The invention relates to methods for immobilizing microvesicles, means and methods for detecting them, and uses thereof, a microfluidic chip for use therein and a chip mould for the preparation of a microfluidic chip. Microvesicles (MVs) or microparticles (MPs) are fragments of plasma membrane ranging from 10 nm to 800nm shed from almost all cell types during activation and/or apoptosis. They are thought to originate directly from the plasma membrane of the cell or from cellular material and comprise the antigenic content of the cells which they originate from. It is shown in vitro that microvesicles are released from cells upon activation and apoptosis (1-5). Blood microvesicles are a population of microvesicles and may originate from different cell types and from different cellular compartment origins. As a consequence, different types of microvesicles are discerned. Different types of microvesicles comprise apoptotic bodies, exosomes and ectosomes. These different types of microvesicles originate from different cell types and also differ in content of lipids and proteins (Boulanger et al. Hypertension 2006; 48: 180-86.). The majority of blood microvesicles consists of platelet-derived microvesicles, characterized by their surface antigens, such as CD41 and CD61 (6, 7). The precise mechanism by which microvesicles are released has not yet been elucidated; however an increase in cytosolic calcium concentration may be necessary to trigger vesicle release (8). Some specific enzymes (floppase, scramblase) are thought to help in remodelling the plasma membrane by inducing the translocation of anionic phospholipids, such as phosphatidylserine (PS) from the inner membrane to the outer membrane (9; 10). This event is followed by microvesicle release and cytoskeleton degradation by Ca2+- dependent proteolysis (11 ). Several studies point to a biological role of microvesicles as an inducer of angiogenesis and cancer metastasis (12-13). Microvesicles bearing active FasL have also been reported to induce lymphocyte apoptosis (14, 15). Our group and others have addressed the pro-coagulant features of microvesicles (7, 16-18).

In recent years blood microvesicles have increasingly received attention as a potential biomarker in the diagnosis and prognosis of disease. However, such studies have been hampered by the lack of accurate methods for the detection and quantification of microvesicles. Different methods and combinations of methods have been used including enzyme-linked immunoassays (ELISA) capturing microvesicles with immobilized annexin V or cell-specific antibodies (19-24) and flow cytometry (4, 5, 7, 16, 25). A drawback of ELISA methods is that it does not provide information on the actual numbers of microvesicles (27). Other disadvantages of ELISA methods are interference of soluble antigens and lack of information on size distribution. A disadvantage of flow cytometry (FACS) is that the size of microvesicles challenges the lowest detection limits of FACS and FACS is therefore inaccurate for determining a number of parameters of microvesicles (size, roughness etc.). The reason for the limited accuracy is that the laser light employed in the conventional flow cytometers which excites at a wavelength of 488 nm makes the measurement of the microvesicles smaller than 488 in diameter not reliable (28). There are at least two reasons why the detection of small microvesicles is not reliable employing methods using light scattering, such as FACS. In the first place, when using light scattering, the angular dependence of the scattering vanishes for small particles: small particles scatter light equally in all directions. Therefore, the intensity is only indirectly related to the size. Secondly, the number of fluorophores that can be bound to a small particle is less as the available surface is less. In both methods smaller particles contribute less to the total signal: the fluorescence signal is proportional to r ² and the light scattering is proportional to r ⁶ for small particles. Therefore, a FACS is not ideal for detecting microvesicles smaller than 488 nm (29). Other potential detection methods require the immobilisation of microvesicles on a flat surface to enable further detection steps. One of the problems is that immobilisation of a sufficient number of microvesicles on a surface for further detection purposes is difficult. High speed centrifugation is often used to achieve a sufficient yield, but a major drawback of high speed centrifugation is that it induces fusion of microvesicles (Langmuir 2007, 23, 9646- 9550). Fusion affects number counts and it prevents establishing the origin of membrane bound proteins present in a microvesicle.

Surprisingly, the inventors have established a method which addresses one or more of the above-mentioned problems. The invention provides a method to immobilize natural microvesicles (which are considered to range from 10-800 nm in diameter, as indicated above) comprising steps of: coating a surface with molecules having an affinity for said microvesicles to obtain a coated surface; allow a fluid comprising said microvesicles to contact said coated surface by applying a laminar flow to said fluid to immobilize said microvesicles to said coated surface. An advantage of this method is considered to be that the number of microvesicles that is immobilized on the surface is higher than when using existing methods and therefore results in preparations with a higher density of microvesicles. Another advantage is that this method is considered not to induce fusion of microvesicles. Another advantage is that a larger variety of different microvesicles can be obtained using this method. Another advantage is that larger microvesicles can be obtained when compared to other techniques. It is considered that the method can be used almost ex vivo, removing the need for ultracentπ ^' fugation and washing, which can potentially disturb the microvesicles and cause systematic errors. Thus, only minimal processing of the fluid may be necessary. Another advantage is that the capture process is considered to be more efficient. Another advantage is that the method according to the invention allows for multiple passes (of the fluid comprising the microvesicles) over said coated surface. In a preferred embodiment, said method comprises a preprocessing step eliminating interfering material, preferably cells and/or proteins. An advantage thereof is considered to be the enhancement of the detection sensitivity and/or selectivity. In another embodiment, said method comprises fluid subfractionation. Preferably, subfractionating is performed using a microfluidic device. Thus, for example, one example of sub-fractionation is the direction (using the microfluidic device) of the fluid flow containing microvesicles to areas of the surface that are covered with different antibodies (and, therefore, with different binding affinities). The resulting split flows would be depleted differently as a result of the combined effect of the binding affinity differences and the ligands that are present on the surface of the microvesicles. Another advantage is considered to be that also smaller microvesicles are obtained when compared to other techniques. Another advantage is that the method is considered to result in preparations containing less aspecific signals.

The term microvesicles refers to particles having at least a bilipid layer and proteins. The term radius (r) and diameter refer to the radius/diameter determined with respect to the smallest length of a straight line through the center of a microvesicle from side to side of a microvesicle. Preferably, microvesicles are natural particles of a cellular origin. Microvesicles may originate from any cell type, i.e. animal cells, plant cells, yeast and bacteria. Any fluid comprising said microvesicles can be used as a source of microvesicles. Preferred sources are bodyfluids, such as blood, plasma, serum, cell suspensions, urine, synovial fluids, saliva, sputum, milk, pleural fluid, ascites, cerebrospinal fluid, lymphoid fluids or eye fluids. It is considered that microvesicles can be released into any bodyfluid, for example from endothelial tissue in contact with that body fluid. An advantage thereof is that microvesicles can easily be isolated from fluids. Preferably, blood plasma or a product thereof is used as a source of microvesicles, because the presence of microvesicles or the number of microvesicles in blood plasma are indicative of the health status of an animal, as noted above. See also Burnier et al 2009; Pap et al 2009, which discuss the important physiological and pathological roles of blood microparticles in coagulation, inflammatory disease and tumor progression..

In a preferred embodiment, platelet poor plasma is used as a source of microvesicles. Preferably, platelet poor plasma comprises fewer platelets than normal plasma. More preferably, platelet poor plasma comprises fewer than 10 * 10 ⁹ platelets per liter. An advantage thereof is that preparations containing few platelets are obtained, as presence of platelets complicates analysis. Examples of methods for preparing platelet poor plasma are described in the Examples and will be well known to those skilled in the art. Typically platelet poor plasma is prepared by double centrifugation of blood (in the presence of an anticoagulant, as will be well known to those skilled in the art, for example sodium citrate) at 2,000xg or similar (typically between 1 ,800xg and 2,500xg), for example for 10 minutes at 20° C without brake. See also Example 7. An example of a method for isolation of microvesicles from blood plasma is described in example 1 , though it considered that such a method involving concentration of the microvesicles, for example by high speed or ultra- centrifugation (for example at 18,890xg, for 30 minutes, with minimum brake as in Example 1), is not necessary with the method of the present invention, which allows microvesicles to be immobilised from a biological fluid such as plasma without the need to concentrate the microvesicles first. In another embodiment, preferably less than 2 ml of said fluid is used in the method according to the invention. An advantage thereof is that this limits the amount of sample material required. More preferably, said fluid has a volume of less than 100 microliter, preferably less than 10 microliter. A suitable aliquot of platelet poor plasma to analyse may be around 250μl, 200μl or 150μl. If performing microparticle isolation, 750μl of PPP may be needed.

In a preferred embodiment said source of microvesicles is a source wherein the number of cells is depleted. For example, platelet poor plasma is an example of a fluid in which the number of cells is depleted, as will be well known to those skilled in the art. The number of cells can be depleted by centrifugation, as will be well known to those skilled in the art, but it is desirable to avoid high speed or ultra centrifugation in order to avoid altering the properties of the microvesicles. In another preferred embodiment said source is a source, wherein the amount of proteins, preferably soluble proteins is depleted. An advantage of depleting cells and/or proteins is that analysis of microvesicles is facilitated, because cells and/or proteins complicate analysis and may lead to blocking of a microfluidic flow system. Methods of depleting proteins or cells in fluids are known in the art. In a preferred embodiment said fluid comprising microvesicles is concentrated by means of filtration prior to use. In a preferred embodiment, microvesicles comprise exosomes. The term "exosome" refers to externally released vesicles originating from the endosomic compartment of cells. In preferred embodiments, said cells comprise tumor cells, endothelial cells or immune cells. Preferred is that said immune cells comprise antigen presenting cells, dendritic cells, macrophages, mast cells, T lymphocytes or B lymphocytes. Preferably, exosomes are of endosomal origin. Preferably, they are secreted in the extracellular milieu following fusion of late endosomal multivesicular bodies with the plasma membrane. Preferably exosomes have a size of 60-100 nm in diameter, or a density of 1 ,13 to 1 ,21 g/dL in a sucrose gradient. Preferred exosomes are characterized by an enrichment in HSP 70, tetraspanins, Tsg101 , Alix and/or MHC molecules. An advantage of using exosomes is that (tumor-derived) exosomes are sometimes used as tumor antigen bearing vehicles. In addition, Toxoplasma gondii antigens pulsed DC-derived exosomes can be used for immunoprophylaxis. There is therefore a need in the art for improved methods of exosome detection.

In another preferred embodiment, microvesicles comprise ectosomes. The term "ectosome" refers to small right-side out vesicles released by cells by direct budding from the cell surface. Preferably, ectosomes are released by actived polymorphonuclear leukocytes (PMNs). Preferably, they bind complement. Preferred ectosomes are immune adherent similarly to large immune complexes or certain microorganisms. Preferably, ectosomes are characterized by their capability to down-modulate monocytes/macrophages activation in vitro similarly to apoptotic cells. Preferably, ectosomes are used as characterized according to Gasser et al. (Experimental Cell Research.Volume 285, Issue 2, 1 May 2003, Pages 243-257).

In another preferred embodiment, microvesicles comprise microvesicles which are released during apoptosis or other processes that compromise the integrity of the outer cell membrane. In another embodiment, microvesicles comprise blood lipoprotein particles such particles are indicative of obesity and diabetes. Any molecule which has an affinity for microvesicles suitable for coating the selected surface material can be used. With a molecule having an affinity for microvesicles is meant that such molecule is capable of binding covalently or non-covalently to a molecule present on a microvesicle. Preferably said molecule present on a microvesicle is a membrane-bound molecule. Preferably, molecules having a high affinity for a microvesicle are used. Preferably, affinity is expressed as a dissociation constant. Preferably, molecules having a dissociation constant lower than 0.1 nM for microvesicles are used, more preferably lower than 10 nM. More preferably, molecules having a dissociation constant lower than 10 ^{" 15} M for microvesicles are used. In another preferred embodiment, an affinity for a microvesicle is used with a dissociation constant in a range between 0.1-10 nM. Methods of determining affinity are known in the art. Preferably, a method is used as described in Johnson et al. Journal of Molecular Biology 368 (2): 434-449. Any surface that is suitable for immobilization using coatings having an affinity for microvesicles can be used. Preferred surfaces are made of material comprising glass, mica, plastic, metal or ceramic materials. There are various methods known for coating surfaces having affinity for (glyco)-proteins, cell membranes or biomolecules in general. Typically, these methods use a reactive group which binds covalently or non-covalently to a certain biomolecule. For example, slides coated with aminoproplylsilane are used for non-covalent adsorption of protein. Epoxysilane coated slides are reactive with lysine, arginine, cysteine and hydroxyls at pH 5-9. Aldehyde coated slides are reactive with lysine and arginine where pH 7-10 drives Schiff's base reaction. A skilled person will know how to select the right coating suitable for use in combination with the selected surface material and test the affinity for microvesicles.

The surface may be smooth enough that microvesicles can be identified using AFM, as discussed further below. It may be desirable that the surface does not interact negatively with the proteins of the blood or other body fluid (for example does not lead to clotting, as discussed further below). It may also be desirable, particularly when using AFM, as discussed further below, that the surface is not disturbed by the removal of a microfluidics chip. It may also be desirable to have a surface that does not autofluoresce in an optical detection system. Typically the surface is a mica surface or polished optical wafer surface. At least part of the mica surface or polished optical wafer surface may have a hydrophilic coating, which may be useful in aiding removal of a microfluidics chip. The hydrophilic coating may comprise, for example, PEG (polyethylene glycol), ethanolamine, DETA (Diethylenetriamine), APDM (3-aminopropyldimethylethoxysilane) or poly-lysine. A coating may be applied to the surface that comprises one or more components to render the surface hydrophilic and one or more different components to facilitate binding of molecules having an affinity for microvesicles to the surface.

The resulting coated surface is put into contact with microvesicles by applying a laminar flow to a fluid comprising said microvesicles. Said fluid can be any fluid that is compatible with microvesicles. With compatible is meant that the integrity of the microvesicles remains intact, which means that at least phospholipids and/or membrane proteins of the microvesicles are present. Preferably said fluid comprises plasma, cell culture medium, phosphate buffered saline (PBS), phosphate buffered potassium, or 4-(5 2-hydroxyethyl)- 1-piperazineethanesulfonic acid (HEPES). Thus, the fluid comprising the microvesicles that contacts the coated surface may be or may comprise platelet poor plasma, for example as described in Examples 7 and 8. The fluid may comprise one or more anticoagulants, as known to those skilled in the art. For example, the fluid (particularly blood plasma or platelet poor plasma) may comprise sodium citrate or EDTA or EGTA. For example, collection tubes containing citrate or EDTA are readily available and a body fluid may suitably be collected into such a tube. The fluid may be platelet poor plasma diluted with EDTA-enriched HEPES buffer, for example as set out in the Examples.

It may be desirable for the time between the drawing of the blood (or other biological fluid) and the microvesicle test should be as short as possible (for example less than 2 hours or more preferably 1 hour) in order further to minimise the likelihood of clotting occurring particularly during the contact between the fluid and the coated surface.

It may also be desirable to expose an anticoagulant to surfaces contacted by the fluid comprising microvesicles (for example, to the channels formed by the microfluidic chip and surface and tubing connected to the microfluidic chip) before contact between those surfaces and the fluid comprising microvesicles. For example, exposing of those surfaces to a fluid comprising EGTA, for example 10OmM EGTA in PBS or a saturated solution of EGTA in PBS (1 M 7.6pH) , for example, is considered to be particularly useful.

A laminar flow is a flow regime characterized by high momentum diffusion, low momentum convection, pressure and velocity independent from time. Said laminar flow is characterized by a Reynolds number of less than 2000 and higher than 0. Preferably, laminar flows with a Reynolds value between 0 and 1000, more preferably between 0 and 500 and most preferably between 0 and 100. A skilled person will know how to achieve a laminar flow. Any method capable of applying a fluid in a laminar flow to said coated surface can be used. Preferred is a laminar flow which is linear in one direction. Preferred is a laminar flow that optimizes the contact area, contact time and flow speed to the functionality of the fiuidic device. Further preferred is a laminar flow through a channel comprising a functionalized wall.

Preferably, a method is used wherein the affinity for the microvesicles is specific. An advantage of a specific affinity is that binding of undesired molecules or particles is reduced. Preferred methods are methods in which an affinity linker is bound covalently or non-covalently to the surface. Binding an affinity linker to a surface may require a chemical treatment of said surface, depending on the material of said surface. Examples for different treatments of mica are provided in example 2 and 3. An affinity linker is a molecule that is capable to covalently or non-covalently bind to a binding partner, resulting in a complex between said affinity linker and said binding partner. Said binding partner can be a molecule capable of binding to a microvesicle or it can be a molecule present on the microvesicle that can directly interact with said affinity linker. Examples of affinity linkers and their binding partners are: Streptavidin or avidin and biotin; an antibody and antigen; a ligand and receptor, lectin and saccharide, protein A and/or protein G-immunoglobulin constant region, and Tag peptide sequence and Tag antibody. The terms affinity linker and binding partner refer to their function. Therefore, depending on how the above mentioned affinity linker-binding partner combinations are used, the terms are exchangeable. For example a biotin can be an affinity linker if it is bound to the surface or it can be a binding partner when it is bound to the microvesicle. Any method to bind an affinity linker to a surface can be used. Methods to bind an affinity linker to a coated surface differ depending on the material of surface and the nature of the affinity linker. A skilled person will be able to select the correct method suitable for the type of surface material and affinity linker of choice. Methods to bind an affinity linker to a surface are known to a skilled person. Methods to bind antibodies to metal or silicon surface are well known in the art. Preferred methods are described in Bioelectrochemistry Volume 66, Issues 1-2, April 2005, Pages 11 1-115. Methods to bind antibodies to glass surface are also known and described in J Colloid Interface Sci. 2002 Aug 1 ;252(1):50-6. Preferably, the affinity linker is an antibody. Preferred methods of binding an affinity linker to a surface are provided in example 2 and 3. Preferred methods to attach antibodies to glass, silica or quartz are described in Pierce Tech Tip #5, 1/2006 on www.piercenet.com (downloaded 16 December 2008). A preferred method of binding an antibody to a surface comprises coating a surface with a DNA using methods for DNA immobilisation as known in the art, followed by allowing a fluid comprising said antibody fused with a nucleotide fragment having a complementary nucleotide sequence capable of hybridizing to said DNA immobilized to said surface to hybridize with said DNA. An advantage of said method is that it is significantly cheaper and faster.

Preferably said surface is atomically flat. This is an advantage, because surface roughness is difficult to distinguish from signals caused by height of a microvesicle when using an Atomic Force Microscopy. For applications other than AFM and preferably for optical applications it is preferred that said surface is a flat surface, preferably an optically flat surface. In a more preferred embodiment, said laminar flow is applied using microfluidics. The term microfluidics refers to devices, systems, and methods for the manipulation of fluid flows with characteristic length scales in the 10 micrometer range up to 1 millimeter (see for a more complete overview: Manz, A. and Becker. H. (Eds.), Microsystem Technology in Chemistry and Life Sciences, Springer-Verlag Berlin Heidelberg New York, ISBN 3-540-65555-7). An advantage is that microfluidic systems possess the capability to execute operations more quickly than conventional, macroscopic systems, while consuming much smaller amounts of chemicals and fluids. A preferred method for applying a laminar flow using a microfluidic chip is provided in example 4.

In a more preferred embodiment, said laminar flow is created using a microfluidic chip. Preferably said microfluidic chip comprises at least one microfluidic channel with an inlet and an outlet, wherein said at least one microfluidic channel has at least one gap at a surface of said microfluidic chip enabling contact between the contents of said at least one channel and a surface placed next to the gap. An advantage of said microfluidic chip is that it enables direct contact between a fluid in said microfluidic channel and a surface. Preferably said inlet and/or said outlet are positioned at a different surface of said microfluidic chip than the surface comprising said gap in said microfluidic channel. An advantage thereof is that this enables clamping between said microfluidic chip and said surface, while maintaining access to said inlet and/or outlet. In a preferred embodiment, holes are provided in said microfluidic chip, enabling screws or other means to attach a surface to said microfluidic chip. An advantage thereof is that a surface can be attached to said microfluidic chip to achieve a contact which prevents leakage of a fluid from said microfluidic channel. An advantage of using a microfluidic channel is that the geometry of the channel enables a controllable inducement of a laminar flow. Preferably, said channel has a height which is less than 1 mm. An advantage thereof is that the surface to volume ratio of said fluid over said coated surface is optimized. In another preferred embodiment, the channel height above the coated area is less than the channel height in other parts of the microfluidic device. An advantage thereof is that the surface to volume ratio of said fluid over the coated area is optimized, while maintaining a flow through. More preferably, said channel height is less than 0.5 mm. An advantage thereof is that this height is optimal for fluids having a viscosity of plasma. Preferably, said channel has a width of less than 1 mm. An advantage thereof is that this results in a minimal contact area of said coated surface which is in contact with said fluid. Preferably, said minimal contact area is smaller than said coated surface area. Preferably, said minimal contact area is between 100 and 10000 square micrometer. In a preferred embodiment, said minimal contact area is less than 1 square millimeter. An advantage thereof is that this limits the surface area inspection time and increases the concentration of collected vesicles per surface area. More preferably, said minimal contact area is between 1 square micrometer and 0.1 square millimeter.

In another preferred embodiment, said channel comprises a filter which allows microvesicles to pass through. A filter is defined as a structure having at least an opening allowing a particle with a size smaller than said opening to pass through, whereas larger particles cannot pass said opening. More preferably, said filter comprises micro pillars. A micropillar is a structure comprising substantially perpendicular projections, such as the micropillars disclosed in WO 03/103835. Said projections or micorpillars are preferably made of a non-porous substrate, and form projections substantially perpendicular to said channel, said projections having a height (H), diameter (D) and a distance or distances between the projections such, that a microvesicle can pass through between said projections. In addition to optimizing the above-mentioned height, diameter and a distance or distances between the projections, the projections may be given a desired chemical, biological or physical functionality, e.g. by modifying the surface of said projections.

In another preferred embodiment is a method wherein said channel comprises a filter which allows particles smaller than a microvesicle to pass through. In this method, microvesicles are collected on the surface or said filter, a change of flow direction is then applied to re-suspend said microvesicles. This method further comprises a step of resuspending said microvesicles in a fluid before allowing said fluid comprising said microvesicles to contact said coated surface.

In another preferred embodiment, said microfluidic chip comprises a pumping system. Any system suitable of pumping fluid inside a microfluidic circuit can be used. Examples are described in PHYSICS AND APPLICATIONS OF MICROFLUIDICS IN BIOLOGY David J. Beebe, Glennys A. Mensing, Glenn M. Walker Annual Review of Biomedical Engineering, August 2002, Vol. 4, Pages 261 -286. In a preferred embodiment, said microfluidic chip comprises a deformable material to prevent leakage by means of self sealing. Such materials comprise, but are not limited to elastomers, preferably PDMS. In a preferred embodiment, said microfluidic chip comprises more than one channel. An advantage of a microfluidic chip comprising more than one channel is that different fluids can easily be applied to a (coated or uncoated) surface. In a preferred embodiment, said microfluidic chip has a dead volume of less than 10 microliter, more preferably less than 1 microliter. Dead volume is defined as the volume of the inner circuit which is not in direct surface connection with said coated surface and any of the volume of the inner circuit which is passed by said fluid after contacting said coated surface. Preferably, the volume of the residing capillary is excluded. In another embodiment the dead volume is defined as the volume in the inlet excluding the volume of the residing injected capillary.

In a more preferred embodiment said surface comprises mica. An advantage of mica is that it creates a molecular flat surface and can easily be cleaved.

As discussed further below it may be desirable to be able to separate the microfluidic chip from the surface. If the microfluidic chip comprises hydrophobic polymer, for example PDMS ₁ then it may be desirable for at least some of the surface that is in contact with the microfluidic chip to be hydrophilic, in order to aid separation of the chip from the surface. Pressure may be needed in order to secure the microfluidic chip in the desired place on the surface during use, for example as described in the Examples. A coating may be applied to the surface to render it sufficiently hydrophilic. The hydrophilic coating may comprise PEG (polyethylene glycol), ethanolamine, DETA (Diethylenetriamine). APDM or poly-lysine. A coating may be applied to the surface that comprises one or more components to render the surface hydrophilic and one or more different components to facilitate binding of molecules having an affinity for microvesicles to the surface. For example, an example with a si lane-treated surface would be to mix 3 parts carboxysilane with 1 part amino-silane. This results in a surface that is mostly hydrophilic (carboxy) but can have sufficient attachment points (amino). Phase separation is considered to arise only on a very small scale and is not considered to be detrimental. The skilled person can readily apply these principles to other systems.

Said molecules having an affinity for said microvesicles may comprise antibodies or Annexin-V. An advantage thereof is that this results in more specific binding of microvesicles. Any antibody specific for microvesicles can be used. Preferably an antibody that is specific for an antigen on microvesicles is used. Preferably, said antibodies comprise antibodies which bind to membrane bound antigens present on cells or cellular parts thereof associated with the release of microvesicles including but not limited to haematopoietic cells, endothelial cells and/or malignant cells, More preferred antibodies comprise antibodies capable of binding to membrane bound antigens present on and/or antigens present in haematopoietic cells, preferably platelets, leukocytes, monocytes and/or erythrocytes. Other more preferred antibodies comprise antibodies binding to membrane bound antigens present on platelets, preferably anti-CD41 , anti- CD41 a, anti-CD42a, anti-CD42b, anti-CD40, anti-CD40L, anti-CD62P, anti-CD61 and/or anti-CD63. Other more preferred antibodies comprise antibodies capable of binding to membrane bound antigens present on or antigens present in leukocytes or cellular parts thereof, more preferably anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD11a, anti- CD1 1 b, anti-CD20, anti-CD45, anti-CD66b, anti-CD66e, anti-CD68 and/or anti-CD162. Other more preferred antibodies comprise antibodies capable of binding membrane bound antigens present on or antigens present in erythrocytes or cellular parts thereof, more preferably, anti-CD235a. Other more preferred antibodies comprise antibodies capable of binding membrane bound antigens present on or antigens present in endothelial cells of cellular parts thereof, more preferably anti-CD31 , anti-CD51 , anti- CD54, anti-CD62e, anti-CD106, anti-CD144, 5 anti-CD146, anti-CD105 or anti-vWF. Other more preferred antibodies comprise antibodies capable of binding membrane bound antigens present on or antigens present in malignant cells or cellular parts thereof, more preferably anti-MUCI , anti-CD133, anti-Fas, anti-FasL, anti-EGFR. Other preferred antibodies comprise antibodies capable of binding G- coupled proteins. Other more preferred antibodies comprise antibodies capable of binding membrane bound proteins present on or proteins present in MV and which are involved in a coagulation cascade. More preferred antibodies comprise antibodies capable of binding tissue factor (preferably membrane bound tissue factor), more preferably anti-CD142. Other more preferred antibodies comprise antibodies capable of binding to proteins or factors involved in cell signalling. Other preferred antibodies comprise antibodies capable of binding to proteins present on cellular parts of cells. Other more preferred are antibodies capable of binding antigens present on exosomes, preferably anti-CD63 and/or anti- CD81. In another preferred embodiment, said antibody comprises an anti-VE Cadherin or an anti-glycophorin A. Other preferred antibodies comprise antibodies capable of binding to antigens susceptible to viral particles (HIV-1 ), more preferably said antibodies comprise anti-CXCR4 and/or anti-CCR5. Other more preferred antibodies are capable of binding to antigens derived from a bacteria, a parasite, and/or a cellular prion protein (PrP(C)).

An advantage of these antibodies is that they capture certain subtypes of microvesicles and therefore their use results in low aspecific binding.

It is considered that a covalent bonding of the antibodies to the surface may be desirable. For example, an antibody that has been specifically modified with a succinimide to preserve its function, may be used with an amine-comprising surface (for example with a coating comprising ethanolamine, APDM (3- Aminopropyldimethylethoxysilane), APTES (3-Aminopropyltriethoxysilane) or DETA). In another preferred embodiment, said surface is coated with an antibody directed against a constant domain of an antibody. An advantage thereof is that coated surfaces are obtained which can easily be coated with any antibody having an affinity for microvesicles. Preferred antibody directed against a constant domain of an antibody are antibodies directed against constant domains of IgG.

In a more preferred embodiment said method further comprises the detection of microvesicles using imaging. An advantage of this is that this allows measurements of one or more parameters comprising but not limited to particle geometry, shape, roughness, light scattering or dimensions of microvesicles or the presence of molecules on the surface of or inside microvesicles can be determined. Any method of imaging can be used in the method. Preferred methods of imaging comprise fluorescence microscopy, electron microscopy (EM), confocal microscopy, Raman spectroscopy, infrared spectroscopy or atomic force microscopy (AFM), or combinations thereof.

More preferably, said method comprises the detection using AFM. An advantage of AFM is that it is more accurate in detecting the size of microvesicles.

In another preferred embodiment of the invention, the detection is performed using an optical method. Any optical method may be used. Preferred optical methods comprise confocal microscopy, internal reflection fluorescence microscopy, ellipsometry/reflectometry, light scattering or surface plasmon microscopy. An advantage of said methods is that it can be implemented in high throughput detection methods. Even more preferably, these optical methods are calibrated using AFM measurements. An advantage thereof is that more reliable results are obtained.

Preferably, said calibration comprises determining the fluorescence light intensity relative to the microvesicle surface. Preferably, the absolute fluorescence light intensity of a surface comprising microvesicles with attached fluorescent antibodies is measured as well as the microvesicle surface concentration of said surface comprising microvesicles using AFM measurements. This calibration results in a specific fluorescence intensity per microvescicle. When using a method to produce said surface comprising microvesicles with attached fluorescent antibodies the concentration of microvescicles can be predicted by measuring said fluorescence intensity using said optical method. If detection using AFM is required then it is considered necessary to separate the microfiuidic chip from the surface so that the surface on which the microvesicles are immobilised is accessible for AFM analysis, as will be apparent to those skilled in the art and as described in the Examples. If optical detection is used then it is not considered necessary to separate the microfiuidic chip from the surface so long as the surface on which the microvesicles are immobilised is accessible to the wavelengths of light required for the optical detection method. Thus, if either the microfiuidic chip or the surface is formed from an optically transparent material it may not be necessary to separate the microfiuidic chip from the surface. Examples of suitable materials will be well known to those skilled in the art. For example, the surface may be formed from glass. Mica is not as transparent as glass, but could be used for some optical methods. PDMS is optically transparent but occludes mechanical contact. After calibration of optical methods a mica/PDMS device can be used for detection without detachment. Glass is a replacement for either mica or PDMS as channels can be made in glass and antibodies can be bound to glass. The combination of PDMS and mica is considered to be optimal for AFM detection and does not exclude optical detection for a broad range of wavelengths.

Also preferred is detection combining use of AFM and optical methods or other imaging techniques. A preferred combination comprises the combination between AFM with

LSCM. When AFM is combined with another type of optical detection method, measurements may be done separately for each detection method. A preferred combined use of said techniques has been described in Modern Research and

Educational Topics in Microscopy by Adams and Czymmek Modern Research and Educational Topics in Microscopy, page 68-76, A Mendez-Vilas and J.Diaz (Eds.).

Results of the measurements performed can be linked by storing the data together with position coordinates of the analysed microvesicles.

A preferred embodiment of the method further comprises a step wherein at least one species of antibodies is bound to the microvesicles. Said species of antibodies can be applied during the coating step before immobilization of microvesicles, in a step simultaneously together with the microvesicles or in a separate step after immobilization of said microvesicles. With a species of antibody is meant a detectable amount of antibodies directed against a single epitope, preferably said antibody is a monoclonal antibody. An advantage is that the presence of said species of antibodies on a microvesicle can be detected, which provides information about the presence of an epitope against which the antibody is directed on said microvesicle. Any species of an antibody directed against an epitope that is present on the microvesicle can be used. In another preferred embodiment, antibodies labeled with gold particles or other labels that can be detected with AFM or with fluorescence labels for fluorescence detection or any other label for these or any other detection methods are used. It is also possible to use antibodies labeled with multiple labels. An advantage thereof is that this enables detection of said antibody using different detection methods. In another preferred embodiment, said antibody is labeled with a molecule with an affinity linker for a special application of AFM, wherein the cantilever of the AFM is labeled with a binding partner of said affinity linker. An advantage thereof is that this application can be used to determine the specific interaction between the affinity linker and its binding partner. The adhesion force upon separation is then a measure of the binding strength. The detection of a positive adhesion force is therefore an alternative for detection techniques using labels that can be visualized by light or by size. An antibody can be used to capture microvesicles to a surface of the microfluidic chip and/or to label microvesicles that are bound to said surface.

In view of the results set out in the Examples, it is considered that the methods of the invention show that the full range of natural microvesicles particles are hard to detect with other methods such as FACS, which is considered by the inventors to detect only larger microvesicles. Smaller microvesicles (for example of around 65nm diameter) are found using the methods of the invention to be unexpectedly be very abundant.

As the detection area determines the detection time needed, counting smaller particles on a small surface is more efficient than scanning a large surface with larger particles. A low particle height limit allows for faster scanning in AFM as tip retraction time can be minimized. However, an advantage of AFM is that it can sense small microvesicles, as well as 1 micron microvesicles at the same time.

A more preferred embodiment is a method wherein a first section of said surface with the microvesicles immobilized thereto is exposed to a first species of antibodies, whereas the remaining section is not exposed. An advantage of this method is that a surface is obtained with a section wherein vesicles are labeled with antibodies and a section that is not labeled and thereby serving as a negative control.

More preferably, said method further comprises a second section which is exposed to a second species of antibodies. An advantage is that a further section is obtained which is labeled with a second species of an antibody. Therefore, a negative control a positive labeling for said first species, a positive labeling for said second species is obtained on a single surface. This enables easy comparison. A skilled person will understand that in the same manner also further species of antibodies can be used on a single surface.

In another preferred embodiment, said fluid is allowed to contact a first and a second section labeled with a first and a second species of antibodies. Preferably, the length of said first section and said second section is more than twice the width of said channel. The length of a section is defined as the length between the beginning of said first section or said second section which is contacted first by said fluid and the end of said first section or said second section, which is contacted the last by said fluid. An advantage thereof is that in this embodiment not all microvesicles are collected in said first section. Therefore, said second section can be used to determine the affinity of a subset of said vesicles in said fluid for said second species of antibodies.

In another preferred embodiment, said fluid is allowed to contact at least a first and a second section labeled with a first species of antibodies. An advantage thereof is that microvesicles in said fluid are contacted to said first section, thereby capturing a proportion of said microvesicles on said first section and further capturing another proportion of said microvesicles in said second section. By determining the difference in the number of microvesicles between said first and said second section captured, the concentration of microvesicles in said fluid can be determined.

The invention further provides a microfluidic chip, comprising at least one microfluidic channel with an inlet and an outlet, wherein said at least one microfluidic channel has at least one gap at a surface of said microfluidic chip enabling contact between said at least one channel and a surface. An advantage of said microfluidic chip is that it enables direct contact between a fluid in said microfluidic channel and a surface.

The invention further provides a method for constructing a mould for a microfluidic chip, said method comprising providing:

- a first microfluidic chip mould comprising a bottom, and surrounding sides to form a first receptacle for fluid,

- wherein at least one side, preferably the top is at least partly open to the exterior of the first mould to allow filling of the first receptacle of the first mould, - said bottom containing an image of a channel, (optionally a fluid inlet and a fluid outlet), and preferably one or more chambers, wherein said fluid inlet, fluid outlet and said one or more chambers are in fluid connection with said channel, - wherein said image is present on a surface of the bottom that is exposed to the fill side of said first receptacle,

- wherein said image comprises material that is in immediate contact with the surface of the bottom that is exposed to the fill side of said first receptacle, said method further comprising

- filling said first mould with a first microfluidic chip casting material,

- curing the first casting material to create a first cast comprising a first microfluidic chip,

- separating the thus formed first microfluidic chip from the first mould, providing the first microfluidic chip with surrounding sides to form a second receptacle for fluid,

- wherein at least one side of the second receptacle, preferably the top is at least partly open to the exterior to allow filling of the second receptacle,

- wherein the surface of the first microfluidic chip containing said channel, (optional fluid inlet and fluid outlet) and preferably one or more chambers is exposed to the fill side of the second receptacle,

- filling said second receptacle with a second mould casting material,

- curing the second casting material thereby creating a second cast comprising a second mould for said microfluidic chip, and separating said second mould from said first microfluidic chip,

- wherein said second mould comprises a copy of the image of the first mould.

In some embodiments, only a subset of the above steps may be required, depending upon specific requirements for the microfluidics chip and/or mould.

In this way, a first microfluidics chip can be cast from a first mould, and a second mould can then be cast from the first microfluidics chip. As an example, the first mould may be manufactured by lithography for silicon. Silicon moulds can be considered as advantageous as they do not require milling, and they can be used to provide smaller channels than brass moulds, for example. However, it can be difficult to use lithography of silicon moulds to provide channels with suitably large receiving chambers for receiving glass capillaries, as is known in the art. Also, silicon can be brittle and therefore not suitable for drilling and insertion of pins as is possible with brass moulds.

An advantage of the above method is that a second mould can be manufactured from the first mould (such as a first silicon mould), whereby the second mould is made from a material that is more suitable for being shaped in a desired way to provide the required receiving chambers. For example, the second mould may be made from polyurethane, so that it can have holes drilled in it for receiving pins, in order to provide a portion of the image that will subsequently provide the receiving chambers. Such an example of processing a mould is described below with reference to Figure 2. Also, it may be considered difficult to produce receiving chambers with a desired depth using known lithographic techniques.

The second mould, which has been cast from the first microfluidics chip, can then be used to cast a second, final, microfluidics chip that can be used with embodiments of the invention.

In a preferred embodiment the top of said image is essentially flat. An advantage thereof is that said top can be polished. Polished images result in atomically flat surfaces of a channel, which is important in achieving a laminar flow with low Reynold values.

In some embodiments, said first microfluidic chip mould is made of a metal, such as brass, or silicon, the image of a channel is produced by lithographic means. This allows the generation of a channel suitable for the production of a microfluidic chip.

An image is the material inversion of the open space within the outer surfaces of said microfluidic chip. Preferably, an image is formed by all material protruding from the flat sides of the mould exposed to the fill side a receptacle. An image can be material protruding from all sides of said mould. Said first microfluidic chip casting material can be any material which can be polymerized and is compatible with microvesicles. Preferably, the first cast (the first microfluidic chip) and the third cast are made of deformable material. Preferably, said material comprises PDMS. Said second cast (the second mould) is made of a material which can be solidified. Preferably said material can be polymerized. In a more preferred embodiment said method further comprises adapting said second mould and/or by removing material from said second mould by adding material to said second mould. An advantage of a method according to the invention is that adapting a mould which can be polymerized is easier than adapting microfluidic chip moulds known in the art. Moulds made using lithographic methods typically produce height differences of typically 30 microns. Producing greater differences in height is very time consuming and not easy. Removing material from a mould made of a material which can be polymerized to create a structure requiring a height difference of more than 30 microns can easily be produced by adapting said second microfluidic chip mould. Another advantage is that drilling into a mould made of a material which can be polymerized is possible from all directions, without risking damage of the structure of said image.

Preferably the material of said second cast comprises polyurethane. In another embodiment the material of said second cast comprises a thermoplast. In another embodiment the material of said second cast comprises ABS, PMMA (perspex), Telfon, polycarbonate and/or polysulfone. Preferably said first cast and said second cast are made of material that can be separated from each other after curing. Preferably said second and said third cast (the second microfluidic chip) are made of material that can be separated from each other after curing.

Preferred is a method, wherein said image comprises material that protrudes from said surface of the bottom that is exposed to the fill side of the first receptacle.

In a preferred embodiment said first cast and said second cast are made from polymers that can be separated from each other after curing. Preferably said first cast comprises PDMS. In a preferred embodiment said second cast comprises polyurethane.

A preferred embodiment is a method comprising providing said second mould with surrounding sides to form a third receptacle for fluid,

- wherein at least one side, preferably the top of said third receptacle is at least partly open to the exterior to allow filling of the third receptacle, and

- wherein the side of said second mould that comprises said image is exposed to the fill side of said third receptacle.

A preferred embodiment is a method further comprising filling said third receptacle with a third microfluidic chip casting material and curing the third casting material to create a third cast comprising the second microfluidic chip, wherein said second cast and said third cast are made from polymers that can be separated from each other after curing.

An advantage associated with a method disclosed herein is that small channels can be used with large receiving chambers in a single chip mould, and this can be achieved with only one moulding step and a single layer of polymer material for the chip. This is an advantage as there are no aligning or bonding steps required according to embodiments of a chip manufacturing procedure disclosed herein. Using a chip with only one layer of polymer material, can enable chips to be more easily/cheaply produced. The invention further provides a mould for a microfluidic chip cast of a deformable material, obtainable by a method according to the invention. The mould preferably comprises sides surrounding said mould to form a receptacle for fluid, wherein at least one side, preferably the top of the receptacle is at least partly open to the exterior to allow filling of the receptacle. The side that is exposed to the fill side of the receptacle is the side that contains the image of the channel, fluid inlet, fluid outlet and preferably one or more chambers, wherein said fluid inlet, fluid outlet and said one or more chamber (if present) are in fluid connection with said channel.

Preferred is a mould for a microfluidic chip, wherein said deformable material is PDMS. The invention provides the use of said chip mould for the preparation of a microfluidic chip. An advantage of using a chip mould is that it allows easy production of microfluidic chips.

There may be provided a method of manufacturing a mould for a microfluidics chip comprising: forming a first mould (optionally in silicon) using lithography; casting a first microfluidics chip (optionally out of PDMS) from the first mould; casting a second mould (optionally from polyurethane) from the first microfluidics chip; and modifying the second mould so as to enlarge one or more portions of an image of the second mould that will provide receiving chambers in a microfluidics chip cast from the second mould.

Modifying the second mould may comprise drilling holes through the second mould at location corresponding to ends of an image that will correspond to channels in the microfluidics chip, and then locating pins in the holes, in order to provide an enlarged portion of the image that will subsequently provide the receiving chambers in the microfluidics chip. Such an example of processing a mould is described below with reference to Figure 2.

The method may further comprise casting a microfluidics chip from the second mould. Such a microfluidics chip can have relatively small channel dimensions due to the lithography used on the first mould, and can have relatively large receiving chambers due to the modification of the second mould. Receiving chambers that can be manufactured using lithography can be too small for receiving glass capillaries, as described below, and can provide no, or little, margin for error when locating the glass capillaries in the receiving chambers. It can be considered easier and more convenient to locate a glass capillary in a receiving chamber of a microfluidics chamber that has been manufactured according to an embodiment of the invention.

The method may further comprise any other method sub-steps disclosed herein.

The invention further provides a method for the preparation of a chip mould according to the invention, comprising steps of covering a microfluidic chip with a polymer, allow said polymer to polymerize and separate said polymerized polymer from said microfluidic chip. Any microfluidic chip suitable for a method of the invention can be used. Preferably, said microfluidic chip comprises a microfluidic chip comprising PDMS. Any polymer can be used which can be separated from said microfluidic chip after setting of said polymer. Preferably, said polymer is polyurethane.

In another aspect the invention provides a coated surface suitable for use of the method. Preferably, said coated surface is prepared using a microfluidic chip of the invention.

Preferably, said coated surface is used for determining a parameter of microvesicles in a biological sample of an animal, wherein the parameter comprises the number, the dimensions and/or the protein factors of said microvesicles. An advantage of this use is that a coated surface can easily be used for determining said parameters. Any biological sample can be used wherein microvesicles are present. Preferably, said biological sample is obtained from a body fluid, for example synovial fluid, whole blood, plasma, serum, lymph, spinal fluid, pleural fluid, milk, ascites, lymphoid fluid, eye fluid or urine, because said samples can easily be obtained. A sample of any animal can be used. Preferably said animal is a human.

More preferably, said coated surface is used for determining a parameter for aiding in determining whether said individual has or is at risk of suffering or developing a disease associated with the presence of microvesicles. An advantage of this use is that it enables easy determination of a parameter for aiding in determining whether said individual has or is at risk of suffering or developing a disease associated with the presence of microvesicles. Another advantage of using said coated surface is that the number of microvesicles immobilized to said coated surface is proportional to the concentration of microvesicles in said fluid. Therefore, a person skilled in the art is able to calculate said concentration based on said number of microvesicles. Of course, a skilled person can also determine based on such parameter a degree of suffering of or recovering from a disease associated with the presence of microvesicles. Preferably, a disease associated with the presence of microvesicles comprises a disease wherein said parameter is different in said animal suffering from a disease than a healthy individual. Preferred diseases are characterized by the presence of an increased number of microvesicles, other diseases are characterized by the presence of specific types of microvesicles. For example, in Tesselaar et al, Journal of Thrombosis and Haemostasis 2007, is described that the presence of MUC1 bearing microvesicles is associated with a poor survival in patients with metastatic breast and pancreatic cancer patients.

More preferably, said disease comprises an inflammatory disease, more preferably a systemic inflammatory disease including an autoimmune disease, diabetes mellitus or immune-mediated thrombosis, diabetes, more preferably diabetes mellitus, a kidney disease, a cardiovascular disease, more preferably an acute coronary syndrome, diabetes mellitus, a neoplasm, more preferably cancer or a malignancy, more preferably a solid tumor or a leukemia, an infectious disease, more preferably a viral infection, a bacterial infection or a parasitic disease, a haematological disease, more preferably a sickle cell disease, a respiratory disease, a nutritional or metabolic disorder, an endocrine disease, an immunological diseases, a neurodegenerative disease or a neurological disorder. More preferably, said disease comprises diseases with vascular involvement and hypercoagulability such as disseminated intravascular coagulation.

In another aspect the invention provides the use of a microfluidic chip for the immobilization of microvesicles to a coated surface according to the invention. An advantage of using a microfluidic chip is that it is a single device ready for use in the method and enables a high yield of immobilized microvesicles while using only a small volume of fluid. Said microfluidic chip can also be used to apply a coating to a surface or to apply antibodies to said microvesicles. A preferred use of said microfluidic chip is described in example.

In another aspect, the invention provides a kit suitable for use of the method, comprising of a microfluidic chip and a coated surface. The kit may be suitable for use in a method of the invention using AFM. The coated surface may be a mica surface or a polished optical wafer. Polished optical wafers may be needed to be able to detect smaller particles using AFM. See for instance www.compart-tech.co.uk/glass.html. The coated surface may also have a hydrophilic coating, as noted above. In another aspect the invention provides a kit of parts comprising a microfluidic chip comprising a hydrophobic polymer, wherein the hydrophobic polymer optionally is PDMS; and a mica surface or polished optical wafer surface, wherein at least part of the mica surface or polished optical wafer surface has a hydrophilic coating. The hydrophilic coating may comprise, for example, PEG (polyethylene glycol), ethanolamine, DETA (Diethylenetriamine). APDM (3-aminopropyldimethylethoxysilane) or poly-lysine. A coating may be applied to the surface that comprises one or more components to render the surface hydrophilic and one or more different components to facilitate binding of molecules having an affinity for microvesicles to the surface as discussed above. In addition the surface must be smooth enough that the microvesicles can be identified, and must not interact negatively with the proteins of the blood. Last the surface must not be disturbed by the removal of the microfluidics chip. It may also be desirable to have a surface that does not autofluoresce in an optical detection system.

The surface may be further exposed to EGTA which may act as a cleansing agent for the next steps. The surface may be further coated with a coating comprising molecules having an affinity for microvesicles as discussed above. The EGTA or molecules having affinity for microvesicles may be applied to the surface after assembly of the surface with the microfluidic chip. Typically the EGTA may be applied to the surface before the molecules having affinity for microvesicles.

The invention is now described in more detail by reference to the following, non-limiting,

Figures and Examples.

20.

Figure 1 : Surfaces tested against the efficiency of capture and prevention of plasma clotting.

Figure 2: Fabrication of the chip mould, the chip and the fluidic connection.

Figure 3: Movie stills of connecting a microfluidic channel.

Figure 4: Left column shows the reaction of the various surfaces with anti-CD41 and purified microparticles. On the right, we see the reaction of platelet poor plasma with IGg. A and D are EGTA treated mica. B and E show APTES and gluteraldyhyde treated surfaces. C and F show APTES and PEG coated surfaces. Figure 5: Left shows plasma that was dripped onto EGTA-anti-CD41 surface and allowed to sit undisturbed for 30 minutes. Right shows plasma that was flowed over the surface with microfluidics over a period of 30 minutes. Large blobs are thought to be activated platelets which are very rare in this plasma.

Figure 6: Example of the preparation of a coated surface using a microfluidic chip comprising 3 channels. In step A, a chip is positioned on a surface. The channels are used to apply a coating onto the surface. In step B the chip is repositioned and used to apply fluids comprising microvesicles onto the coated surface.

Figure 7 A-F: Example showing the effects of the microfluidics on concentrating the microvesicles by improving exposure to the surface. This figure shows the microvesicles extracted from the platelet poor plasma (PPP) using microfluidics (Figure A) compared with a number of controls. Figure B is included to show the number of CD41 positive microvesicles that are typically pulled from the PPP without the concentration of the microfluidics. In figure B the PPP was applied by setting a drop of PPP 50 μl on the surface and then allowing it to incubate for one hour. The difference in the number of microvesicles that are extracted between figure A and B is clear. The plasma drop experiment shows a few particles that have been captured (small white dots). Figure B shows a smaller number of microvesicles. In figure C we see a surface before any plasma has been applied. This surface is only coated with the mouse antihuman CD41 antibody. This shows that there are no spots on this surface that would be mistaken for microvesicles. Figure D shows a surface that is prepared in the same manner as the other surfaces, only the anti-CD41 antibody is replaced with a mouse IgGI isotype control in the same concentration and this negative control has no action against any of the objects in the plasma. The lack of microvesicles on this surface shows that the surface does not nonspecifically capture the CD41 positive microvesicles. As specificity is even more important than the raw numbers, the comparison of figure D to figure A shows that this technique is able to purify and sort only the CD41 positive microvesicles from the blood plasma. (Streaking is an artifact of the atomic force spectroscopy technique, z scales have not been normalized to facilitate comparison of the images)

Figures 7E to H. Image collection of cells and microvesicles collected from flowing the PPP through the microfluidics. The microfluidics method is more efficient than the plasma drop experiments in capturing all the different types of microvesicles in the blood. These unusual microvesicles are not seen in the plasma drop experiments, shown in figure 7 F and G, and most likely are activated platelets, the precursors of platelet microvesicles, and fibrin. Figure F also shows uncommon features of the mica surface. The figure in the lower left side (H) shows the more common image of microvesicles that can be caught with the microfluidics method, showing a variation of sizes.

Figure 71 shows the CD41 positive microvesicles that are collected from concentrated, isolated microvesicle preparation that has been run through the microfluidics (left). The figure on the right (J) shows the same type of microvesicle preparation dropped on the surface.

Figure 8

The size standard polystrene beads with diameters of 0.2, 0.5, and 1 μm were measured by FACS. The light scatter patterns of these beads were plotted in one histogram and compared with the light scatter pattern of MVs.

Figure 9

AFM image showing the three-dimensional surface topography of mica (a), modified mica (b) anti-CD41 antibody-coated mica (c) and IgGI isotype control coated mica (d). The inserts show the results of the height analysis (z value) of the surfaces.

Figure 10

Three-dimensional AFM topography of CD41 ⁺ MVs (a). The insert at the bottom represents the region denoted by the dashed box in the centre. The imaging of isotype control (IgGI )-coated mica hardly shows any MPs attached (b). The scale bar in these images is 1 μm.

Figure 1 1

AFM detection of CD41 ⁺ MVs isolated from fresh PPP showing the size distribution and amount of MVs found on a 100 μm ² scan (a). A scatter plot of the apparent height of CD41 ⁺ MPs as a function of their corresponding diameter is presented (b).

Figure 12

FACS plots representing the measurement of the absolute count beads (a), the detection of CD41 ⁺ MVs (b), and IgGI -isotype control (c).

Figure 13. Plasma drop experiments with variable plasma incubation times. The solid bar shows plasma that is applied to anti-lgG control surface forming a functional control. Normalized count refers to the number of particles per 10*10 Dm ² area. There are too few particles per image for these measurements to be statistically relevant.

Figure 14. A thin layer of clotting that formed on an anti-CD41 surface in response to the plasma that flowed through the microfluidics. The left side of the image shows the place where the edge of the microfluidic channel was before its removal. The maximum height in this image is 200 nm.

Figure 15: Matrix showing the performance of the various experimental methods. The reaction of dropped plasma with anti-lgG and anti-CD41 surfaces are shown at the top. The interactions with the same surfaces against the microfluidics is shown at the bottom. It is clear that the only surface that captures the mciroparticles is the anti-CD41 surface with the microfluidics technique.

Figure 16: Comparison of number of particles captured in 100 μm ² . All samples other than the drop experiment were collect by the microfluidics system.

Figure 17. Distributions of the effective radius of the particles. Black bar show sizes of particles detected from purified MPs. Hashed bars show particles from Hepes diluted plasma while particles detected in EDTA Hepes plasma are shown in white. All counts have been normalized to 1 by the max value. Drop experiments were not examined since there were too few particles in those experiments to do statistics on.

Figure 18 Example of a surface with large amounts of protein contamination on the surface. Height scale is set to 25 nm (white is maximum height). Texture on the lower half of the image is thought to be protein contamination.

Examples

Preparation of a chip mould, and use thereof for the preparation of a microfluidic chip will now be described with reference to figure 2. Figures 2A to 2D illustrate cross-sectional views of the preparation of the chip mould, and figures 2E to 2L illustrate cross-sectional views of the preparation of the microfluidics chip using the mould. A first step in preparing a mould for creating a microfluidic chip is shown as figure 2A and comprises milling an image of one or more channels 2 into the top surface of a suitable material 1. The material 2 may be a metal, such as brass. The milling is performed such that the image of the channel 2 is raised above a top surface of the brass 1 , thereby defining a floor in the top surface of the brass 1 around the channel image 2. The channel image 2 may comprise a plurality of straight ridges with dimensions 10mm x 300μm x 100μm. An automated milling machine can be used to provide the image of the one or more channels 2.

The top surface of the brass 1 that surrounds the milled away image is shown with reference 3 in figure 2B. The height of the channel ridges 2 can be kept at the same level as a top surface of the brass 1 surrounding the milled away image, and this can be convenient for polishing the top of the channel image 2 by providing a frame around the channel image 2 that can be used to more effectively guide the location of a polisher. The top surface of the channel ridges 2 should be smooth to enable optical viewing techniques to be performed through the microfluidics chip that will be made from the mould.

As a next step, the frame 3 around the channel image 2 is removed by milling, and holes 4 are drilled through the brass 1. The holes 4 are drilled at locations that correspond to one or more sections of the channel image 2, and a hole can be drilled at each end of a channel ridge 2. This is shown in Figure 2C. The holes 4 can be drilled from the bottom surface of the brass thereby completely removing the material of the bottom of the mould at these positions. An advantage to drilling the holes from the bottom can be a reduction in the likelihood that the channels will be damaged by the drilling action. Two holes 4 are shown in Figure 2C, one for each of the channel ridges 2 that are shown.

As shown in figure 2D, pins 6 are inserted in the holes 4 that are shown in figure 2C. The pins can be mounted such that they extend to a height above the floor of the top surface of the brass 1 that corresponds to half the intended height of the chip plus half the diameter of the glass capillary that will be used to connect to the chip, and the capillaries that are used to provide inlets and outlets to the channels are described below. Example pins 6 can have a height of about 1.mm and a diameter of 1 mm.

The pins will form the receiving chambers (9) in the channels of the microfluidic chip (7) once the microchip is cast. Side walls 5 are clamped around the brass material 1 to provide side walls of the mould and define a volume that is suitable for receiving a material for forming the microfluidics chip. A material that is suitable for forming the microfluidics chip is PolyDiMethylSiloxane (PDMS), and the side walls 5 can prevent leakage of the PDMS in steps that are illustrated in figures 2e and 2f.

In this example, the PDMS for the microfluidics chip was fabricated using a Sylgard 184 kit (Dow Corning, UK). Silicone primer and catalyst were mixed in a 10:1 ratio by weight and this mixture was placed in a vacuum chamber for about thirty minutes to an hour to remove air bubbles, thereby degassing the mixture.

As shown in figure 2E, the PDMS mixture 7 is poured into the mould. The PDMS 7 will form the body of the microfluidics chip. The PDMS flows over the channel image/ridges 2 and is constrained by the side walls 5.

The filled mould is carefully covered with a glass plate 8 as shown in figure 2F, and the glass plate 8 can rest on shoulders on the inside of the side walls 5 so that it is retained at a correct height relative to the mould. The glass plate 8 can provide an optically flat surface though which imaging can be performed. In this example, the PDMS 7 is then cured at 70 degrees Celsius for one hour.

The mould, consisting of elements 1-6, is then removed from the PDMS 7 and glass plate 8. The remaining PDMS 7 and glass plate 8 are shown in Figure 2G. The PDMS 7 has channels corresponding to the channel ridges 2 of the mould, with receiving chambers 9 present at each end of each ridge 2. The channels extend into what will now be referred to as a top surface of the PDMS 7. The glass plate 10 can now be considered as being attached to a bottom surface of the PDMS 7.

A second glass plate 10 is attached to the top surface of the cured PDMS 7, over the channels 9, and is shown in figure 2H. The second glass plate 10 is used to protect the channels from dust and can be removed from the PDMS when the chip is to be used. As will be described in more detail below, the microfluidics chip that is illustrated in figures

2E to 2L may be inverted when in-use so that the top surface of the microfluidics chip that is shown in figures 2E to 2L is a bottom surface when the chip is in-use, and vice versa. In some examples, additional PDMS 11 can be applied between the regions of the two glass plates 8, 10 that are exposed around the periphery of the PDMS 7, and this is shown as figure 2I. The additional PDMS 1 1 can provide a stronger bond between the second glass plate 15 and the PDMS microfluidic chip 7.

To create inlets and outlets to the channels of the microfluidic chip, a capillary 12 is connected to the receiving chamber 9 at each end of a microfluidic channel through the PDMS 7 that is exposed on the sides of the microfluidics chip between the two glass plates 8, 10. This is shown in figure 2J, and can be performed in accordance with the teachings of WO2008/072968 (Universiteit Leiden). Glass capillaries 12 that have a sharpened end can be used to perforate the PDMS 7 of the microfluidic chip. The capillaries 12 can be coated with polymide to strengthen them, and make them flexible. An example fo a suitable capillary is TSP-150375-D-10 (www.bgb-shop.com). The capillaries 12 can be cut using a piece of aluminum oxide to create a slight scratch through the polyimide coating. Tips of the capillaries 12 can be beveled by mechanical grinding with a disc containing diamond dust. Before and after the mechanical grinding, the capillaries can be rinsed with water to clean them.

The capillaries 12 are put into fluid communication with the channels so that a fluid that is passed through an inlet capillary 12 flows through the channel before exiting the microfluidics chip through the other, outlet, capillary 12.

As shown in figure 2L, the chip and capillaries 12 are sterilized using an autoclave at 120 degrees Celsius for 25 minutes, and this can also be performed before and/or after connecting the capillaries 12 to the chip

To make the connections between the capillaries 12 and the microfluidic chip more robust, the capillaries 12 can be secured to the existing PDMS 7, 1 1 with a further still layer of PDMS 13. An advantage is that this extra layer provides a stronger bond between the PDMS chip 7 and the glass capillaries 12.

The channels may be coated with an anti-clotting agent such as EGTA, or in some embodiments, heparin. The coating, if required, may be added to the mould and/or the microfluidics chip, or can be passed through a channel of the microfluidics chip so that it applies a coating to the channels. It will be appreciated that a coating can be applied to the channels before or after assembly. . Other materials that can be used to manufacture the chip include glass, rubber and plastic, as long as these materials do not react with the fluid to be tested, for example blood plasma. A coating of the channel may be used so that materials that would otherwise react with fluid to be tested can be used. As an example, glass may need to be coated. For embodiments of the invention that use AFM, the material of the chip should be suitable for separating the chip from the coated surface so that the coated surface can be tested. Such separation may not be necessary for embodiments that use optical detection. In examples where the chip is not made from glass, a mould may not be required.

Use of a microfluidic chip that has been created by the process illustrated in figure 2 will now be described with reference to figure 6, which shows a surface 7 that will be used with a microfluidics chip having three channels (not shown in figure 6).

Initially, a surface 7 that will be used with the microfluidics chip is prepared. Preparation of the surface in this example comprises preparing a surface of mica (Electron Microscopy Sciences, Washington) for MP attachment as described by Yuana et al. (accepted in JTH 2009) with a slight modification. In brief, a freshly cleaved mica (diameter= 10 mm) was immersed in DMSO containing 55% (w/v) Ethanolamine for overnight at 70° C. Subsequently, the mica surface was rinsed twice with dry DMSO at 70°C, rinsed with HLPC grade ethanol to remove the DMSO. Next, the mica surface was put into a solution of 30 ml PBK (pH 7.6 1M) containing 100 mg EGTA for 10 minutes. The surfaces were then rinsed with Hepes buffer (10 mM Hepes (Merck, Germany), 137 mM NaCI (Merck, Germany), 4 mM KCI (Merck, Germany), 0.1 mM Pefabloc® SC (Fluka, Germany), pH 7.4), and then 20 μl of 0.05 mg/ml mouse anti-human CD41 antibody clone P2 (Beckman coultrer, Fullerton, CA) was applied for 3 hours. Excess of anti-CD41 was removed by washing with Hepes buffer. Anti-CD41 antibody coated-mica surface was stored in Hepes buffer until used. As a negative control, mouse IgGI pure clone X40 (Becton Dickinson, San Jose, CA) was used (0.05 mg/ml). All chemicals were purchased from Sigma Aldrich unless otherwise indicated.

As a first step, a chip is positioned on the surface 7 so that the three channels run across the surface in a first direction, which in figure 6A is a side-to-side direction. The second glass plate is removed from the microfluidics chip shown in figure 2, and the microfluidics chip is inverted and placed on the surface so that its open channels are closed by the surface 7. The chip is pressed against the surface 7 to prevent leakage of the fluid that will flow through the channels. The chip and surface can be pressed together using a simple holder device which consists of two parts screwed together with the chip and surface in between. Two capillaries (as shown in figure 2) are then connected to a fluid source at an inlet and suitable vessel for the outlet, and can be attached to the holder using scotch tape. The screws, for example four screws, are used to press the chip onto the mica surface, finally the mica can be glued onto a metal disc for use in loading the sample into the AFM.

A coating material having an affinity with microvesicles is then passed through the channels of the microfluidics chip, and the deposited coating is shown as side-to-side regions in figure 6A.

The microfluidics chip is then removed from the surface 7. The rubbery nature of the PDMS elastomer used for the microfluidics chip can allow convenient removal of the brittle mica surface without breaking the mica. The PDMS can be carefully lifted from one side to remove/peel it from the mica surface. The fluid confinement is not glued and therefore no exogeneous residues can be left on the surface that is to be studied. The microfluidics chip is then repositioned on the surface 7 so that the three channels run across the surface in a second direction that can be perpendicular to the first direction.

In figure 6B the second direction is a top-to-bottom direction. In this way, a fluid to be tested can flow through the microfluidics chip across a region of the surface 7 that has been coated, and measurements can be taken from the surface by removing the microfluidics chip and using AFM, or by using optical methods through the microfluidics chip. In this example the fluid to be tested is plasma.

The channels of the microfluidics chip can enable a laminar flow of fluid through the channels, and the associated lack of turbulence can enable a desired interaction between the fluid and the surface to which the microfluidics chip is attached. In some examples, the small scale of the microfluidics chip, with channel lengths of the order of 10 μm to 1 mm, can avoid, or reduce the likelihood of, turbulence occurring.

Example 1 : Blood collection and platelet poor plasma isolation

Blood was collected from seven donors between 8.00-10.00 A.M. and processed within 10-15 minutes. Venipucture was performed using a 21 gauge needle (BD vacutainer,

San Jose, CA) and minimal stasis. After discarding the first four ml of blood, blood was collected in 1/10 volume of sodium citrate (3.2%, 0.105 M) using a 4.5 ml vacutainer tube (Becton Dickinson, San Jose, CA). Blood was centrifuged at 2,000xg for 10 min at 20 ⁰ C, without brake. Plasma was carefully collected and centrifuged again at 2,000xg for 10 min, 20 ⁰ C, without brake, to obtain platelet poor plasma (PPP). PPP was aliquotted in 250 μl portions. For the isolation of microvesicles (MVs) from fresh PPP, PPP was directly subjected to the next centrifugation steps. Otherwise, PPP was snap frozen in liquid N2 and stored at -80 ⁰ C until used for MP isolation.

Isolation of Microvesicles

For AFM measurement, 750 μl PPP was pooled and centrifuged at 18,890xg, 20 ⁰ C for 30 minutes with minimum brake. The supernatant was removed carefully except for 25 μl containing the MV pellet. This pellet was resuspended with 1 ml Hepes buffer (10 mM Hepes (Merck, Germany), 137 mM NaCI (Merck, Germany), 4 mM KCI (Merck, Germany), 0.1 mM PefablocOSC (Fluka, Germany), pH 7.4), vortexed, and centrifuged as before. The supernatant was removed leaving a volume of 25 μl containing the MV pellet. This 25 μl was vortexed and then used for AFM measurement.

For FACS measurement, 250 μl PPP was centrifuged at 18,890xg, 20 ⁰ C for 30 minutes with minimum brake and then the supernatant was carefully removed leaving a volume of 25 μl containing the pellet. To wash MVs, 225 μl of Hepes buffer was added and then this mixture was centrifuged again at 18,890xg, 20 ⁰ C for 30 minutes with minimum brake. The supernatant (225 μl) was removed and the residual 25 μl was diluted to 100 μl with Hepes buffer. This MV preparation was directly used for staining with antibodies.

Example 2: Modification of mica for immobilizing anti-CD41 antibody The surface of mica was modified as follows: a solution was made containing 55% (w/v) ethanolamine (Sigma Aldrich, Germany) in DMSO (99.7% purity; Biosolve BV, Netherlands). Molecular sieve beads (0.3 nm; Sigma Aldrich, Germany) were added to adsorb the water formed later during the amine reaction on the mica surface. This solution was heated at 70 ⁰ C until ethanolamine was completely dissolved. Next, freshly cleaved muscovite mica sheets (Electron Microscopy Sciences, Washington) having a diameter of 10 mm were incubated overnight in the ethanolamine solution. Subsequently mica sheets were rinsed twice in DMSO (99.7% purity, 70 ⁰ C) and twice in ethanol absolute (Biosolve BV, Netherlands) at room temperature (RT). These mica sheets were dried under N2 flow. At this point, the amine modified mica can be used immediately for the next modification step or stored in a desiccator for up to several weeks. Prior to protein coupling, the modified mica was incubated three hours with 1 mg/ml Ethylene glycol-bis-(2-aminoethyl)-N,N,N', N'-tetraacetic acid (EGTA) (Sigma Aldrich, Germany) in chloroform (Merck, Gemany) containing 0.5% v/v triethylamine (Merck, Germany). Next, the mica sheets were washed in chloroform and dried under N2 flow, then glued on a steel disc (diameter = 12 mm) with a silicon based glue (Rhodia silicon, Germany). Fifty μl of mouse anti-human CD41 antibody clone P2 (Beckman Coulter, Fullerton, CA) (0.01 mg/ml) was applied to the modified mica and incubated for 30 minutes at RT. Excess of anti-CD41 was removed by washing with Hepes buffer. As a negative control, mouse IgGI pure clone X40 (Becton Dickinson, San Jose, CA) was used (0.01 mg/ml).

Example 3: AFM analysis of CD41 +microvesicles Twenty μl of MV fraction was applied to anti-CD41 -coated mica surface and incubated for 30 minutes to allow the binding of MVs to anti-CD41. To remove unbound MVs mica was washed twice with Hepes buffer. During the incubation step and before imaging, the mica surface was kept in a closed container.

Before use, the AFM glass fluid cell (Veeco, NY) was sonicated for 5 minutes in Sodium Dodecylsulfate (SDS) (Serva Electrophoresis, Germany), rinsed with distilled water, and allowed to dry under N2 flow. A micro cantilever silicon tip (Olympus, Japan) with a spring constant of 2 N/m and 70 kHz resonant frequency was attached to the fluid cell. After mounting the steel disc containing the sample on the piezoelectric scanner of AFM, 80 μl of imaging buffer containing 20 mM Tris (Roche, Switzerland) and 150 mM KCI (pH 7.4) was added. The fluid cell with the cantilever attached was then seated against the sample. AFM (Veeco, NY) operated in fluid-tapping mode was performed to scan the topography of the mica and attached MVs thus acquiring threedimensional images of the MVs. With an imaging rate of 1.5 Hz the surface was scanned at different positions to obtain 4 to 10 images with a scan size of 100 μm ² per image. Images were captured and recorded using Nanoscope software version 5.30r2.

AFM data was analysed using SPMediator version 6.1 software (courtesy of Dr. S. J. T. van Noort) that evaluates the centre of mass of particles in comparison with the surface background. This software automatically calculates the volume and z dimensions (height) of MVs. From the MV volume, the MV diameter was calculated assuming a spherical shape.

To calculate the number of CD41 +MVs/ L PPP detected by AFM, the following formula was used: (10 ⁶ /750) x (25/20) x (78.5x10 ⁶ /100) x NAFM, in which 750 (μl) is the original volume of PPP for MVs isolation, 25 (μl) is the volume of MV suspension, 20 (μl) is the volume of the MVs suspension added on the mica surface, 78.5x10 ⁶ (Rm ² ) is the surface area of the mica, 100 (Rm ² ) is the surface area of an AFM image, and NAFM is the mean of the total number of CD41 +MVs per image after correction for the number of MVs found on IgGI control-coated mica.

FACS analysis of CD41 +microvesicles

FACS analysis was performed using a FACS Calibur flow cytometer with CellQuest pro software (Becton Dickinson, San Jose, CA). To calibrate the flow cytometer and to set the scatter parameters for MVs analysis, size standard polystrene beads of 0.2, 0.5, and 1 Rm (Fluka, Germany) were used. When MVs were measured, the forward size scatter of MVs showed that MVs with a size of 0.2 μm and above could be detected (Figure 8).

Five μl MV fraction was incubated with 5 μl phycoerythrin (PE) labelled mouse anti- human CD41 clone P2 (1 :100 dilution) (Beckman coulter, Fullerton, CA) in 40 μl of Hepes buffer (containing 137 mM NaCI, 4 mM KCI, and 0.1 mM Pefabloc®) for 30 minutes at RT in the dark. Next, 200 μl Hepes buffer was added and the MV suspension was centrifuged at 18,890xg, 20 ⁰ C for 30 minutes with minimum brake. The supernatant was removed carefully and the residual 50 μl was diluted to 350 μl with Hepes buffer. As negative control, mouse IgGI PE clone X40 (Becton Dickinson, San Jose, CA) was used at the same concentration as the anti-CD41 antibody. For quantification of CD41 +MVs per L, we used FACS absolute count standard beads with a mean diameter of 7.58 μm/bead and a known concentration (Cbeads) (Bang Laboratories, Fishers, IN) as an external standard. Two hundred μl of these beads (Vbeads) was mixed with Hepes buffer to a volume of 600 μl (Vtot) and then measured in triplicate (Nbeads). For measuring counting beads as well as MVs, the flow cytometer was programmed to count events in one minute. To calculate the volume (Z) measured by FACS, the following formula was used: Vtot/Vbeads x (Nbeads/Cbeads). The amount of CD41+MV/ L of PPP detected by FACS was calculated as: (3507Z) x (100/5) x 10 ⁶ /250 x NFACS, in which 350 (μl) is the total volume of MVs before analysis, 100 (μl) is the total volume of the original MV suspension, 5 (μl) is the volume of MV used for antibody labelling, 250 (μl) is the original volume of PPP for MV isolation, and NFACS is the mean number of CD41 + events counted by FACS, after correction for IgGI control background events.

Statistical analysis

Statistical evaluation was performed using SPSS 14.0. Correlation of the amount of MVs in PPP measured by AFM and FACS was tested with the Pearson and Spearman correlation tests. RESULTS AFM

The surface of cleaved mica scanned with AFM operated in fluid-tapping mode was flat with an apparent height of ~0.1 nm (Figure 9a). After modification of this surface, the surface topography was still nearly flat with a measured height of ~0.2 nm (Figure 9b).

Following the immobilization of mouse antihuman CD41 antibody to the modified mica, an apparent height of ~2.6 nm of the anti-CD41 antibody was detected (Figure 9c). As a negative control, a mouse IgGI isotype control was used at the same concentration as the mouse anti-human CD41 antibody. The surface topography scan showed an apparent height of -0.9 nm of the mouse IgGI isotype control (Figure 9d).

Imaging with AFM demonstrated specific binding of MVs isolated from fresh PPP to anti- CD41-coated mica. IgGI isotype control coated mica was used to control for non-specific MVs binding. Three dimensional AFM topography showed that there were spherical vesicles, defined as MVs, attached to the anti-CD41 -coated mica while these were virtually absent on the IgGI isotype control-coated mica (Figure 10a,b).

Variation of the incubation time of MVs with the anti-CD41 -coated mica (2, 15, 30, and 45 min) showed that the total number and size distribution of specifically attached MVs had reached a plateau after 15 minutes (323 counts/100 μm ² ).

Figure 11 shows the results of a representative experiment. Figure 11a shows that the MVs attached to the anti-CD41 -coated mica have diameters ranging from 10-325 nm. The diameter of the majority of these MVs was about 50 nm. In figure 11 b the apparent height of CD41 ⁺ MVs is plotted on the x axis as a function of their corresponding diameter on the y axis. The plot showed that the diameter size of the CD41 ⁺ MVs exceeded the height approximately tenfold. Therefore to define the size distribution of MVs, the analysis of AFM data with the SPMediator version 6.1 software was based on the volume of MVs assuming a spherical shape.

To investigate the relationship between MV concentration and number of CD41 ⁺ MVs detected by AFM ₁ different dilutions (D) (D= 1 , 2, and 4) of a MV preparation isolated from fresh PPP were incubated with anti-CD41 -coated mica and imaged by AFM. A linear relationship (Y = 286.88 X + 34.91 counts/100 μm ² , R2 = 0.98) was obtained between the number of CD41 ⁺ MVs (Y) detected per 100 μm ² mica and the MV concentration (X= 100/D). From this linear relationship, it could be predicted that about 35 MVs attach nonspecifically to 100 μm ² of IgGI isotype control-coated mica. To determine the reproducibility of the measurement of CD41 ⁺ MVs with AFM, 25 MVs were isolated from frozen-thawed PPP on three consecutive days and analysed by AFM for the presence of CD41 ⁺ MVs. From this experiment, we estimate that the between assay variation for MVs isolation and AFM measurement (including mica modification) was 16% (mean 528x10 ⁶ CD41 ⁺ MVs/L; SD 83x10 ⁶ CD41 ⁺ MVs/L).

To investigate the effect of freezing and thawing on the number and size distribution of CD41 ⁺ MVs measured by AFM, MVs were isolated from PPP of three donors before and after freezing-thawing. For two donors there was no significant difference in size distribution of the CD41 +MVs measured by AFM before and after freezing-thawing, although the number CD41 ⁺ MVs decreased (2- and 7-fold, respectively). For one donor we found a two-fold increase in number and a slight shift in size distribution of the CD41 +MVs in MVs isolated after freezing and thawing of the PPP (from 50-250 nm to 10-200 nm).

Flow cytometry

The CD41 ⁺ MVs were quantified by FACS using a known concentration of counting beads as external standard in the measurement. The singlet population of these beads could be detected as shown in the forward scatter plot of Figure 12a. By counting the number of beads, the volume used for the measurement could be calculated. In all experiments the mean volume analyzed by FACS was 80±4 μl. MVs were characterized by their CD41 expression (Figure 12b, c). The number of CD41 ⁺ MVs was calculated after correction for the number of MVs staining with the IgGI isotype (negative control). The reproducibility of MVs measurement with FACS was assessed on three consecutive days by one single operator performing the CD41 ⁺ MVs measurement in triplicate. For these experiments, MVs were isolated from aliquots of frozen PPP. The variation in the number of CD41 ⁺ MVs was 10% (mean 0.40x10 ⁶ MVs/L, SD 0.04x10 ⁶ M Vs/L).

The effect of freezing and thawing on the number of CD41 ⁺ MVs measured by FACS was also assessed. Flow cytometric analysis of MVs isolated from PPP of 4 donors before and after thawing revealed that depending on the donor 2-10 fold more CD41 ⁺ MVs were detected in the MVs fraction prepared from frozenthawed PPP than in the MVs isolated from fresh PPP.

AFM versus flow cytometry

MVs were isolated from fresh PPP of 7 donors (MVs1-7). From one donor, PPP for MV isolation was obtained on three different days (MVs 1a, b, c). All 10 MV preparations were analysed simultaneously by AFM and FACS for numbers and size distribution of CD41 ⁺ MVs.

The numbers of CD41+MVs obtained by AFM and FACS are shown in Table 1. Depending on the MV preparation 4-10 images of 100 μm2 were scanned. About 255 CD41 ⁺ MVs were detected by AFM per 100 μm ² of the anti-CD41 -coated mica surface whereas about 19% of these MVs (mean 48 counts/100 μm ² of MVs) attached non- specifically to IgGI isotype control-coated mica. We found that the number of CD41 ⁺ MVs detected by AFM (32-702 x 10 ⁹ /L PPP) was about 1000-fold higher than the number of CD41 ⁺ MVs detected by FACS (11-291 x 10 ⁶ /L PPP). In all seven donors the size of the CD41 ⁺ MVs measured by AFM ranged from 10-475 nm. The majority of these MVs have a diameter of about 67.5±26.5 nm. On the IgGI isotype control-coated mica particles were found ranging from sizes 10-200 nm, with the majority of MVs having a measured diameter of 53.8±13.4 nm.

The seven donors have a quite high variation in their number of CD41+MVs (75% for AFM measurements and 86% for FACS measurements). There was only a weak correlation between the number of CD41 ⁺ MVs detected with AFM and FACS (r=0.17, P>0.05).

DISCUSSION

In this example, we report a method for the detection of CD41 ⁺ MVs with AFM. We have demonstrated that the number of CD41 ⁺ MVs detected by AFM is about 1000-fold higher than the number detected by FACS. Our method of mica modification, and detection of CD41 ⁺ MVs on anti-CD41 -coated mica with AFM resulted in reproducible numbers of CD41 ⁺ MVs (CV= 16%). The high interindividual variation in the number of CD41 ⁺ MVs isolated from fresh PPP (75% for AFM and 86% for FACS) mainly reflects biological variation. Immobilization of mouse anti-human CD41 antibody and of mouse IgGI isotype as a control on modified mica was successful, and a linear relationship between the MV concentration and the number of CD41 ⁺ MVs as detected by AFM was demonstrated. Binding of MVs to anti-CD41 -coated mica seemed specific since we only found much less MVs bound to IgGI isotype control coated mica in repeated experiments (see table 1). These non-specifically bound MVs were found to have a slightly smaller diameter range (53.8±13.410 nm). At this moment we do not know what these objects are but they might be contaminants from the environment or non specifically bound (aggregates of) plasma proteins which still present in the MV preparation. With AFM, we not only obtained the information of the numbers but also on the shape and size distribution of C D41 ⁺ MVs. Using AFM we counted CD41 ⁺ particles with diameter sizes ranging from 10 to 475 nm, while with FACS these nanosized CD41 ⁺ MVs are not detectable. This might, in part, explain why the numbers of CD41 ⁺ MVs isolated from fresh PPP and detected by AFM were about 1000-fold higher than those detected by FACS. A similar observation was made by Zwicker (35) who reported that the number of tissue factor-bearing microvesicles observed by light scatter FACS was 10, 000-fold less than what they observed using an impedance-based flow cytometer. Apparently conventional FACS is not suitable for detection of small sized-MVs. Indeed statistical analysis indicated that the number of CD41 ⁺ MVs detected by AFM correlated only weakly with the number detected by FACS (r=0.17, P>0.05).

With AFM we found that the diameter of CD41 ⁺ MVs isolated from fresh PPP ranges from 10-475 nm, with the majority clustering at 67.5 ± 26.5 nm. Siedlecki et al (36) also used AFM to analyse PMPs and used Nanoscope III software to calculate the size distribution of these MVs. They reported that the majority of the PMPs have a diameter of 125±21 nm and a height of 5.2±3.6 nm. They produced the PMPs by contact activation of isolated platelets on glass and on low density polyethylene, while we isolated MVs directly from PPP. This might explain the difference in MVs diameter found in both studies. Similar to their finding we observed that the height of MVs 5 was much smaller than their diameter (z=17.4±8.6 nm), indicating that MVs bound to mica have a non- spherical shape which may be related to the process of binding to the antibody-coated mica surface. The shape of adhering (or bound) vesicles is apparently governed by the balance between bending and adhesion energies (37). Richter and Brisson (38) observed that the apparent inner height of attached vesicles corresponds to only 20% of the vesicle's original inner diameter.

As we and others have shown that about 80% of plasma MVs express platelet antigens as determined by FACS (6, 7), we characterized MVs based on their CD41 expression using a mouse anti-CD41 antibody. However, MVs from other cells may have different characteristics. Salzer et al (39) isolated microvesicles and nanovesicles from calcium ionophore treated-erythrocytes and reported that the maximum size of these vesicles is

179 nm and 81 nm, respectively. MV size may depend on the parental cell and probably also on the mechanism by which MVs are formed. So far, there is no generally accepted protocol for the isolation of MVs from plasma (27). In this study fresh PPP obtained after double centrifugation (2,000xg for 10 min) of citrated plasma was used for the isolation of MVs to overcome artifacts of freeze-thaw procedure (22). In the present study we have compared MV preparations isolated from fresh and frozen-thawed PPP and found that there were changes in the number and the characteristics of MV. Surprisingly, after freezing and thawing a two-fold decrease in the numbers of CD41 ⁺ MVs was found by AFM, whereas an increase in numbers of MV was found by FACS. Aggregation of microparticles during freeze-thaw procedure might create larger particles which leads to an increase of events counted by FACS.

Recently the question has been raised (40) whether MVs preparations might contain exosomes, secreted microvesicles formerly present inside large multivesicular endosomes (41). Interestingly, exosomes have been reported to have a diameter ranging from 30 to 90 nm (41), whereas the diameters of the CD41 ⁺ MVs in our study ranged from 10-475 nm. Further experiments, which are beyond the scope of the present study, using exosome specific antibodies like anti-CD63 and anti-CD81 (25, 41), and density-gradient differential ultracentrifugation are needed to investigate whether part of the MVs isolated from PPP consist of exosomes.

In this study we were able to demonstrate that AFM is a suitable method to sensitively and reproducibly detect and quantify CD41+MVs and that AFM detects 1000-fold more CD41 ⁺ MVs than conventional FACS.

Table 1

AFM and FACS of CD41 ⁺ MVs isolated from fresh PPP. AFM quantification is presented as the number of MVs per 100 μm ² scanned image. For both AFM and FACS measurements the number of CD41 ⁺ MVs was calculated per L PPP after correction for IgG control-bound MPs.

Example 4: Surface Tests using Mica

EGTA:

Freshly cleaved mica surfaces were immersed in 1 M Hepes buffer at pH 7.6 with 10 mM Calcium Dichloride for 10 minutes to deposit a layer of calcium on the surface. Dry EGTA (ethylene glycol tetraacetic acid) was 5 then added to the buffer until saturation occurred and the EGTA began to precipitate. This solution was allowed to incubate on the mica surface for 10 minutes whereupon the mica was removed from the solution and then rinsed with the calcium enriched buffer twice. This resulted in a functionalized, EGTA covered 10 surface suitable for the binding of antibodies. The functionalized mica surfaces were then used immediately for binding antibodies. Antibodies are now applied as described in the antibody section below.

Glutaraldehyde: 15 Mica is cleaved and then immersed in absolute ethanol. APTES (3- AMINOPROPYLTRIETHOXYSILANE) is added to the ethanol solution which contained the mica until a 3% v/v mixture is obtained and then stirred rapidly for 3 minutes, resulting in the functionalized mica. The surface of the functionalized mica was then rinsed with ethanol and then with milli-Q water. The mica surface is then placed in a solution of 10% v/v gluteraldehyde in milli-Q water for 10 minutes. The mica surface is then rinsed well with milli-Q water. This functionalized surface is extremely reactive and must be used immediately for antibody fixation. Antibodies are now applied as described in the antibody section below.

PEG: Mica is cleaved and then immersed in absolute ethanol. APTES was added to the ethanol solution to make a concentration of 3% ATPES v/v and stirred rapidly for 3 minutes. The surface was then rinsed with ethanol and then with milli-Q water. The surface is then placed in a small container of PBS buffer at pH 7.5 and BsPEG is added to make a 100 mM solution. The mica surface is allowed to react for 20 minutes and then it is quickly rinsed with milli-Q water. Antibodies are added as described below. Antibody application:

Antibodies were diluted to 0.05 mg/ml with the Hepes buffer and 20 μl of the antibody solution was placed on the functionalized mica surface and allowed to incubate for 1 hour. The surfaces were then well rinsed with the Hepes buffer to remove all unfixed antibodies. Antibody-mica surfaces were stored in Hepes buffer until they were used for the microvesicle experiments. Possible Antibodies comprise: anti- CD41 , anti-P- Selectin, anti- CD62e, anti-CD142, anti-CD 144, and anti-MUC1

Attachment of microvesicles using a microfluidic chip: Prepared coated mica surfaces were carefully kept wet for the remainder of the experiment with Hepes buffer. A microfluidic chip according to the invention was placed on a mica surface and then clamped down to prevent leaks. Using a Harvard Apparatus PicoPlus (http://www.harvardapparatus.com) syringe pump with 1ml Becton Dickinson S.A. (http://www.db.com) Syringe set at a constant flow speed of 0.01 ml/min the channels of the microfluidic chip were subsequently rinsed with 1.5 ml Hepes buffer to make sure there were no bubbles in the lines and to ensure that the channels were clean. Said Syringe was connected to inlets and outlets made of glass capillaries using Luer- LockTM Adapters and One-Piece Fittings from LabSmith (http://www.labsmith.com). Said glass capillaries were connected to said microfluidic chip as has been described in WO2008072968. Approximately 1.5 ml blood plasma was allowed to flow through the channel in the microfluidic chip, thereby contacting the coated mica surface for a period of approximately 30 minutes. The channels were then rinsed with 1.5 ml Hepes buffer to remove any microvesicles not bound to the antibodies. Subsequently, the microfluidic chip was removed and the coated surface with the attached microvesicles rinsed well with Hepes buffer. The coated surfaces were stored in the Hepes buffer until they were imaged. Example 5: use of a microfluidic chip with 3 channels for the immobilization of microvesicles to a coated surface according to the invention (see figure 6). In this method a microfluidic chip according to the invention comprising three channels applied to a mica surface (7). Subsequently, the channels are connected to a fluid comprising coating material via inlets (1), (2) and (3). Outlets (4), (5) and (6) are connected to the channels to a container, wherein the fluid is collected. Subsequently, a fluid suitable for coating a surface is flown through the channels. Subsequently, the microfluidic chip is removed from the coated surface (7) and the coated surface (7) is rotated 90 degrees and another microfluidic chip is reapplied to the coated mica surface (7) in position (6B). The 3 channels now cross the coating at positions (8), (9) and (10). Each channel is subsequently flown with a fluid comprising microvesicles. Microvesicles are now attached at positions (8), (9) and (10).

Example 6

Microfluidic application of the sample to the surface

Figure 7 A-F: Example showing the effects of the microfluidics on concentrating the microvesicles by improving exposure to the surface. This figure shows the microfluidics as extraced from the platelet poor plasma (PPP) (Figure A) compared with a number of controls. Figure B is included to show the number of CD41 positive microvesicles that are typically pulled from the PPP without the concentration of the microfluidics. In figure B the PPP was applied by setting a drop of PPP 50 μl on the surface and then allowing it to incubate for one hour. The difference in the number of microvesicles that are extracted between figure A and B is clear. The plasma drop experiment shows a few particles that have been captured (small white dots). Figure B shows a smaller number of microvesicles. In figure C we see a surface before any plasma has been applied. This surface is only coated with the mouse antihuman CD41 antibody. This shows that there are no spots on this surface that would be mistaken for microvesicles. Figure D shows a surface that is prepared in the same manner as the other surfaces, only the anti-CD41 antibody is replaced with a mouse IgGI isotype control in the same concentration and this negative control has no action against any of the objects in the plasma. The lack of microvesicles on this surface shows that the surface does not nonspecifically capture the CD41 positive microvesicles. As specificity is even more important than the raw numbers, the comparison of figure D to figure A shows that this technique is able to purify and sort only the CD41 positive microvesicles from the blood plasma. (Streaking is an artifact of the atomic force spectroscopy technique, z scales have not been normalized to facilitate comparison of the images) Image collection of cells and microvesicles collected from flowing the PPP through the microfluidics. The microfluidics method is more efficient than the plasma drop experiments in capturing all the different types of microvesicles in the blood. These unusual microvesicles are not seen in the plasma drop experiments, shown in figure 7 E and F, and most likely are activated platelets, the precursors of platelet microvesicles, and fibrin. The figure in the lower right side (G) shows the more common image of microvesicles that can be caught with this method. These cells are extremely rare in this plasma and show the power of this method to capture these targeted microvesicles. This shows another relevant sorting parameter available with this technique as the AFM allows sorting of the microvesicles by size, number and form.

Figure 7I shows the CD41 positive microvesicles that are collected from concentrated, isolated microvesicle preparation that has been run through the microfluidics (left). The figure on the right (J) shows the same type of microvesicle preparation dropped on the surface. In this case there are so many microvesicles in both preparations that the surface is simply saturated in a manner of minutes. It is clear that the microfluidics seem to capture larger and more varied microvesicles. The high centrifugation speed applied during microvesicle isolation helps to purify and concentrate the microvesicles from other plasma proteins, but also might change the characteristics of the microvesicles. On the other hand, the microfluidics method allows concentration and purification of the plasma in one step without any manipulation of the plasma which causes fusion, fission, and activation.

Example 7: Using microfluidics to allow the ex-vivo study of blood microvesicles

We describe a method to detect MPs directly from blood plasma by using a microfiuidic chip and performing subsequent analysis using AFM in liquid tapping mode. The term microfluidics refers to devices, systems, and methods for the manipulation of fluid flows with characteristic length scales in the 10 micrometer range up to 1 millimeter. Laminar flow patterns within the microfiuidic device ensure complete fluid turnover in a controlled manner. As the microfiuidic format is inherently miniaturized, it allows experimentation with very small sample volumes and potentially for high throughput application. In this study, a detachable microfiuidic chip was developed in order to enable direct contact between the fluid in microfiuidic channel and the surface, which is later to be analyzed by AFM. Plasma was flown through the microfiuidic channel with a constant laminar flow and made a direct contact with anti-CD41 antibody-coated mica. MPs with CD41 antigen were captured on this surface and subsequently imaging was done for this surface by AFM. We employed the AFM method for MP detection as described in earlier Examples.

Clotting of the plasma poses a problem in such small volumes and with such sensitive detection as AFM. A variety of methods have been developed by us to prevent the clotting of the plasma including coating of the microfluidics using EGTA, dilution of the plasma with EDTA enriched Hepes buffer and specific control of the coating of the microfluidics channels and tubing leading up to the microfluidics system.

With these measures, we show that this method allows ~100 times more particles to be captured per unit area than can be done without the microfluidics. In conclusion, the application of microfludic chip allows the AFM measurement of MPs directly from blood plasma which shows that high throughput analyses of MPs in a clinical setting are possible, especially using optical detection.

Methods

Blood collection and plasma preparation

After giving their informed consent, venous blood of three healthy volunteers was drawn by using a 21-gauge needle (BD vacutainer, San Jose, CA) and with minimal stasis. Blood, except the first four ml, was collected in 1/10 volume of sodium citrate (3.2%, 0.105 M) using a 4.5 ml BD Vacutainer tube (Becton Dickinson, San Jose, CA). Within 10-15 minutes after withdrawal, blood was centrifuged at 2,000xg for 10 min at 20 ⁰ C, without brake. Plasma was carefully collected and centrifuged again at 2,000xg for 10 min, 2O ⁰ C, without brake, to obtain platelet poor plasma (PPP). PPP was aliquotted in 250 μl portions, snap frozen in liquid N2, and stored at -8O ⁰ C until used.

Microfluidic chip mold fabrication

A microfluidic chip mold was fabricated from brass. This brass was milled so that ridges with dimensions of 10 mm X 300μm X 100μm were maintained. The top surface of the ridges was polished to allow for viewing through the channel from bottom to top after molding. Then, at the end of the ridges small holes were drilled and little pins were inserted with a diameter of 1 mm and a height of about 1.5 mm. Further details are given above.

Microfluidic chip fabrication

Polydimethylsiloxane (PDMS) was fabricated using a Sylgard 184 kit (Dow Corning, UK). Silicone primer and catalyst were mixed in a 10:1 ratio by weight and this mixture was placed in a vacuum chamber for an hour to remove air bubbles. Next, the mixture was slowly poured into the mold and then the mold was carefully closed with a glass plate.

The mold containing the polymer solution was placed in an oven at 70 ⁰ C for one hour.

Afterwards, the glass slide with the PDMS chip was released from the mold and covered with a clean glass slide to keep the chip channel area dust free.

Microfluidic chip setup preparation

The microfluidic chip to create the laminar flow was made by use of injection molding. The chip was pressed against the surface to prevent leaking of the plasma out of the microfluidic channel using a simple holder device which constitutes of two parts screwed together with the chip and surface in between. Two capillaries were connected and attached to the holder using scotch tape. The four screws pressed the chip onto the mica surface which was glued onto a metal disc. The microfluidic channel was filled with a blue solution for illustration purposes.

Mica Surface preparation for attachment of mouse anti-human CD41 antibody

The surface of mica (Electron Microscopy Sciences, Washington) for MP attachment is prepared as described in previous Examples with a slight modification. In brief, a freshly cleaved mica (diameter= 10 mm) was immersed in DMSO containing 55% (w/v) Ethanolamine for overnight at 70° C. Subsequently, the mica surface was rinsed twice with dry DMSO at 70 ⁰ C, rinsed with HLPC grade ethanol to remove the DMSO. Next, the mica surface was put into a solution of 30 ml PBK (pH 7.6 1M) containing 100 mg EGTA for 10 minutes. The surfaces were then rinsed with Hepes buffer (10 mM Hepes (Merck, Germany), 137 mM NaCI (Merck, Germany), 4 mM KCI (Merck, Germany), 0.1 mM Pefabloc® SC (Fluka, Germany), pH 7.4), and then 20 μl of 0.05 mg/ml mouse anti- human CD41 antibody clone P2 (Beckman Coulter, Fullerton, CA) was applied for 3 hours. Excess of anti-CD41 was removed by washing with Hepes buffer. Anti-CD41 antibody coated-mica surface was stored in Hepes buffer until used. As a negative control, mouse IgGI pure clone X40 (Becton Dickinson, San Jose, CA) was used (0.05 mg/ml). All chemicals were purchased from Sigma Aldrich unless otherwise indicated. Attachment of microparticles

Plasma drop experiment

100 ul of PPP was placed directly on the prepared mica surface and incubated at room temperature for 30 minutes. The surface was then rinsed with Hepes buffer and imaged immediately.

Attachment of microparticles using a microfluidic chip

Prepared coated mica surfaces were carefully kept wet for the remainder of the experiment with Hepes buffer. The open microfluidic channel surface was attached to a mica surface reducing the applied surface area and then clamped down to prevent leaks. Using a constant laminar flow, the 150 μl of PPP was flown over a smaller area concentrating attached MPs per surface area.

Using a Harvard Apparatus PicoPlus (http://www.harvardapparatus.com) syringe pump with 1ml Becton Dickinson S.A. (http://www.db.com) Syringe set at a constant flow speed of 0.01 ml/min, the channels of the microfluidic chip were subsequently rinsed with 200 μl Hepes buffer to ensure no bubbles in the lines and to clean channels. The syringe was connected to the fused quartz glass capillaries using Luer-Lock Adapters and One-Piece Fittings from LabSmith (http://www.labsmith.com). The glass capillaries were connected to the microfluidic chip as described above. 150 μl blood plasma was allowed to flow through the channel in the microfluidic chip, thereby contacting the coated mica surface for a period of approximately 15 minutes. The channels were then rinsed with 200 μl Hepes buffer to remove any unbound MPs. Subsequently, the microfluidic chip was removed and the coated surface with the attached MPs was rinsed well with the Hepes buffer. The coated surfaces were stored in the Hepes buffer until they were imaged by AFM. All steps were performed at room temperature.

AFM imaging

AFM imaging was performed with a Digital Instruments Multi-mode atomic force microscope(Veeco, NY) using the E scanner. Olympus cantilevers with force constant of 2 N/m and a resonant frequency of 70 kHz. The liquid cell was rinsed with ethanol and milli-Q water between each sample to prevent MP transference. Each image was scanned at 10x10 μm and 10 representative images were taken at a variety of locations on the surface.

AFM images were processed to count the number of particles and to extract the sizes of the particles using a custom software developed in the lab. This software is a combination of C# (Microsoft Corp, Bellview, Washington) and Labview (National

Instruments, ni.com/labview) with the numerical calculations being performed by

ILNumerics.Net (http://ilnumerics.net/) The automated counting was performed with a enhanced version of the simple watershed method. Some background subtraction is required to allow AFM images to be used with the watershed method. Both functions can be performed by either gwyddion or ImageJ. The program can be automated for batch processing and automatic output.

Results

For plasma drop experiments and microfluidics experiments PPP of three healthy volunteers were used. Anti-lgG and anti-CD41 surfaces were prepared and plasma was dropped onto the surface and the surfaces were allowed to rest for 2 min, 30 min, and 60 minutes. These surfaces were then scanned with the AFM to determine how many microparticles were captured. 0.6, 0.2, 2 MPs per μm ² with a variance of +/- 3 for the 2, 30 and 60 min surface respectively. The IgG control, which was incubated for 60 minutes showed 1 MPs per μm ² This surface showed the same variance. These numbers do not correspond to those reported in Example 1 as purified MPs were used in those experiments, while platelet poor plasma (PPP) was used here.

Surface chemistry in all cases was carefully checked to make sure that the resultant coated surface had both antibodies as well as a minimum of spurious nonspecific particles. The chemistry is as described in previous Examples except for a number of modifications that help to improve the anti clotting performance.

Severe clotting will prevent flow of plasma through the microfluidic channel. Figure 14 demonstrates the effect of mild clotting. Snowflake shaped crystals have formed all the way to the edge of the channel. We believe the particles to the left of the line delineated by the crystals are plasma proteins that diffuse underneath the wall of the microfluidic channel when it is lifted by the pressure applied to flow the plasma through the channel. They are also present when the mica is IgG coated and are not absent when purified MPs are used. To prevent clotting of the plasma in the microfluidic experiment we tested several anticoagulants to prevent clotting, either in the microfluidic channel or on the mica surface. Clotting was very severe with undiluted plasma. Clotting continued to occurwith both citrated plasma and EDTA plasma (citrate and EDTA blood collection protocol) and this problem was not completely solved by diluting the plasma with Hepes buffer containing 3.2% citrate.

Clotting was reduced by diluting EDTA plasma, adding 5 parts EDTA Hepes (20 mM EDTA in Hepes at 7.6 pH) to 1 part plasma . Clotting was reduced further by rinsing the microfluidics and channels with a saturated solution of EGTA in PBS. With this rinse, experiments have been able to run at 1 :1 dilutions of plasma without clotting problems.

Isolated MPs were used to compare with MPs detected directly in plasma. The isolated MPs were diluted 16 times in Hepes and were run through the microfluidics to give a comparison to the drop experiments of Example 1. The number of isolated MPs detected were compared to both the numbers from Hepes dilution, EDTA Hepes dilution and finally a comparison was made to the numbers that could be detected from the plasma drop experiment. Numbers collected are 520, 290, 200, 3 MPs per μm ² for the anti-CD41 surface with Purified MPs, Hepes dilution, EDTA Hepes dilution, and the drop experiment. We believe these values reflect the maximum number of particles that can be captured by a surface because Example 1 found in titration experiments that the maximum number of particles was approximately 500 per 100 Dm ² . The saturation values of surfaces interacting with MPs in plasma may very well be lower because plasma proteins may prevent MPs from reaching their binding sites. For the anti-lgG surfaces (in the same order) we found 1 , 5, 1 , 1 MPs per μm ² . This shows demonstrates that the microfluidics method captures at least ~100 times more particles per unit area.

The AFM does not measure volume, but only the height at each spot. This means that some manipulation must be made to convert an AFM image into a MP size. First an estimate of the size of the particle is made by summing the heights of each pixel. Then it is assumed that the particle was spherical when it was free of the surface. From this spherical particle, the radius and diameter can be derived. For the experiments all plasma and purified samples were diluted with Hepes. The most probable size, as shown by the peak on the frequency histogram, of the MPs for the purified MPs is 45 nm

(±10) diameter, while the most likely size of both Hepes dilued and EDTA Hepes diluted samples is 55 nm(±10). The difference is size is stable against bin size changes from twice as large as shown to 1/3 as small. Drop experiments were not analyzed as there were not enough particles to form a histogram.

Discussion and Conclusion

The microparticles that are collected in this experiment were captured using CD41 antibody. This implies they originate from platelets. These are very active biomarkers that could continue to interact with their environment throughout the collection and detection phases. Therefore, it is expedient that the MPs are examined as much ex-vivo as possible. Figure 13 and 14 show the most intractable problems of detecting these particles ex-vivo. It is obvious that even after an hour, the number of particles collected in the plasma drop experiment fail to rise above the noise floor. This means that in contrast to the results of Example 1 , in which purified MPs were examined, the drop method can not be used as a detection method with unpurified plasma.

With microfluidics, however, we considered that it is possible to reduce the area of the surface that interacts with a reasonably large volume of the plasma, while ensuring that almost of the microparticles in the plasma have a chance to interact with the surface.

We demonstrate that by using a removable microfluidic circuit, MPs can be detected directly by AFM from plasma. By using the microfluidics system, at least 100 times more microparticles per unit area are immobilized. The microfluidics system has been specifically designed to work with the AFM and to be accommodating to blood plasma. This system required balancing many demands.

The AFM is a very sensitive system that is able to detect the many microparticles that are below the detection limit of other methods, as shown here. However, the AFM requires an atomically flat background in order to function best and the AFM cantilever must have access to the whole top of the sample in order to scan. Mica is the preferred background material because mica has the unique material property that it will peal apart in atomically flat layers when it is pulled. PDMS, the material of the microfluidics, bonds strongly to mica and therefore, the mica surface was functionalized over the whole surface. This ensures that the PDMS and the mica were not able to bond together, resulting in a microfluidics system that can be removed from the surface of the sample without damaging the microparticles on the mica nor the mica itself.

Once the physical microfluidics system was prepared, as well as the surface chemistry determined, the plasma was run through the microfluidics system. It became immediately clear that the blood plasma was strongly activated by the microfluidics system, resulting in blood clotting. This can be due to the glass in the tubing, interactions between the plasma and the PDMS and this can also include shear flow and trapped air. In addition, MPs may carry active tissue factor on their surface. Tissue factor (TF), a 47 kDa transmembrane glycoprotein, is a cell surface glycoprotein responsible for initiating the extrinsic pathway of coagulation of plasma protein as well as microparticles. When MPs carry active TF, clotting may occur on the surface of the microfluidic chip and on the mica surface, as shown in figure 14, if clotting factors (fVII and fX) and calcium are present in the plasma. Clotting has been the single most difficult problem in producing a highly reliable device.

This clotting still persisted even though plasma was isolated from blood withdrawn in tubes with anticoagulants such as sodium citrate and EDTA. Diluting PPP with Hepes buffer supplemented with 20 mM EDTA (ratio 1 :5) helps to prevent clotting to some degree, while dilution of the PPP with Hepes did not produce as strong a protective effect. When EDTA is used, there is less clotting found in the microfluidic channel and on the mica surface. We believe this is because EDTA is a strong chelator of calcium ion and its binding is strong.

Just dilution of the plasma helped as presumably, the clotting proteins are less likely to find each other, but in the small confines of the microfluidics channel, clotting still occurred.

The EDTA and dilution helps control the clotting in the channel, but it is clear the PDMS can produce clotting. In an attempt to ensure that the microfluidics disturbs the sample as little as possible, a solution of 100 mM EGTA in PBS is flowed through the channel before application of the plasma. With the EGTA rinsing, even low dilution plasma experiments can be performed without the threat of clotting. The mechanism of the

EGTA is not fully understood, but has been tested for both plasma on the surface as well as these clotting experiments. It is thought that the EGTA either produces a very clean surface (as a chelator) or that it may physisorb on the surface providing a similar static action as EDTA.

The microfluidics seem to act as a MP signal amplifier. All amplifiers have the problem of saturation. In this case, saturation means that every possible MP binding site on the surface has a MP. At this point, the count of MPs no longer scales with the number of MPs in solution and the count becomes meaningless in all situations except those that involve checking for the presence or absence of MPs or the shapes. Each application will require a dilution series to be made to determine what dilutions of plasma, or other body fluid, are required to ensure that the MP count falls in the dynamic range of this device, as will be readily apparent to those skilled in the art.

Figure 16 shows a slight difference in the size distribution of particles found by the microfluidics method between purified microparticles and microparticles originating from plasma. This size distribution is persistent through both Hepes and EDTA Hepes dilutions. While we have no explanation for this difference in size, it suggest that it is desirable to detect microparticles directly in plasma.

We believe the particle numbers detected in Figure 15 reflect the maximum number of particles that can be collected by the surface. The maximum number of particles is influenced by plasma proteins that are attracted to the surface as shown in figure 17. These proteins prevent the MPs from reaching the surface. While the experiments employing purified MPs display the lowest amount of these additional proteins, they show the highest MP saturation number and this number is indeed similar to the saturation number shown in Example 1. For both the plasma experiments, there is an enormous amount of reactive protein in solution, specifically designed to stick to everything. It seems plausible that this would result in a decrease of the number of particles that can be captured. EDTA plasma appears to have the lowest number but this falls within the error margin for the saturation number. It is considered that the variability of this technique can be reduced by using a glass/silicon Polymer system. Cleaved mica, a mineral surface, can not always be covered homogeneously with active antibodies, which can result in antibody surface density differences.

We consider that the method can be used to detect MPs from other origins (endothelial cells, monocytes, tumor cells, etc) by using different antibodies. This will help in monitoring certain types of MPs which may play role in certain diseases. The power of the ex-vivo method has already been demonstrated in these Examples. In an effort to help develop a diagnostic tool, high speed AFM methods are being developed and automatic particle counting and analysis have been optimized for this technique. Additionally, we believe the number of particles captured also allows optical detection of these particles. We find that on average the particles are spaced by 1 to 2 μm. While this does not allow the separation of particles with ordinary wide field or confocal microscopy, and therefore one can no longer count the number of particles and the size of the particles, one would be able to infer from the fluorescence intensity what the total number of antigens is. The increase in density of particles should aid in increasing the signal to background ratio of such a fluorescence experiment. Although many particles are smaller than the resolution limit of conventional microscopy, the average distance between the particles, still allows the discrimination of individual particles. The fluorescence intensity reflect the abundance of bound fluorescently labelled anti-bodies and therefore the surface density of the ligand on the microparticles. This type of measurement should be accompanied with a negative control e.g. MV-free plasma.

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