WO2009150583A1

WO2009150583A1 - Diagnostic device

Info

Publication number: WO2009150583A1
Application number: PCT/IB2009/052369
Authority: WO
Inventors: Gardiye H. P. K. C. Punyadeera; Ron M. L. Van Lieshout; Ko Hermans; An M. Prenen; Cornelis W. M. Bastiaansen; Dirk J. Broer
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2008-06-10
Filing date: 2009-06-04
Publication date: 2009-12-17

Abstract

The present invention relates to a device for detecting an analyte in a fluid or for performing a reaction in a fluid comprising a capture probe module, wherein the capture probe module comprises a microchannel comprising one or more surface area elements confined by a porous barrier, and wherein particles are trapped by said porous barrier within the one or more surface area elements, and wherein said particles may not pass said porous barrier but the fluid may pass said porous barrier. Also methods for the production of the devices and the use of the devices is subject of the present invention.

Description

Diagnostic device

FIELD OF THE INVENTION

The present invention relates to a flow-through diagnostic device.

BACKGROUND OF THE INVENTION Devices for molecular diagnostics are generally built around substrates that contain patterns of biosensors for detecting the presence of specific analytes in a sample. The biosensors generally comprise receptor molecules that are capable of binding with the biomolecular analytes to be detected, such as DNA, RNA, proteins, cells or drugs present within the sample. For example, for detecting whether an antigen is present in the sample, antibody binding may be used. For detecting the presence of DNA, protein interactions may be applied or a piece of a complimentary single-strand DNA.

Various detection methods have been developed to sense whether specific binding has occurred. Commonly, an optical detection method is used that is based on, for example, fluorescence, refractive index changes or spectral changes. Also localized changes in magnetic or dielectric behavior are being used. In a particular example of an optical biosensor, a membrane substrate is provided with an array of capture targets of different receptor molecules, for example, applied via inkjet printing and/or contact printing. The receptor molecules are brought into contact with the sample containing the analytes via a direct contact in a well plate or in a micro-channel using a so-call flow-over principle. In order to enhance the sensitivity of the detection device and for reducing the assay time needed to diagnose the sample, a flow-through principle may alternatively be used.

In order to detect the biomolecules of interest, i.e. the analytes in a sample, these biomolecules have to bind specifically to the corresponding capture probes. This step is often referred to as either hybridization, in case of a DNA target, or incubation, in case of a protein analyte. For determining whether the specific capturing took place, fluorescent labeled single strand DNA (ssDNA) fragments or antibodies may be used which will bind to the capture probe: analyte complex. The presence of the specific biomolecules can then be detected by means of an optical detection device, such as a CCD camera if the fluorescent probes are excited to emit light. As indicated above, the analyte of interest can be brought to the capture site of the diagnostic device either by flow-through or by flow-over. These two types of sample introduction give rise to two types of devices that mainly differ in the flow direction of the analyte towards the capture sites. In a flow-over device, the analyte flows parallel to a solid substrate. The substrate comprises an array of nanowells in which the capture molecules are located. The flow-over concept exhibits a low-pressure drop of the analyte flowing through the device. It is further possible to arrange the different biosensors in spots of a small size. A device of the flow-over type may be manufactured in such a way that the different spots are in series and the analyte may hit every capture probe. In practice, this often is not the case as the flow in a micro fluidic device normally is laminar so that the transport of the target molecules, that is, the analytes, to the capture sites should occur by diffusion. This results in long assay times if the channel depth is large.

WO 2006/062312 describes a biosensor for an on-the-spot analysis where commercial membranes are incorporated into microfluidic channels engraved on the surface of a plastic chip. WO 2007/012975 describes a hybrid device that combines a microfluidic component and a porous component.

SUMMARY OF THE INVENTION

There is a need for an improved detection device for detecting an analyte in a fluid. It is thus an object of the present invention to provide a diagnostic device which overcomes at least some of the limitations of the known devices. In particular, a preferred diagnostic device of the present invention provides a high signal-to-noise ratio while only requiring small sample volumes and a high surface/volume area.

The present invention provides a device for detecting an analyte in a fluid or for reacting a starting material in a fluid comprising a capture probe module, wherein the capture probe module comprises a microchannel comprising one or more surface area elements confined by a porous barrier and wherein particles are trapped by said porous barrier within the one or more surface area elements and wherein said particles may not pass said porous barrier but the fluid may pass said porous barrier. Preferably, the particles comprise capture probes.

The present invention also relates to a method for making the device of the present invention, comprising the steps of: providing a microchannel with porous barriers by using a replicating technique, dispensing particles in between said porous barrier within the one or more surface area elements.

Preferably, the particles are pre-functionalized with capture probes. The present invention also relates to the use of a device of the present invention for diagnosis or as research tool for detecting an analyte in a fluid.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates a schematic representation of a device manufactured via the method according to the invention. Left: Micro fluidic channel and localized elements fabricated with a replication technique. Right: device after dispensing microspheres in the space confined by the localized elements.

Fig. 2 shows a microscopic photograph of an example of a device according to the invention

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention provides a device for detecting an analyte in a fluid or for performing a reaction in a fluid comprising a capture probe module, wherein the capture probe module comprises: a microchannel comprising one or more surface area elements confined by a porous barrier, and wherein particles are trapped by said porous barrier within the one or more surface area elements, and wherein said particles may not pass said porous barrier but the fluid may pass said porous barrier.

In a preferred embodiment of the invention, the particles comprise capture probes. If the dispensed particles do not comprise the capture probes said dispensed particles may at least be capable to immobilize capture probes. Examples of methods to immobilize capture probes on particles are described below. For example, if antibodies are used as capture probes, these can be covalently coupled to dispensed particles (e.g. microspheres) via standard EDC/NS chemistry, wherein the dispensed particles may be chemically modified with e.g. COOH functional groups. In general, the "capture probe module" is a part of the device in which binding of analytes from a fluid or starting materials for a reaction to capture probes may occur. The device may comprise one or more capture probe modules.

A "microchannel" (or "micro-channel") in the context of the present invention is a channel within the device and/or the capture probe module of the device allowing for the flow of fluid through the device and having has a width of less than 1000 μm, preferably a width of less than 300 μm and most preferably less than 150 μm. In a particular embodiment said channel has a length of preferably less than 1000 μm, more preferably less than 500 μm, most preferably less than 200 μm but at least more than 1 μm and preferably more than 10 μm. However, the length of the channel is not limited in the context of this invention. The device and/or the capture probe modules may in some embodiments comprise more than one microchannel. Preferably the microchannels are arranged in parallel to each other and in parallel to the flow direction.

In a preferred embodiment the channels may be closed from their top side in order to avoid transport of analyte along unwanted voids left between the capture probe modules by adhering a cover plate covered with soft compliant material facing the channels and the capture probe modules. In a preferred embodiment the compliant material is loosely crosslinked polydimethyl-siloxane.

The design of the device and the channels of the device is such that the pressure drop over the capture probe module is relatively small, allowing the placement of a large number of such capture probe modules in series without the need of using large pressure to transport the analyte through the channel. Also, the design of the capture probe module comprising elements, is such that the average diffusion distance of target molecules to reach the capture probe at the surfaces of the particles is relatively small, which translates into short assay times.

The device may have a certain flow path (also termed "flow channel"), i.e. the route an applied fluid flows through said device. The capture probe module(s) are arranged in the flow path, such that the fluid is passed through said capture probe modules. They may for example be arranged in series or in parallel, e.g. allowing for multiplexing. When arranged in series, the pore size of the porous barrier may decrease over the flow path to avoid clogging. The capture probe modules may comprise different or the same capture probes. When comprising different capture probes, simultaneous detection of different analytes is possible.

The present invention thus combines properties of flow-through and flow-over devices so that the specific advantages of both types of devices are provided. "Surface area elements" in the context of the present invention are parts of the microchannel where capture probes are immobilized on a surface or where immobilization of capture probes to a surface is possible, i.e. in the context of the present invention the surface are elements are those areas in which the particles are present. The particles may have a random shape or a regular shape. It is preferred that the dispensed particles have a regular shape. A regular shape may for be an ellipsoid, particularly a sphere, an oblate spheroid (disk-shaped), a prolate spheroid (cigar-shaped) or a scalene ellipsoid. Also egg-shaped or cylindrical particles and the like may be used. Preferably, the dispensed particles have dimensions in the range of from about 50μm to 50 nm. However, the size of the particles must in any way be larger than the pores in the porous barrier, in order to trap the particles within the surface area elements. The dispensed particles are produced by dispensing the particles into the surface area element as described herein below. The dispensed particles are preferably made from polymeric materials such as polystyrene, polymethylmethacrylate or melamine resin, ceramic materials such as glass or titaniumoxide or metallic materials such as silver or gold. The surface of the dispensed particles may preferably comprise carboxylic acid or amino groups. Preferably, the particles are transparent for the certain excitation and emission wavelength.

In a particular embodiment the dispensed particles are magnetic particles, e.g. magnetic microspheres. Said magnetic microspheres may be moved within the area confined by the porous membrane present in the microfluidic channel via an external magnetic field. This can be used for active mixing of the liquid sample flowing through the microfluidic channel and increase the contact between the functionalized microspheres and the sample. It would be possible to use the magnetic microspheres purely as an active but localized mixing element in a microfluidic channel. In addition the magnetic microspheres can be up- concentrated creating a higher signal density.

"Pre- functionalized" particles means that the dispensed particles may either comprise surface-immobilized capture probes or may at least comprise functionalities that allow for the attachment of capture probes.

The porous barrier may for example be a sieve-like structure, preferably made of vertical protrusions or may be a membrane with pores of a size allowing for the passing of the fluid but not of the passing of the dispensed particles. In some embodiments, the porous barrier comprises parallel pillars or vertical protrusions.

The protrusions may preferably by lamellar. The porous barrier is preferably made of a polymeric material such as polystyrene, polymethylmethacrylate, polycarbonate, polyamide or polyethylene terephthalate. Alternatively, the porous barrier may be for example made from aluminum oxide. The device may comprise a microfluidic system with microchannel(s) provided with placeholders, in the form a micro-sieving elements (e.g. equidistant parallel pillars or vertical protrusions), between which the dispended particles are trapped.

Capture probes according to the present invention may be capable to bind to the targeted analytes specifically in a said fluid. Capture probes may be nucleic acids such as a DNA, RNA, aptamers, antibodies, Fab fragments, Fc tails. Capture probes may be proteins, such as e.g. receptors, antibodies. Antibodies may be used in form of polyclonal or/and monoclonal antibodies. A capture probe may be a drug or a cell or other chemical compounds.

Capture probes may be bound to the dispensed particles already before the test, for example by adsorptive or covalent interactions. Also specific high affinity interactions may be used, e.g. streptavidin or avidin/biotin or antibody/antigen interactions. As another preferred alternative the surface of the dispensed particles may be functionalized with specific chemical end-groups, for example by graft polymerization. In a specific example the surface is modified with N-hydroxysuccinimide groups which react with primary amino groups in many protein antibodies. Thus, the device can be tuned for any desired application. The biomolecules of interest (i.e. analytes) bind specifically to the corresponding capture probes. Various detection methods have been developed to detect the bound analyte whether the specific binding has occurred. The detection method according to the present invention may preferably be an optical detection method based on e.g. fluorescence; it may be based on refractive index changes, spectral changes, but also changes in localized magnetic or dielectric behavior. In principle, all labeling techniques which can be used of the type described are employed, including labeling with radio isotopes, enzymes, fluorescent, chemo luminescent or bio luminescent labels and directly optically detectable colour markers, such as, for example, gold atoms and dye particles, magnetic particles.

So-called sandwich assays in which two capture probes, e.g. two receptors or two antibodies, directed against different epitopes of the analytes to be determined, are frequently used for the quantitative determination of analytes in a sample. In this method a first soluble capture probe, e.g. receptor / antibody, is preferably directly or indirectly coupled with a signal- generating system i.e. with a label, whereas a second capture probe, e.g. receptor/antibody, is present coupled to a solid phase or is provided with a binding component such as e.g. biotin which is able to bind to a suitably coated solid phase.

In case of a sandwich immunoassay it is preferable to mark both antibodies with parts of a detection system which, when both antibodies are integrated into a single sandwich, permit signal generation or signal triggering. Such techniques can be designed in particular as fluorescence extinction detection methods.

Exemplary, a sandwich immunoassay may be conducted with a first antibody immobilized on the dispensed particle. The sample containing the analyte is incubated on the dispensed particle. Excess analyte is removed by washing. A biotinylated second antibody is added. In order to remove unspecific binding another washing step is performed. Cy5/Alexa 647 labeled streptavidin is then added which will bind to the antibody-analyte-antibody complex and will give the signal which may be measured via a CCD camera.

Alternatively the immunoassay may be performed in a one-step assay with the help of an antibody or another capture probe which is immobilized on a substrate, the dispensed particle, whereas the analyte is labeled.

Alternatively, the immunoassay may be based on the known principle of competitive immunoassay, the most typical member of which in turn is a radioimmunoassay (RIA) and an enzyme immunoassay (Hartter et al.; Clin Chem Lab Med 2000, 38 (1) 27 -32). In the case of a competitive immunoassay both the sample containing the unlabeled analyte and a labeled analyte are incubated together on the dispensed particle. The analyte may be labeled e.g. via biotinylation. After incubation unbound analytes are removed during a washing step. Streptavidin-Cy5 solution is added. In this exemplified assay there is an inverse relationship between signal intensity and the amount of analyte present. Higher signal would mean that the analyte of interest is present in minute quantities. The signal intensity may be measured via a CCD camera.

Further immunoassay embodiments are known to the person skilled in the art and may be performed according to the present invention.

It is preferred that the device further comprises a detection device ("detector"), preferably an optical read out. A detection device may be selected from the group consisting of a photodiode light sensor for measuring absorption or emission of light, CCD camera for measuring optical signals from an array of probes, confocal microscopy, GMR (giant magneto resistance) sensor - for magnetic particles, gamma detector (radio-isotopes), a capacitance bridge - for measuring changes in dielectric properties. Preferably, the detector may be a fluorescence detector for detecting fluorescence, e.g. of analytes comprising fluorescence labels. All optical detectors in this context may be termed "optical read out". In one embodiment for each capture probe module one detecting device is present in another embodiment two or more capture probe modules share a common detector.

The microchannel comprises preferably more than one surface area element bounded by a porous barrier with trapped dispensed particles. It is also possible that the different surface area elements comprise confined particles with different capture probes.

The surface area element with the capture probes may also be a reaction zone for the reaction of starting materials. In this embodiment the capture probes may act as catalysts or reaction partners for captured starting materials, e.g. by modifying or cleaving the bound starting material.

A subject of the present invention is also to provide a method for making the devices of the present invention, comprising the steps of: providing a microchannel with porous barriers by using a replicating technique, dispensing particles in between said porous barrier within the one or more surface area elements.

The particles can be pre- functionalized with capture probes. Preferably, the "replicating technique" is selected from the group comprising photolithography hot-embossing, injection molding and micromolding. Alternatively, the porous barrier is fabricated by phase-separation of polymer/solvent mixture.

In a preferred embodiment of the method, dispensing is carried out with an inkjet printer. Apart from inkjet printing, various other methods can be chosen from simple pipetting by hand to complex offset printing.

A person skilled in the art will understand that there are further possibilities to manufacture the inventive device. Direct-write technologies as ion beam writing, electron beam lithography, replication techniques by molding of a liquid polymer precursor, etc.

Subject of the present invention is further a method for detecting an analyte in a fluid using a device according to the present invention comprising the following steps: applying a fluid containing an analyte to the device; making the fluid flow through the capture probe modules; and detecting the captured analyte on the carrier substrate.

In order to detect whether in the capture probe modules the specific binding of analyte to the capture probe has occurred, the known methods may be used, such as optical methods based on fluorescence, refractive index changes, spectral changes, etc., but also changes in localized magnetic or dielectric behavior may be used.

The present invention also relates to a method for processing starting materials in a device according to the present invention comprising the following steps: applying a fluid containing a starting material to the device; making the fluid flow through the capture probe modules under conditions allowing a reaction of the starting material bound to capture probes to take place; and detecting the reaction product.

The present invention also relates to the use of a device of the present invention for diagnosis or as research tool for detecting an analyte in a fluid or for reacting starting materials.

The method of the invention can be used in the production of devices for the efficient and sensitive scanning of fluids on the present and/or amount of DNA, proteins, antibodies, tissue, cells, drugs and other chemical compounds. By functionalizing the surface of the dispensed particles with specific chemical end-groups, for example by graft polymerization, the device can be tuned for any desired application. In addition to minimize a specific binding, the sensor can be blocked before performing the immunoassay e.g. 5% BSA, gelatin or casein.

The fluid in the context of the present invention may particularly be selected from a group comprising patient fluid, buffered fluid, inorganic and organic solvents. The fluid may also be selected from a group comprising whole blood, plasma/serum, urine, saliva, bal fluid, stomach fluid, wounds, environmental substances.

The analyte in the context of the present invention may for example be selected from a group comprising proteins, DNA, RNA, microRNA, DNA methylated, cells, metabolites, drugs, peptides, toxins, pathogens, small molecules and chemical compounds.

"An analyte" in the context of the present invention may also refer to more than one analyte.

The "starting material" in the context of the present invention may for example be selected from the group comprising whole blood, plasma/serum, urine, saliva, bal fluid, stomach fluid, wounds, environmental substances. When bound to said capture probes, in some embodiments a reaction might occur that modifies, e.g. cleaves, the starting material, wherein the capture probe serves as a catalyst or a reaction partner, e.g. the capture probe may be an enzyme or ribozyme or comprise an enzyme or ribozyme. Further examples for reactions include (but are not limited to) DNA aptamer, RNA aptamer, peptide, oligonucleotide, sDNA molecule. The particles with the capture probes are in such an embodiment thus forming a "reactor"-like entity. The detection of the reaction products from the reaction of the starting material on said capture probe must not necessarily take place within the surface area element of the device. It is preferred that in such an embodiment the detection may take place elsewhere in the device. The reaction products may or may not be bound to the capture probes or parts of the capture probes after the reaction. The reaction products may or may not be retained on the surface area element after reaction. Examples for a starting material include DNA, RNA, proteins, environmental toxins, small molecules, drugs, chromogenic substrate and chemo luminescence substrates .

Claims

CLAIMS:

1. Device for detecting an analyte in a fluid or for performing a reaction in a fluid comprising a capture probe module, wherein the capture probe module comprises: a microchannel comprising one or more surface area elements confined by a porous barrier, and wherein particles are trapped by said porous barrier within the one or more surface area elements, and wherein said particles may not pass said porous barrier but the fluid may pass said porous barrier.

2. Device according to claim 1, wherein the particles comprise capture probes.

3. Device according to any of claims 1 and 2, wherein the device further comprises a detector.

4. Device according to any one of claims 1 to 3, wherein the particles are transparent.

5. Device according to any one of claims 1 to 4, wherein the porous barrier comprises parallel pillars or vertical protrusions.

6. Device according any one of claims 1 to 5, wherein different surface area elements comprise particles with different capture probes.

7. Device according to any one of claims 1 to 6, wherein the particles are magnetic microspheres.

8. Method for making a device according to any one of claims 1 to 7, comprising the steps of: providing a microchannel with porous barriers by using a replicating technique, dispensing particles in between said porous barrier within the one or more surface area elements.

9 A method according to claim 8, wherein the particles are pre-functionalized.

10. Method according to any one of claims 8 or 9, wherein dispensing is carried out with an inkjet printer.

11. Use of a device according to claims 1 to 7 for detecting a diagnostic analyte.

12. Use of a device according to claims 1 to 7 as research tool for detecting an analyte in a fluid.

13. Use according to claim 11 or 12 wherein the fluid is selected from a group comprising whole blood, plasma/serum, urine, saliva, bal fluid, stomach fluid, wounds and environmental substances.

14. Use according to claim 13 wherein the analyte is selected from a group comprising proteins, DNA, RNA, small molecules, drugs, toxins, pathogens and peptides.