WO2011090441A1

WO2011090441A1 - A process for making a microarray

Info

Publication number: WO2011090441A1
Application number: PCT/SG2011/000033
Authority: WO
Inventors: Daniel Lubrich; Dieter Trau
Original assignee: National University Of Singapore
Priority date: 2010-01-25
Filing date: 2011-01-25
Publication date: 2011-07-28

Abstract

There is provided a process for making a microarray. The process comprises the steps of: a) providing a plurality of holes that extend into a substrate; b) providing a population of microparticles that are chemically or biologically active; and c) forcing the microparticles at least partially into said holes, wherein the holes and microparticles are dimensioned relative to each other to enable locking engagement of said microparticles at least partially within said holes.

Description

A PROCESS FOR MAKING A MICROARRAY

Technical Field

The present invention generally relates to a process of forming a microarray. The present invention also relates to the microarray.

Background

Microarrays are widely used for diagnostic applications in research and clinical settings. Patterning biomolecules such as proteins and DNA on a solid support in a controlled way is the basic method behind the fabrication of microarrays. In traditional microarrays, such biomolecules are carried directly on their solid surface using spotting technology. However, poor precision and low reproducibility are downfalls of such technology.

On the other hand, bead microarrays carry microbeads which in turn are conjugated to biomolecules. Bead microarrays have significant advantages such as consistency, flexibility and faster kinetics. Bead microarrays are flexible because the surface chemistry of the beads can be tailored to suit the biomolecule to be conjugated on the bead. Further, bead microarrays ensure consistent results since the dimensions of the beads can be made identical.

However, the two main problems with bead microarrays are with encoding and decoding of the beads and with efficient ways of depositing the beads onto the array surface. Currently, bead protein arrays typically come in the form of "liquid arrays" where the beads remain in suspension and are read out and decoded via physical tags during flow cytometer based analysis, such as VeraCode™ assays of Illumina Inc., San Diego,. California, United States of America and xMAP™ referencing of Luminex Corporation of Austin, Texas, United States of America.

The current method of depositing the beads onto the array surface is by capturing the microbeads with structured surfaces having micro-wells. Typically employed techniques for microstructuring of solid surfaces include lithography and imprinting. However in current bead deposition processes, a significant portion of the beads is usually lost because random bead deposition outside of the wells requires a washing step that removes these beads. Additionally, only a fraction of the beads is deposited and the exact number of beads deposited or the bead surface coverage can not be controlled.

Therefore, there is a need to provide a microparticle deposition technique that overcomes, or at least ameliorates, one or more of the disadvantages described above .

There is a need to provide a microarray that overcomes, or at least ameliorates, one or more of the disadvantages described above.

Summary

According to a first aspect, there is provided a process for making a microarray comprising the steps of: a) providing a plurality of holes that extend through a substrate;

b) providing a population of microparticles that are chemically or biologically active; and

c) forcing the microparticles at least partially into said holes, wherein the holes and microparticles are dimensioned relative to each other to enable locking engagement of said microparticles at least partially within said holes. Advantageously, the disclosed process may ensure that a hole has only one microparticle lockingly engaged therein. More advantageously, the disclosed process may ensure that a desired number of microparticles are deposited onto the substrate. By defining the number of microparticles to be deposited onto the substrate, a defined surface coverage or deposition density may be obtained. The disclosed process may allow for a 100% deposition yield of microparticles on the substrate.

Advantageously, because the holes extend through the substrate, they allow fluid carrying the particles to flow through the holes and allow the carriage of the microparticles at a speed which allows rapid deposition and firm locking engagement into the holes of the substrate. It has been observed that such rapid deposition significantly reduces the manufacture time of a microarray .

Even more advantageously, the disclosed process may allow the control of exact microparticle deposition numbers. Hence, the disclosed process may overcome problems associated with the prior art such as uncontrolled deposition density resulting from random microparticle deposition in which only a fraction of the microparticles is deposited, inability to control the exact number of microparticles to be deposited and inability to control the microparticle surface coverage.

The disclosed process may not require a washing step in order to remove excess microparticles. This is in comparison to prior art methods of depositing microparticles such as microbeads in wells in which excess microbeads that are not deposited have to be removed by washing. Hence, in one embodiment, the process may exclude a washing step to remove excess microparticles. Further, a washing step is not necessary because the deposition of a desired number of microparticles can be controlled adequately. The deposition number or density can be controlled by ensuring that a hole has one micropart icle lockingly engaged therein, due to the force applied during micropart icle deposition.

In one embodiment, the deposition process is imaged at intervals in order to determine if the desired deposition density is achieved. In one embodiment, the deposition process is imaged continuously in order to determine if the desired deposition density is achieved. If the desired deposition density has not been achieved, then more microparticles can be deposited until the desired deposition density is reached.

In another embodiment, if an imaging apparatus is not available and the number of holes is known, in order to achieve a 100% deposition density, the exact number of microparticles equivalent to the number of holes can be deposited .

The disclosed process may allow several microparticle batches to be deposited sequentially or concomitantly.

According to a second aspect, there is provided a microarray comprising a substrate having a plurality of holes that extend through the substrate and a plurality of chemically or biologically active microparticles that are lockingly engaged at least partially within said holes, wherein said locking engagement is due to the relative dimensions of the microparticles and the holes.

According to a third aspect, there is provided a microarray for use in a system for identifying the presence of one or more target analytes in a sample, the microarray comprising a substrate having a plurality of holes that extend through the substrate and a plurality of chemically or biologically active microparticles that are lockingly engaged at least partially within said holes, wherein said locking engagement is due to the relative dimensions of the microparticles and the holes.

According to a fourth aspect, there is provided a microarray system for identifying the presence of one or more target analytes in a sample comprising:

a microarray comprising a substrate having a plurality of holes that extend through the substrate and a plurality of chemically or biologically active microparticles that are lockingly engaged at least partially within said holes, wherein said locking engagement is due to the relative dimensions of the microparticles and the holes; a memory recording the location of each microparticle, said location of each microparticle having been determined by imaging of the substrate;

a detector to detect changes in the microparticles upon contact with the sample; and

a processor responsive to program instructions to interrogate the memory and to compare data received from the detector to identify the presence of the one or more target analytes in the sample based on the location of each microparticle.

Definitions

The following words and terms used herein shall have the meaning indicated:

The terms "microarray" or "array" as used herein refers to an array of microparticles on a solid support, wherein each microparticle has a selected, active agent capable of binding with one or more target analytes.

The term "microparticle" refers to a particle having a particle size in the micron-sized range, or from 0.1 microns to about 1000 microns. In one embodiment, when the microparticle is substantially spherical in shape, the particle size refers to the diameter of the microparticle, which is in the micron-sized range. Where the microparticle is spherical shaped, the "microparticle" is then termed as a "microbead". In another embodiment where the microparticle does not have a spherical shape, the particle size may refer to the equivalent diameter of the particle relative to a spherical particle or may refer to a dimension (length, breadth, height or thickness) of the non-spherical particle.

The term "through-hole" refers to a hole that extends through one dimension, typically the height, of the substrate such that the hole connects a first surface of the substrate to a second surface of the substrate, the second surface being opposite to the first surface. In one embodiment, the through-hole can be envisaged as extending through the height of the substrate such that the through- hole connects the top surface to the bottom surface of the substrate, with no intervening substrate material therebetween .

The term "population" as used herein refers to the total universe of microparticles deposited on the microarray. The terms "subpopulation" as used herein refers to a group of microparticles within the population that all comprise the same active agent.

The terms "lockingly engage", "lockingly engaged" or grammatical variants thereof, refers to .physical engagement of the microparticles with the sidewalls of their respective through-holes such that the microparticles are at least partially embedded or wedged into the holes. Due to their physical contact with the holes, the microparticles may not be dislodged easily from the holes unless they are subjected to a force that overcomes the physical interaction between the microparticles and their respective holes. Typically, the microparticles will not dislodge from the holes under the application of gravity (ie if the microarray were to be held such that the microparticles would not fall from the holes under action of gravity) . Further, a lockingly engaged microparticle has a stable position within the hole and on the substrate during the performance of bioassays, imaging or deposition of other particle subpopulations , but may be intentionally dislodged by applying a strong force, e.g. using a fluid or gas flow.

The term "active agent" may refer to any chemical agent that is chemically active or biological agent that is biologically active and which is capable of reacting^' with a target analyte or an intermediary bound to the target analyte. The active agent may exhibit chemical activity and may include an environmental contaminant such as organic materials (for example, aliphatic hydrocarbon compounds, aromatic-containing compounds and chlorinated compounds) or inorganic materials (for example, metals and nitrates); a chemical warfare agent (for example, nerve agents such as sarin, soman, tabun and cyclosarin, blood agents such as arsines and hydrogen cyanide, or lachrymatory agents such as tear gas and pepper spray) ; a herbicide; a pesticide; a metabolite; a drug; or a chemical catalyst. The active agent may exhibit biological activity and may be referred to in the specification as a "bioactive agent". Exemplary bioactive agents include proteins, oligopeptides, small organic molecules, coordination complexes, aptamers, cells, cell fragments, virus particles, antigens, polysaccharides and polynucleotides, which can be attached to or bonded to a microparticle. Accordingly, the term "biologically active microparticle" refers to a microparticle as defined above which has an active agent that has biological activity or by itself is biologically active. The term "chemical active microparticle" refers to a microparticle as defined above which has an active agent that has a chemical activity. The term "active agent" may also refer to an agent exhibiting physical activity, such as responding to a physical stimulus in a predetermined way including processes such as emission of light upon excitation, emission of heat upon absorption of electromagnetic radiation or microwaves.

The term "target analyte" refers to a substance to be detected that is capable of binding to the active agent. A target analyte may also be a substance to be detected for calibration purposes. Exemplary target analytes include,^" but are not limited to, nucleic acids, polynucleotides, drugs, hormones, proteins, enzymes, antibodies, carbohydrates, and antigens. The target analytes may be labelled with a fluorescent tag to create a fluorescent signal.

The term "specific binding substance" may refer to a substance which has a specific affinity for a certain substance. For example, a target analyte in a sample may be capable of undergoing a specific binding reaction with the active agent. Examples of combinations of the specific substance with the specific binding substance include: antigens with corresponding antibody molecules, a nucleic acid sequence with its complementary sequence, effector molecules with receptor molecules, enzymes with inhibitors, activators or substrates, sugar chain- containing compounds with lectins, aptamers with its binding partners, an antibody molecule with another antibody molecule specific for the former antibody, receptor molecules with corresponding antibody molecules and the like combinations. Other examples of the specific binding substances include a compound which has been chemically modified to such a degree that its specific binding activity still remains intact and a complex body of a compound bound to other components. Examples of combinations of such types of specific binding substances with the specific substances include: a chemically biotin- modified antibody molecule or polynucleotide with avidin, an avidin-bound antibody molecule with biotin and the like combinations.

The term "protein" as used herein may be defined as two or more covalently bonded amino acid, which includes proteins, polypeptides, oligopeptides and peptides.

The terms "amino acid" and "peptide", as used herein refer to both naturally occurring and synthetic amino acid and amino acid chains respectively.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically + /- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Certain embodiments may also be described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Detailed Disclosure of Embodiments

Exemplary, non-limiting embodiments of a process for making a microarray will now be disclosed.

The process comprises the steps of a) providing a plurality of holes that extend through a substrate; b) providing a population of microparticles that are chemically or biologically active; and c) forcing the microparticles at least partially into said holes, wherein the holes and microparticles are dimensioned relative to each other to enable locking engagement of said microparticles at least partially within said holes.

In one embodiment, the holes extend through the substrate. Hence, these holes can be considered as through-holes. Accordingly, these holes are distinguished from wells or other microstructures such as channels, pillars or lines.

The holes may be generally circular in cross-section. The holes may not be completely circular in cross-section and may be oval-shaped. The holes may be of any shape such as a cylindrical shape in which the side-walls of the holes are substantially parallel with respect to each other, or the holes may be of a conical shape, in which the sidewalls of the holes are generally tapered from a first circumference at a first surface of the substrate to a second smaller circumference at a second surface of the^' substrate, the second surface being opposite to the first surface.

The holes may be sized depending on the particle size of the microparticle to be lockingly engaged therein. In one embodiment, the circumference of the hole on the side that first contacts with the microparticle may be substantially larger than or equal to the diameter (or equivalent diameter) of the microparticle. Accordingly, the size of the holes may be in the micron-sized range, or from 0.1 micron to about 1000 microns.

In one embodiment, where the hole is of a conical shape such that the first circumference of the hole at the first surface of the substrate is, larger than the second circumference at the second surface of the substrate, the cross-sectional circumference of the microparticle may be of a value between the first and second circumferences such that the cross-sectional circumference of the microparticle is greater than the second circumference. Hence, the largest cross-sectional area of the microparticle may be greater than the area defined by the second circumference of the hole. Accordingly, at least part of the microparticle may protrude from the hole · or the microparticle may reside completely within the hole. The plurality of holes may be of different shapes and sizes on the same substrate in order to accommodate the microparticles which can be of varying shapes, sizes, colours and types. Hence, by controlling the shapes and sizes of the holes, the deposition density and positions of a specific subpopulation of microparticle can also be controlled accordingly.

The population of microparticles may be provided onto the substrate as a suspension of microparticles in a liquid carrier or in an aerosolized form. The movement of the liquid carrier or aerosol gas cloud may aid in^' ensuring that each microparticle is provided at the entry of a suitably shaped and sized hole. The microparticle may then be forced into its respective hole to be lockingly engaged therein.

The microparticle may be forced into its respective hole by applying at least one of the following steps: (i) applying a pressure differential between the first and second surfaces of the substrate; (ii) applying a magnetic field to the microparticles; (iii) applying an electric field ^' to the microparticles; (iv) subjecting the microparticles to a centrifugal force; (v) subjecting the microparticles to a gravitational force; (vi) providing a fluid flowing in a direction perpendicular to the surface of the substrate to move the microparticles into the respective holes; and (vii) providing a fluid flowing in a direction parallel to the surface of the substrate to move the microparticles into the respective holes.

In a particular embodiment, the pressure differential can result from applying a transversal flow of the suspension or an inert fluid through the substrate in order to force the microparticles into their respective holes. The transversal fluid flow is applied in a direction that is generally normal to the surface of the substrate and hence is a generally transverse fluid flow with respect to a planar substrate surface so that preferably the fluid goes directly along a longitudinal axis that extends through the holes of the substrate. It is important to note that the transverse fluid flow of the fluid with respect to the planar substrate surface does not have to be completely transverse but should be generally transverse in that the fluid flow is not a parallel fluid flow with respect to the planar surface of the substrate. Typically, the fluid flow is at least 30° with respect to the planar surface of the substrate, preferably at least 45°, more preferably at least 60°, and advantageously about 70 to 90°. The generally transverse fluid flows through the holes, thereby transporting the microparticles directly into the holes. The more transverse fluid flow, the more rapid will be the microparticle deposition of the microparticles into the holes of the substrate. In a more specific embodiment, the number of particles per volume element of the fluid is known and by measuring and regulating the fluid volume flow, a predetermined number of particles is deposited per time. Advantageously, a transversal flow forces the microparticles into their respective holes at a speed which may be about 10 to about 100 fold faster than a parallel flow. In a parallel flow, each batch of microparticles may require a few minutes to be deposited into their respective holes. However, the time taken to deposit the microparticles in a transversal flow may be a few seconds. Rapid deposition is important because it significantly speeds up the manufacture time of the micro- array.

The transversal flow applied may be a suction of a fluid through these holes during manufacturing, where the particles can be actively guided into their capturing sites or holes. A pump or a suction pad using capillary forces can be used to create the transversal flow. Other ways to force the microparticle into their respective holes can include buoyancy force or capillary flow. In the embodiment where capillary flow is used, the holes are regarded as capillaries themselves and when contacted with a fluid carrying suspended particles, a capillary flow is created to carry the suspended particles into the hole. In the embodiment where buoyancy force is used, the suspended particles have a lower density than the suspension liquid and the floating particles are directed into a hole via- capillary flow. Steps b) and c) of the disclosed process may be repeated to enable locking engagement of at least two populations of microparticles.

The number of microparticles deposited within the same hole may be controlled. In one embodiment, a single microparticle is deposited in one hole by using a smaller average hole diameter relative to the particle diameter. The microparticle may^ be lockingly engaged in the hole by the forcing step. In another embodiment, more than one microparticle resides within one hole. For example, two microparticles may be deposited into the same , hole. In this embodiment, conically shaped holes are used and a first population of microparticles with a diameter larger than the smaller diameter of the holes is applied and lockingly engaged in the holes by the forcing step. A second subpopulation of microparticles with a larger diameter or a diameter relatively closer to the diameter of the larger diameter of the holes may be subsequently applied and lockingly engaged in the occupied holes, thereby leading to the deposition of two microparticles in the same hole.

The number of microparticles deposited per unit area, also termed as "microparticle density", may be controlled. The microparticle density may be controlled by continuous or non-continuous imaging during or after the forcing step in the microparticle deposition process.

Hence, the number of holes may be more than the number of microparticles deposited. In an embodiment where 100% deposition of microparticles is desired, all the microparticles deposited and lockingly engaged.

In an embodiment where 100% fill rate for holes is desired, the number of holes is the same as the number of microparticles to be deposited.

Imaging the substrate during or after each forcing step may allow for precise control and/or counting of the number and positions of deposited particles and microparticle density. Imaging may also aid in determining the positions of the microparticles in the holes. Imaging may also enable the possibility of depositing several batches of microparticles without using a physical characteristic of the microparticle. Imaging during or after the forcing step may allow for the identification of each microparticle via its position on the substrate.

The deposition of microparticles may be stopped whenever a defined number of microparticles are deposited or when a defined surface coverage or deposition density is reached. For example, the deposition density may be typically about less than 1% to 20% per batch to deposit a plurality of batches or 20% to 100% to deposit a single batch. Several microparticle batches may be deposited sequentially or concomitantly; wherein a batch may be composed from the same microparticles or it may contain sub-batches with different microparticles. To distinguish microparticles in holes from microparticles not trapped within a hole, a specific interaction between microparticles and the interior of holes to create a signal is possible. Such an interaction is for example, but not limited, a FRET based reaction at which a donor dye on the microparticle transmits energy to an acceptor dye on the substrate at the interior of the holes to create a signal. Other possibilities are detector dyes and quencher combinations. In general, all reactions or interactions which generate a signal if two interaction partners come in very close proximity are possible.

Accordingly, the disclosed process may allow the control of exact microparticle deposition numbers and deposition density for a single batch or sequential batches of microparticles . Alternatively, or in addition to the imaging, the flow rate as the function of the applied pressure difference is recorded. The flow rate as a function of pressure difference is a measure for the number of microparticles assembled within the through- holes and is reduced with increasing number of microparticles captured. The flow rate may be kept constant by controlling and increasing the pressure difference to assemble a predetermined number of microparticles within the holes per unit time by using microparticle suspensions (or aerosols) with exactly known microparticle number per volume element.

In one embodiment, the microparticle may be a microbead or a biological entity such as a cell, a bacteria or a virus particle. In embodiments where the microparticle is a microbead, the microbead may be an irregularly shaped microbead or a regularly shaped microbead. The microbead may also have a shape selected from the group consisting of microsphere, microcapsule, microrod, microcube and microtube. Most preferably, the microbead is a microsphere.

An exemplary microbead may be formed of a material selected from plastic, ceramic, glass, metal, silicon dioxide, polystyrene, methylstyrene, acrylic polymer, paramagneticmaterial , thoria sol, carbon graphite, titanium dioxide, latex or cross-linked dextran such as sepharose, cellulose, nylon, cross-linked micelle and teflon or with the similar compositions used in peptide, nucleic acid and organic moiety synthesis or mixtures thereof, e.g. metal filled polymer particles.

In embodiments where the microparticle is a cell, the cell may be a living cell or a dead cell. The cell particles may be applied as a cell suspension containing single cells or cluster of cells and deposited by any methods described above to produce cell microarrays. The^' holes of the substrate may be selected in size and shape to selectively capture only one particular cell type of a cell suspension containing a plurality of cell types or selectively capture single cells or cell clusters of a suspension containing a mixture of single cells and cell clusters. Captured cells may be cultured in the holes of the substrate and allowed to multiply. The morphological analysis of cells by means of microscopic methods may be performed, including cell staining methods.

If a large surface area is desired, the microparticle may be at least partially porous. For porous microparticles , reactions to perform a physical, chemical, biochemical, enzymatic or immunoassay can be carried out both on the surface of the microparticle and in the interior of the microparticle. Porous microparticles may have diffusion properties controlled by their porosity and permeability to exclude unwanted or interfering molecules from diffusion into the interior. Porous microparticles may also have diffusion properties to entrap active agents such as enzymes, antibodies, DNA, cells or reagents from diffusing out and thereby entrapping or immobilizing them into the interior. Accordingly, the microparticle may be at least partially porous or has a porous capsule to allow the passage of desired analytes into the interior of the microparticle.

The microparticle may have a particle size in the range from about 0.1 micron to about 500 microns, or from about 1 micron to about 10 microns. Each microparticle may comprise at least one active agent that is attached to, or incorporated within, the microparticle structure that is capable of specific binding with at least one target analyte. In one embodiment, the microparticle comprises a single type of active agent. In another embodiment, the microparticle may comprise at least two active agents, each agent may be independently from each other be a chemical agent or a bioactive agent.

The active agent that may be attached to the microparticles may be an organic compound or an inorganic compound. The organic active agent may be selected from the group consisting of peptides, proteins, nucleic acids, metabolites, carbohydrates, enzymes, antibodies, hormones, lectines, drugs, pesticides, allergens, antigens, receptors, fatty acids and mixtures thereof.

The protein may be a naturally occurring protein or a synthetically synthesized protein. The protein may be obtained from cellular extracts or from random or directed digests of proteinaceous cellular extracts.

The , nucleic acid may be naturally occurring or synthetically synthesized. The nucleic acid may be single stranded or double stranded or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid.

The active agents may be modified through conventional chemical, physical and biochemical means prior to attachment on the microparticles.

The .active agents may either be synthesized directly on the microparticles, or they may be made and then attached after synthesis. In one embodiment, linkers are employed to attach the active agents to the microparticles , to provide better attachment, improve interaction with the target molecule due to the increased flexibility, and to reduce undesirable or non-specific binding. The attachment of the active agent onto the microparticle may be dependent on chemical interactions selected from the group consisting of electrostatic interaction, ionic bonds, covalent bonds, hydrogen bonds and dipole-dipole interaction. Prior to attaching of the active agent to the microparticle, the microparticle may^' be functionalized with chemically reactive groups to facilitate binding.

The microparticles deposited onto the substrate may belong to different subpopulations in which the microparticles in the respective subpopulations differ from each other in at least one of shape, size, colour, type of microparticle, type of active agent attached to the microparticle and type of identifier (if any) attached to the microparticle.

The substrate material may be selected from synthetic or naturally occurring polymeric materials, organic materials, inorganic materials, metals, ceramics, plastic, rubber, glass, fibrous materials, graphite or silicon. Exemplary substrates are selected from the group consisting of silicon, silicon dioxide, silicon nitride, modified silicon, glass and modified or functionalized glass, inorganic glasses, plastics, acrylics, polystyrene and copolymers of styrene, polypropylene, polyethylene, polybutylene , polyurethanes , Teflon, polysaccharides, nylon, nitrocellulose, resins, silica, silica-based materials, carbon and metals. In one embodiment, the substrate does not auto-fluoresce . The substrate material may be a composite of two or more of the above materials. In one embodiment, the substrate may be composed of an upper material containing the through-holes sandwiched with a second material which is permeable to the microparticle liquid carrier or aerosol gas cloud. The second material may be mechanically stable and forms a sandwich composite with the upper material. The additional substrate materials may be sandwiched on one of two sides of the upper substrate material. The additional substrate materials may be selected from the group selected from a porous substrate, a non-flexible and planar substrate, a substrate having a plurality of holes that extend through the substrate and a mixture thereof. A non-flexible and planar substrate may be more flexible relative to a porous substrate and are typically mechanically stiff.

In one embodiment, the through-hole of a first substrate may be aligned with the through-hole of a second substrate wherein the two through-holes may have different dimensions to facilitate the lockingly engagement of a particle .

The substrate may comprise a surface that is hydrophobic or hydrophilic. Advantageously, the hydrophobic or hydrophilic surfaces can be utilized to attract sample fluids containing said target analytes and possibly to repeal unwanted fluids. For example, if the target analyte is in an aqueous fluid, the substrate may be hydrophilic to promote attraction of the target analyte to the microparticles .

The surface of the substrate or the interior of the through-holes or a second substrate material below the first substrate may be chemically modified to facilitate a stronger adhesion of microparticles into the holes. I

The target analyte may be organic or inorganic molecules. The target analyte may be selected from the group consisting of environmental pollutants (including pesticides, insecticides, toxins, etc.); a chemical (including solvents, polymers, organic materials, etc.); therapeutic molecules (including therapeutic and abused drugs, antibiotics, etc.); biomolecules (including hormones, cytokines, proteins, nucleic acids, lipids, carbohydrates, enzyme, antibodies, antigens, cellular membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands,^' etc); whole cells (including procaryotic (such as pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells); viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); and spores. The target analytes may be nucleic acids and proteins (including immunoglobulins; enzymes, hormones and cytokines). The specific binding of the target analyte to the bioactive agent may be dependent on chemical interactions selected from the group consisting of electrostatic interaction, ionic bonds, covalen^'t bonds, hydrogen bonds and dipole-dipole interactions.

When the microarray is being used to analyse a sample potentially containing a target analyte, the microparticles may be tagged with an identifier selected from the group consisting of a fluorescent tag, a bar code, a chemical identifier, a quantum dot, a microstructure , a nucleic acid identifier, an engraving and a radio frequency tag. The identifier may also be added to the target analyte.

Advantageously, the identifier may be used to enhance the identification of the location of the microparticles and subsequently the active agents on the microparticles. The identifier may exhibit a change when the active agent binds to the target analyte. This change may be viewed optically under an imaging apparatus or colorimetric apparatus. In one embodiment, the identifier may be applied after the sample containing the target analytes have bound to the active agents of the microparticles .

The identifier may be conjugated on the target analyte which in turn binds to the corresponding active agent on the microparticle. The identifier may be directly conjugated to the target analyte or indirectly conjugated^' to the target analyte by means of a linker molecule.

In an embodiment where the identifier is a fluorescent tag, the fluorescent tag may be a mixture of reporter dyes. The variation of the composition of the mixture of reporter dyes may change the output optical signal intensity, providing a large possible range of unqiue optical signatures.

The optical signature may be detected with a detector, such as an optical detector. The optical detector may send a signal to the computer memory ^' which is then accessed by a computer processor for generating an image file. Data associated with the image file such as the position and type of microparticle which exhibits the optical signature is then compared with the data obtained after each microparticle batch deposition, i.e. the encoding/decoding data table, to identify the identity or the batch of the microparticle. Those microparticles which exhibit the optical signature are those which have bound with the target, analyte. As the active agent for the subpopulation of the microparticles is known from the encoding/decoding data table, it is possible to identify the target analytes in the sample. The plurality of microparticles may be differentiated from each other by way of a visual identifier selected from the group consisting of colour, shape and size. The plurality of microparticles may also be differentiated from each other by way of spatial position.

After the microparticles are deposited onto the substrate, the microparticles may be immobilized onto the substrate by applying a porous sealant such as a gel or hydrogel film or layer on top of the substrate material to entrap the microparticles. The gel or hydrogel may be permeable to small molecules and macromolecules , including^" viruses and bacteria but can effectively entrap the much larger microparticles. Alternatively, the substrate carrying the microparticles may be sandwiched with another substrate above and/or below to thereby cover and entrap the microparticles.

After deposition and optional immobilization of the microparticles, the substrate may be cut into smaller pieces and used individually or integrated into a system or device. The substrate may be further treated by chemicals or dried; for example, the particles may be chemically crosslinked to the substrate to facilitate adhesion .

The disclosed process may enable microparticle arrays with very high multiplexing capabilities to be produced.

The microparticle array made according to the disclosed process may be used for analytical purposes. Here, a sample is brought into contact with the microparticle array. The sample may be a liquid or a gas. The sample may be applied via a pipette or a similar device, or the sample may be applied via a vertical flow through the substrate and/or a horizontal flow over it. The microparticle array may be placed into a fluidic device or into a well. Several such fluidic devices or wells may form part of a larger device carrying many microparticle arrays.

When the sample contains a target analyte that binds to the active agent on the microparticles lockingly engaged in the holes, a signal would be generated as a result of this binding. For example, the active agent may be an antibody and the target analyte may be an antigen that binds to the antibody active agent. The antigen may be tagged with a fluorescent tag such that a fluorescent signal is generated upon antibody-antigen binding. The analyte may be directly labeled by a chemical reaction^" with the fluorophore or by using a second fluorescent labeled detector antibody.

The disclosed process may provide an alternative method to fabricate bead microarray device for analysis and quantification of biological, chemical or physical parameters.

The disclosed process may ensure that a 100% deposition yield of the microparticles on the substrate can be achieved. The disclosed process may deposit the microparticles on the substrate in a short period of time, from about 1 millisecond to about 10 seconds, or about 500 milliseconds to about 10 seconds, or about 1 second to about 10 seconds or about 5 seconds to about 10 seconds, or about 1 millisecond to about 5 seconds, or about 1 millisecond to about 1 second. In one embodiment, the disclosed process may deposit the microparticles on the substrate from about 1 second to about 10 seconds.

There is also provided a microarray for use in a system for identifying the presence of one or more target analytes in a sample, the micro-array comprising a substrate having a plurality of holes that extend through the substrate and a plurality of chemically or biologically active microparticles that are lockingly I engaged at least partially within said holes, wherein said locking engagement is due to the relative dimensions of the microparticles and the holes.

Further, there is provided a microarray system for identifying the presence of one or more target analytes in a sample comprising: a micro-array comprising a substrate having a plurality of holes that extend through the substrate and a plurality of chemically or biologically active microparticles that are lockingly engaged at least partially within said holes, wherein said locking engagement is due to the relative dimensions of the^" microparticles and the holes; a memory recording the location of each microparticle, said location of each microparticle having been determined by imaging of the substrate; a detector to detect changes in the microparticles upon contact with the sample; and a processor responsive to program instructions to interrogate the memory and to compare data received from the detector to identify the presence of the one or more target analytes in the sample based on the location of each microparticle.

The image of the location of the microparticle may be recorded in the encoding/decoding data table in the memory of a computer. The image can therefore be readily accessed by the processor acting under instructions of a computer program to decode the microarray by determining the position of the microparticle and the associated target analyte relative to the fudicial. The computer readable memory may comprise an acquisition module that may comprise a computer code that can receive a data image from a random array and a registration module comprising a computer code that can register the data image using at least one fiducial to generate a registered data image. The registered data image may be stored in a storage module as needed. The same computer code, or different code, may be used to receive additional data images and generate additional registered data images, which may also be stored. Preferably, the computer readable memory may further comprise a comparison module comprising computer code that can compare the registered data images to determine the differences between them, to allow both decoding of the array and target analyte detection. The comparison of at least two registered data images allows the identification of the location of at least two unique bioactive agents on the array.

Brief Description Of Drawings

The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

Fig. 1 schematically illustrates the micropart icles being assembled on a substrate to produce a microarray in accordance with an embodiment of the disclosure.

Fig. 2 is a schematic cross-sectional view of microparticles captured on a substrate with through-holes.

Fig. 3 is a schematic of microparticles being directed into the substrate holes via the application of a vertical flow through the substrate holes.

Fig. 4 is an optical micrograph showing deposited microparticles on a substrate with 9 μιτι holes, lying on top of another substrate with about 100 m holes.

Fig. 5 is an optical micrograph of the substrates in

Fig. A on a glass surface showing the deposited microparticles. Fig. 6 is a side view optical micrograph of a substrate with 9 μπι holes with deposited microparticles.

Fig. 7 is a series of quasi-continuous imaging during particle deposition to deposit a predetermined number of microparticles.

Fig. 8 is a process flow diagram to produce the microparticle microarray.

Figs. 9-1 to 9-3 show a series of micrographs showing the array before and after a bioassay.

In the figures, like numerals denote like parts.

Detailed Description of Drawings

The current disclosure provides a process to control microparticle deposition density and deposit an exact pre- determined number of nano or microparticles onto a substrate material. Referring to Fig. 8, an overview of the process flow to produce the microparticle microarray in accordance with an embodiment of the present disclosure is shown.

In step 101, a porous substrate is provided. The porous substrate is produced for example by laser micromachining conical through-holes on the substrate material. The porous substrate is then mounted into a transparent flow chamber of a flow through system connected to a pump. In step 103, a suspension of microparticles or multiple batches of microparticle suspensions are provided and introduced into the flow chamber. In step 105, a pressure difference is established in the flow chamber to drive a transversal flow of the suspension through the substrate. The deposition ■ of microparticles onto the substrate surface is observed through an imaging system and continuous or non-continuous imaging of the microparticle deposition process is performed in step 107. The number of deposited microparticles is controlled by imaging the deposited microparticles and capturing the images then counting them. In this way, the number and distribution including the spatial position of microparticles is determined and may be recorded for later use. The surface of the porous substrate is continuously or non-cont inuously imaged and captured as microparticle deposition continues and the images are processed in step 109 to determine the exact^' number of microparticles and corresponding spatial positons of the microparticles. If desired, additional batches of microparticle suspensions may be introduced to achieve the desired number of batches or number of microparticles deposited or the desired deposition density. Once the exact number of microparticles and exact predetermined surface coverage of microparticles on the substrate is achieved, the flow of the microparticle suspension is stopped. Accordingly, the particle microarray produced achieves 100% deposition yield in a shorter time as compared to prior art bead microarrays.

Alternatively, non real-time imaging can be used to make the microarrays by non-precise control of particle deposition. In this method, the flow of the microparticle suspension is stopped before steps 107 and 109, i.e. the imaging, capturing and processing of the images, take place. If required, additional batches of microparticle suspensions may be introduced to achieve the desired number of batches or number of microparticles deposited.

Yet in another alternative method, the substrate surface is continuously or non-continuously scanned during the microparticle deposition process in step 107a to determine the physical characteristics of the microparticles , for example their colour. At intervals, the substrate surface is scanned to recognize and register the physical characteristics of the microparticles. An example of such a scan is a photograph or a LASER scan. In this way, the number, nature and distribution including the spatial position of microparticles is determined and may be recorded for later . use. Microparticles with different physical characteristics may carry different molecules on their surface or inside the microparticles. The scans are then processed in step 109 to determine the nature, exact number of microparticles and corresponding^' spatial positions of microparticles. If desired, additional batches of microparticle suspensions may be introduced to achieve the desired number of batches or number of microparticles deposited. In this method, a chemical or biological sensor can be fabricated. The cell or virus particle arrays may be used to perform biological, chemical or physical assays. In addition, morphological analysis of cells by means of microscopic methods may be performed, including cell staining methods.

In the foregoing methods, microarrays of different types can be fabricated, such as DNA microarrays, protein microarrays, peptide microarrays, antibody microarrays, metabolite microarrays, drug microarrays, lipid microarrays, carbohydrate microarrays, aptamer microarrays, cell microarrays, immunogen microarrays and allergen microarrays.

Referring to Fig. 1, an assembly 200 to produce particles arrays in accordance with an embodiment of the disclosure is shown. Microparticles 204 were deposited into the through-holes 205 of substrate 206 in the direction of arrows 202 by the application of a differential pressure through the substrate 206. Substrate 206 was supported by substrate support 208. During the application of a differential pressure, each microparticle 204 was sucked into hole 205 where it remained stuck. As shown in Fig. 3, the fluid flow 202 created by the differential pressure directed the microparticles 204 into the through-holes of substrate 206. The positioning of microparticles 204 on the substrate surface is shown in Fig. 2. As shown, not all of the holes are necessarily filled with a microparticle. An optical micrograph of the microparticles 204 lying in the holes 205 on the surface of substrate 206 is shown in Fig. 6.

Referring to Fig. 4, an optical micrograph showing deposited beads on a substrate 206 with holes of 9 μηα in diameter lying on top of another substrate 208 with holes of about 100 μπι in diameter is shown. The substrate 208 served as a support for the substrate 206. A microparticle suspension with microparticles of 10 μπι in diameter was sucked through both substrates with a vertical flow. It is shown that the microparticles (shown as an assembly of white dots 204) were stuck in the 9 m holes of the first substrate 206. It is clearly visible that only above the large holes of the second substrate 208, microparticles were deposited into the 9 μπι holes because only above the larger holes, a vertical flow was present, thereby demonstrating that a flow was necessary for microparticle deposition.

Referring to Fig. 5, an optical micrograph of substrates 206 and 208 in Fig. 4 is shown on a background of a glass surface. The deposited microparticles 204 (now marked as black dots due to a difference in lighting) were still visible. Examples

Non-limiting examples of the invention will be further described in greater detail by reference to specific examples, which should not be construed as in any way limiting the scope of the invention.

Example 1

This example demonstrates continuous bead deposition with continuous and discontinuous imaging.

MF-Microbeads with a diameter of 10 μπι were obtained^" from microParticles GmbH in Berlin, Germany and used in dilutions of 100 to 10000 fold in PBS buffer. Polycarbonate and polyethylene substrate materials were laser micromachined by the Laser Micromachining Laboratory (LML) at Denbighshire, United Kingdom, for the generation of conical through-holes with a diameter of 10 μπι, specifically 11 m at the entry and 9 pm at the exit, with a pitch of 20 μιη between holes. Accordingly, a through- hole array of 400 \im x 400 μιη containing 27 x 28 through- holes was fabricated.

The array was mounted into a transparent flow chamber of a transverse flow through system connected to a peristaltic pump with adjustable flow rate from 0.05 to 5 ml/min. Beads were injected into the flow with a pipette. The deposition of beads was observed through the transparent flow chamber by using a fluorescence microscope (Olympus CX-41, Japan) operated in bright field mode, allowing for photographic recording of the bead deposition process using a CCD camera (Q-imaging Retiga 4000, Canada) .

The surface of the through-hole array substrate material was imaged every 30 seconds to determine the number of captured beads. When the desired number of 60 beads was deposited, the flow of the bead suspension was stopped .

Referring now to Fig. 7, the series of quasi- continuous images during bead deposition is shown. Fig. 7i shows that 40 beads were deposited. Next, 10 beads were deposited as shown in Fig. 7ii, followed by the deposition of 2 beads, 4 beads, 2 beads and lastly 2 beads in Figs. 7iii-vi. The flow was stopped after Fig. 7vi captured an image of a total of exactly 60 beads in the through-holes of the array.

Example 2

This example demonstrates a bioassay on a bead microarray manufactured by using the disclosed method.

Three batches of 10 micrometer beads (microParticles

GmbH in Berlin, Germany) were used. The first batch of beads was coated with Streptavidin, the second batch of beads was coated with IgG antibody and the third batch of beads was coated with Alexa 594. The three batches were sequentially deposited as described in the first example. The deposition was performed on a laser micromachined (Laser Micromachining Laboratory (LML) at Denbighshire, United Kingdom) polycarbonate substrate material with conical through-holes of a diameter of 10 micrometers, specifically 11 micrometers at the entry and 9 micrometers at the exit and with a pitch of 20 micrometers between holes. As seen in Fig. 9-2, the Streptavidin beads 204a served as a negative control and the Alexa 594 beads 204b served as a fluorescent standard to normalize other fluorescent intensities to the intensity of the Alexa 594 beads 204b. The anti-IgG Antibody beads were used to perform a bioassay and to detect a target, i.e. the anti- IgG antibody, in a serum sample. The bioassay was performed by incubation of a 10 microliter serum sample, spiked with 1 microgram per ml of anti-IgG antibody Alexa 594 conjugate target analyte for 3 minutes at room temperature. After incubation, the non- bound target was washed off with lxPBS buffer and imaged with a fluorescent microscope.

Referring to Figs. 9-1 to 9-3, a series of micrographs showing the array before and after the bioassay is shown. Specifically, Fig. 9-1 shows a bright field image of the deposited beads on the through-hole substrate, while Fig. 9-2 shows the same deposited beads on the substrate but as a fluorescent micrograph before incubation with the target analyte. It is clearly seen that the Alexa 594 standard beads 204b are visible in Fig. 9-2. Fig. 9-3 shows the same deposited beads on the substrate after performance of the bioassay. It can be seen that several new beads 204c appear in the fluorescent micrograph of Fig. 9-3 after binding of the fluorescing anti-IgG antibody Alexa 594 target to the beads with IgG antibody. A magnified area of the array is shown below each image for better visualization. The image size is 420 micrometer x 420 micrometer. Images were made with an Olympus CX-41 (Japan) microscope with a Q-imaging Retiga 4000 (Canada) CCD camera.

Applications

Advantageously, the method of the present disclosure provides an improved method of making bead microarrays where the microparticles do not get substantially lost during the process.

Advantageously, the exact number of beads to be deposited on the bead microarray can be determined. Advantageously, the disclosed bead microarray can achieve 100% deposition yield in a shorter time as compared to prior art bead microarrays.

Advantageously, microarrays of different types can be fabricated by the disclosed method, such as DNA microarrays, protein microarrays, peptide microarrays, antibody microarrays, metabolite microarrays, drug microarrays, cell microarrays, virus particle microarrays, immunogen microarrays and allergen microarrays. Typically, the disclosed microarrays are applied in technical fields such as biomolecular analysis, diagnostics, environmental^' analysis, molecular biology, drug screening and point-of- care testing devices for field and the bedside.

It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.

Claims

1. A process for making a microarray comprising the steps of:

a) providing a plurality of holes that extend through a substrate;

b) providing a population of microparticles that are chemically or biologically active; and c) forcing the microparticles at least partially into said holes, wherein the holes and microparticles are dimensioned relative to each other to enable locking engagement of said microparticles at least partially within said holes.

2. The process as claimed in claim 1, wherein the holes are generally circular in cross-section.

3. The process as claimed in claim 2, wherein the sidewalls of the holes are generally tapered from a first circumference at a first surface of the substrate^' to a second smaller circumference at a second surface of the substrate, the second surface being opposite to the first surface.

4. The process as claimed in claim 2, wherein the largest cross-sectional area of the microparticle is greater than the area defined by the second circumference of the hole.

5. The process as claimed in any one of the preceding claims, wherein the forcing step comprises at least one of the following: (i) applying a pressure differential between the first and second surfaces of the substrate;

(ii) applying a magnetic field to the microparticles ;

(iii) applying an electric field to the micropart icles ;

(iv) subjecting the microparticles to a centrifugal force;

(v) subjecting the microparticles to a gravitational force;

(vi) providing a fluid flowing in a direction perpendicular to the surface of the substrate to move the microparticles into the respective holes; and

(vii) providing a fluid flowing in a direction parallel to the surface of the substrate to move the microparticles into the respective holes.

6. The process as claimed in claim 1, wherein steps b) and c) are repeated to enable locking engagement of at more than one population of microparticles in said holes .

7. The process as claimed in any one of the preceding claims, comprising the step of imaging the substrate during or after each forcing step in order to record the number and/or positions of the microparticles in the holes.

8. The process as claimed in any one of the preceding claims, wherein the number of holes is more than the number of microparticles.

9. The process as claimed in claim 1, wherein the number of holes is the same or less than the number of microparticles.

10. The process as claimed in any one of the preceding claims, wherein the microparticles reside completely within the holes.

11. The process as claimed in claim 6, wherein more than one microparticle resides within one hole.

12. The process as claimed in any one of the preceding claims, comprising the step of tagging the microparticles with an identifier to identify the micropartices .

13. The process as claimed in claim 1, comprising the step of differentiating between the plurality of microparticles by way of a visual identifier.

14. The process as claimed in claim 1, comprising the step of differentiating between the plurality of microparticles by way of spatial position.

15. The process as claimed in claim 1, wherein the microparticle is at least one of a microbead and a biological entity.

16. The process as claimed in claim 15, wherein the microbead has at least one active agent capable of specific binding with at least one target analyte.

17. The process as claimed in claim 15, wherein the biological entity is selected from the group selected from a cell, a bacteria and a virus.

18. The process as claimed in claim 16, wherein the microparticle is at least partially porous to allow the passage of the target analyte into the interior of the microparticle .

19. The process as claimed in any one of the preceding claims comprising the step of immobilizing the the microparticles in their respective holes.

20. A microarray comprising a substrate having a plurality of holes that extend through the substrate and a plurality of chemically or biologically active microparticles that are lockingly engaged at least partially within said holes, wherein said locking engagement is due- to the relative dimensions of the microparticles and the holes.

21. The microarray as claimed in claim 20, wherein the holes extend through the substrate.

22. The microarray as claimed in claim 20 or claim

21, comprising at least one additional substrate sandwiched on one or two sides of the substrate.

23. The microarray as claimed in claim 22, wherein the additional substrate is selected from the group selected from a porous substrate, a non-flexible and planar substrate, a substrate having a plurality of holes that extend through the substrate and a mixture thereof.

24. The microarray as claimed in any one of claims 20 to 23, wherein a porous sealant covers said microparticles.

25. The microarray as claimed in any one of claims

20 to 24, wherein the sidewalls of the holes are generally tapered from a first circumference at a first surface of the substrate to a second smaller circumference at a second surface of the substrate, the second surface being opposite to the first surface.

26. The microarray as claimed in any one of claims 20 to 25, wherein the number of holes is more than the number of microparticles.

27. The microarray as claimed in any one of claims 20 to 26, wherein the number of holes is the same or less than the number of microparticles.

28. The microarray as claimed in any one of claims

20 to 27, wherein the microparticles reside completely within the holes.

29. The microarray as claimed in any one of claims 20 to 28, wherein the microparticles are tagged with an identifier to identify the micropartices.

30. The microarray as claimed in any one of claims 20 to 29, wherein the microparticles are differentiated by means of a visual identifier.

31. A microarray for use in a system for identifying, the presence of one or more target analytes in a sample, the microarray comprising a substrate having a plurality of holes that extend through the substrate and a plurality of chemically or biologically active microparticles that are lockingly engaged at least partially within said holes, wherein said locking engagement is due to the relative dimensions of the microparticles and the holes.

32. A microarray system for identifying the presence of one or more target analytes in a sample comprising:

a microarray comprising a substrate having a plurality of holes that extend through the substrate and a plurality of chemically or biologically active microparticles that are lockingly engaged at least partially within said holes, wherein said locking engagement is due to the relative dimensions of the microparticles and the holes;

a memory recording the location of each microparticle, said location of each microparticle having been determined by imaging of the substrate;