Microfluidic system Number:7,148,476 from the United States Patent and Trademark Office (PTO) owispatent

Title: Microfluidic system

Abstract: The present invention relates to a method for presenting an analyte of a liquid sample as an MS-analyte to a mass spectrometer. More particularly, the method comprises the steps of applying a liquid sample containing the analyte to a sample inlet port of a microchannel structure of a microfluidic device, said structure also comprising an outlet port (MS-port) that is capable of being interfaced with a mass spectrometer, passing the analyte to the MS-port thereby transforming it to an MS-analyte, and presenting the MS-analyte to mass spectrometer via the MS-port.

Patent Number: 7,148,476 Issued on 12/12/2006 to Andersson,   et al.

---

Inventors:	Andersson; Per (Uppsala, SE), Derand; Helene (Taby, SE), Gustafsson; Magnus (Solna, SE), Palm; Anders (Uppsala, SE), Wallenborg; Sussanne (Uppsala, SE)
Assignee:	Gyros Patent AB (Uppsala, SE)
Appl. No.:	10/867,893
Filed:	June 15, 2004

---

Related U.S. Patent Documents

---

	Application Number	Filing Date	Patent Number	Issue Date
	10621868	Jul., 2003	6812457
	09811741	Mar., 2001	6653625

---

Current U.S. Class:	250/288 ; 250/281; 250/282; 422/100; 422/68.1; 422/70
Current International Class:	B01D 59/44 (20060101); B01D 15/08 (20060101)

---

References Cited [Referenced By]

---

U.S. Patent Documents

	4279862		July 1981		Bretaudiere et al.
	4879458		November 1989		Brunfeldt et al.
	5115131		May 1992		Jorgenson et al.
	5197185		March 1993		McCoy et al.
	5310523		May 1994		Smethers et al.
	5376252		December 1994		Ekstrom et al.
	5637469		June 1997		Wilding et al.
	5705813		January 1998		Apffel et al.
	5716825		February 1998		Hancock et al.
	5773488		June 1998		Allmer
	5866345		February 1999		Wilding et al.
	5869830		February 1999		Franzen et al.
	5872010		February 1999		Karger et al.
	5962081		October 1999		Ohman et al.
	5969353		October 1999		Hsieh
	5995209		November 1999		Ohman
	6063589		May 2000		Kellogg et al.
	6110343		August 2000		Ramsey et al.
	6126765		October 2000		Ohman
	6143247		November 2000		Sheppard, Jr. et al.
	6144447		November 2000		Ohman et al.
	6191418		February 2001		Hindsgaul et al.
	6192768		February 2001		Wallman et al.
	6203291		March 2001		Stemme et al.
	6302134		October 2001		Kellogg et al.
	6319468		November 2001		Sheppard, Jr. et al.
	6319469		November 2001		Mian et al.
	6322682		November 2001		Arvidsson et al.
	6326083		December 2001		Yang et al.
	6454970		September 2002		Ohman et al.
	6499499		December 2002		Dantsker et al.
	6620478		September 2003		Ohman
	6632656		October 2003		Thomas et al.
	6653625		November 2003		Andersson et al.
	6717136		April 2004		Andersson et al.
	6728644		April 2004		Bielik et al.
	6811736		November 2004		Ohman et al.
	6812456		November 2004		Andersson et al.
	6812457		November 2004		Andersson et al.
	2003/0044322		March 2003		Andersson et al.
	2003/0047823		March 2003		Ohmn et al.
	2003/0053934		March 2003		Andersson et al.
	2003/0054563		March 2003		Ljungstrom
	2003/0082075		May 2003		Agreh
	2003/0094502		May 2003		Andersson et al.
	2003/0129360		July 2003		Derand
	2003/0156763		August 2003		Zeller
	2003/0211012		November 2003		Bergstrom
	2003/0213551		November 2003		Perand
	2003/0231312		December 2003		Sioberg et al.
	2004/0058408		March 2004		Thomas
	2004/0096867		May 2004		Andersson et al.
	2004/0099310		May 2004		Andersson
	2004/0120656		June 2004		Andersson et al.
	2004/0202579		October 2004		Larsson et al.
	2005/0042770		February 2005		Derand

Foreign Patent Documents

	0073513		Mar., 1983		EP
	0073513		Jul., 1985		EP
	0073513		Feb., 1992		EP
	WO/97/04297		Feb., 1997		WO
	WO 97/21090		Jun., 1997		WO
	WO 00/40750		Jul., 2000		WO
	WO 01/47637		Jul., 2001		WO
	WO/01/54810		Aug., 2001		WO
	WO 01/63241		Aug., 2001		WO

Other References

Zhang, B. et al. Microfabricated Devices for Capillary Electrophoresis-Electrospray Mass Spectrometry; Analytical Chemistry, vol. 71, No. 15, Aug. 1, 1999 pp. 3258-3264. cited by other . Kido, H. et al, Disc-based Immunoassay Microarrays; Analytica Chimica Acta 411 (2000). cited by other . U.S. Appl. No. 10/999,532, Gyros. cited by other . U.S. Appl. No. 11/017,252, Gyros. cited by other . U.S. Appl. No. 10/111,822, Gyros. cited by other . U.S. Appl. No. 10/030,297, Gyros. cited by other . U.S. Appl. No. 11/010,869, Gyros. cited by other . U.S. Appl. No. 11/010,870, Gyros. cited by other . U.S. Appl. No. 10/169,056, Gyros. cited by other . U.S. Appl. No. 10/450,177, Gyros. cited by other . U.S. Appl. No. 10/402,138, Gyros. cited by other . U.S. Appl. No. 10/402,137, Gyros. cited by other . U.S. Appl. No. 10/244,667, Gyros. cited by other . U.S. Appl. No. 10/182,792, Gyros. cited by other . U.S. Appl. No. 10/069,827, Gyros. cited by other . U.S. Appl. No. 10/168,942, Gyros. cited by other . U.S. Appl. No. 10/957,452, Gyros. cited by other . U.S. Appl. No. 10/276,282, Gyros. cited by other . U.S. Appl. No. 10/867,893, Gyros. cited by other . U.S. Appl. No. 11/010,977, Gyros. cited by other . U.S. Appl. No. 11/038,712, Gyros. cited by other . U.S. Appl. No. 11/010,956, Gyros. cited by other . U.S. Appl. No. 10/070,912, Gyros. cited by other . U.S. Appl. No. 09/937,533, Gyros. cited by other . U.S. Appl. No. 10/924,151, Gyros. cited by other . U.S. Appl. No. 10/513,084, Gyros. cited by other . U.S. Appl. No. 09/674,457, Gyros. cited by other . U.S. Appl. No. 09/830,475, Gyros. cited by other . U.S. Appl. No. 09/869,554, Gyros. cited by other . U.S. Appl. No. 10/129,032, Gyros. cited by other . U.S. Appl. No. 09/958,577, Gyros. cited by other.

Primary Examiner: Wells; Nikita
Attorney, Agent or Firm: Fulbright & Jaworski LLP

---

Parent Case Text

---

This application is a continuation of U.S. application Ser. No. 10/621,868 filed Jul. 17, 2003 issued as U.S. Pat. No. 6,812,457, which is a continuation of U.S. application Ser. No. 09/811,741 filed Mar. 19, 2001 issued as U.S. Pat. No. 6,653,625.

---

Claims

---

The invention claimed is:

1. A method for presenting an analyte of a liquid sample as an MS-analyte to a mass spectrometer comprising the steps of: (i) applying the liquid sample containing the analyte to a sample inlet port of a microchannel structure of a microfluidic device, said microchannel structure comprises an outlet port (MS-port) that is interfaced with a mass spectrometer and inertia force is used for liquid transportation within at least a part of said microchannel structure; (ii) passing the analyte to the MS-port thereby transforming the analyte to an MS-analyte, and (iii) presenting the MS-analyte to the mass spectrometer via the MS-port.

2. The method of claim 1, wherein the analyte is a biopolymer comprising a carbohydrate structure, a nucleic acid structure or a peptide structure.

3. The method of claim 1, wherein the analyte comprises a peptide structure.

4. The method of claim 1, wherein transformation of said analyte to an MS-analyte comprises digestion of the analyte into fragments.

5. The method of claim 4, wherein said digestion is a chemical digestion or an enzymatic digestion.

6. The method of claim 1, wherein transformation of said analyte to an MS-analyte comprises chemical derivatization of the analyte.

7. The method of claim 1, wherein transformation of said analyte to an MS-analyte comprises mass tagging of said analyte or of a derivative formed during the transformation.

8. The method of to claim 7, wherein said derivative to be mass tagged is a fragment of the analyte.

9. The method of claim 1, wherein the inertia force is centrifugal force.

10. The method of claim 1, wherein said MS-port comprises an opening permitting release of the MS-analytes into the mass spectrometer.

11. The method of claim 1, wherein said microfluidic device comprises a planar substrate having at least one microchannel structure that extends radially in the plane of the substrate with said MS-pen being located at an outer position and said sample inlet port at an inner position.

12. The method of claim 11, wherein said planar substrate alter or simultaneously with the application of the sample is spun around its central axis which is perpendicular to said plane thereby forcing liquids present in the microchannel structure to move outwards.

13. The method of claim 11 wherein said microfluidic device comprises a plurality of said microchannel structures.

14. The method of claim 11, wherein the plurality of microchannel structures are annularly arranged around a central axis.

15. The method of claim 14, wherein each EDI area comprises a conducting layer (layer I) with a conductive connection.

16. The method of claim 15, wherein said separation zone comprises a separation medium which is capable of selectively capturing said derivative when a liquid containing said derivative is allowed to pass through the zone.

17. The method of claim 15, wherein transformation of an analyte to said MS-analyte occurs in a reaction zone in said microchannel.

18. The method of 15, wherein said separation zone separates, concentrates, or purifies the analyte, MS-analyte or derivative thereof.

19. The method of claim 15, wherein said separation zone comprises separation medium which exhibits ligand structures that are capable of binding to said analyte or said MS-analyte or derivative by affinity or reversible covalent bonds.

20. The method of claim 15, wherein the separation zone is located downstream the sample inlet port and upstream the MS port.

21. The method of claim 15, wherein the separation zone is located within the MS-port.

22. The method of claim 1, wherein the MS-port comprises an electro spray arrangement.

23. The method of claim 22, wherein there are two or more EDI areas on the microfluidic device and that layer I in at least two of said EDI areas are present in a common continuous conducting layer comprising the connection for electricity.

24. The method of claim 1, wherein the MS-port comprises an EDI arrangement with an EDI area.

25. The method of claim 1, wherein the microchannel structure further comprises a separation zone.

26. The method of claim 25, wherein said separation zone and said reaction zone coincide.

27. The method of claim 1, wherein the MS-analyte is a derivative of the analyte.

---

Description

---

TECHNICAL FIELD

The present invention relates to a microfluidic device, which can be interfaced to a mass spectrometer (MS). The device comprises a microchannel structure having a first port (inlet port) and a second port (outlet port). A sample to be analysed is applied to the first port and presented to the mass spectrometer in the second port. This second port will be called an MS-port. There may be additional inlet and outlet ports. During passage through the microchannel structure the sample is prepared to make it suitable for analysis by mass spectrometry.

The sample presented in an MS-port will be called an MS-sample. An analyte in an MS-sample is an MS-analyte. "Sample" and "analyte" without prefix will primarily refer to a sample applied to an inlet port.

One important aspect of the present invention concerns mass spectrometry in which the MS-samples are subjected to Energy Desorption/Ionisation from a surface by input of energy. Generically this kind of process will be called EDI and the surface an EDI surface in the context of the invention. Typicallly EDIs are thermal desorption/ionisation (TDI), plasma desorption/ionisation (PDI) and various kinds of irradiation desorption/ionisation (IDI) such as by fast atom bombardment (FAB), electron impact etc. In the case a laser is used the principle is called laser desorption/ionisation (LDI). Desorption may be assisted by presenting the MS analyte together with various helper substances or functional groups on the surface. Common names are matrix assisted laser desorption/ionisation (MALDI) including surface-enhanced laser desorption/ionisation (SELDI). For MALDI see the publications discussed under Background Publications below. For SELDI see WO 0067293 (Ciphergen Biosystems).

The surface from which desorption/ionisation is intended to take place is called an EDI surface.

By microformat is meant that in least a part of the microchannel structures the depth and/or width is in the microformat range, i.e. <10.sup.3 .mu.m, preferably <10.sup.2 .mu.m. In the most typical microformat structures either the width and/or the depth are in principle within these ranges essentially everywhere between the sample inlet port and the MS-port.

BACKGROUND PUBLICATIONS

For some time there has been a demand for microfluidic sample handling and preparation devices with integrated MS-ports. This kind of devices would facilitate automation and parallel experiments, reduce loss of analyte, increase reproducility and speed etc. WO 9704297 (Karger et al) describes a microfluidic device that has an outlet port that is claimed useful when conducting electrospray ionisation mass spectrometry (ESI MS), atmospheric pressure chemical ionisation mass spectrometry (APCI MS), matrix assisted laser desorption/ionisation mass spectrometry (MALDI MS) and a number of other analytical principles. U.S. Pat. No. 6,110,343 (Ramsey et al) describes an electrospray interface between a microfluidic device and a mass spectrometer. U.S. Pat. No. 5,969,353 (Hsieh) describes an improved interface for electrospray ionization mass spectrometry. The interface is in the form of an electrospray tip connected to a microchannel structure of a chip. U.S. Pat. No. 5,197,185 (Yeung et al) describes a laser-induced vaporisation and ionization interface for directly coupling a microscale liquid based separation process to a mass spectrometer. A light-adsorbing component may be included in the eluting liquid in order to facilitate vaporisation. U.S. Pat. No. 5,705,813 (Apffel et al) and U.S. Pat. No. 5,716,825 (Hancock et al) describe a microfluidic chip containing an interface between a microfluidic device and an MALDI-TOF MS apparatus. The microfluidic device comprises (a) an open ionisation surface that may be used as the probe surface in the vaccum gate of an MALDI-TOF MS apparatus (column 6, lines 53 58 of U.S. Pat. No. 5,705,813) or (b) a pure capture/reaction surface from which the MS-analyte can be transferred to a proper probe surface for MALDI-TOF MS (column 12, lines 13 34, of U.S. Pat. No. 5,716,825).

These publications suggest that means, such as electrical connections, pumps etc, for transporting the liquid within a microchannel structure of the device are integrated with or connected to the device. This kind of transporting means imposes an extra complexity on the design and use, which in turn may negatively influence the production costs, easiness of handling etc of these devices.

U.S. Pat. No. 5,705,813 (Apffel et al) and U.S. Pat. No. 5,716,825 (Hancock et al) are scarce about the proper fluidics around the MALDI ionisation surface, the proper crystallisation on the MALDI ionisation surface, the proper geometry of the port in relation to crystallisation, evaporation, the incident laser beam etc, the proper arrangement of conductive connections to the MALDI ionisation surface for MALDI MS analysis.

WO 04297 (Karger et al) and WO 0247913 (Gyros AB) suggest to have microchannel structures in radial or spoke arrangement.

A number of publications referring to the use of centrifugal force for moving liquids within microfluidic systems have appeared during the last years. See for instance WO 9721090 (Gamera Bioscience), WO 9807019 (Gamera Bioscience) WO 9853311 (Gamera Bioscience), WO 9955827 (Gyros AB), WO 9958245 (Gyros AB), WO 0025921 (Gyros AB), WO 0040750 (Gyros AB), WO 0056808 (Gyros AB), WO 0062042 (Gyros AB) and WO 0102737 (Gyros AB) as well as WO 0147637 (Gyros AB), WO 0154810 (Gyros AB), WO 0147638 (Gyros AB), and WO 0146465.

See also Zhang et al, "Microfabricated devices for capillary electrophoresis--electrospray mass spectrometry", Anal. Chem. 71 (1999) 3258 3264) and references cited therein.

Kido et al., ("Disc-based immunoassay microarrays", Anal. Chim. Acta 411 (2000) 1 11) has described microspot immunoassays on a compact disc (CD). The authors suggest that a CD could be used as a continuous sample collector for microbore HPLC and subsequent detection for instance by MALDI MS. In a preliminary experiment a piece of a CD manufactured in polycarbonate was covered with gold and spotted with a mixture of peptides and MALDI matrix.

OBJECTS OF THE INVENTION

A first object is to provide improved means and methods for transporting samples, analytes including fragments and derivatives, reagents etc in microfluidic devices that are capable of being interfaced with a mass spectrometer. A second object is to provide improved microfluidic methods and means for sample handling before presentation of a sample analyte as an MS-analyte. Sub-objects are to provide an efficient concentration, purification and/or transformation of a sample within the microfluidic device while maintaining a reproducible yield/recovery, and/or minimal loss of precious material. A third object is to provide improved microfluidic methods and means that will enable efficient and improved presentation of the MS-sample/MS-analyte. This object in particular applies to MS-samples that are presented on a surface, i.e. an EDI surface. A fourth object is to enable reproducible mass values from an MS-sample that is presented on a surface, i.e. on an EDI surface. A fifth object is to provide improved microfluidic means and methods for parallel sample treatment before presentation of the analyte to mass spectrometry. The improvements of this object refer to features such as accuracy in concentrating, in chemical transformation, in required time for individual steps and for the total treatment protocol etc. By parallel sample treatment is meant that two or more sample treatments are run in parallel, for instance more than five, such as more than 10, 50, 80, 100, 200, 300 or 400 runs. Particular important numbers of parallel samples are below or equal to the standard number of wells in microtiter plates, e.g. 96 or less, 384 or less, 1536 or less, etc A sixth object is to provide a cheap and disposable microfluidic device unit enabling parallel sample treatments and having one or more MS-ports that are adapted to a mass spectrometer.

SUMMARY OF THE INVENTION

The present inventors have recognized that several of the above-mentioned objects can be met in the case inertia force is used for transportation of a liquid within a microfluidic device of the kind discussed above. This is applicable to any liquid that is used in the microfluidic device, for instance washing liquids and liquids containing at least one of (a) the analyte including derivatives and fragments thereof, (b) a reagent used in the transformation of the sample/analyte, etc.

The present inventors have also recognized that one way of optimizing an EDI area within a microfluidic device is related to (a) the design and/or positioning of a conducting layer in the EDI area, and/or (b) the importance of a conductive connection to the EDI area for MS analysis. This kind of connection supports the proper voltage and/or charge transport at the EDI area, for instance.

Improper conductive properties may interfere with the mass accuracy, sensitivity, resolution etc.

Conductive and non-conductive properties shell refer to the property of conducting electricity.

A first aspect of the invention is thus a method for transforming a liquid sample containing an analyte to an MS-sample containing an MS-analyte and presenting the MS-sample to a mass spectrometer. The method is characterized in comprising the steps of: (a) applying the liquid sample to an inlet port of a covered microchannel structure of a microfluidic device, (b) transforming the liquid sample to an MS-sample containing the MS-analyte within the microchannel structure, and (c) presenting the MS-analyte to the mass spectrometer.

A further characteristic feature of this aspect is that transport of liquid within the microchannel structure is performed by the application of inertia force. Inertia force may be the driving force in only a part of the microchannel structure or the whole way from an inlet port to an MS-port and/or to any other outlet port. It is believed that the most general and significant advantages of using inertia force will be accomplished in so called transporting zones, i.e. between zones having predetermined functionalities, or for overcoming or passing through valve functions within a microchannel structure (capillary junctions, hydrophobic breaks etc). See below. The MS-port typically has a conductive connection for MS analysis.

At the priority date the most important inertia force for microfluidic devices is centrifugal force. In other words a force that causes outward radial transportation of liquid by spinning a disc in which the liquid is located within microchannel structures that are oriented radially (spinning is around an axis that is perpendicular to the plane of the disc). Inertia force caused by other changes of direction and/or magnitude of a force can be utilized.

The first aspect also includes the corresponding mass spectrometric method, i.e. the same method together with the actual collection of a mass spectrum and analysis thereof, for instance in order to gain molecular weight and structure information about the analyte.

The first aspect is further defined as discussed below for the microfluidic device as such and for the individual steps.

A second aspect of the invention is a microfluidic device containing one, two or more microchannel structures containing an inlet port, an MS-port and a flow path connected to one or both of the ports. The device may be disc-formed or otherwise provide a planar form. The characteristic feature is that the microchannel structures are oriented radially in an annular/circular arrangement. Thus each microchannel structure extends in a radial direction with an inlet port at an inner position and an outlet port such as an MS-port, at an outer peripheral position. The MS-port typically has a conductive connection as discussed above. The features discussed below further define this aspect of the invention.

A third aspect of the invention is a microfluidic device comprising a plurality of covered microchannel structures as defined herein and with each microchannel structure having an MS-port comprising an EDI area in which there is a conducting layer (layer I). This aspect of the present invention comprises a number of subaspects having the common characteristic feature that there may be a conductive connection to layer (I) of each individual EDI area, as discussed above. There are also features that are distinct for each subaspect. A first subaspect is further characterized in that layer (I) of each EDI area is part of a continuous conducting layer that is common for two or more up to all of the EDI-areas. A second subaspect is further characterized in that in each EDI area there is a non-conducting layer (layer II) between layer (I) and the surface of the EDI area. Layer (II) in each EDI area may be part of a continuous non-conducting layer that is common for two or more up to all of the EDI-areas. A third subaspect is further characterised in that each MS-port has an opening that is restricted by a lid which is common for and covers a number of microchannel structures. The lid may have a conducting layer that at least embraces the openings that are present in the lid. The conducting layer may be continuous in the sense that it covers at least the areas around and between the openings of two or more up to all of the MS-ports. This layer may have a conductive connection as discussed above. A fourth subaspect is similar to the third subaspect in the sense that there is a lid covering at least a part of each microchannel structures. In this subaspect the lid also covers or restricts the openings of the MS-ports and is removable to an extent that enables exposure of the opening in each MS-port, for instance exposing the surfaces of EDI areas. For EDI ports the removal will facilitate irradiation and the desorption/ionisation of the MS-analyte. The removal may also facilitate evaporation of volatile components. The Sample

The sample applied to an inlet port may contain one or more analytes, which may comprise lipid, carbohydrate, nucleic acid and/or peptide structure or any other inorganic or organic structure. The sample treatment protocol to take place within the microchannel structure typically means that the sample is transformed to one or more MS-samples in which (a) the MS-analyte is a derivative of the starting analyte and/or (b) the amount(s) of non-analyte species have been changed compared to the starting sample, and/or (c) the relative occurrence of different MS-analytes in a sample is changed compared to the starting sample, and/or (d) the concentration of an MS-analyte is changed relative the corresponding starting analyte in the starting sample, and/or (e) sample constituents, such as solvents, have been changed and/or the analyte has been changed from a dissolved form to a solid form, for instance in a co-crystallised form.

Item (a) includes digestion into fragments of various sizes and/or chemical derivatization of an analyte. Digestion may be purely chemical or enzymatic. Derivatization includes so-called mass tagging of either the starting analyte or of a fragment or other derivative formed during a sample treatment protocol, which takes place in the microchannel structure. Items (b) and/or (c) include that the sample analyte has been purified and/or concentrated. Items (a) (d), in particular, apply to analytes that are biopolymers comprising carbohydrate, nucleic acid and/or peptide structure.

The sample is typically in liquid form and may be aqueous.

The sample may also pass through the microchannel structure without being changed. In this case the structure only provide a proper form for dosing of the analyte to the mass spectrometer.

FIGURES

FIGS. 1 3 illustrate various microchannel structures that have an MS-port.

FIG. 4 illustrates an MS-port in form of an electrospray (sideview).

FIGS. 5a f illustrate various design and positions of the conducting layer (I) in MS-ports containing an EDI area (cross-sectional sideview of two ms ports). The microfluidic device is fabricated in a planar substrate.

FIG. 6 illustrates an arrangement around EDI MS-ports with layer (I) and conductive connections (transparent lid, seen from above).

FIGS. 7a b illustrate a variant of an EDI-port with a transparent lid (seen from above and in a cross-sectional sideview, respectively).

DETAILED DESCRIPTION OF THE INNOVATIVE MICROFLUIDIC DEVICE

The Microfluidic Structure

The microfluidic device comprises one or more microchannel structures having an inlet port for application of a liquid sample and an MS-port for release and presentation of an MS-analyte to a mass spectrometer. These kinds of ports may coincide in a microchannel structure. There may also be separate inlet ports for application of solvents and reagents and separate outlet ports or waste chambers/cavities for withdrawal of other components that are added and/or produced in the structure. Two or more microchannel structures may have common inlet ports. Depending on the particular design of the device some of the ports may be closed during the sample treatment but opened later on, for instance in order to enable proper release and presentation of the MS-analyte.

The distance between two opposite walls in a channel is typically .ltoreq.1000 .mu.m, such as .ltoreq.100 .mu.m, or even .ltoreq.10 .mu.m, such as .ltoreq.1 .mu.m. Functional channel parts (chambers, cavities etc) typical have volumes that are .ltoreq.500 .mu.l, such as .ltoreq.100 .mu.l and even .ltoreq.10 .mu.l such as .ltoreq.1 .mu.l. In important variants these volumes may be .ltoreq.500 nl such as .ltoreq.100 nl or .ltoreq.50 nl. The depths of these parts may be in the interval .ltoreq.1000 .mu.m such as .ltoreq.100 .mu.m such as .ltoreq.10 .mu.m or even .ltoreq.1 .mu.m. The lower limits (width and depth) are always significantly greater than the largest of the reagents and analytes (including fragments and derivatives) that are to be transported within the microchannel structure. The lower limits of the different channel parts are typically in the range 0.1 0.01 .mu.m. The aspect ratio (depth to width) may be .gtoreq.1 or .ltoreq.1 in all parts or in only a part of a microchannel structure.

Preferred microfluidic devices typically comprise one, two or more, preferably more than 5, microchannel structures fabricated wholly or partly in the surface of a planar substrate. In the preferred microfluidic devices of the invention, the side of the substrate in which the microchannels are located (microchannel side) may be covered by a lid comprising remaining parts, if any, of the microchannel structure. When the lid is properly mated to the upper side of the substrate, parts of the microchannel structures in the lid, if any, match the structures in the microchannel side thereby completing the microchannel structures of the device. The lid will prevent or minimise undesired evaporation of liquids as well as facilitate transport of liquids.

Each microchannel structure preferably extends in a common plane of the planar substrate material. In addition there may be extensions in other directions, primarily perpendicular to the common plane. Such other extensions may function as sample or liquid application areas or connections to other microchannel structures that are not located in the common plane, for instance.

The microfluidic devices may be disc-formed and have various geometries, with the circular form being the preferred variant (CD-form). Other variants of discs may have an axis of symmetry that is at least 3- or at least 6-numbered.

On devices having circular forms or an axis of symmetry as mentioned in the previous paragraph, each microchannel structures may be oriented radially around a central axis with an intended flow direction for each structure from an inner application area (inlet port) towards the periphery of the disc. The arrangement may be in form of one or more concentric circles (annular/circular arrangements). According to the first aspect of the invention the liquid is transported by inertia force, for instance centrifugal force, in at least a part of a microchannel structure. Examples of other ways of transportation are by capillary action, hydrodynamically, electrokinetically etc. These alternatives may also be combined with inertia force in line with what has been discussed for the first aspect of the invention.

Each microchannel structure comprises one or more channels in the microformat. The channels may comprise chambers/cavities that are in the microformat. Different parts of a structure may have different discrete functions. In addition to the channel parts mentioned above (inlet port, MS-port, transportation conduit/channel), there may be one or more channel parts that function as (a) application zone/port for reagents and liquids other than sample liquid (second inlet port), (b) additional MS-ports, (c) reaction zone, for instance for derivatization of an analyte discussed above (digestion, tagging etc). (d) pressure creating zone (for instance hydrostatic pressure), (e) volume defining zone, (f) mixing zone, (g) zone for separating and/or concentrating and/or purifying the analyte or a derivative or fragment thereof, for instance by capillary electrophoresis, chromatography and the like, (h) waste conduit/chamber/cavity (for instance in the form of an outlet port), (i) zone for splitting a liquid flow, etc.

These kinds of zones may be present as distinct chambers or conduits that may have a cross-sectional dimension that differs from a preceding and/or a subsequent part of the microchannel structure.

Splitting may be located at the inlet so that a starting sample is divided in several aliquots, each of which is processed in parallel within a structure.

Except for the presence of an MS-port this kind of microchannel structures have been described in a number of previous patent publications. See the background publications discussed above.

Between parts having different functions there may be valves that can be overcome by increasing the force driving the liquid. For variants utilizing spinning, this may for instance be accomplished by increasing the spinning and/or utilizing pressure built up within the structure due to addition of a new portion of liquid combined with spinning. See for instance WO 0040750 (Gyros AB) and WO 0146465 (Gyros AB). Valves may be based on capillary junctions (WO 9807019 (Gamera Bioscience)) or hydrophobic breaks (WO 9958245 (Gyros AB) or on thermic properties of the valve material. The latter kind of valves may be illustrated by so called sacrificing valves (WO 9853311 (Gamera Bioscience)) for instance containing a plug of wax-like material, or reversible valves, for instance containing a thermoreversible polymer in the form of a plug (WO 0102737 (Gyros AB)).

One kind of versatile microchannel structures used according to the invention comprise a zone in which separation and/or concentration and/or a purification of the analyte or an analyte-derived entity can take place. This zone is located either before or in the MS-port. Examples of analyte-derived entities are fragments and derivatives of the analyte. This kind of functionality may be particularly important for samples containing low concentrations of analytes, complex mixtures of analytes or high concentrations of interfering substances that may negatively affect the resolution and/or sensitivity of the MS-analyte when analyzed by mass spectrometry. Separation and/or concentration and/or purification of the sample analyte or analyte-derived entities can be accomplished according to similar principles as typically employed in the life science area, i.e. separations based on size exclusion and/or on differences in binding to a ligand structure are applicable. Accordingly, this kind of channel part may contain a separation medium that is capable of binding the analyte or an analyte-derived entity but not to the contaminants, or vice versa. The separation medium is typically in particle/bead form, or attached on the surface of the separation zone or in the form of a monolithic plug that permits through flow. If the analyte or the analyte-derived entity becomes bound, a liquid having the proper desorption characteristics for the bound entity is subsequently allowed to pass through the chamber whereupon the bound entity is released and transported downstream. This transport may be directly to the MS-port or to a zone in which a further preparation step is accomplished. Washing steps may be inserted between the sample liquid and the desorption liquid. The separation medium may be soluble or insoluble during the binding step. Soluble separation media are typically insolubilized after binding according to principles well-known in the field of macrosopic separations.

Binding to the separation medium may involve formation of covalent bonds or encompass affinity binding. Binding of covalent nature for this purpose is typically reversible, for instance by thiol-disulfide exchange, such as between a thiol-containing analyte or analyte-derived entity and a separation medium containing a so called reactive disulfide, or vice versa. Affinity binding, including adsorption, can be illustrated with: (a) electrostatic interaction that typically requires that the ligand and the entity to be bound have opposite charges, (b) hydrophobic interaction that typically requires that the ligand and the entity to be bound comprises hydrophobic groups, (c) electron-donor acceptor interaction that typically requires that the ligand and the entity to be bound have an electron-acceptor and electron-donor group, respectively, or vice versa, and (d) bioaffinity binding including other kinds of binding in which the interaction is of complex nature, typically involving a mixture of several different kinds of interactions and/or groups.

Ion exchange ligands may be cationic (=anion exchange ligands) or anionic (=cation exchange ligands). Typical anion exchange ligands have a positively charged nitrogen, the most common ones being primary, secondary, tertiary or quarternary ammonium ligands, and also certain amidinium groups. Typical cation exchange ligands are negatively charged carboxylate groups, phosphate groups, phosphonate groups, sulphate groups and sulphonate groups.

Bioaffinity binding includes that the analyte or the analyte-derived entity is a member of a so-called bioaffinity pair and the ligand is the other member of the pair. Typical bioaffinity pairs are antigen/hapten and an antibody/antigen binding fragment of the antibody; complementary nucleic acids; immunoglobulin-binding protein and immunoglobulin (for instance IgG or an Fc-part thereof and protein A or G), lectin and the corresponding carbohydrate, etc. The term "bioaffinity pair" includes affinity pairs in which one or both of the members are synthetic, for instance mimicking a native member of a bioaffinity pair.

If the analytes in a sample have peptide structure or nucleic acid structure or in other ways have a pronounced hydrophobicity, the separation medium may be of the reverse phase type (hydrophobic) combined with using desorption liquids (eluents) that are organic, for instance acetonitrile, isopropanol, methanol, and the like. Depending on the particular sample and the presence of analytes or analyte-derived entities, which have a common binding structure, a group-specific separation medium may be utilized for the kind of separations discussed above. The separation medium may thus, like a reverse phase adsorbent, result in an MS-sample that has a reduced concentration of salt, i.e. in desalting.

In each microchannel structure there may be two or more separations zone. In this case the zones typically seaparate according to different principles such as size and charge. For amphoteric substances such as proteins and peptides the latter principle may be illustrated with isoelectric focusing.

By using a separation zone it is possible to concentrate the sample such that the concentration of an analyte or an analyte-derived entity in the desorption liquid after passage of the separation medium is higher than in the starting sample. The increase be be with a factor >10.sup.0 and may typically be found in the interval 10.sup.1 10.sup.6, such as 10.sup.1 10.sup.4.

As already mentioned a separation zone may be combined with zones for derivatization. There may also be microchannel structures that have a derivatization zone but no separation zone.

FIG. 1 illustrates a microchannel structure that comprises (a) an inlet port (1) for liquids including the sample liquid, (b) an MS-port (2) comprising for instance an EDI area that may be opened or closed, (c) a flow conduit (3) between the inlet port (1) and the MS-port (2). The flow conduit (3) may have a zone (4) containing an adsorbent for separation/concentration. If there are several microchannel structure in a device there may be a common application area/channel with openings for the inlet ports (not shown). The MS-port may be an EDI MS-port, an eletrospray MS-port.

The use of the structure of FIG. 1 is as indicated. By using spinning for liquid transportation into an open form of the MS-port, aqueous liquids, for instance the sample or the washing liquids, may leave the port as small drops by the centrifugal force while liquids having a lower vapour pressure may evaporate leaving the MS-analyte in the port, e.g. at a lower spinning rate.

FIG. 2 illustrates another variant of a suitable microchannel structure. It has two inlet ports (5,6) that may be used for application of sample, washing liquids and desorption liquid. One of the inlet ports (5) is connected to an application area/channel (7) that may be common to several microchannel structures in the same device. This first inlet port (5) is connected to one of the shanks (8) in a U-shaped channel part via the application area/channel (7). The other inlet port (6) is connected to the other shank of the U. In the lower part of the U there is an exit conduit (9) leading to an MS-port (10). In the channel (11) between the exit conduit (9) from the U and the MS-port (10) there may be a zone (12) containing a separation medium. From the MS-port (10) there may be a waste channel (13) leading to a waste space (14) that may be common for several microchannel structures in the same device. There may be a valve function, for instance in the form of a hydrophobic break, in the exit conduit (9).

FIG. 3 illustrates another alternative of a microchannel structure which comprises a separate sample inlet port (14), an MS-port (15) and therebetween a microchannel structure that may be used for sample preparation. In this variant there is a volume-defining unit (16) between the two ports (14,15) with an over-flow conduit (17). At the lower part of the volume-defining unit (16) there is a first exit conduit (18) leading to one of the shanks (19) of a U-shaped channel part. The other shank (20) of this U may be connected to an inlet port (21) for washing and desorption liquids. At the lower part of the U-shaped channel part there may be a second exit conduit (22) leading into one of the shanks (23) of a second U-shaped channel part. The other shank (24) may be connected to a waste channel (25b) that after a bent (26) may end in a waste chamber (25a). At the lower part of this second U-formed channel part there may be a third exit conduit (27) leading into the MS-port (15) that may contain an EDI area or an electrospray unit. In order to control the flow in the structure, valve functions are preferably located in the first exit conduit (18), for instance immediately downstream the volume-defining unit (16), possibly also in the second exit conduit (22), for instance immediately after the first U-shape, and in the third exit conduit (27), for instance immediately after the second U-shaped channel part. The valves may be of the types discussed above with preference for hydrophobic breaks. A suitable adsorbent (28) as discussed above may be placed in the shank (23) of the second U-shaped channel part and may also function as a valve. In case the adsorbent is in the form of particles they are preferably kept in place by a constriction of the inner walls of the conduits.

The structure presented in FIG. 3 is adapted for transporting the liquid with centrifugal forces, i.e. with the structure present in a disc and oriented radially outwards from the centre of the disc. At start the volume-defining unit (16) is filled up somewhat above the over-flow channel (17). By overcoming a valve function located in the first exit conduit (18), the liquid will pass into the first U-shaped channel part and down through the adsorbent where the analytes are captured. The remaining liquid containing non-analyte components will pass out into the waste channel (25b). In the next step, washing liquids may be applied through the inlet port (21), i.e. through the second shank (20) of the first U-shaped channel part or via the same inlet port (14) as the sample. Also these liquids will pass out into the waste channel (25b). Subsequently, a desorption liquid is applied through either of the two inlet port (14,21) and allowed to pass through the valve function in the third exit conduit (27). The desorption liquid containing released analyte or analyte-derived entities is passed downstream, for instance into the MS-port (15). The operations are preferably carried out while spinning the disc. If the valves are in the form of hydrophobic breaks they can be passed by properly adapting the g-forces, i.e. by the spinning. By properly balancing the hydrophilicity/hydrophobicity of a liquid, passage or non-passage through a valve may be controlled without changing the spinning speed. This is illustrated by utilizing a hydrophobic break as the valve in the third exit conduit (27) combined with utilizing water-solutions as samples and as washing liquids and liquids containing organic solvents as desorption liquids. In the alternative, valves that are opened by external means can be used. By placing the outlet to the first exit conduit (18) at a distance above the lowest part of the volume-defining unit (16) particulate matters, if present in the sample, will sediment and be retained in the volume-defining unit (16) when it is emptied through the first exit conduit (18).

Calibrator areas (29) are shown in each of FIGS. 1 3. Each calibrator area may be connected to a common area for application of calibrator.

The size of a volume-defining unit depends on the sample, reagents used, washing etc. and the requirement the sensitivity of the mass spectrometer sets for concentrating an analyte or an analyte-derived entity. Typical volumes of channel parts that have specific functions are in the range of 1 nl to 1000 .mu.l, mostly below 1 .mu.l such as below 500 nl or even below 100 nl such as below 25 or 10 nl (volume defining unit, reactor part, separation part etc). Application of aliquots of a sample to the same inlet port may replace the need for a larger volume defining unit.

These kind of flow systems has been described in WO 0040750 (Gyros AB) and WO 0146465 (Gyros AB) which are hereby incorporated by reference.

In certain variants the inlet port for the sample and the MS-port may coincide. In this case the MS-port preferably comprises the surface on which the analyte can be collected (adsorbed). Remaining liquid and washing liquids, if used, are