WO2023111587A1

WO2023111587A1 - Improvements in or relating to microarray fabrication

Info

Publication number: WO2023111587A1
Application number: PCT/GB2022/053265
Authority: WO
Inventors: Callum Robertson SMITH; David R. Klug; Marko Dorrestijn; Stefan Leo VAN WORKUM
Original assignee: Vidya Holdings Ltd
Priority date: 2021-12-17
Filing date: 2022-12-16
Publication date: 2023-06-22

Abstract

A method of fabricating a plurality of substrates, wherein each substrate comprises at least one array of capture agents and at least one microfluidic channel, the method comprising the steps of: depositing multiple arrays of capture agents onto a substrate; bonding the substrate with an additional material layer to form a plurality of microfluidic channels; and dividing the substrate to produce a plurality of substrates; wherein each of the plurality of substrates contains at least one array of capture agents and at least one microfluidic channel; and wherein depositing multiple arrays of capture agents, comprises printing the capture agents.

Description

IMPROVEMENTS IN OR RELATING TO MICROARRAY FABRICATION

FIELD OF THE INVENTION

The present invention relates to a method for fabricating a plurality of microarrays, wherein each microarray includes a plurality of capture agents for one or more bioassays.

BACKGROUND TO THE INVENTION

Microarrays enable multiplexed and high-throughput analysis of biological analytes. DNA microarrays, which consist of an array of fragments of DNA immobilised on a solid support, can be used for analysing gene expression levels. Protein microarrays are used for diagnostics and proteomics. They typically consist of an array of capture proteins which interact with the proteins in a sample. The interaction results directly, or indirectly, in the emission of a signal which can be read by a scanner. Commonly, standard glass microscope slides are used as supports for microarrays, owing to their wide-scale availability, low cost, excellent flatness and chemical inertness.

To facilitate the adhesion of microarrays on glass slides, the glass slides are typically coated or functionalised to provide a uniform surface prior to the printing or in situ synthesis of the microarray. The coating is typically applied to glass slides by dip coating or thermal vapour deposition. In order to obtain a highly uniform, robust and reliable coating, the surface preparation, substrate chemistry and deposition conditions must be carefully controlled.

Microfluidic technology can be combined with microarrays to control and enhance the exposure of a sample to a microarray. This typically involves bonding the glass microarray slide to a microfluidic chip, and aligning the microarray with the microchannels in the microfluidic device such that running the sample through the microchannels exposes the sample to the microarray.

The utility of microarrays supported on standard glass microscope slides to high throughput, miniaturised assay devices or 'lab-on-a-chip' systems, is limited by challenges in reproducibly and efficiently fabricating supported microarrays. Producing microarrays on an individual basis is inefficient, and results in a lack of suitability for scale-up and manufacturing. A method in which a plurality of supported microarrays can be produced simultaneously from a single substrate would improve the efficiency of fabricating supported microarrays and would increase the cost savings in the manufacture of 'lab-on-a- chip' systems.

Methods of fabricating a plurality of substrates simultaneously are known: for example, in the fields of semiconductors and Micro Electro-Mechanical Systems (MEMS). In such fields, high temperature processing steps such as firing and sintering are required to produce the substrate material.

Similar methods have been adopted to produce microfluidic systems. For example in US 6544734 Bl, which relates to a multi-layered microfluidic DNA analysis system including a cell lysis chamber, a DNA separation chamber, a DNA amplification chamber, and a DNA detection system. The multi-layered microfluidic DNA analysis system is provided as a substantially monolithic structure formed from a plurality of green-sheet layers sintered together. The multi-layered substrate is fired at 250 to 500°C to remove organic material and sintered at 950 to 1600°C. The multi-layered substrate is then subsequently diced into separate individual devices. The device can subsequently be coupled to a molecular probe array for use in DNA detection.

Such a method is incompatible with forming individual supported microarrays from a single substrate because the high-temperature firing and sintering steps would destroy the delicate organic material and biological analytes of which the microarray comprises.

There is therefore a requirement for a highly efficient method of producing a plurality of microarrays supported on a substrate. The method should be suitable for scaling up and manufacturing such that it is compatible for use with high-throughput micro-scale devices.

It is against this background that the present invention has arisen.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a method of fabricating a plurality of substrates, wherein each substrate comprises at least one array of capture agents and at least one microfluidic channel. The method comprises the steps of: depositing multiple arrays of capture agents onto a substrate; bonding the substrate with an additional material layer to form a plurality of microfluidic channels; and dividing the substrate to produce a plurality of substrates; wherein each of the plurality of substrates contains at least one array of capture agents and at least one microfluidic channel; and wherein depositing multiple arrays of capture agents, comprises printing the capture agents.

In some embodiments, there is provided a method of fabricating a plurality of substrates, wherein each substrate comprises at least one array of capture agents and at least one microfluidic channel, the method comprising the steps of: depositing multiple arrays of capture agents onto a substrate; bonding the substrate with an additional material layer to form a plurality of microfluidic channels; and dividing the substrate to produce a plurality of substrates; wherein each of the plurality of substrates contains at least one array of capture agents and at least one microfluidic channel.

The method of the present invention provides an efficient process for producing a plurality of substrates, each comprising at least one microfluidic channel and each with an array of capture agents deposited thereon. By carrying out the processing steps prior to dividing the substrate, it is possible to deposit multiple arrays of capture agents, and form a plurality of channels at one time, which increases the efficiency of manufacturing microfluidic device testing consumables comprising individual supported microarrays, compared to processing them one-by-one. The method of the present invention can be used to produce substrates for use in microscale devices with a high-throughput and scalability, optimising effort, cost and time by performing processes across multiple individual test units simultaneously. Furthermore, the method of the present invention can be used to produce a plurality of substrates without any high temperature processing steps. As such the method is suitable for producing a plurality of substrates each comprising an array of capture agents deposited thereon. The multiple arrays of capture agents may be deposited onto the substrate prior to dividing therefore maximising the efficiency with which the supported microarray substrates can be produced.

Within the context of the present invention, the term "capture agent" should be understood to include any agent which captures an analyte of interest. A capture agent may include one or more capture components and/or one or more detection reagents. Capture components may bind onto a target component of interest such as a biomarker. Various biomarkers include, but are not limited to, immunoglobulins, CRP, NGAL, Leptin, Adiponectin, PIGF, DNAs and/or microRNAs. The capture component may be an antibody. Alternatively or additionally, the capture component could be a nucleic acid such as DNA, RNA, mRNA or microRNA, or chemically modified nucleic acid; it could be a protein, or a modified protein; or a peptide; or a polymer. It could be a hormone; or a tethered small molecule configured to capture a protein. In some embodiments, the capture component may be a non-specific capture component such as saliva or polylysine. In some embodiments, the capture agents may comprise organic material.

Within the context of the present invention, the term 'multiple arrays of capture agents' should be understood to include any capture agent arrangement and is not limited to multiple copies of the same array. In some embodiments, each array in the multiple arrays of capture agents may be substantially different. In some embodiments, each array in the multiple arrays of capture agents may be substantially the same.

The detection reagent may be a secondary antibody, and can be bound with a label. Optionally, the label may be a fluorophore, a nanoparticle or a quantum dot. The label can be attached to the detection reagent. The detection reagent can bind to the target component to form a detection reagent-target component complex. The detection reagenttarget component complex can then bind to the capture component to form a sandwich complex. The detection reagent can either have inherent light emitting or scattering properties or the detection reagent may comprise a label with light emitting or scattering properties The detection reagent may be, but is not limited to, one or more of the following: a peptide, a protein, a protein assembly, an oligonucleotide, a polynucleotide, a modified oligonucleotide, a modified polynucleotide, an aptamer, a morpholino, a small molecule, a cell, a cell membrane, a viral particle, a glycan, a conjugated solid particle, a conjugated solid bead or a cofactor.

The label may be, but is not limited to, one or more of the following: a luminescence molecule; a fluorescent molecule; a phosphorescence molecule; a chemiluminescent molecule; a molecule that exhibits Rayleigh scattering or Raman scattering; an upconversion particle; an enzyme and its substrate that produces a colorimetric signal; a metallic or inorganic particles e.g. nanoparticles, a polycyclic aromatic hydrocarbon, a metalized complex, a quantum dot or an ion. The ion may be an atomistic ion or a salt of an organic molecule.

The label can be attached to the detection reagent. Additionally or alternatively, the detection reagent may comprise an antibody. In some instances, the detection antibody can be fluorescently labelled.

The capture agent may form part of a competition assay or sandwich assay.

In some embodiments, prior to dividing the substrate, the method may further comprise polishing at least one edge of the substrate.

In some embodiments, by polishing the substrate prior to the dividing step, the entirety of the relevant surface can be polished in a single step, therefore increasing the efficiency of the polishing step compared to polishing the divided substrates one-by-one.

In some embodiments, wherein the substrate is an optically transmissive substrate, at least one edge of the substrate may be polished such that it has optical properties sufficient to cause a predetermined proportion/substantially all of the incident light to undergo TIR.

In some embodiments, the dividing step may be sufficiently precise that the substrate does not require polishing.

In some embodiments, one of the common faces of the substrates may be cut or cleaved to provide at least one edge of optical quality. The cleaving may be mechanical or laser cleaving. In this context, the term optical quality of a surface means that the surface is sufficiently free of defects to avoid the distortion of the TIR beam. This includes both scratch and dig surface defects and the overall flatness of the surface.

In some embodiments, prior to depositing multiple arrays of capture agents, the method may comprise the step of coating the substrate.

In some embodiments, the array of capture agents may be deposited onto a substantially uncoated surface of the substrate. In some embodiments, prior to dividing the substrate and the array of capture agents being deposited thereon, the top surface of the substrate may be treated with a specific surface chemistry. In some embodiments, it may be beneficial or required to prepare the surfaces of the substrate through functionalisation. The surface of the substrate may be coated to facilitate the linking of the biomolecules in the array with the inorganic substrate. The coating may act as an adhesion promoter or molecular bridge between the array of capture agents and the substrate.

In some embodiments, the coating may be an organic coating, such as a silane coating, in particular an organofunctional silane. In some embodiments, the organic coating could include polymers, proteins, nucleic acids, small molecules or combinations thereof. In some embodiments, the coating may be an inorganic coating such as a thin gold film or metal oxide thin film.

In some embodiments, the surface of the substrate may be coated with a suitable coating for preventing the various elements of the microarray from merging after deposition. An example of a suitable coating may include an anti-wetting treatment.

In some embodiments, prior to dividing the substrate, the method may comprise bonding the substrate with an additional material layer to form a plurality of microfluidic channels. In some embodiments, the additional material may be glass. In some embodiments, the additional material may be plastic or may be a polymer. In some embodiments, the additional material may comprise reagents deposited thereon.

In some embodiments, subsequent to the capture agents being deposited onto the surface of the substrate and prior to dividing the substrate, the top surface of the substrate may be bonded to an additional material layer to form a plurality of microfluidic channels between the two substrates. In some embodiments, the additional substrate may also have relevant reagents deposited thereon. In some embodiments, prior to bonding the substrate with an additional material layer, the surface of the substrate may be etched. In some embodiments, the surface of the substrate may be etched with a laser prior to bonding with the additional material. In some embodiments, the surface of the substrate may be chemically etched prior to bonding with the additional material. In some embodiments, increasing the surface roughness of the substrate may facilitate the adhesion of the additional material.

In some embodiments, subsequent to the dividing step, the plurality of substrates may each comprise at least one microfluidic channel. In some embodiments, adding microfluidic channels to the substrate prior to dividing can facilitate the high-throughput manufacture of microfluidic device testing consumables.

In some embodiments, adding microfluidic channels to the substrate prior to dividing the substrate can protect the microfluidic channels from being exposed to contaminants which may be produced during the dividing step.

In some embodiments, adding microfluidic channels to the substrate prior to dividing the substrate can facilitate their alignment. In some embodiments, by forming the microfluidic channels prior to dividing the substrate, the microfluidic channels and reagents deposited thereon are easier to align with their opposing part. Therefore, by adding the microfluidic channels prior to dividing the substrate, the finish and positional accuracy of the divided substrates may be improved.

In some embodiments, the microfluidic channels may be free flow channels. In some embodiments, the microfluidic channels may have any flow regime in which the interfacial tension is sufficiently high to drive the flow through the entire channel. In some embodiments, the microfluidic channels may have substantially laminar, turbulence free flow. In some embodiments, the microfluidic channels may have momentum transfer that occurs in the inertial flow regime.

In some embodiments, subsequent to depositing multiple copies of an array of capture agents onto the substrate, the substrate may be passivated to prevent or reduce the attachment of either reagents or elements of the sample to the substrate. In some embodiments, the surface of the substrate that does not have capture agents deposited thereon may be passivated.

In some embodiments, the substrate may be passivated by washing to remove excess capture agent, followed by provision of a passivation layer which can be deposited on the surface from a solution, which may be an aqueous or a non-aqueous solution. The passivation layer that is deposited may be formed of polymers, proteins, nucleic acids, lipids, small molecules and surfactants or combinations thereof. In some embodiments, the deposition may not be solution based, but may be, for example, chemical vapour deposition. In some embodiments, dividing the substrate may comprise scribing and breaking the substrate.

Scribing and breaking is a commonly used process for cutting materials such as silicon wafers and glass, however it is typically applied to materials less than 1 mm thick. In some embodiments, the method of the present invention may be used to divide substrates which a thickness greater than 1 mm. For example, when the divided substrate is intended to be used in a TIR microscope, a substrate thickness of 3.8 mm may be preferable.

In some embodiments, testing of the individual test units may be carried out prior to the dividing step. In some embodiments, a shallow and clean linear defect scribe line is made on the substrate prior to division, initiating a vertical crack and defining the physical dimensions of the individual test units. In some embodiments, the surface of the substrate may be scribed using a mechanical scribe such as a tungsten carbide or a diamond scribe tool. In some embodiments, a laser beam may be used to scribe the surface of the substrate and impart a linear defect. In some embodiments, laser filamentation may be used to produce a defect which permeates through the entire thickness of the substrate. In some embodiments, a scribing and breaking method using laser filamentation may be advantageous because by imparting defects through the entire thickness of the substrate, less energy may be required to break the substrate in the breaking step. In some embodiments, it may be advantageous to use laser filamentation to scribe a substrate because by imparting defects through the entire thickness of the substrate, chipping along the scribe line may be prevented during the breaking step.

In some embodiments, the substrate is subsequently stressed such that a vertical crack propagates from the defect line through the substrate, breaking it apart. In some embodiments, the substrate may be broken using a mechanical tool which provides a moment force either side of the scribe line resulting in crack propagation through the substrate, and substrate separation. In some embodiments, the substrate may be thermally stressed resulting in breaking along the scribe line. In some embodiments, the substrate may be thermally stressed using a CO₂ laser.

In some embodiments, scribing the substrate may take place prior to depositing multiple arrays of capture agents onto the substrate. In some embodiments, the scribe lines may be made on the substrate as the initial step in the method, prior to depositing the capture agents thereon. In some embodiments, the breaking of the substrate may be the final step in the method, after all other processing steps have been carried out on the substrate. In some embodiments, the substrate may be scribed and broken in successive steps. In some embodiments, the scribing and breaking of the substrate may be the final steps of the method, after all other processing steps have been carried out on the substrate.

In some embodiments, dividing the substrate may comprise making two or more divisions along a single axis. In a preferred embodiment, dividing the substrate may comprise making more than two divisions along a single axis such that a plurality of substrates is produced from the initial substrate. In some embodiments, producing more than two substrates from the single substrate limits the size and shape of the additional material layer bonded to the substrate. For example, in an embodiment in which dividing the substrate comprises making two divisions along a single axis, the additional material layer can extend over one of the ends of the substrate and is therefore less limited to a specific size and/or shape. In contrast, when dividing the substrate comprises making more than two divisions along a single axis, the additional material layer bonded to the substrate is limited to a repeating and/or an interlocked pattern. In some embodiments, producing greater than two substrates from a single substrate can result in increased cost savings in the manufacture of individual substrates.

In some embodiments, dividing the substrate via scribing and breaking may be advantageous. In some embodiments, a scribe and break process for dividing the substrate may result in less contaminants being produced and contaminating the substrate. In some embodiments, a scribe and break process for dividing the substrate may be faster than dicing and result in a higher throughput.

In some embodiments, dividing the substrate may comprise dicing the substrate which may include cutting the substrate with a mechanical saw. In some embodiments, many passes of the saw may be required to cut through the substrate causing heating such that coolant may have to be applied to the area of the substrate being diced. In some embodiments, dicing may produce an increased amount of contaminants compared to a scribing and breaking process. In some embodiments, dicing may remove material from the substrate, such as glass fragments, which may contaminate the substrate. In some embodiments, because the saw must be passed over the area to be cut multiple times, the method of dicing the substrate may be slower compared to scribing and breaking, and result in a lower throughput.

In some embodiments, dividing the substrate may comprise cutting the substrate with a laser.

In some embodiments, cutting the substrate with a laser may require multiple passes of the laser over the area to be cut in order to divide the substrate. In some embodiments, dividing the substrate using a laser may be slower compared to scribing and breaking, and result in a lower throughput. In some embodiments, the substrate may be cut with a more powerful laser in order to reduce the number of passes over the substrate required to divide the substrate. In some embodiments, cutting the substrate with a high powered laser may produce a higher level of contaminants compared to a scribing and breaking process.

In some embodiments, by dividing the substrate with a laser, physical contact with the substrate may be avoided, which may be advantageous compared to mechanical methods of cutting such as dicing, as it may reduce the level of contamination.

In some embodiments, dividing the substrate using a laser may be less cost effective compared to a mechanical method of cutting the substrate or scribing and breaking.

In some embodiments, dividing the substrate may comprise etching the substrate. In some embodiments, the substrate may be etched with a laser or chemically etched as part of the dividing process.

In some embodiments, depositing multiple arrays of capture agents may comprise printing the capture agents.

In some embodiments, the capture agents may be deposited onto the surface of the substrate by contact printing methods such as dip-pen lithography, capillary tubes, spilt pins or ink stamps. In some embodiments, the capture agents may be deposited onto the surface of the substrate by non-contact printing methods such as inkjet, piezo or acoustic dispensing. In some embodiments, the capture agents are in liquid form. In some embodiments, the capture agents are in a dry form. In some embodiments, the capture agents, which may be in liquid form, can be deposited onto the surface of the substrate and dry out.

In some embodiments, the capture agents may be printed onto the surface of the substrate by a lithographic printing process. This may be particularly advantageous because it can improve the resolution of the capture agents during detection.

In some embodiments, the substrate is any optically transmissive material suitable for use in a total internal reflection microscope. In some embodiments, the substrate may be any cleavable crystalline or amorphous optical material which is suitable for creating total internal reflection at the test site.

In some embodiments, the supported arrays of capture agents may be suitable for use in a total internal reflection (TIR) microscope. The TIR microscope comprises a light source such as a laser beam for illuminating the capture agents printed onto the surface of the substrate. As the light from the laser beam reaches the surface of the substrate, the light can be configured to excite the capture agents that may cause the capture agents to emit light at a specific wavelength or to scatter light. The light may undergo a single reflection or it may undergo multiple reflections at the surface of the substrate. The emissions from the capture agents may be luminescence for example, fluorescence or phosphorescence. In some embodiments, detection reagents provided on the additional material layer may be positioned directly above or slightly upstream of the capture agents provided on the substrate. In some embodiments, TIR can be used to selectively excite the supported arrays of capture agents without the detection reagents interfering with the read-out.

In some embodiments, the substrate may be float glass. In some embodiments, the substrate may be fused quartz, or may be borosilicate glass. In some embodiments, the substrate may be glass produced using a float process because it is cost effective to manufacture in high volumes, and can be produced to a high optical quality in terms of inclusions, scratches, bubbles and impurities.

In some embodiments, the substrate is produced in a scalable manner such that large quantities can be obtained cost effectively for use in the method of the present invention. In some embodiments, the substrate may be Borofloat® 33, a high quality float glass produced by Schott.

In some embodiments, the substrate may be silicon, single crystal silicon wafer or sapphire.

In some embodiments, in which the substrate is silicon, the supported array of capture agents may be used in an electrochemical device.

In some embodiments, the substrate may be 100 pm to 5 mm thick. In some embodiments, the substrate may be more than 100 pm, 500 pm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm or 4.5 mm thick. In some embodiments, the substrate may be less than 5 mm, 4.5 mm, 4.0 mm, 3.5 mm, 3.0 mm, 2.5 mm, 2.0 mm, 1.5 mm, 1.0 mm, 500 pm or 100 pm thick. In some embodiments, the substrate must have a minimum thickness such that it is suitable for use in a TIR microscope. Thicker substrates may be more difficult to divide. In some embodiments, the dividing may require more force to be used than would be used to break a conventional glass coverslip for example.

In some embodiments, the method may be carried out under clean conditions.

In some embodiments, the method may be carried out in a clean room in order to avoid particulate contaminant, gaseous contaminants or contamination from lubricants.

FIGURES

The present invention will now be described, by way of example only, with reference to the accompanying figures in which:

Figures 1A to ID show schematically the scribe-and break process for dividing a substrate, wherein the substrates are viewed side-on;

Figure 2A to 2C show schematically the scribe and break process for dividing a substrate, wherein the substrates are viewed from above;

Figure 3A shows an example of a substrate prior to division, and the relevant dimensions;

Figure 3B shows an example of a divided substrate, and the relevant dimensions; Figure 4 shows a pair of breaking pliers used to mechanically break the substrate after scribing;

Figure 5A shows substrates after division using a scribe line pitch of 12 mm;

Figure 5B shows substrates after division using a scribe line pitch of 10 mm;

Figure 5C shows substrates after division using a scribe line pitch of 8 mm;

Figure 6 shows a plurality of substrates fabricated within a frame;

Figure 7 shows a plurality of substrates with the frame of Figure 6 removed; and

Figure 8 shows the plurality of substrates from Figure 7 after a single substrate has been divided from the plurality of substrates.

DETAILED DESCRIPTION

The method of the present invention may be used to fabricate a plurality of substrates, wherein each substrate comprises at least one array of capture agents and at least one microfluidic channel.

The method comprises depositing multiple arrays of capture agents onto a substrate, bonding the substrate with an additional material layer to form a plurality of microfluidic channels, and dividing the substrates to produce a plurality of substrates. The depositing may be achieved using any suitable method including, but not limited to, printing. The surface of the substrate may be passivated. Prior to the deposition of the capture agents, the surface of the substrate may be coated. Alternatively, the array of capture agents may be deposited onto an uncoated substrate. Any suitable coating may be used to coat the surface of the substrate, including, but not limited to, silane coatings and anti-wetting treatments. Prior to the division step, the substrate may be bonded with an additional material layer, which may have relevant reagents supported thereon, to form a plurality of microfluidic channels between the two substrates. The reagents supported on the microfluidic channels are provided within the diffusion distance of those placed on the substrate. The phrase "within the diffusion distance" within this specification is intended to mean that, within the required time frame of the completion of the assay, for example, within 1 minute, 5 minutes or 20 minutes, the reagents supported on the microfluidic channels can diffuse across the distance that separates them from the reagents deposited on the substrate and can therefore interact with those reagents.

The surface of the substrate may be etched using plasma, a laser, or may be etched chemically to enhance the surface roughness of the substrate and promote the adhesion of the additional material layer. At least one edge of the substrate may be polished, prior to division. The division step is such that the plurality of divided substrates each contains at least one array of capture agents and/or at least one microfluidic channel.

The substrate may be divided using a scribe and break process, a dicing process or using a laser.

Referring to Figure 1, a side-view schematic of the scribe and break process for dividing the substrate 12 is shown. Figure 1A shows the original substrate 12, prior to the division step. At this stage, the substrate 12 may have undergone processing steps including the depositing of capture agents, etching, coating and/or and bonding with another substrate to form microfluidic channels. Alternatively, the substrate 12 may not yet have undergone any processing, and the scribing step shown in Figure IB, may be the initial step in the method, with the processing steps to follow. Figure IB shows a scribe line 14 made in the surface of the substrate 12. The scribe line may be made by a mechanical scribe tool such as a tungsten carbide or a diamond scribe tool. Alternatively, a laser beam may be used to scribe the surface of the substrate or laser filamentation may be used to produce a defect which permeated through the entire thickness of the substrate. As shown in Figure 1C, the substrate 12 may be stressed along the scribed defect line 14, such that a crack 16 propagates vertically along the scribed line 14. The crack 16 may be propagated by mechanical stress such as by applying force either side of the scribe line 14, or may be propagated by thermal stress, such as with a CO₂ laser. The crack 16 propagates until the substrate 12 breaks, and smaller divided substrates 18 are formed, as shown in Figure ID. When the scribing step shown in Figure 1A is carried out after the processing steps, the breaking steps shown in Figures 1C and ID may immediately follow the scribing step. Alternatively, the scribing step may take place before the processing steps, and the breaking steps may be the final steps in the method, after the processing steps.

Similarly to Figure 1, Figure 2 illustrates the scribe and break process with the substrates viewed from above. Figure 2A shows the original substrate 12, with a rectilinear geometry. A rectilinear geometry may be preferable to minimise wastage of materials and maximising efficiency. However, the method of the present invention is suitable for use with any substrate geometry. Figure 2B shows the substrate 12 with scribe lines 14 made on the surface. Figure 2C shows the divided substrates 18 after breaking has taken place along the scribe lines 14.

Figure 3A shows a substrate 12 prior to division. In the example shown in Figure 3A, the substrate 12 has a rectilinear geometry with the dimensions of 3.8 mm x 20 mm x 120 mm. The substrate may be any thickness between 100 pm to 5 mm. In the example shown in Figure 3, the substrate has a thickness of 3.8 mm, which may be preferable when the divided substrate 18 is intended for use in a TIR microscope. The largest surface 20 of the substrate 12 corresponds to the surface with the dimensions 20 mm x 120 mm. In order for the substrates to be suitable for use in optical set-ups after division, the largest surface 20 must have a high-quality and be substantially free from defects such as inclusions, scratches, bubbles and impurities. Consequently, the substrate is typically a borosilicate glass or a fused quartz. The substrate may be produced in a float process, or any other process which can produce the substrate with sufficiently high quality. The substrate may be any cleavable crystalline or amorphous material with transmissive properties sufficient to enable total internal reflection at the test site. If the divided substrates are intended for use in an electrochemical device, then the substrate may be silicon. The next largest surface 22 corresponds to the surface with dimensions of 3.8 mm x 120 mm in Figure 3A. At least one of these surfaces 22 may form part of the input face in a microscope and as such must have a high optical quality. Therefore, it may be necessary to polish at least one of the surfaces 22, prior to division. The smallest surfaces 24, corresponding to the dimensions 3.8 mm x 20 mm in Figure 3A, do not have requirements in terms of optical quality, and can be cut without requirement for polishing. Referring to Figure 3B, an example of a divided substrate 18 fabricated by dividing the substrate 12 of Figure 3A is shown. After division, in the example shown, the dimensions of the divided substrates are 3.8 mm x 20 mm x 120 mm. In some embodiments, the substrate 12 shown in Figure 3A may have an edge 24 with dimensions which are larger than the desired dimensions of the corresponding edge in the divided substrate 18. For example, the substrate 12 may have the dimensions 3.8 mm x 40 mm x 120 mm prior to division. The substrate may be divided such that the divided substrates 18 have the dimensions 3.8 mm x 20 mm x 10 mm, as shown in Figure 3B. In this embodiment, the surface 22 which is divided along would form the output face of the unit when inserted into a microscope.

Figure 4 shows three-point jaws pliers 36 which is an example of a mechanical tool which can be used to break the substrate 12 along the scribe lines 14.

Figure 5 shows borosilicate float glass substrates which were divided using a manual scribe and break method, as illustrated by Figure 5. Different scribe line pitches were used. Figure 5A shows the divided substrates 18 produced with a scribe line pitch of 12 mm. Figure 5B shows the divided substrates 18 produced with a scribe line pitch of 10 mm. Figure 5C shows the divided substrates 18 produced with a scribe line pitch of 8 mm. Figure 5 illustrates the reasonably good performance of the manual scribe and break process using substrates 12 with dimensions of 3.3 mm x 20 mm x 120 mm. A similar performance was noted for 3.8 mm thick borosilicate glass substrates, with a slight degradation in consistency for 5 mm thick borosilicate glass substrates.

Figure 6 shows a plurality of substrates 18 that have been fabricated within a frame 50. A single piece injection moulded additional layer 51 that provides a lid 52 for each substrate 18. The additional layer 51 is provided across all of the substrates 18 prior to the singulation of the substrates 18. Each lid 52 has reagents deposited on the surface and the positioning of the additional layer 51 comprising the lids 52 on the plurality of substrates 18 is finely controlled using a plurality of alignment features on the frame 50. In Figure 6 the alignment features are round holes 53. The provision of the alignment features 53 ensures that the reagents deposited on the lid 52 are within a predetermined distance of the reagents that are deposited on the substrates 18. The predetermined distance is selected such that the reagents on the lid 52 and the reagents on the substrate 18 will diffuse into contact within a time frame which is reasonable to expect a user to wait for an assay to be completed. This time may be 5 minutes, 10 minutes, 15 minutes or up to 30 minutes. The separation between the reagents is therefore controlled to be within the diffusion distance within this time frame. The control of the application of the additional layer 51 as a whole is possible with much greater accuracy than would be attainable once the substrates 18 have been separated.

The additional layer 51 can be bonded to the plurality of substrates 18 using either a pressure sensitive adhesive or a laser weld. The additional layer 51 is fabricated from a plastic material such as a machined acrylic. The application of the additional layer 51 to the plurality of substrates 18 precedes the scribing process.

In an alternative embodiment, not shown in the accompanying drawings, alignment features may be provided on the substrates so that each lid can be individually aligned. Each lid is provided with a corresponding alignment feature, typically a round hole, and the alignment features in the substrate and lid are brought into alignment using a pin that passes through both holes to ensure alignment.

Figure 7 shows the plurality of substrates 18 after the frame has been removed and the scribing step has occurred such that the location of the separation between the substrates 18 is determined. It will be readily apparent that there are still witness marks on the substrates 18 at the locations where the frame 50 was formerly attached. This means that the end surfaces of the substrate are not free of imperfections. However, this is not critical as the critical alignment between the lid and the substrate is already complete.

Figure 8 shows the plurality of substrates 18 after one of the substrates 18 has been broken from the end of the strip of substrates 18. It is be readily apparent that the break is not perfectly aligned with the scribe line. A perfect break would have resulted in a rectilinear piece with the break line at 90° to the end surface at which the substrate 18 was formerly attached to the frame. However, as with the imperfections resulting from interface with the frame, any imperfections in the angle of the break are not critical as the lid and substrate are already aligned and bonded.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments. It is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.

Claims

1. A method of fabricating a plurality of substrates, wherein each substrate comprises at least one array of capture agents and at least one microfluidic channel, the method comprising the steps of: printing multiple arrays of capture agents onto a substrate; bonding the substrate with an additional material layer to form a plurality of microfluidic channels; and dividing the substrate to produce a plurality of substrates; wherein each of the plurality of substrates contains at least one array of capture agents and at least one microfluidic channel.

2. The method according to claim 1, wherein the capture agents comprise organic material.

3. The method according to claim 1 or claim 2, wherein prior to dividing the substrate, the method further comprises polishing at least one edge of the substrate.

4. The method according to claim 1, wherein prior to dividing the substrate, the method further comprises cutting the substrate to provide at least one edge of optical quality. The method according to any one of the preceding claims, wherein prior to depositing multiple arrays of capture agents, the method comprises the step of coating the substrate. The method according to any one of the preceding claims, wherein the additional material comprises reagents deposited thereon. The method according to any one of the preceding claims, wherein prior to bonding the substrate with the additional material layer, the surface of the substrate is etched. The method according to any one of the preceding claims, wherein subsequent to depositing multiple arrays of capture agents onto the substrate, the substrate is passivated. The method according to any one of the preceding claims, wherein dividing the substrate comprises scribing and breaking the substrate. The method according to claim 8, wherein scribing the substrate takes place prior to depositing multiple arrays of capture agents onto the substrate. The method according to any one of claims 1 to 7, wherein dividing the substrate comprises dicing the substrate. The method according to any one of claims 1 to 7, wherein dividing the substrate comprises cutting the substrate with a laser. The method according to any one of the preceding claims, wherein the substrate is any optically transmissive material suitable for use in a total internal reflection microscope. The method according to any one of the preceding claims, wherein the substrate is float glass. The method according to any one of claims 1 to 12, wherein the substrate is silicon. The method according to any one of the preceding claims, wherein the substrate is 100 pm to 5 mm thick. The method according to any one of the preceding claims, wherein the method is carried out under clean conditions. The method according to any one of the preceding claims, wherein the plurality of substrates are provided within a frame and wherein the method further comprises the step of removing the frame.