METHOD AND A SYSTEM FOR QUANTITATIVE OR QUALITATIVE DETERMINATION OF A TARGET COMPONENT

Abstract

A method and a system for quantitative or qualitative determination of a target component in a liquid sample. The method includes i) providing a plurality of magnetic particles including capture sites for the target component on their respective surfaces; ii) providing a plurality of fluorophores configured to bind to the capture sites of the magnetic particles; iii) bringing the liquid sample into contact with the fluorophores and the magnetic particles in a flow channel of a micro fluidic device including a transparent window; and iv) at least temporally immobilizing the magnetic particles adjacent to the transparent window using a magnet, emitting exciting electromagnetic beam towards the immobilized magnetic particles, reading signals emitted from fluorophores and performing a quantitative or qualitative determination of the target component based on the read signal. A suitable micro fluidic device for use in the method/system and a kit for preparing a liquid sample.

Claims

1. A system for quantitative or qualitative determination of a target component in a liquid sample, the system comprises a micro fluidic device comprising at least one flow channel with a transparent window and an inlet for the liquid sample; a plurality of magnetic particles comprising capture sites for the target component on their respective surfaces; a plurality of fluorophores configured to bind to said capture sites of the magnetic particles or to said target component; a magnet arranged to at least temporally immobilize said magnetic particle adjacent to said transparent window; an emitter for exciting said fluorophores, and a reader for reading signals emitted from said fluorophores, wherein the micro fluidic device comprises a flexible wall section of the flow channel or of a sink section in fluid connection with the flow channel and the system comprises an actuator, which is adapted to move the flexible wall section for feeding a liquid sample into the flow channel.

2. The system of claim 1, wherein the micro fluidic device comprises polymer and comprises a substrate with a groove for the flow channel and a foil covering the flow channel.

3. The system of claim 1, wherein the inlet of the micro fluidic device is an inlet for suction in a liquid sample.

4. The system of claim 1, wherein the actuator is a step motor driven actuator.

5. The system of claim 1, wherein the inlet and the actuator are arranged such that the upon activation of the actuator, the flexible wall section will be moved and air will be pressed out of the flow channel where after the flexible wall will return to its initial position and a liquid sample will be sucked into the flow channel.

6. The system of claim 1, wherein the micro fluidic device comprises two or more flow channels, wherein the two or more flow channels have a common inlet.

7. The system of 1, wherein the micro fluidic device comprises an excitation and a read out zone provided in the form of the transparent window, the window is transparent for at least the exciting and emitting wavelengths of the fluorophores.

8. The system of claim 1, wherein the system comprises a temperature regulator for regulating the temperature of the liquid sample in the flow channel.

9. The system of claim 1, wherein the magnetic particles are coated magnetic particles comprising a coating comprising the captures sites.

10. The system of claim 1, wherein the fluorophores are configured to bind to said capture sites of the magnetic particles by being coupled to a component, which can bind to the capture sites of the magnetic particles.

11. The system of claim 10, wherein the component is identical to or homolog to said target component so that the component is also able to bind to said capture sites of the magnetic particles.

12. The system of claim 1, wherein the fluorophores comprising at least one capture site for the target component to provide that the target component may bind to at least one of the fluorophores.

13. The system of claim 1, wherein said fluorophores and said magnetic particles are temporally immobilized in said flow channel of the micro fluidic device such that they cannot bind to each other prior to feeding of a liquid sample to said flow channel.

14. The system of claim 1, wherein said magnet is adapted to at least temporally immobilize said magnetic particle adjacent to said transparent wall section for a sufficient time to excite at least a part of possible fluorophores captured by said capture sites of the magnetic particles by the emitter and to read out any emitted signal from such excited captured fluorophores.

15. The system of claim 1, wherein said emitter comprises at least one optical fiber with an output end for emitting said electromagnetic radiation and said reader comprises at least one optical fiber with an input end for receiving said signals emitted from fluorophores captured by magnetic particles, said optical fiber of the emitter and said optical fiber of the reader are arranged adjacent to each other in at least respective length sections adjacent to respectively the output end and the input end.

16. The system of claim 14, wherein said emitter comprises a plurality of optical fibers, each fiber having an output end for emitting said electromagnetic radiation and said reader comprises a plurality of optical fibers, each fiber having an input end for receiving said signals emitted from fluorophores captured by magnetic particles, said optical fibers of the emitter and said optical fibers of the reader are arranged adjacent to each other in at least respective length sections adjacent to respectively the output end and the input.

17. A micro fluidic device for use in preparing a liquid sample for optical analysis for quantitative or qualitative determination of a target component in a sample, the micro fluidic device comprising at least one flow channel with a transparent window and an inlet for the liquid sample, the micro fluidic device further comprises in its flow channel a plurality of magnetic particles comprising capture sites for the target component on their surfaces; and a plurality of fluorophores configured to bind the capture sites of the magnetic particles or to said target component wherein said fluorophores and said magnetic particles are temporally immobilized in said flow channel of the micro fluidic device such that they cannot bind to each other prior to feeding of a liquid sample to said flow channel and wherein the micro fluidic device comprises a flexible wall section of the flow channel or of a sink section in fluid connection with the flow channel, said flexible wall section being capable of being moved by an actuator for feeding a liquid sample into the flow channel.

18. A micro fluidic device of claim 17, wherein the micro fluidic device comprising a substrate with a groove for the flow channel and a foil covering the flow channel, wherein the flexible wall section is movable to provide that air will be pressed out of the flow channel where after the flexible wall will return to its initial position.

19. A micro fluidic device for use in quantitative or qualitative determination of target component in a sample, the micro fluidic device comprising at least one flow channel with a transparent window and an inlet for the liquid sample, wherein the micro fluidic device comprises a flexible wall section of the flow channel or of a sink section in fluid connection with the flow channel, said flexible wall section being capable of being moved by an actuator for feeding a liquid sample into the flow channel, wherein the micro fluidic device comprising a substrate with a groove for the flow channel and a foil covering the flow channel, wherein the flexible wall section provides said flexible wall section and is movable to provide that air will be pressed out of the flow channel where after the flexible wall will return to its initial position.

Description

BRIEF DESCRIPTION OF DRAWINGS AND EXAMPLES

[0249] The invention will be explained more fully below in connection with examples and preferred embodiments and with reference to the drawings in which:

[0250] FIG. 1a is a schematic top view of a micro titer plate suitable for performing the method of the invention.

[0251] FIG. 1b is a schematic cross sectional view seen in the line A-A of FIG. 1.

[0252] FIG. 2 is a schematic top view of a micro fluidic device suitable for performing the method of the invention.

[0253] FIG. 3 is a schematic sectional side view seen in the line B-B of FIG. 2.

[0254] FIG. 4 is a schematic top view of micro fluidic device suitable for performing the method of the invention and with temporally immobilized magnetic particles and temporally immobilized fluorophores.

[0255] FIG. 5 is a schematic illustration of the system of the invention comprising a micro fluidic device, an emitter and a reader.

[0256] FIG. 6 is a schematic illustration of a fluorophore in the form of a quantum dot suitable for use in the invention.

[0257] FIGS. 7a, 7b and 7c are schematic illustrations of a performance of the method of the invention.

[0258] FIGS. 8a, 8b and 8c are schematic illustrations of another performance of the method of the invention.

[0259] The figures are schematic and may be simplified for clarity. Throughout, the same reference numerals are used for identical or corresponding parts.

[0260] FIGS. 1a and 1b show a test plate suitable for being applied in the present invention. The shown test plate is a micro titer plate with 128 wells 1.

[0261] FIG. 9 is a schematic side view of an emitter-reader assembly.

[0262] Micro titer plates are well known in the art under many names, such as well plates and micro plates. A micro titer plate is a generally flat plate with multiple wells used as small test tubes. The shown micro titer plate comprises a thin cover film 2, which is peeled of prior to use of the titer plate. The cover film 2 can be divided into sections, such that it can be peeled off in sections, e.g. such that only one or only a number less than all wells are uncovered by removal of a section of the cover film 2. The micro titer plate has an edge 4 for reducing spill.

[0263] Each well 1 of a micro plate typically holds somewhere between tens of nanolitres to several millilitres of liquid. Wells of a suitable micro titer plate can in principle have any shape, such as circular or square, and their respective bottom parts can be rounded or plane. In the shown micro titer plate, the wells 1 are round and with plane bottom parts 3. The round bottom parts 2 of the respective wells 1 constitute the transparent window usable for exciting and reading out. In use the fluorophores and the magnetic particles can be pre-arranged in the wells e.g. in dry form and e.g. in temporally immobilized form. Alternatively the fluorophores and the magnetic particles can be added to the well immediately before, simultaneously with or after adding the liquid sample. After a selected incubating time e. g. on a shaking board, the micro titer plate is placed on a magnet for temporally immobilizing the magnetic particles adjacent to the transparent window, namely at the bottom part 3. An emitter is arranged to emitting exciting electromagnetic beam towards the immobilized magnetic particles, and a reader is arranged to read signals emitted from fluorophores captured by the immobilized magnetic particles. The read signals are used to perform a quantitative or qualitative determination of the target component. For reducing noise, the liquid can be removed from the respective wells, and optionally the wells are washed e.g. with water prior to reading out signals. The incubating time is usually very short e.g. a few minutes.

[0264] FIGS. 2 and 3 show a test plate suitable for being applied in the present invention. The shown test plate is a micro fluidic device. Although any micro fluidic devices in principle could be applied in the present invention, the micro fluidic device shown is particularly designed for the purpose and provides additional benefits to the present invention as described herein.

[0265] The micro fluidic device comprises a substrate 12 with three flow channels 11. The channels 11 are provided in the form of grooves covered with a foil 11a. Each channel 11 comprises an inlet 13 and the channels 11 is in fluid connection with a common sink 14.

[0266] The inlet 13 is in the form of a well-shaped inlet.

[0267] The common sink 14 of the micro fluidic device comprises a flexible wall section 15. The flexible wall section 15 can be moved e.g. using a not shown actuator as described above.

[0268] By pressing the flexible wall section 15 it will be moved and air will be pressed out of the channels 11 where after the flexible wall section 15 will return to its initial position and a liquid sample arranged in the inlet will be sucked into the channel to a desired position. By further manipulating the flexible wall section the liquid sample can be drawn further into the channels 11 or it can be pulsated in the channels. Finally the flexible wall section 15 can be manipulated to collect the sample in the sink and to reflush the sample into the channels, if desired. The flexible wall section 15 thereby provides a simple and cheap method of controlling the liquid sample in the micro fluidic device.

[0269] The micro fluidic device also comprises an indent which provides a read out section 16 for the channels 11. In the read out sections 16 of the channels 11, the channels comprise a transparent window and the magnetic particles can be temporally immobilized using a not shown magnet.

[0270] FIG. 4 shows another preferred micro fluidic device suitable for use in the invention.

[0271] The micro fluidic device comprises a substrate 22 with five flow channels 21. Each channel 21 comprises an inlet 23 and is in fluid connection with a sink 24 with a not shown flexible wall section.

[0272] The micro fluidic device also comprises an indent which provides a read out section 26 for the channels 21, where the channels comprise a transparent window and the magnetic particles can be temporally immobilized using a not shown magnet.

[0273] Each channel 21 comprises temporally immobilized magnetic particles and temporally immobilized fluorophores. The micro fluidic device is divided into zones comprising zone 0 which is the inlet zone, zone 1 and zone 2 which comprise temporally immobilized fluorophores and magnetic particles 17 arranged such that they do not react until they are in contact with the liquid sample, zone 3 which is the read out zone and zone 4 which is the sink zone.

[0274] In an embodiment zone 1 comprises temporally immobilized fluorophores and zone 2 comprises temporally immobilized magnetic particles.

[0275] In an embodiment zone 1 comprises temporally immobilized magnetic particles and zone 2 comprises temporally immobilized fluorophores.

[0276] The micro fluidic device could comprise several subzones of zone 1 and zone 2, if desired.

[0277] In use the liquid sample is fed to the inlet 23, the sample is sucked into zone 1 of the channels using the flexible wall section. Optionally the liquid sample is pulsated in zone 1 to dissolve or resuspend the immobilized elements 17 in zone 1. Thereafter the liquid sample is drawn further into the channels 21 to zone 2 for dissolving or resuspending the immobilized elements 17 in zone 2. After a preselected incubation time the liquid sample is drawn fully into the sinks 24. The magnetic particles are immobilized in the read out zone 3. If desired, the liquid sample can be reintroduced into the channels 21 by using the flexible wall of the sinks 24 and the immobilized magnetic particles can be flushed using the liquid sample to remove not immobilized fluorophores and other elements that could potentially provide noise.

[0278] FIG. 5 shows a system of the invention comprising a support element 32 supporting a micro fluidic device 31, an emitter 38 and a reader 39 coupled to a computer 34. The micro fluidic device comprises a read out section 36. The support element 32 comprises a temperature control element 35 for maintaining the liquid sample at a desired temperature during the test. The support element 32 further comprises a magnet 33. The micro fluidic device is arranged such that the magnet is located adjacent the read out section 36 to thereby temporally immobilize the magnetic particles in the read out section 36. The emitter 38 is configured to emit electromagnetic radiation directed at the read out section 36 to thereby excite fluorophores on the immobilized magnetic particles. The reader 39 is configured to read signals emitted from fluorophores captures by the immobilized magnetic particles and the read signals are transmitted to the computer 34 for processing to quantitative and/or qualitative determination of target compound(s).

[0279] FIG. 6 shows a fluorophore in the form of a quantum dot suitable for use in the invention. The quantum dot comprises a core 41 of a binary semiconductor alloy covered by a transparent shell 42 which is at least transparent for the wavelength emitted by the core. The shell 42 is further covered by an organic coating 43, such as a polymer coating which is coupled to one or more not shown components which can bind to the capture sites of the magnetic particles e.g. such as described above.

[0280] FIGS. 7a, 7b and 7c show a performance of the method of the invention in three steps. Step 1 is illustrated in FIG. 7a. Sample with the target component 51 is mixed with fluorophores 52 coupled to homologue target component 53. The relative amount of target component 51 to fluorophores 52 coupled to homologue target component 53 is relatively low. Step 2 is illustrated in FIG. 7b. The mixture of target component 51 and fluorophores 52 coupled to homologue target component 53 is further mixed with magnetic particles 54 carrying capture sites 55 for the target component 51 and the homologue target component 53. Step 3 is illustrated in FIG. 7c. Target component 51 and the homologue target component 53 are captured by the capture sites 55 carried by the magnetic particles 54. In the illustration shown, only the homologue target component 53 is captured by the capture sites 55. This is shown to illustrate that the amount of captured homologue target component 53 is relatively high and accordingly the amount of immobilized fluorophores 52 is relatively high. When the magnetic particles 54 are immobilized using a magnet arranged adjacent to the transparent window, and the fluorophores 52 are excited, the emitted signal from the fluorophores 52 is relatively high, and the amount of target component 51 can be determined.

[0281] FIGS. 8a, 8b and 8c show another performance of the method of the invention in three steps. Step 1 is illustrated in FIG. 8a. Sample with the target component 61 is mixed with fluorophores 62 coupled to homologue target component 63. The relative amount of target component 61 to fluorophores 62 coupled to homologue target component 63 is relatively high. Step 2 is illustrated in FIG. 8b. The mixture of target component 61 and fluorophores 62 coupled to homologue target component 63 is further mixed with magnetic particles 64 carrying capture sites 65 for the target component 61 and the homologue target component 63. Step 3 is illustrated in FIG. 8c. Target component 61 and the homologue target component 63 are captured by the capture sites 65 carried by the magnetic particles 64. In the illustration only the target component 61 captured by the capture sites 65 is shown to illustrate that the amount of captured target component 61 is relatively high and accordingly the amount of immobilized fluorophores 62 is relatively low or there may be none at all and when the magnetic particles 64 are immobilized using a magnet adjacent to the transparent window and the fluorophores 62 have been excited, the emitted signal from the fluorophores 62 is relatively low or absent, and the amount of target component 61 can be determined.

[0282] The emitter-reader assembly shown in FIG. 9 is comprises a casing 90 comprising a plurality of not shown diodes with respective center wavelengths for exciting the respective wavelengths of the fluorophores. The emitter-reader assembly further comprises an emitter fiber bundle 91 comprising a plurality of optical fibers in light connection with the respective diodes for guiding the light towards not shown fluorophores bound to temporally immobilized magnetic particles in a micro fluidic device. The emitter fiber bundle 91 has a length section 92 adjacent to emitter output ends 93 of the optical fibers from where the light 99 is emitted.

[0283] In the length section 92 the emitter bundle 91 is merged with a reader fiber bundle 96 such that the length section is a common emitter-reader length section 92. The common emitter-reader length section 92 is held together by a sleeve 94. The reader fiber bundle 96 comprises a plurality of optical fibers having reader input ends 95 arranged to receive the light signal 99 from the fluorophores. The reader fiber bundle 96 is fixed to a connector 97 where it is connected to a not shown reading unite.g. a spectroscope, via a waveguide 98 e.g. in form of another fiber bundle.

[0284] The emitter output ends 93 and the reader input ends 95 are advantageously arranged in a predetermined pattern. The predetermined pattern is advantageously selected such as to obtain high exciting rate and high reading rate. The emitter output ends 93 and the reader input ends 95 are advantageously positioned immediately adjacent to the transparent window, e.g. where the magnet was arranged when immobilizing the magnetic particles and/or immediately adjacent to the magnet.

EXAMPLES

Example 1

[0285] Screening Tests

[0286] Milk samples are screened for the target analyte Ampecillin.

[0287] A system as shown in FIG. 5 is used. The micro fluidic device is in the form of a cartridge similar to the micro fluidic device of FIG. 4, but with the difference that the 5 flow channels each have their respective inlet with an inlet-well. The magnet applied is a permanent magnet arranged to immobilize magnetic particles in the reading zone.

[0288] The channels are in fluid connection to sink sections 4 and have together with the sink section 5 zones, an inlet zone 0, a zone with temporally immobilized magnetic particles 1, a zone with temporally immobilized fluorophores 2, a reading zone with a transparent window 3 and a zone with flexible wall and sink sections 4.

[0289] By having 5 separate flow channels with separate inlets it is possible to screen 5 different samples simultaneously.

[0290] The temporally immobilized magnetic particles are 1.5 m Biomag Protein G magnetic particles from Qiagen with Ampicillin antibody loaded onto Protein G. 1 L of 0.4% by weight of the magnetic particles solution in buffer is deposited in the channel (zone 1) and dried down.

[0291] The temporally immobilized fluorophores are Qdot 655 Biotin Conjugate from Invitrogen loaded with Ampicillin. 1 L of 15 nM buffer solution of the Qdot 655 is deposited in the channel (zone 2) and dried down.

[0292] As an internal reference signal Bio-Adembeads Streptavidin magnetic beads from Ademtech are labeled with Qdot 605 biotin conjugate from Invitrogen. The Bio-Adembeads Streptavidin magnetic beads are deposited in the fluorophores zone (zone 2).

[0293] The tests are performed as follows:

[0294] 5 different milk samples are loaded in the 5 inlet-wells on the cartridge. Each sample is drawn into the respective channel of the cartridge and re-suspends the magnetic particle in zone 1. Incubation is done by cycling the flow for 20 seconds over the site comprising the immobilized magnetic particles to re-suspend these and allow the magnetic particles to catch target analytes in the exposed sample volume. The sample is then drawn further into the channels of the cartridge to zone 2 and re-suspends the Qdots. Again incubation is done by cycling the flow for 20 seconds. Finally the sample is drawn into the sink section 4 whereby the magnetic particles approaching the magnet while the sample is passing are immobilized in the reading zone.

[0295] The magnetic particles are subjected to exciting wavelength(s) and the emitted signal is recorded.

[0296] The signals recorded at 655 nm can be normalized with the signal recorded at 605 nm. The resulting signal will show whether the respective sample comprises the target analyte.

Example 2

[0297] Quantitative Determination of One Target Analyte

[0298] Mouse serum is tested for Mouse IgG. The samples are prepared by dilution of the Mouse serum in buffer.

[0299] A system as shown in FIG. 5 is used. The micro fluidic device is in the form of a cartridge similar to the micro fluidic device of FIG. 4 but with the difference that the micro fluidic device comprises 2 flow channels with a common inlet with an inlet-well and the micro fluidic device comprises a common sink section in fluid connection with the flow channels. The micro fluidic device further comprises a flexible wall section which is common for the flow channels. In this example it is important that the flow channels and the deposition in the flow channels are essentially identical.

[0300] The magnet applied is a permanent magnet arranged to immobilize magnetic particles in the reading zone.

[0301] The channels in flow connection with the sink sections 4 have 5 zones, a common inlet zone 0, a zone with temporally immobilized fluorophores 1, a zone with temporally immobilized magnetic particles 2, a reading zone with a transparent window 3 and a common zone with flexible wall and sink section 4. It should be observed that the magnetic particles zone and the fluorophores zone in this example are reversed compared to the order thereof in example 1.

[0302] By having 5 separate flow channels with separate inlet it is possible to screen 5 different samples simultaneously.

[0303] The temporally immobilized magnetic particles are 1.5 m Biomag Protein G magnetic particles from Qiagen with mouse IgG loaded onto Protein G. 1 L of 0.4% by weight of the magnetic particles solution in buffer is deposited in the channel (zone 2) and dried down.

[0304] The temporally immobilized fluorophores are Qdot 655 Goat F(ab)2 anti-Mouse IgG Conjugate (H+L) from Invitrogen. 1 L of 15 nM buffer solution of the Qdot 655 is deposited in the channel (zone 1) and dried down.

[0305] Additionally a surfactant in the form of a detergent is applied in the sink section.

[0306] The tests are performed as follows:

[0307] Sample is applied in the well and drawn into the channels of the cartridge and re-suspends Qdots in zone 1. Incubation is done by cycling the flow for 20 seconds over the site for the immobilized Qdots to re-suspend these. The sample is then drawn further into the channels of the cartridge and re-suspends the immobilized magnetic particles in zone 2 and simultaneously the magnetic particles will catch analytes and Qdots. The analytes and Qdots will compete about the capture sites of the magnetic particles. Again incubation is done by cycling the flow for 20 seconds. Finally the sample is drawn into the sink section whereby the magnetic particles approaching the magnet while the sample is passing are immobilized in the reading zone. In the sink section the dried down detergent is dissolved and thereby the surface tension of the sample is lowered. To reduce background noise, the sample is finally pushed back into the channels where it is flushing the reading zone of non-immobilized sample but leaving the magnetic particles with the signal at the reading site. The detergent improves the flushing of the fluidic system.

[0308] The magnetic particles are subjected to exciting wavelength(s) and the emitted signal is recorded.

[0309] By comparing the obtained signals by a reference schedule as described above, e.g. a calibration curve, the quantitative determination can be obtained.

Example 3

[0310] Quantitative Determination of Two Target Analytes

[0311] Milk sample tested for the target analyte Ampecillin and the target analyte Tetracyclin.

[0312] A system as shown in FIG. 5 is used. The micro fluidic device is in the form of a cartridge similar to the micro fluidic device of FIG. 4 but with the difference that the micro fluidic device comprises 2 flow channels with a common inlet with an inlet-well, a common flexible wall section and in fluid connection with a common sink section. In this example it is desired that the flow channels and the deposition in the flow channels are essentially identical for improved precision.

[0313] The magnet applied is a permanent magnet arranged to immobilize magnetic particles in the reading zone.

[0314] The channels in fluid connection with the sink section 4 have 5 zones, a common inlet zone 0, a zone with temporally immobilized magnetic particles 1, a zone with temporally immobilized fluorophores 2, a reading zone with a transparent window 3 and a common zone with flexible wall and sink 4.

[0315] The temporally immobilized magnetic particles are 1.5 m Biomag Protein G magnetic particles from Qiagen with Ampicillin antibody loaded onto Protein G and 1.5 m Biomag Protein G magnetic particles from Qiagen with Tetracyclin antibody loaded onto Protein G. 1 L of 0.2% by weight of each of the magnetic particles solution in buffer is deposited in the channel (zone 1) and dried down.

[0316] The temporally immobilized fluorophores are Qdot 655 Biotin Conjugate from Invitrogen loaded with Ampicillin and Qdot 605 Biotin Conjugate from Invitrogen loaded with Tetracyclin. 1 L 7.5 nM buffer solutions of both Qdots are deposited in the channel (zone 2) and dried down.

[0317] The tests are performed as follows:

[0318] Sample is applied in the well and drawn into channels of the cartridge and re-suspends magnetic beads in zone 1. Incubation is done by cycling the flow for 20 seconds over the site for the immobilized magnetic particles to re-suspend these and allow the magnetic particles to catch target analytes in the exposed sample volume. The sample is then drawn further into the channels of the cartridge and re-suspends the Qdots in zone 2. Again incubation is done by cycling the flow for 20 seconds. Finally the sample is drawn into the sink section whereby the magnetic particles approaching the magnet while the sample is passing are immobilized in the reading zone. The magnetic particles are subjected to exciting wavelength(s) and the emitted signal is recorded.

[0319] The recorded signal at 655 nm is related to the content of Ampicillin in the sample. The recorded signal at 605 nm is related to the content of Tetracyclin in the sample.

Example 4

[0320] Quantitative Determination of One Target Analyte in Whole Blood

[0321] Whole blood is tested for CRP. The sample is undiluted.

[0322] A system as shown in FIG. 5 is used. The micro fluidic device is in the form of a cartridge similar to the micro fluidic device of FIG. 4 but with the difference that the micro fluidic device comprises 2 flow channels with a common inlet with an inlet-well and with a common flexible wall section and in fluid communication with a common sink section. In this example it is important that the flow channels and the deposition in the flow channels are essentially identical.

[0323] The magnet applied is a permanent magnet arranged to immobilize magnetic particles in the reading zone.

[0324] The channels in fluid connection with a sink section 4 have 5 zones, a common inlet zone 0, a zone with temporally immobilized fluorophores 1, a zone with temporally immobilized magnetic particles 2, a reading zone with a transparent window 3 and a common zone with flexible wall and sink section 4. By having 5 separate flow channels with separate inlets it is possible to screen 5 different samples simultaneously.

[0325] The temporally immobilized magnetic particles are 1.5 m Biomag Protein G magnetic particles from Qiagen with CPR loaded onto Protein G. 1 L of 0.4% by weight of the magnetic particles solution in buffer is deposited in the channel (zone 2) and dried down. The temporally immobilized fluorophores are Qdot 655 Biotin Conjugate from Invitrogen loaded with CRP antibody. 1 L of 15 nM buffer solution of the Qdot 655 is deposited in the channel (zone 1) and dried down.

[0326] The tests are performed as follows:

[0327] Sample is applied in the well and is drawn into cartridge and re-suspends Qdots in zone 1. Incubation is done by cycling the flow for 40 seconds over the site for the immobilized Qdots. The sample is then drawn further into channels of the cartridge and re-suspends the immobilized magnetic beads and simultaneously the magnetic particles will catch analytes and Qdots. The analytes and Qdots will compete about the capture sites of the magnetic particles. Again incubation is done by cycling the flow for 40 seconds. Finally, the sample is drawn into the sink section whereby the magnetic particles approaching the magnet while the sample is passing are immobilized in the reading zone.

[0328] The magnetic particles are subjected to exciting wavelength(s) and the emitted signal is recorded.

[0329] By comparing the obtained signals by a reference schedule as described above, e.g. a calibration curve, the quantitative determination can be obtained.

Example 5

[0330] Example 1 is repeated using an extract of crushed beef diluted with water. Samples with different degree of dilution are applied.

Example 6

[0331] Example 2 is repeated with the difference that the sample is mixed with the magnetic particles and the fluorophores before applying the sample to the well and drawing it into the channels of the cartridge.

[0332] The mouse serum is diluted in a buffer and mixed with magnetic particle solution and q-dot solution in a vial and is incubated for 5 minutes prior to application in the well and introduction into the channels.

[0333] The sample can immediately be drawn into the sink section whereby the magnetic particles approaching the magnet while the sample is passing are immobilized in the reading zone.

Example 7

[0334] Example 2 is repeated with the difference that the system is flushed with the sample by pushing the sample from the sink section into the channels to flush the reading zone from non-immobilized sample but leaving the magnetic particles with the signal at the reading site.

[0335] When the system has been flushed, a read-out module is positioned above one channel. The Qdots are excited using a 420 LED and the emitted spectrum is recorded. An algorithm running on a PC finds and records the peak light intensity at 655 nm and 605 nm. The read-out module is then positioned above the next channel.

[0336] Some preferred embodiments have been shown in the foregoing, but it should be stressed that the invention is not limited to these, but may be embodied in other ways within the subject-matter defined in the following claims.

EMBODIMENTS

[0337] 1. A method for quantitative or qualitative determination of a target component in a liquid sample, the method comprises

[0338] providing a plurality of magnetic particles comprising one or more capture sites for the target component on their respective surfaces;

[0339] providing a plurality of fluorophores configured to bind to said capture sites of the magnetic particles;

[0340] bringing the liquid sample, said fluorophores and said magnetic particles into a flow channel of a micro fluidic device comprising a transparent window into the flow channel; and

[0341] at least temporally immobilizing said magnetic particles adjacent to said transparent window using a magnet, emitting exciting electromagnetic beam towards said immobilized magnetic particles, reading signals emitted from fluorophores captured by said immobilized magnetic particles and performing a quantitative or qualitative determination of said target component based on the read signal.

[0342] 2. The method of embodiment 1, wherein the liquid sample comprises a biological fluid or a fraction of a biological fluid, such as human or animal or vegetable fluids for example blood, saliva, urine, milk, cytosol (intracellular fluid), interstitial fluid (tissue fluid) and/or one or more fractions and/or mixtures thereof, and/or suspended biological solids, such as tissue or solid food e.g. meat or vegetable.

[0343] 3. The method of embodiment 1 or embodiment 2, wherein the target component comprises a biomolecule, such as a single organic molecule or a structure of organic molecules.

[0344] 4. The method of any of the preceding embodiments, wherein the target component comprises a microorganism such as at least one of bacterial, viral, or fungal pathogens, e.g. E-coli E. coli, Citrobacter spp, Aeromonas spp., Pasteurella spp., non-serogroup DI Salmonella, Camphylobacter Staphylococcus spp. and combinations thereof.

[0345] 5. The method of any of the preceding embodiments, wherein the target component comprises a cell, such as a blood cell, a stem cell or a tumor cell.

[0346] 6. The method of any of the preceding embodiments, wherein the target component comprises proteins, nucleotides, carbohydrates, or lipids, in particular an enzyme, an antigen or an antibody.

[0347] 7. The method of any of the preceding embodiments, wherein capture sites are specific for said target component.

[0348] 8. The method of any of the preceding embodiments, wherein capture sites are specific for a group of components comprising one or more target components.

[0349] 9. The method of any of the preceding embodiments, wherein the magnetic particles are coated magnetic particles comprising a coating comprising the captures sites, wherein the capture sites are selected to be capture sites for the target component, the coating for example comprises the capture sites in the form of antigen, antibody, avidine, biotin or Goat anti-Mouse IgG.

[0350] 10. The method of any of the preceding embodiments, wherein the fluorophores are quantum dots or aromatic probes and/or conjugated probes, such as fluorescein, derivatives of benzene and metal-chalcogenide fluorophores.

[0351] 11. The method of any of the preceding embodiments, wherein the fluorophores are configured to bind to said capture sites of the magnetic particles by being coupled to a component which can bind to the capture sites of the magnetic particles, the component is preferably identical or homolog to said target component.

[0352] 12. The method of any of the preceding embodiments, wherein the fluorophores are quantum dots that emit one or more discrete frequencies of light when stimulated by a light source.

[0353] 13. The method of embodiment 12, wherein each quantum dot comprises a core of an excitable material, such as a semiconductor nanoparticle or a rare earth doped oxide colloidal nanoparticle.

[0354] 14. The method of any of the preceding embodiments 12-13, wherein the quantum dots each comprise a core with a size of up to about 25 nm, such as from 2-10 nm, the quantum dots preferably comprise an organic coating, such as a polymer coating, the coating is coupled to a component which can bind to the capture sites of the magnetic particles.

[0355] 15. The method of any of the preceding embodiments 12-14, wherein the quantum dots each comprise a core of a binary semiconductor alloy, such as cadmium-selenide, cadmium-sulphide, indium-arsenide or indium-phosphide, covered with a transparent shell optionally comprising or consisting of Zinc sulphide.

[0356] 16. The method of any of the preceding embodiments, wherein the liquid sample is brought into contact with one or more of said fluorophores and one or more of said magnetic particles outside the flow channel of the micro fluidic device and thereafter the liquid sample is fed to the flow channel of the micro fluidic device, preferably the liquid sample is brought into contact with one or more of said fluorophores and one or more of said magnetic particles outside the flow channel of the micro fluidic device and thereafter the liquid sample is fed to the flow channel of the micro fluidic device.

[0357] 17. The method of any of the preceding embodiments, wherein the liquid sample is brought into contact with one or more of said fluorophores and said magnetic particles in the flow channel of the micro fluidic device.

[0358] 18. The method of embodiment 15, wherein said fluorophores and said magnetic particles are arranged in said flow channel of the micro fluidic device, the method comprises feeding said liquid sample into said flow channel, said fluorophores and said magnetic particles are preferably arranged in said flow channel at a distance from each other.

[0359] 19. The method of embodiment 18, wherein said fluorophores and said magnetic particles are temporally immobilized in said flow channel of the micro fluidic device such that they cannot bind to each other prior to the feeding of said liquid sample to said flow channel.

[0360] 20. The method of any of the preceding embodiments, wherein the liquid sample is fed into the flow channel of the micro fluidic device by being sucked into the flow channel, the suction is provided by an actuator.

[0361] 21. The method of embodiment 20, wherein the actuator is arranged to move a flexible wall section of the flow channel or a sink section in fluid connection with the flow channel to provide a suction to suck the liquid sample into the flow channel, the flow channel preferably being a flow channel comprising a first feeding end and a second actuator end comprising the flexible wall section.

[0362] 22. The method of any of the preceding embodiments, wherein the method comprises allowing the capture sites of the magnetic particles to capture possible target component in the liquid sample and/or fluorophores, the method preferably comprises pulsating said liquid sample in said flow channel optionally using an actuator, where after the magnetic particles are at least temporally immobilized adjacent to said transparent window using a magnet.

[0363] 23. The method of any of the preceding embodiments, wherein said magnet has a magnetic field sufficiently strong to at least temporally immobilize said magnetic particles adjacent to said transparent window.

[0364] 24. The method of any of the preceding embodiments, wherein said at least temporally immobilized magnetic particles are subjected to said electromagnetic beam such that at least a part of possible fluorophores captured by said capture sites of the magnetic particles are excited, where after the emitted signal from any captured fluorophores is read and a quantitative or qualitative determination of said target component based on the read signal is performed.

[0365] 25. The method of any of the preceding embodiments wherein said at least temporally immobilized magnetic particles are released from magnetic forces applied by the magnet prior to being subjected to said electromagnetic beam.

[0366] 26. The method of any of the preceding embodiments, wherein said plurality of fluorophores is substantially identical.

[0367] 27. The method of any of the preceding embodiments 1-25, wherein said plurality of fluorophores comprise two or more groups of fluorophores, wherein the two or more groups of fluorophores differ from each other with respect to type, size, coating, shape and/or amount.

[0368] 28. The method of any of the preceding embodiments, wherein said magnetic particles are substantially identical with respect to capture sites and optionally with respect to number of capture sites and/or size.

[0369] 29. The method of any of the preceding embodiments 1-28, wherein said plurality of magnetic particles comprise two or more groups of magnetic particles, wherein said two or more groups of magnetic particles differ from each other with respect to e.g. with respect to size, capture site, number and/or type(s).

[0370] 30. The method of any of the preceding embodiments, wherein said method comprises quantitative or qualitative determination of two or more target components in a liquid sample, the magnetic particle comprises one or more types of capture sites for the two or more target components, the capture sites for one target component preferably differ from the capture sites for another target component.

[0371] 31. The method of embodiment 30, wherein the plurality of fluorophores comprise at least one group of fluorophores configured to bind to capture sites for one target component and at least another group of fluorophores configured to bind to the capture sites for another target component.

[0372] 32. The method of any of the preceding embodiments, wherein said method comprises performing two or more parallel assays on the liquid sample for quantitative or qualitative determination of the target component(s), each assay comprises: [0373] bringing a part of the liquid sample into contact with said fluorophores and said magnetic particles in a micro fluidic device comprising a transparent window; and [0374] at least temporally immobilizing said magnetic particles adjacent to said transparent window using a magnet, emitting exciting electromagnetic beam towards said immobilized magnetic particles, and reading signals emitted from fluorophores captured by said immobilized magnetic particles.

[0375] 33. The method of embodiment 32, wherein the fluorophores used in one of the two or more parallel assays differ from the fluorophores used in another one of the two or more parallel assays, preferably the fluorophores used in one of the two or more parallel assays differ from the fluorophores used in another one of the two or more parallel assays with respect to type, size, coating, shape and/or amount.

[0376] 34. The method of embodiment 32 or embodiment 33, wherein the magnetic particles used in one of the two or more parallel assays differ from the magnetic particles used in another one of the two or more parallel assays, preferably the magnetic particles used in one of the two or more parallel assays differ from the magnetic particles used in another one of the two or more parallel assays with respect to type, size, coating, shape and/or amount.

[0377] 35. The method of any of the preceding embodiments, wherein said two or more parallel assays are performed simultaneously in the same micro fluidic device, the two or more parallel assays are performed in respective flow channels, such as in parallel flow channels of the same micro fluidic device.

[0378] 36. The method of any of the preceding embodiments, wherein the quantitative or qualitative determination of target component(s) in a liquid sample is performed by comparing the read signal(s) with a reference schedule.

[0379] 37. The method of any of the preceding embodiments, wherein the quantitative or qualitative determination of target component(s) in a liquid sample is performed by comparing the read signal(s) with signals obtained from liquid samples of known composition preferably using an artificial intelligent processor.

[0380] 38. The method of any of the preceding embodiments, wherein the quantitative or qualitative determination of target component(s) in a liquid sample is performed by multiplexing the read signal(s) from different groups of fluorophores e.g. from the same assay, from fluorophores from parallel assays and/or from fluorophores in reference tests of known or unknown liquid samples.

[0381] 39. A system for quantitative or qualitative determination of a target component in a liquid sample, the system comprises [0382] a micro fluidic device comprising at least one flow channel with a transparent window and an inlet for the liquid sample; [0383] a plurality of magnetic particles comprising one or more capture sites for the target component on their respective surfaces; [0384] a plurality of fluorophores configured to bind to said capture sites of the magnetic particles; [0385] a magnet arranged to at least temporally immobilize said magnetic particle adjacent to said transparent window; [0386] an emitter for exciting said fluorophores, and [0387] a reader for reading signals emitted from said fluorophores.

[0388] 40. The system of embodiment 39, wherein the micro fluidic device is of polymer and or glass.

[0389] 41. The system of embodiment 40, wherein the micro fluidic device comprises a substrate with a groove for the flow channel and a foil covering the flow channel.

[0390] 42. The system of embodiment 41, wherein the micro fluidic device comprises an inlet to the flow channel, the inlet is preferably an opening for suction, a capillary inlet or a membrane covered inlet.

[0391] 43. The system of any of embodiments 41-42, wherein the micro fluidic device comprises an inlet for suction in the liquid sample.

[0392] 44. The system of any of embodiments 41-43, wherein the micro fluidic device comprises a flexible wall section of the flow channel or a sink section in fluid connection with the flow channel, the system preferably comprises an actuator, preferably the actuator is arranged to move the flexible wall section, the actuator optionally being a step motor driven actuator.

[0393] 45. The system of embodiment 44, wherein the inlet and the actuator are arranged such that the upon activation of the actuator the flexible wall section will be moved and air will be pressed out of the flow channel where after the flexible wall will return to its initial position and the liquid sample will be sucked into the flow channel.

[0394] 46. The system of any of embodiments 41-45, the flow channel being in fluid communication with a sink section of the micro fluidic device.

[0395] 47. The system of any of embodiments 41-46, wherein the micro fluidic device comprises two or more flow channels optionally for performing parallel test, the two or more flow channels optionally have a common inlet.

[0396] 48. The system of any of embodiments 39-47, wherein the micro fluidic device comprises an excitation and read out zone referred to as a reading zone in the form of the transparent window, the window is transparent for at least the exciting and emitting wavelengths of the fluorophores.

[0397] 49. The system of embodiment 48, wherein the micro fluidic device is a micro fluidic device and the micro fluidic device comprises an excitation and read out section with a length dimension, preferably of at least about 1 mm, such as at least about 3 mm, such as at least about 5 mm.

[0398] 50. The system of any of embodiments 39-49, wherein the micro fluidic device is of a transparent material.

[0399] 51. The system of any of embodiments 39-50, wherein the system comprises a temperature regulator for regulating the temperature of the liquid sample in the flow channel, the temperature regulator optionally comprises a peltier element, a thin film heating element and/or other resistive heating elements.

[0400] 52. The system of any of embodiments 39-51, wherein the magnetic particles are coated magnetic particles comprising a coating comprising the captures sites, wherein the capture sites are selected to be capture sites for the target component, such as a biomolecule.

[0401] 53. The system of any of embodiments 39-52, wherein the fluorophores are quantum dots or aromatic probes and/or conjugated probes, the fluorophores are preferably quantum dots.

[0402] 54. The system of any of embodiments 39-53, wherein the fluorophores are configured to bind to said capture sites of the magnetic particles by being coupled to a component which can bind to the capture sites of the magnetic particles, the component is preferably identical or homolog to said target component.

[0403] 55. The system of any of embodiments 39-54, wherein said fluorophores and said magnetic particles are temporally immobilized in said flow channel of the micro fluidic device such that they cannot bind to each other prior to the feeding of a liquid sample to said flow channel.

[0404] 56. The system of any of embodiments 39-55, wherein said magnet is arranged to at least temporally immobilize said magnetic particle adjacent to said transparent wall section for a sufficient time to excite at least a part of possible fluorophores captured by said capture sites of the magnetic particles by the emitter and to read out any emitted signal from possibly captured fluorophores.

[0405] 57. The system of any of embodiments 39-56, wherein said emitter is a light emitting diode or a laser which is capable of emitting electromagnetic radiation comprising the exciting wavelength of the fluorophores.

[0406] 58. The system of any of embodiments 39-59, wherein said emitter is configured to emit electromagnetic radiation directed at the transparent window, the window has a planar surface, the emitter is preferably configured to emit electromagnetic radiation directed at the transparent window perpendicular to the surface of the window or with an angle to the surface of the window which is from about 20 to about 170, such as from about 30 to about 150. 59. The system of any of embodiments 39-58, wherein said reader is configured for reading signals emitted from fluorophores captured by magnetic particles which are temporally immobilized adjacent to said window.

[0407] 60. The system of any of embodiments 39-59, wherein said emitter comprises at least one optical fiber with an output end for emitting said electromagnetic radiation and said reader comprises at least one optical fiber with an input end for receiving said signals emitted from fluorophores captured by magnetic particles, preferably said optical fiber of the emitter and said optical fiber of the reader are arranged adjacent to each other in at least respective length sections adjacent to respectively the output end and the input end.

[0408] 61. The system of embodiment 60, wherein said emitter comprises a plurality of optical fibers, each with an output end for emitting said electromagnetic radiation and said reader comprises a plurality of optical fibers, each with an input end for receiving said signals emitted from fluorophores captured by magnetic particles, preferably said optical fibers of the emitter and said optical fibers of the reader are arranged adjacent to each other in at least respective length sections adjacent to respectively the output end and the input, more preferably the output ends and the input ends are arranged in a predetermined pattern.

[0409] 62. The system of any of embodiments 39-61, wherein said system comprises a computer for performing the quantitative or qualitative determination of target component(s) in a liquid sample based on the read signal(s).

[0410] 63. The system of any of embodiment 62, wherein the computer comprises a memory for storing of read signal(s) and/or quantitative or qualitative determinations performed.

[0411] 64. The system of any of embodiments 62 or 63, wherein the computer comprises a memory comprising a reference schedule for comparing the read signal(s) to perform the determination, the reference schedule preferably comprises sets of a quantitative or qualitative determination with read signal(s).

[0412] 65. The system of any of embodiments 62-64, wherein the computer is an artificial intelligent processor programmed to compare read signal(s) with stored signals obtained from liquid samples with known compositions.

[0413] 66. The system of any of embodiments 62-65, wherein the system comprises a signal processor comprising the computer wherein the signal processor is configured to multiplex signals from different groups of fluorophores, from fluorophores from parallel assays and/or from fluorophores in reference tests of known or unknown liquid samples.

[0414] 67. The system of any of embodiments 62-66, wherein the system comprises a signal processor comprising the computer wherein the signal processor is configured to multiplex signals from different groups of fluorophores applied in the same assay.

[0415] 68. A kit for preparing a liquid sample for optical analysis for quantitative or qualitative determination of a plurality of target components in the sample, the kit comprises [0416] a plurality of magnetic particles comprising a type of capture sites for each of the target components on their surfaces; and [0417] a plurality of groups of fluorophores, each group of fluorophores is configured to bind to one of the types of capture sites of the magnetic particles.

[0418] 69. The kit of embodiment 68 for preparing a liquid sample for optical analysis for quantitative or qualitative determination of N different target components in the sample, where N is an integer of 2 or more, the kit comprises [0419] a plurality of magnetic particles comprising N types of capture sites, including one type of capture sites for each of the target components; and [0420] N groups of fluorophores, each group of fluorophores is configured to bind to one of the types of capture sites of the magnetic particles.

[0421] 70. The kit of embodiment 68 for preparing a liquid sample for optical analysis for quantitative or qualitative determination of N different target components in the sample, where N is an integer of 2 or more, the kit comprises [0422] N groups of magnetic particles, each group of magnetic particles comprises one type of capture site for one target component; and [0423] N groups of fluorophores, each group of fluorophores is configured to bind to one of the types of capture sites of the magnetic particles.

[0424] 71. A kit of any of embodiments 68-70, wherein two or more groups of fluorophores are provided in one single solution, preferably the magnetic particles are provided in the form of one solution or suspension.

[0425] 72. A kit of any of embodiments 68-71, wherein two or more groups of fluorophores are quantum dots capable of being excited by electromagnetic waves of the same wavelength.

[0426] 73. A kit of any of embodiments 68-71, wherein the kit further comprises a micro fluidic device and/or a magnet.

[0427] 74. A kit of any of embodiments 68-73, wherein N is an integer from 2 to 10.

[0428] 75. A micro fluidic device for use in preparing a liquid sample for optical analysis for quantitative or qualitative determination of a of target component in the sample, the micro fluidic device comprising at least one flow channel with a transparent window and an inlet for the liquid sample, the micro fluidic device further comprises in its flow channel [0429] a plurality of magnetic particles comprising capture sites for the target component on their surfaces; and [0430] a plurality of fluorophores configured to bind the capture sites of the magnetic particles,

[0431] the micro fluidic device preferably being a micro fluidic device.

[0432] 76. A micro fluidic device for use in preparing a liquid sample for optical analysis for quantitative or qualitative determination of a of target component in the sample, the micro fluidic device comprising a substrate with a groove for a flow channel and a foil covering the flow channel, the flow channel comprises a transparent window and an inlet for suction in the liquid sample, the micro fluidic device comprises a flexible wall section of the flow channel or of a sink section in fluid connection with the flow channel, the flexible wall section can be moved such that air will be pressed out of the flow channel where after the flexible wall will return to its initial position, preferably the flow channel being in fluid communication with a sink section of the micro fluidic device.

[0433] 77. A sandwich-type assay for quantitative or qualitative determination of a target component in a liquid sample, the assay comprises

[0434] providing a plurality of magnetic particles comprising one or more capture sites for the target component on their respective surfaces;

[0435] providing a plurality of fluorophores comprising one or more capture sites for the target component;

[0436] bringing the liquid sample, said fluorophores and said magnetic particles into a flow channel of a micro fluidic device comprising a transparent window into the flow channel; and

[0437] at least temporally immobilizing said magnetic particles adjacent to said transparent window using a magnet, emitting exciting electromagnetic beam towards said immobilized magnetic particles, reading signals emitted from fluorophores captured by said immobilized magnetic particles via said target components and performing a quantitative or qualitative determination of said target component based on the read signal.

[0438] 78. The sandwich-type assay of embodiment 77, wherein the micro fluidic device is a micro fluidic device as in embodiment 76.