μl, even more preferred less than 80μl, even more preferred less than 70μl, such as less than 60μl, less than 50μl or even less than 40μl. In an even more preferred aspect the first part of the capillary channel has a volume of less than 30μl, even more preferred less than 25μl, even more preferred less than 20μl, such as less than 15μl, less than lOμl or even less than 5 μl.

In another embodiment at least the lower part of the internal surface of the first capillary channel facing the liquid is made of a surface treated plastic material. The surface treatment may be an oxidation, preferably a corona treatment.

In an further embodiment the device comprises an upper part and a lower part, where the two parts when assembled form a separation chamber (2), a first capillary channel (3), and a physical barrier (10) preventing flow of residue retentate from a lower part of the chamber into the first capillary channel, said upper part having an application well (1 ) leading to the separation chamber.

In a further aspect the invention relates to the use of the device according to the invention for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase, where the liquid phase is substantially free of suspended matter. The suspension might be blood, the liquid phase plasma/serum and the retentate blood cells.

In a further aspect the invention relates to a method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the sus- pended matter, and a liquid phase substantially free of suspended matter; the method comprising the steps of:

a. optionally applying a suspension to a prefilter and leading the suspension through the prefilter for the retention of suspended matter and substantially uni- form transfer the liquid to the filter material of step b; b. applying less than 200 μl of a sample suspension, or the liquid of step a,, to a filter material; c applying the filter material comprising the suspension to a separation chamber, which is connected to a first capillary channel; d. over saturating the filter to feed the first capillary channel; e. preventing flow of residue retentate from the lower part of the separation chamber into the first capillary channel, thereby sedimenting the suspended matter on the lower part of the of the separation chamber to separate the suspension into a retentate phase and a liquid phase; and f. directing the liquid phase into the first capillary channel

In a further aspect of the method the liquid phase is directed into the first capillary channel solely by the combined action of capillary forces provided by the first capillary channel and hydrostatic pressure generated by the applied sample.

In the experimental development leading to the present invention the inventors further found that critical parameters for obtaining a highly sensitive, reproducible and full quantitative assay for quantitatively detecting presence or absence of analytes in small samples are to increase the signal to noise ratio by lowing the background noise. Fur- ther, efficient mixing procedures between the target analyte and tracer/capture antibodies are preferred, as well as efficient washing procedures for lowing background noise. Even further it was found that a large reaction surface between target analyte and tracer/capture antibodies is preferred. Further preferred features are efficient amplification reagent such as HRP or ALP enzyme conjugated tracer antibodies and the possi- bility of using temperature controlled assays.

By combining microfluid and magnetic particle technology in a special constellation the present inventors found that it was possible to fulfil the critical parameters and at the same way obtaining a relative small handheld instrument (below 500 gram), capable of analysing samples of less than 200μl.

Accordingly in a preferred aspect of the invention it relates to a device for quantitative detecting the presence or absence of a target analyte in a liquid sample, the device comprising a reaction chamber in the form of a capillary channel having a volume of less than 200μl, the reaction chamber comprising: a. a first part (fig ref.) comprising a sample inlet (fig ref.) for the introduction of a sample containing an analyte, and a discharge outlet (fig. ref.) for the discharge of waste products;

b a second part (fig. ref.) comprising means for detection (fig. ref.) of the target analyte, and a solution inlet (fig. ref.) for introduction of washing solutions and reaction mixtures; and

c. means for transferring an immobilised analyte from the first part to the second part of the chamber and vice versa;

where the first and second parts are separated such that other liquid sample material may not enter the second part of the chamber.

In a further aspect the invention relates to the use of a device according to the invention for the quantitative detection of the presence or absence of a target analyte in a sample

In a further aspect the invention relates to a method for quantitative detecting the pres- ence or absence of a target analyte in a sample consisting of less than 200 μi liquid, comprising the steps of:

a) providing an analyte containing liquid sample consisting of less than 200 μl liquid; b) supplying the liquid sample to a first reaction part of a chamber, the chamber comprising a first reaction part and a second detection part, the two parts being physically separated such that liquid sample material cannot enter into contact with the second detection part c) contacting the sample in the first reaction part of a chamber with an immobilisa- tton matrix capable of capturing the analyte; d) immobilising the immobilisation matrix comprising the captured analyte; e) transferring the immobilisation matrix comprising the captured analyte to the second part of the chamber; f) remobilising and washing the immobilisation matrix comprising the captured analyte with a washing solution; g) immobilising the immobilisation matrix comprising the captured analyte; h) optionally, discarding the washing solution i) optionally, remobilising the immobilisation matrix comprising the captured ana- lyte and repeating steps f) to h); j) transferring the immobilisation matrix comprising the captured analyte to the de- tector part of the second part of the chamber; and k) detecting the presence or absence of a target analyte using conventional detection means.

In a further aspect the invention relates to a kit of parts comprising a device according to the invention and a magnetic material

Brief Description of the Drawings

The invention is explained in detail below with reference to the drawings, in which

Fig. 1 illustrates a schematic presentation of a sample device comprising a microfluid channel having three chambers (3, 5, 6), an application zone (1), a separation chamber (2), a first capillary channel (3), a collection chamber (4a), a waste outlet (4b), a washing chamber (5), a detection chamber (6), magnetic particles location in washing chamber (7), an inlet channel for washing and detector solution (8), a physical barrier (10 (vertical), 10' (incline)) between the separation chamber and the first capillary channel, capillary micro channels (11) in the first capillary channel (3), corona treatment (12) (symbolised by the grey shade) of the first capillary channel, and a detector unit (14).

Fig 2 illustrates the same principle as in Fig 1 with a three dimension illustration. A sample device comprising a microfluid channel having three chambers (3, 5, 6), an application well (1'), a separation chamber (2), a hydrophilic filter material (17) for blood filtration, a first capillary channel (3), a collection chamber (4a), a waste outlet (4b), a washing chamber (5), a detection chamber (6), magnetic particles location in washing chamber (7), an inlet channel for washing and detector solution (8), a physical barrier (10, 10') between the separation chamber and the first capillary channel (3), capillary micro channels (11) in the first capillary channel (3), corona treatment (12) of the first capillary channel (3) and a detector unit (14). Fig. 3 illustrates a schematic site view of a separation device comprising a microfluid channel (3), an application well (V), a separation chamber (2), a first capillary channel (3), a physical barrier (1C) between the separation chamber and the first capillary channel, a hydrophilic filter material (17), and a prefilter (15).

Fig. 4a illustrates a schematic site view of an integrated separation and detection device comprising a microfluid channel (3,5,6), an application well (1), a separation chamber (2) and the hydrophilic filter (17), a first capillary channel (3), serum/plasma (18) in the first capillary channel,, signal solution (19) in washing (5) and detector chamber (6), light trap version A (20) in connecting junction between the first capillary channel (3) and the washing chamber (5), and a detector unit (14)

Fig. 4b illustrates a schematic site view of an integrated separation and detection device comprising a microfluid channel (3,5,6), a application well (1), a separation cham- ber (2) and hydrophilic filter (17), a first capillary channel (3), serum/plasma (18) in the first capillary channel, signal solution (19) in washing (5) and detector chamber (6), a light trap version B (20') in connecting junction between the first capillary channel (3) and the washing chamber (5), and a detector unit (14).

Fig. 5 illustrates a prototype picture of fig. 2 presentation of a separation device comprising a microfluid channel having three chambers (3, 5, 6), a application well ( T), a separation chamber (2), a first capillary channel (3), a washing chamber (5), a detection chamber (6), a physical barrier (10') between the separation chamber and the first capillary channel, and a hydrophilic filter (17)

Fig. 6 illustrates a prototype picture of fig. 4 (backside), presentation of an integrated separation and detection device comprising a microfluid channel having three chambers (3, 5, 6), an application well (V) backside, a separation chamber (2) backside, a first capillary channel (3), a washing chamber (5), a detection chamber (6), a physical barrier (10') between the separation chamber and the first capillary channel, and a hydrophilic filter (17). Left circle is a magnified view of the physical barrier (10') between the separation chamber and the first capillary channel in order to illustrate the capillary microchannels (1 1 ) in the first capillary channel Right circle is a magnified view of the first capillary channel at the collection chamber in order to illustrate the capillary micro- channels. Fig. 7 illustrates same principle as in fig. 1 with a three dimension illustration including more features. A integrated separation and detection device comprising a microfluid channel having three chambers (3, 5, 6), an application well (1 '), a separation chamber (2), a first capillary channel (3), a collection chamber (4a), a waste outlet (4b), a wash- ing chamber (5), a detection chamber (6), magnetic particles location in washing chamber (7), an inlet channel for washing and detector solution (8), a physical barrier (10, 10') between the separation chamber and the first capillary channel, capillary micro channels (11) in the first capillary channel (3), a detector unit (14), a first compartment for detection solution A (9), a second compartment for detection solution B (15), a washing solution compartment (16), and a blood lid (12a).

Fig. 8A - Fig. 8 J illustrate a target analyte fluid detection cycle

Fig. 8A The fluid inlet lid (12a) are pushed to the left.

Fig. 8B

Between 10 - 100 uL human full fluid (blood) is taken from a finger tip (13) and directly placed in the fluid inlet hole (1).

Fig. 8C

The fluid inlet lid (12a) is pushed over the fluid inlet hole (1 )

Fig. 8D After the blood separation process the plasma/serum enters the first part of the reaction chamber (3) via special design micro structures and treatment for enhancement of capillary forces.

Fig.. 8E Washing solution (16) is injected via Inlet channel for washing and detector solution (8) and the detection part of the second part of the reaction chamber (6) into the washing part of the second part of the reaction chamber (5).

Fig. 8F Magnetic particles in washing part of the second part of the reaction chamber (7) are dissolved and moved into the first part of the reaction chamber (3) for incubating. Rg. 8G

Magnetic particles located in first part of the reaction chamber (3) are moved back into washing part of the second part of the reaction chamber (5) for washing

Fig. 8H

Detector solution (9, 21) is injected via Inlet channel for washing and detector solution (8) and the detection part of the second part of the reaction chamber (6) into the washing part of the second part of the reaction chamber (5).

Fig. 8I

Same as illustrated in Fig, 8H. The washing chamber (5) is now complete filled with detector solution form detector compartments (9,,21).

Fig. 8J

Magnetic particles (7) located in the washing part of the second part of the reaction chamber (5) are moved forward to the detection part of the second part of the reaction chamber (6) where presence or absence of target analyte are detected via a sensor unit (14).

Fig. 9 A - Fig. 9C

Illustrates the magnetic particles motor system that moves the magnetic particles between the different chamber locations as illustrated in Fig 8.

Fig. 9 A

Fig. 9A illustrates a magnetic motor system that moves the magnetic particles located in the microfluid channel. The sample device (22) containing the magnetic particles (7) are located under the magnetic particle motor system (23) driven by an electromotor (24). The magnet (26) is located in the magnet holder (25). The magnet can move be- tween all three chambers as illustrated in fig. 8A - 8J

Fig. 9 B

Same as fig, 9A, the magnet are positioned on top of the magnetic particles in washing chamber as illustrated at fig. 8B. Rg. 9C

Same as fig. 9A₁ the magnet have now completed a cycle and moved away from the microfluid channel comprising the three chambers.

Fig. 10

Sensor data illustrating 3 x 8 measurement using three different concentrations of the analyte Brain Natriuretic Peptide (BNP) in serum. Green lines: BNP concentration 7.2 pg/mL; red lines: BNP concentration 64 pg/mL and blue lines: BNP concentration 205 pg/mL

Definitions

In the context of the present invention, by "capillary channel" is meant a narrow tube or channel through which a fluid can pass. Preferably the diameter (or with) of a capillary channel according to the invention is less than 10 mm Even more preferred the diameter of a capillary channel according to the invention is less than 5mm, such as less than 4 mm, or less than 3 mm or even less than 2 mm. In a most preferred aspect the capillary channel has a diameter of 1 mm or less, e g 0,2-1.0 mm. The channels may also be formed of non-circular shapes, e.g. rectangular or triangular, in which case the "diameter" refers to the mean distance from the center of the cannel to the periphery.

The terms "capillary channel" and "first capillary channel" are used interchangeable

In the context of the present invention, by "micro channels" or "capillary micro channel" is meant a very small narrow tube or channel through which a fluid can pass. Preferably the diameter or with of a micro channel according to the invention is less than 1/5 of the capillary channel. Even more preferred the diameter of a micro channel according to the invention is less than 1mm, such as less than 0.5 mm, or less than 0.2 mm or even less than 0.1 mm. In a most preferred aspect the mircro channel has a diameter of 0,1 mm or less, e.g 0,02-0.1 mm The channels may also be formed of non-circular shapes, e.g. rectangular or triangular, in which case the "diameter" refers to the mean distance from the center of the cannel to the periphery.

In the context of the present invention, by "lower part" is meant the part of a device when in use, which is closest to the center of the earth. By "upper" is meant the oppo- site, namely, the part furthest away from the centre of the earth when in use. Accordingly, a liquid would lie on the lower part and not the upper part when in use.

Detailed description of the Invention

An object of the invention was to develop a device and a method capable to separate a suspension into a liquid phase and a retentate phase in a short time, where the liquid phase is substantially free of retentate contamination, and where the separation is driven without the use of an external force.

The inventors of the present invention surprisingly found that surface treated plastic treated such that the hydrophilicity of the material was improved, was highly beneficial to the drag force of the device, such that liquid could flow through the device without the use of external force

Accordingly, in one aspect the invention relate to a device for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase, said device comprising a separation chamber (2) comprising a application zone (1 ) and a hydrophilic filter material (17), said separation chamber being connected to a capillary channel (3), where at least the lower part of the internal surface of the capillary channel facing the liquid is made of a surface treated plastic material In one embodiment the surface treatment is an oxidation. In a further embodiment the oxidation is a corona treatment. Especially when at least the lower part of the internal surface of the first capillary channel facing the liquid is made of a corona treated plastic surface, it was observed by vis- ual inspection that the capillary channel was very efficient in pulling the liquid into the capillary channel.

In a preferred embodiment the plastic material is polystyrene, polymethylmethacrylate, polyethylene, polypropylene, polyacrylates, silicon elastomers or the like.

In one embodiment the device further comprise a collecting chamber (4a) connected to the capillary channel.

In one embodiment the device further comprise an upper part and a lower part, where the two parts when assembled form a separation chamber (2) comprising an applica- tion well (1') and a hydrophilic filter (17), a capillary channel (3), said upper part having an inlet leading to the separation chamber.

It has further been shown during the course of the experiments leading to the present invention that by using or creating microchannels in the capillary channels, these were much more efficient in providing the required drag force on the liquid sample.

Accordingly, In one aspect the invention relates to a device for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase, said device com- prising an application chamber (1 ) comprising a hydrophilic filter material (17), said application chamber being connected to a capillary channel (3) comprises two or more capillary microchannels (11).

In a further aspect the capillary channel comprises two or more capillary microchan- nels.

In a further aspect the capillary channel comprises three or more capillary microchannels.

A further object was to develop a device and a method capable to separate a suspen- sion into a liquid phase and a retentate phase in a short time where the separation is driven without the use of an external force.

This was achieved by the device according to the invention.

Accordingly, in one embodiment the invention relates to a device for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase, the device comprises a separation chamber (2) comprising an application zone (1 ) and a hydrophilic filter material (17), said separation chamber being connected to a first capillary channel (3), where the connecting junction between the separation chamber and the first capillary channel comprise a physical barrier (10) preventing flow of residue retentate from a lower part of the chamber into the first capillary channel. In a preferred aspect the sample to be analysed preferably has a volume of less than 200μl, In an even more preferred aspect the sample to be analysed has a volume of less than 150μl, even more preferred less than 100μl, even more preferred less than 90μl, such as less than 80μl, less than 70μl or even less than 60μl In an even more preferred aspect the sample to be analysed has a volume of less than 50μl, even more preferred less than 45μl, even more preferred less than 40μl.

In a preferred aspect the first part of the capillary channel has a volume of less than 1O0μl. In an even more preferred aspect the the capillary channel has a volume of less than 90μl, even more preferred less than 80μl, even more preferred less than 70μl, such as less than 60μl, less than 50μl or even less than 40μl, In an even more preferred aspect the first part of the capillary channel has a volume of less than 30μl, even more preferred less than 25μl, even more preferred less than 20μl, such as less than 15μl, less than 10μl or even less than 5 μl,

The invention thus further relates to a device according to the above inventions where further the connecting junction between the separation chamber and the capillary channel comprise a physical barrier (10) preventing flow of residue retentate from a lower part of the chamber into the capillary channel

In one embodiment the physical barrier is in the form of a vertical barrier having a height (10) of at least 0.2-1 ,6 mm. In a further embodiment the height of the barrier is at least 0.8-1.6 mm. In a further embodiment the physical barrier (10) in the horizontal plane and in the direction towards the first capillary channel describes an incline extending from the bottom of the separation chamber. In a further embodiment the incline in vertical direction is 0.2-1.6 mm, and in horizontal direction 0-100% of the length of the first capillary channel. In a further embodiment the incline in vertical direction is about 0.8-1,6 mm, and in horizontal direction about 20-80% of the length of the first capillary channel.

In one embodiment the device comprise two parts an upper and a lower part, which, when assembled form the device assording to the invention. Preferably, the interfaces between the upper and lower parts are sealed with a hydrophobic sealant. Even more preferably, the hydrophobic sealant is high grade silicone grease. In a preferred embodiment of the invention the capillary channels are formed as hollows and the capillary channels are covered by a removable top between the upper and lower parts of the device. Preferably the removable top is adhesive, more preferably the adhesive removable top is tape. Preferably, the surface of the top facing the liq- uid is hydrophobic.

Preferably the hydrophilic filter (17) is a glass fibre filter Preferably the hydrophilic filter (17) has a thickness of 0.25-0.50 mm. Even more preferably, the hydrophilic filter (17) has a thickness of 0 35-0.40 mm.

In a preferred embodiment the separation chamber (2) tapers, in the direction towards the capillary channel Thereby an increased separation is achieved.

The device may further comprise a prefilter material (15), which may be made from glass fibre. In one embodiment the prefilter has a thickness of 0.30-0.90 mm. In one embodiment the prefilter has a thickness of 0.60-0.80 mm.

In one embodiment the total volume of the separation chamber is 15-200 μl. In one embodiment the total volume of the separation chamber is 30-100 μl.

In one embodiment the total height of the separation chamber is 1.5-5.0 mm. In one embodiment the total height of the separation chamber is 2 0-3.5 mm. The width and height of the capillary channel is 0.25-2.0 mm and 0,2-1.0 mm, more preferably 0 8-1.2 mm and 0.2-0.5 mm, respectively.

In one embodiment the width and height of the capillary channel is about 1.0 mm and about 0.2 mm, respectively. The length of the capillary channel from the outlet of the separation chamber (3) to the inlet of collection chamber (4a) is 5-50 mm In one embodiment the length of the capillary channel from the outlet of the separation chamber to the inlet of collection chamber is 5-20 mm. In one embodiment the length of the capillary channel from the outlet of the separation chamber to the inlet of collection chamber is about 30 mm.

In one aspect the invention relates to the use of a device according to the above inven- tion, for separating a suspension comprising 200 μl or less into a liquid phase and a re- tentate phase. Thereby, liquid [plasma] phase obtained may be substantially free of suspended matter, such as 99% free of suspended matter.. The liquid phase may even be 99.9% free of suspended matter. Or it may even be 100% free of suspended matter.

Preferably the suspension to be analysed using the device according to the invention comprises 5-100 μl. Even more preferably the suspension comprises 5-40 μl. Preferably, the suspension is blood. Even more preferably the blood is whole blood. Even more preferably the blood is of human origin.

in another aspect the present invention relates to a method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the suspended matter, and a liquid phase substantially free of suspended matter; the method comprising the steps of:

a., optionally applying less than 200 μl suspension to a prefilter and leading the suspension through the prefilter for the retention of suspended matter and substantially uniform transfer the liquid to the filter material of step b; b. applying less than 200 μl of a sample suspension, or the liquid of step a., to a filter material; c. applying the filter material comprising the suspension to a separation chamber, which is connected to a capillary channel; d . over saturating the filter to feed the capillary channel; e. directing the liquid phase into the capillary channel by means of capillary forces generated by a surface-treated capillary channel.

a. optionally applying the less than 200 μl suspension to a prefilter and leading the suspension through the prefilter for the retention of suspended matter and substantially uniform transfer the liquid to the filter material of step b; b. applying less than 200 μi of a sample suspension, or the liquid of step a., to a filter material; c. applying the filter material comprising the suspension to a separation chamber, which is connected to a capillary channel; d. over saturating the filter to feed the capillary channel; e. preventing flow of residue retentate from the lower part of the separation chamber into the capillary channel, thereby sedimenting the suspended matter on the lower part of the of the separation chamber to separate the suspension into a retentate phase and a liquid phase; and f. directing the liquid phase into the capillary channel.

In another aspect the invention relates to a method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the sus- pended matter, and a liquid phase substantially free of suspended matter; the method comprising the steps of:

a. optionally applying less than 200 μl suspension to a prefilter and leading the suspension through the prefilter for the retention of suspended matter and sub- stantially uniform transfer the liquid to the filter material of step b; b- applying less than 200 μl of a sample suspension, or the liquid of step a., to a filter material; c. applying the filter material comprising the suspension to a separation chamber, which is connected to a capillary channel; d. over saturating the filter to feed the capillary channel; e. directing the liquid phase into the capillary channel by means of capillary micro- channels present in the capillary channel.

The invention further relate to a combination of the above methods .

The liquid phase may be directed into the capillary channel solely by the combined action of capillary forces provided by the capillary channel and hydrostatic pressure generated by the applied sample.

The invention further relates to a device for quantitative detecting the presence or absence of a target analyte in a sample, the device comprising a reaction chamber in the form of a capillary channel having a volume of less than 200 μl, the reaction chamber comprising: a. a first part comprising 1 ) a sample inlet for the introduction of a sample containing an analyte, 2) an discharge outlet for the discharge of waste products;

b. a second part comprising 3) means for detection of the target analyte, 4) a solu- tion inlet for introduction of washing solutions and reaction mixtures; and

where the first and second parts are separated such that sample material may not enter the second part of the chamber.

Signal detection in microfluidic systems are often jeopardised by a very low sensitivity requiring large amounts of analyte to generate a reliable and reproducible signal. Much effort has been put into development of more sensitive and sophisticated detection means. However, surprisingly, less has been done in order to remove or reduce the level of unspecific signal (noise). The present inventors surprisingly found that simple measures reducing the noise of the system significantly improved the reproducibility and the sensitivity of the system significantly.

The inventive concept of the present invention may be seen in general as the physical separation, in a microfluidic system, of the steps of binding and immobilising an analyte and the steps of detecting the analyte Preferably, any signal deriving from non-analyte species (background signal) remains in the first part of the device (or the first steps in the method), whereas in the second part of the device (later steps in the method) the signal derived from the analyte, with a minimal background signal, is detected.

Accordingly, in one aspect the invention relates to a device for quantitative detecting the presence or absence of a target analyte in a liquid sample, the device comprising a reaction chamber in the form of one or more capillary channels having a volume of less than 200μl, the reaction chamber comprising:

a. a first part comprising a capillary channels having a volume of less than 200μl, a sample inlet for the introduction of a sample containing an analyte, and a dis- charge outlet for the discharge of waste products; b. a second part comprising means for detection of the target analyte, and a solution inlet for introduction of washing solutions and reaction mixtures; and

where the first and second parts are separated such that other liquid sample material may not enter the second part of the chamber. By other sample material is meant sample material excluding the analyte.

The reaction chamber may contain several compartments or parts. Further each part may be divided into further parts or compartments wherein specific reactions are to occur. By separating the reaction chamber in a first part for binding the analyte and a second part and detecting the analyte, a significant reduction in background signal could be obtained.

In a preferred aspect the sample to be analysed preferably has a volume of less than 200μl. In an even more preferred aspect the sample to be analysed has a volume of less than 150μl, even more preferred less than 100μl, even more preferred less than 90μl, such as less than 80μl, less than 70μl or even less than 60μl. In an even more preferred aspect the sample to be analysed has a volume of less than 50μl, even more preferred less than 45μl, even more preferred less than 40μl, such as less than 35μl, less than 30μl or even less than 25 μl.

In a preferred aspect the first part of the capillary channel has a volume of less than 100μl. In an even more preferred aspect the first part of the capillary channel has a volume of less than 90μl, even more preferred less than 80μl, even more preferred less than 70μl, such as less than 60μl, less than 50μl or even less than 40μl. In an even more preferred aspect the first part of the capillary channel has a volume of less than 30μl, even more preferred less than 25μl, even more preferred less than 20μl, such as less than 15μl, less than 10μl or even less than 5 μL The same preferred volumes apply for the second part of the reaction chamber. The reaction chamber comprises a first and a second part. In a preferred aspect both the first and the second part are made of capillary channels. The first and second part may be separated e.g. by a collection chamber from which residual sample matter and added reagents may be collected and later expelled. Such a collection chamber and the volume thereof is not to be understood as part of the reaction chamber or the preferred volumes thereof.

In a preferred aspect of the invention the means for transferring the immobilised ana- lyte from the first part to the second part of the chamber and vice versa is an external magnetic force generating source, which can apply a magnetic field to the chamber and be moved along the edge of the chamber on demand.

In one aspect of the invention the first and second parts are separated by a collection chamber (fig. ref.). The collection chamber may serve the purpose of separating the first and second parts such that liquid sample material, other then analyte species actively transported between the first and second part, may not enter the second part of the chamber The collection chamber also serves the purpose of an outlet for waste products such as washing solution and residual sample material. The placement of the collection chamber between the first and the second part provides that the collection chamber serves as an outlet for material from both the first and the second part of the chamber.

In a preferred aspect of the invention, in order to move magnetic particles comprising the immobilised analyte most efficiently, a magnetic field is moved along the top edge of the chamber on demand.

In a preferred aspect of the invention the first and second parts are separated such that a significant part of the signal (e.g light) may not be transferred from the first part of the chamber to the detector part of the second part of the chamber. By a significant part is meant more than 50%, such as more than 75% or even more than 90%, or even more than 99%. This may be achieved by placing the exit point from the first part and the entry point of the second part in different levels e.g. by introducing a bend on the path from the first part to the second part of the chamber, such that signal (in the form of light rays) from the first part of the chamber may not enter the detection part of the second chamber. Another possibility is introducing a bend in the second part of the chamber such that the detector part is not in line with the entry point of the analyte to the second part of the chamber. A preferred possibility is the placement of a light- impermeable barrier between the two parts such that a significant part of the light is prevented from entering the second part from the first part. Of course the barrier must not prevent the transfer of analyte (e,g via magnetic particles) from the first and second parts.

Preferably, the surface structure and the colour of the internal surface of the reaction chamber, or at least the second part of the chamber, is non-reflecting and/or light absorbing, respectively. In one aspect of the invention the non-reflecting and/or light absorbing surface is obtained by obscuring and/or darkening of the surface. In a preferred aspect the darkening is blackening. Most preferably the colour of the internal surface of the reaction chamber is black.

In a preferred aspect of the invention the means for detection of the target analyte are selected among surface acoustic wave (SAW) detectors, spectrophotometers, fluoro- meters, CCD sensor chip(s), CCOS sensor chip(s), PMT detector(s), or any suitable light detector.

In a preferred aspect the internal width and height of the reaction chamber, or at least the first part of the reaction chamber, is 0.1-5 mm and 0,05 - 2 mm respectively . More preferably, the internal width and height of the reaction chamber, or at least the first part of the reaction chamber, is 0.25-2 mm and 0.2 - 1 mm, respectively

In a preferred aspect the length of the reaction chamber is 2-30 mm, more preferably 5~ 20 mm,

The device according to the invention may be used for the quantitative detection of the presence or absence of a target analyte in a sample. Preferably, the sample is derived from blood. In one aspect the sample is serum. In one aspect the sample is plasma. Plasma may obtained by applying an anti coagulant to the blood sample to be analysed. Preferred anti-coagulant may be selected among the group comprising K3- EDTA, citrate and heparine.

In a preferred aspect of the invention the sample is of human origin.

In another aspect the invention relates to a method for quantitative detecting the presence or absence of a target analyte in a sample consisting of less than 200 μl liquid, comprising the steps of: a) providing an analyte containing liquid sample consisting of less than 200 μl liquid; b) supplying the liquid sample to a first reaction part of a chamber, the chamber comprising a first reaction part and a second detection part, the two parts being physi- cally separated such that liquid sample material cannot enter into contact with the second detection part; c) contacting the sample in the first reaction part of a chamber with an immobilisation matrix capable of capturing the analyte; d) immobilising the immobilisation matrix comprising the captured analyte; e) transferring the immobilisation matrix comprising the captured analyte to the second part of the chamber; f) remobilising and washing the immobilisation matrix comprising the captured analyte with a washing solution; g) immobilising the immobilisation matrix comprising the captured analyte; h) optionally, discarding the washing solution i) optionally, remobilising the immobilisation matrix comprising the captured analyte and repeating steps f) to h); j) transferring the immobilisation matrix comprising the captured analyte to the detector part of the second part of the chamber; and k) detecting the presence or absence of a target analyte using conventional detection means.

By separating the steps a) - d) of binding the analyte in one compartment and the steps e) - k) of washing and detecting the analyte in a second compartment a signifi- cant reduction in background signal was observed.

In a preferred aspect the method further comprise a step a¹) of contacting the analyte with a biological marker, capable of binding to the analyte. The biological marker may be an antibody e.g. with enzyme horseradish peroxidise (HRP)₁ biotin or alkaline phosphatase (ALP). Thereby the analyte may become more detectable by increasing the signal for detection. In a preferred aspect of the method according to the invention the step a') of contacting the analyte with a biological marker, capable of binding to the analyte is performed prior to step e). Thereby, the presence of unbound biological marker in the detection part of the method is minimised and the background signal is significantly reduced. In a preferred aspect of the invention the biological marker is ca- pable of reaction with a substrate whereby signal may be amplified Accordingly, in one aspect of the invention the method further comprise a step f ) of contacting the immobilisation matrix comprising the captured analyte with a substance capable of reacting with the biological marker.

In a preferred aspect of the invention the biological marker is one [or more] selected from compounds, mono-, oligo- and polyclonal antibodies, antigens, receptors, ligands, enzymes, proteins, peptides and nucleic acids. Preferably the biological marker is one or more selected from the group having the properties of light absorption, fluorescence emission, phosphorescence emission, or luminescence emission

in a preferred aspect the immobilisation matrix comprises magnetic material. In a preferred aspect the step e) is performed by moving a magnetic source along the external edge of the first reaction chamber toward the second detection chamber.

The magnetic material is preferably selected from the group comprising magnetic particles, magnetic nanoparticles and superparamagnetic nanoparticles.

It was further surprisingly observed that using magnetic particles having a non unimo- dal size distribution, such as a bimodal size distribution, a more efficient performance in terms of washing efficiency and time was obtained, Accordingly in a preferred aspect of the invention the magnetic material has an at least bimodal size distribution In another aspect of the invention the magnetic material has a trimodal size distribution.

In a preferred aspect of the invention the conventional detection means are selected among surface acoustic wave (SAW) detectors, spectrophotometers, fluorometers, CCD sensor chip(s), CCOS sensor chip(s), PMT detector(s), or any suitable light detector.

The method according to the invention may be used for the quantitative detection of the presence or absence of a target analyte in a sample. Preferably, the sample is derived from blood. In one aspect the sample is serum. In one aspect the sample is plasma. Plasma may obtained by applying an anti coagulant to the blood sample to be analysed. Preferred anti-coagulant may be selected among the group comprising K3- EDTA, citrate and heparine., In a preferred aspect of the invention the sample is of hu- man origin.

In one aspect, the invention relates to a kit of parts comprising a device as defined above and a magnetic material according to the invention.. Preferably this kit is for use in detection of the presence or absence of a target analyte in a sample.

In one aspect the invention further relate to the use of a device according to the invention for the quantitative detection of the presence or absence of a target analyte in a sample.

Preferably, the sample is derived from blood., Even more preferably, the sample is serum- Even more preferably, the sample is plasma. Plasma may be obtained by applying an anti coagulant to the blood. The anti-coagulant may be one of K3-EDTA, citrate and heparine.

In one preferred aspect the invention relates to a kit of parts comprising a device for quantitative detecting the presence or absence of a target analyte in a liquid sample and a magnetic material according to the above.

In another aspect the invention relates to apparatus comprising a device for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase as described above and a device for quantitative detecting the presence or absence of a target analyte in a liquid sample as described above.

Preferably the capillary channel of the device for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase as described above is the first part of the reaction chamber of the device for quantitative detecting the presence or absence of a target analyte in a liquid sample as described above.

Preferably the apparatus according to the invention is used for analysing blood sam- pies

Accordingly, the invention further relates to a method for quantitative detecting the presence or absence of a target analyte in a sample consisting of less than 200 μl suspension; said the method comprising separating the suspension according to the inven- five methods described above, and detecting the presence or absence of the analyte according to the inventive methods described above.

Examples

Example 1

Investigation of presence of physical barrier, corona treatment and micro channels on the separation into clear plasma in collection channel using blood filtration device

Conclusions

Presence of a physical barrier (10,) at the connecting junction between the separation chamber and the first capillary channel, preventing flow of residue retentate from a lower part of the chamber into the first capillary channel, result in an improved separa- tion of the liquid and the suspended matter

The corona treatment of at least the lower part of the internal surface of the first capillary channel facing the liquid, significantly enhances the filling of the collection chamber with plasma,

The use of micro channels in at least the lower part of the internal surface of the first capillary channel facing the liquid is made of a surface treated plastic decreases the filling time significantly.

Experimental setup The blood filtration device used for the experiments was the milled K2 cartridge in clear polystyrene as illustrated in fig. 2, with capillary stop and hydrophobic film covering the milled channels. The K2 blood inlet was used with oval 5 x 7.5mm pre-filter (vertical flow filter VF1 , Whatman). The lateral flow filter 4x15 mm (Fusion 5, Whatman) was mounted on a hydrophobic adhesive. 100 μl K₃EDTA stabilized human blood (2 weeks old) was used for each experiment.

The volume of the collection chamber was 4.6 μl for the K2 device with the 3 micro channels

(^«0.15x0.15mm).

The volume of the collection channel was measured by slowly filling it with indicator solution with a 1-1 Oμl pipette.

The investigation was done using K2 cartridge as illustrated in fig. 2 with and without the micro channels. For both setups the filling time of collection chamber for non corona treated and corona treated cartridges was measured.

Results

Preliminary investigations on the presence or absence of the physical barrier at the connecting junction between the separation chamber and the first capillary channel, preventing flow of residue retentate from a lower part of the chamber into the first capil- lary channel, showed an improved separation of the liquid and the suspended matter when the barrier was present.

Further investigations on the capillary channels produced the following results: The volume of the collection chamber without the micro channels was measured to 3.1 μl . The volume of collection chamber including the micro channels was 4.6 μl .

Discussion

The results in the table above show it is very beneficial to corona treat the collection chamber in order to get it sufficiently hydrophiiic and filled with plasma by capillary force. Note this is under the circumstances using hydrophobic film covering the milled channels..

The table also shows a shorter filling time by the use of capillary micro channels milled in the capillary channel. The micro channels fills fast by capillary force and then promote the filling of the rest of the channel.

Conclusion

The corona treatment is highly preferable to get the collection chamber filled with plasma.

The use of micro channels decreases the filling time.

Example 2

To illustrate the measuring principle the analyte Brain Natriuretic Peptide (BNP) are measured in plasma using the sample device illustrated in fig, 1.

Materials

Samples: Three different plasma samples are spiked with the BNP analyte in the following concentration.

Plasma sample L: 7,2 pg/mL of BNP Plasma sample M: 64 pg/mL of BNP Plasma sample H: 205 pg/mL of BNP

Antibodies: MP coated BNP monoclonal catching antibody Tracer antibody is a HRP label monoclonal BNP antibody. Tracer antibody was placed direct in blood separation filter.

Blood stabilizing reagent: EDTA are added to the blood separation membrane.

Washing solution: TBS + 0.05% tween and 0.05% BSA

Detector solution: Pierce SuperSignal ELISA Femto Maximum Sensitivity Substrate

Detector: PMT detector

Assay temperature: 23 C

Mechanics and Electronics: All mechanical parts, electronics controllers and software are produces in house by the assignee company.

Assay procedures:

A total of 24 samples (plasma sample L; plasma sample M; plasma sample H) was analysed according the procedure illustrated in table I. The numbers in () correspond to numbers in fig. 1. It can be observed from fig- 5 that the three different BNP concentrations can be detected using the device principle described in this application. The following parameters were important for detection the BNP at low concentrations in a reproducible manner

• Applying a drop of blood (20 - 100 uL) directly into the analysing device.

• Increase the signal to noise ratio by lowing the background noise.

• Efficient mixing procedures between the target analyte and tracer/capture antibodies.

• Efficient washing procedures for lowing background noise • Large reaction surface between target analyte and tracer/capture antibodies. «• Efficient amplification reagent such as HRP or ALP enzyme conjugated tracer antibodies. • Temperature controlled assays

Table I: The BNP assay procedure in sample device, All position numbers is according to Fig, 1

Conclusion

It is possible to detect the analyte BNP in concentration at 7,2 pg/mL All CV values calculated were below 15% at all three BNP concentrations (sample L, sample M and sample H).

Claims

1. A device for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase, said device comprising a separation chamber (2) comprising a application zone (1 ) and a hydrophilic filter material (17), said separation chamber being connected to a capillary channel (3), where at least the lower part of the internal surface of the capillary channel facing the liquid is made of a surface treated plastic material.

2 Device according to claim 1 further comprising a collecting chamber (4a) connected to the capillary channel.

3. Device according to any of the claims 1 or 2 comprising an upper part and a lower part, where the two parts when assembled form a separation chamber (2) comprising an application well (V) and a hydrophilic filter (17), a capillary channel (3), said upper part having an inlet leading to the separation chamber.

4. Device according to any of the preceding claims, where the stable plastic material is polystyrene, polymethylmethacrylate, polyethylene, polypropylene, poiyacrylates, silicon elastomers or the like

5. The device according to any of the preceding claims, where the surface treatment is an oxidation.

6. The device of claim 5, where the oxidation is a corona treatment.

7. Device according to any of the preceding claims, where the lower part of the capillary channel has one or more capillary micro channel(s) (11).

8. Device of claim 7, where the lower of the capillary channel has three or more capillary micro channels (11).

9. Device according to any of the claims 7 or 8, where the width and height of the capillary micro channel(s) is 0.05-02 mm and 0.05-0.2 mm, respectively.

10. Device of claim 9, where the width and height of the capillary micro channel(s) is 0.08-0.12 mm and 0.08-0,12 mm, respectively.

11. Device of claim 10, where the width and height of the capillary micro channels(s) is about 0.10 mm and about 0.10 mm, respectively.

12. Device according to any of the preceding claims, where the connecting junction between the separation chamber and the capillary channel comprise a physical barrier (10) preventing flow of residue retentate from a lower part of the chamber into the capil- lary channel.

13. Device according to claim 12, where the physical barrier is in the form of a vertical barrier (10) having a height of at least 0,2-1.6 mm.

14. Device according to claim 13, where the height is at least 0.8-1.6 mm.

15. Device of claim 12, where the physical barrier in the horizontal plane and in the direction towards the collection chamber describes an incline (10¹) extending from the bottom of the separation chamber

16. Device of claim 15, where the incline in vertical direction is 0 2 - 1.6 mm, and in horizontal direction 0-100% of the length of the capillary channel.

17. Device of claim 16, where the incline in vertical direction is about 0.8.-1,6 mm, and in horizontal direction about 20-80% of the length of the capillary channel.

19. Device according to any of the claims 3-18, where the interfaces between the upper and lower parts are sealed with a hydrophobic sealant.

20. Device of claim 19, where the hydrophobic sealant is high grade silicone grease,

21. Device according to any of the claims 3-20, where the capillary channel is covered by a removable top between the upper and lower parts.

22. Device of claim 21 , where the removable top is adhesive .

23. Device of claim 22, where the adhesive removable top is tape

24. Device according to any of the claims 21-23, where the surface of the top facing the liquid is hydrophobic,

25. Device according to any of the preceding claims, where the hydrophilic filter (17) is a glass fibre filter.

26. Device according to any of the preceding claims, where there with of the separa- tion chamber (2) tapers, in the direction towards the capillary channel.

27 Device according to any of the preceding claims, further comprising a prefilter material (15).

28. Device according to claim 27, where the prefilter is a glass fibre filter.

29. Device according to any of the preceding claims, where the total volume of the separation chamber is 15-200 μl.

30 Device according to claim 29, where the total volume of the separation chamber is 30-100 μl.

31 Device according to any of the preceding claims, where the total height of the separation chamber is 1.5-5.0 mm

32. Device according to claim 31, where the total height of the separation chamber is 2.0-3.5 mm.

33. Device according to any of the preceding claims, where the width and height of the capillary channel is 0 25-2.0 mm and 0.2-1 0 mm, respectively.

34. Device of claim 33, where the width and height of the capillary channel is 0.8-1.2 mm and 0.2-0 5 mm, respectively 35 Device according to any of the preceding claims, where the length of the capillary channel from the outlet of the separation chamber (3) to the inlet of collection chamber (4a) is 5-50 mm

36. Use of a device according to any of the claims 1-35, for separating a suspension comprising 200 μl or less into a liquid phase and a retentate phase.

37. Use according to claim 36, where the liquid phase obtained is substantially free of suspended matter, such as 99% free of suspended matter

38. Use according to claim 37, where the liquid phase is 99.9% free of suspended matter.

39. Use according to claim 38, where the liquid phase is 100% free of suspended matter.

40. Use according to any of the claims 36-39, where the suspension comprises 5-100 μl.

41. Use according to claim 40, where the suspension comprises 5-40 μl

42 Use according to any of the claims 36-41 , where the suspension is blood,

43. Use according to claim 42, where the blood is whole blood

44. Use according to claim 42 or 43, where the blood is of human origin.

45. A method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the suspended matter, and a liquid phase substan- tially free of suspended matter; the method comprising the steps of:

a, optionally applying less than 200 μl suspension to a prefilter and leading the suspension through the prefilter for the retention of suspended matter and substantially uniform transfer the liquid to the filter material of step b; b. applying less than 200 μl of a sample suspension, or the liquid of step a., to a filter material; c applying the filter material comprising the suspension to a separation chamber, which is connected to a capillary channel; d over saturating the filter to feed the capillary channel; e. directing the liquid phase into the capillary channel by means of capillary forces generated by a surface-treated capillary channel.

46. A method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the suspended matter, and a liquid phase substantially free of suspended matter; the method comprising the steps of:

a. optionally applying the less than 200 μl suspension to a prefilter and leading the suspension through the prefilter for the retention of suspended matter and substantially uniform transfer the liquid to the filter material of step b; b. applying less than 200 μl of a sample suspension, or the liquid of step a., to a filter material; c. applying the filter material comprising the suspension to a separation chamber, which is connected to a capillary channel; d. over saturating the filter to feed the capillary channel; e. preventing flow of residue retentate from the lower part of the separation charn- ber into the capillary channel, thereby sedimenting the suspended matter on the lower part of the of the separation chamber to separate the suspension into a retentate phase and a liquid phase; and f. directing the liquid phase into the capillary channel.

47. A method for separating a liquid sample consisting of less than 200 μl suspension, into a retentate phase comprising the suspended matter, and a liquid phase substantially free of suspended matter; the method comprising the steps of:

a. optionally applying less than 200 μl suspension to a prefilter and leading the suspension through the prefilter for the retention of suspended matter and substantially uniform transfer the liquid to the filter material of step b; b. applying less than 200 μl of a sample suspension, or the liquid of step a., to a filter material; c. applying the filter material comprising the suspension to a separation chamber, which is connected to a capillary channel; d. over saturating the filter to feed the capillary channel; e. directing the liquid phase into the capillary channel by means of capillary micro- channels present in the capillary channel.

48. Method according to any of the above claims 45-47 wherein the method comprises any combination of these methods.

49. Method according to any of the claims 45-48, where the liquid phase is directed into the capillary channel solely by the combined action of capillary forces provided by the capillary channel and hydrostatic pressure generated by the applied sample.

50. Method according to any of the claims 45-49, where the suspension is blood.

51. A device for quantitative detecting the presence or absence of a target analyte in a sample, the device comprising a reaction chamber in the form of a capillary channel having a volume of less than 200 μl, the reaction chamber comprising:

a. a first part comprising 1) a sample inlet for the introduction of a sample containing an analyte, 2) an discharge outlet for the discharge of waste products;

b a second part comprising 3) means for detection of the target analyte, 4) a solution inlet for introduction of washing solutions and reaction mixtures; and

c means for transferring an immobilised analyte from the first part to the second part of the chamber and vice versa;

52. Device according to claim 51, where the means for transferring the immobilised analyte from the first part to the second part of the chamber and vice versa is an external magnetic force generating source, which can apply a magnetic field to the chamber and be moved along the edge of the chamber on demand.

53. Device according to any of the claims 51 or 52, where the first and second parts are separated by a collection chamber.

54. Device according to claim 51- 53, where the first and second parts are separated such that light may not be transferred from the first part of the chamber to the detector part of the second part of the chamber.

55. Device according to any of the claims 51-54, where the magnetic field is moved along the top edge of the chamber on demand,

56. Device according to any of the claims 51-55, where the surface structure and the colour of the internal surface of the reaction chamber is light non-reflecting and/or light absorbing, respectively.

57. Device according to claim 56, where the light non-reflecting and/or light absorbing surface is obtained by obscuring and/or darkening of the surface,

58. Device according to claim 57, where the darkening is blackening.

59. Device according to any of the claims 56-58, where the colour of the internal surface of the reaction chamber is black.

60. Device according to any of the claims 51-59, where the means for detection of the target analyte are selected among surface acoustic wave (SAW) detectors, spectrophotometers, fiuorometers, CCD sensor chip(s), CCOS sensor chip(s), PMT detectors), or any suitable light detector,

61. Use of a device according to any of the claims 51-60 for the quantitative detection of the presence or absence of a target analyte in a sample.

62. Use according to claim 61, where the sample is derived from blood.

63. Use according to claim 62, where the sample is serum.

64. Use according to claim 62, where the sample is plasma.

65. Method for quantitative detecting the presence or absence of a target analyte in a sample consisting of less than 200 μl liquid, comprising the steps of: a) providing an analyte containing liquid sample consisting of less than 200μl liquid; b) supplying the liquid sample to a first reaction part of a chamber, the chamber comprising a first reaction part and a second detection part, the two parts being physi- cally separated such that sample material cannot enter into contact with the second detection part prior to step h) below; c) contacting the sample with an immobilisation matrix capable of capturing the analyte; d) immobilising the immobilisation matrix comprising the captured analyte; e) discarding the supernatant; f) washing the chamber with a washing solution; g) optionally, remobilising the immobilisation matrix comprising the captured analyte and repeating step d) to f); h) transferring the immobilisation matrix comprising the captured analyte to the second detection part of the chamber; and i) detecting the presence or absence of a target analyte using conventional detection means.

66 Method according to claim 65, where the first and second parts are separated by a collection chamber

67. Method according to any of the claims 65-66, where the flow of a liquid having been in contact with the second detection part of the chamber is unidirectional towards the first reaction part of the chamber.

68. Method according any of the claims 65-67, further comprising a step b') of contacting the analyte with a biological marker, capable of binding to the analyte

69 Method according to claim 68, where the biological marker is a antibody.

70 Method according any of the claims 68-69, where the step b') is performed after step b) and prior to step h), preferably prior to step T).

71. Method according to any of the claims 65-70, further comprising a step f ) of con- tacting the analyte with a substance capable of reacting with the biological marker. 72 Method according to any of the claims 65-71 , where the immobilisation matrix comprises magnetic material.

73 Method according to any of the claims 65-72, where the step h) is performed by moving a magnetic source along the external edge of the first reaction chamber toward the second detection chamber.

74 Method according to any of the claims 65-72, where the step h) is performed by moving a magnetic source along the external top edge of the first reaction chamber to- ward the second detection chamber.

75. Method according to any of the claims 72-74, where the magnetic material is selected from the group comprising magnetic particles, magnetic nanoparticles and superparamagnetic nanoparticles.

76. Method according to any of the claims 72-75, where the magnetic material has an at least bimodal size distribution.

77. Method according to claim 76, where the magnetic material has a trimodal size dis- tribution.

78. Kit of parts comprising a device according to any of the claims 51-60 and a magnetic material according to the any of the claims 72 and 75-77.

79. Apparatus comprising a device according to any of the claims 1-35 and a device according to any of the claims 51-60.

80. Apparatus according to claim 79 where the capillary channel according to any of the claims 1-35 is the first part of the reaction chamber according to any of the claims 51-60

81. A method for quantitative detecting the presence or absence of a target ana- lyte in a sample consisting of less than 200 μl suspension; said the method comprising separating the suspension according to the method of any of the claims 45-50, and de- tecting the presence or absence of the analyte according to the method of any of the claims 65-77,