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  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
  • G01N33/525—Multi-layer analytical elements
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54386—Analytical elements
  • G01N33/54387—Immunochromatographic test strips
  • G01N33/54391—Immunochromatographic test strips based on vertical flow
• • B—PERFORMING OPERATIONS; TRANSPORTING
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
• • B—PERFORMING OPERATIONS; TRANSPORTING
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• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01L2300/12—Specific details about materials
  • B01L2300/123—Flexible; Elastomeric
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/12—Specific details about materials
  • B01L2300/126—Paper
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces

Definitions

• Protein microarrays can be classified based on the test site biomolecule.
• Forward phase, or capture, arrays are a pattern of several different probes (e.g. antibodies). The array is incubated in the sample solution to simultaneously test for multiple targets.
• a reverse phase array consists of a pattern of samples; a fraction from multiple protein samples is spotted onto the substrate and tested for the presence of one protein.
• Forward phase arrays are typically employed in biomarker discovery[4] or clinical diagnosisQ . , 5], while reverse phase arrays, originally designed for biomarker detection[i, 5], additionally feature in cell pathway analysis for therapy response or disease progression[5, 6].
• both the forward and reverse phase arrays present problems.
• Labels e.g. enzymes, fluorescent dyes, quantum dots, or metallic nanoparticles
• the target is either directly labelled or detected with a second, labelled antibody (i.e. sandwich assay).
• sandwich assay The fragile and complex nature of proteins discourages direct labelling; variations in labelling efficiency hinders quantification and adding a tag could affect protein binding[J_, 3, II] .
• the sandwich assay has higher specificity and sensitivity, but suffers from additional cross reactivity between detection antibodies [i, 3]. As a result, multiplexing in the system is limited to detecting 30-50 antigens in parallel[3, 5J.
• the invention proposes a device comprising a simple and low cost three-dimensional (3D) microarray to simultaneously sort and pattern fractions of biological samples. It addresses the issues present in either forward or reverse phased arrays, alone, by combining forward and reverse phase arrays.
• the samples are distributed into microchannels in the x-y plane and sorted in the z-direction by
• Capture probes are immobilized systematically along the length of the separable columns.
• the columns comprise in z-direction a stack of layers, whereas each layer in the stack captures a specific target component.
• a layered vertical- flow system capable of simultaneously fractionating and spotting several protein samples in parallel.
• the samples could be, but is not limited to, blood, urine, cell lysate or tissue lysate.
• the spotting and/or detection process carried out by the apparatus is not limited to proteins, but with appropriate markers in the corresponding capture layers, it could also be applied to DNA, peptides, other small molecules or a combination of the proposed ligands. Furthermore, it is not limited to assemblies where the sample comprises ligands that bind directly to the capture layer, but the capture or the binding could be achieved by placing specific carriers and/or microparticles in the capture layers.
• the layers could be a non-permeable support containing a microarray of holes. The holes are filled with a porous material (e.g. a hydrogel) with a pore size large enough to let proteins diffuse through and small enough to physically trap biofunctionalised microparticles within the matrix.
• the hydrogel is inserted into the holes and crosslinked (e.g by UV, temperature change, or ions), creating individually functionalised layers.
• the 3D microarray is not limited to affinity-based sorting; target proteins could also be directly attached to the layers. Unlike a traditional reverse phase microarray, the proteins would be sorted by size or charge as they are patterned onto the substrate. This could be achieved by stacking membranes with different pore sizes or by filling the channels with a conductive medium (e.g. a hydrogel) and applying an electric field. The layers would then be separated, as before, and analysed individually.
• a conductive medium e.g. a hydrogel
• each layer in the microarray stack is functionalised with a different capture probe.
• Functionalization can be physical adsorption or covalent coupling.
• the probe can be directly attached to the matrix or linked to a carrier (e.g. a microparticle) which is then embedded in the porous material.
• the patterned and functionalized microarrays are held together in a stack with appropriate non- permanent means, so that they can be peeled apart for analysis.
• the adhesion can be realized, for instance,,by a non-permanent, double-sided adhesive, or just by appropriate pressure over the whole surface by mechanical clamping layers from top and bottom.
• the top and/or bottom mechanical clamping layer contains holes that serve as the entry channel to the multiplexed affinity columns, such as a bottomless wellplate.
• a spacer layer is inserted between the top and/or bottom patterned nitrocellulose layer and the respective top and/or bottom mechanical clamping layer, more preferably each of those spacer layers also containing a hole just above or below each of the channels, that serves as a reservoir to hold the sample and to prevent leakage between the mechanical clamping layer and the capture layer.
• an essentially liquid sample is introduced into an array of separable affinity columns obtained by layering, as described, above.
• the liquid samples are in the order of one or few ⁇ .
• target proteins bind to specific locations throughout the microchannel. Similar to a reverse phase array, this approach analyses small volumes from multiple samples; however, it has the added advantage of testing each 90 sample for multiple targets, corresponding to the multiple layers, as is done in a forward phase array.
• microchannels i.e. a force is exerted onto the sample liquid in parallel to the microchannel axis, most preferably be centrifuging the device. This leads to a reduction in assay time, ensures the entire sample is pulled through the channel, and thus increases signal intensity and reduces variability.
• the layers in the 3D stack are peeled apart or otherwise separated, revealing 2D microarrays that contain only a specific fraction from multiple samples.
• these arrays are then incubated in a labelled antibody specific to the captured protein, which increases the multiplexing by removing cross- reactivity between detection antibodies.
• the capture layers are essentially made from a porous material, most a 100 hydrogel or a polymer, whereas the mechanical clamping and spacer layers are non-porous.
• the capture layers contain furthermore a spacing material, preferable a hydrophobic spacing material to ensure separation between the channels.
• Sorting and capturing the proteins at the same time eliminates loss associated with pre-fractionation.
• the high cost of protein microarray technology contributes to its limited popularity [7].
• the invention aims to make microarrays more accessible by keeping the design flexible, simple and inexpensive.
• the layers in the proposed 3D stack are made of nitrocellulose and the hydrophobic barriers, which isolate the columns in the microarray, are patterned with wax.
• Nitrocellulose is a porous substrate with a high protein binding capacity; it is widely used in protein immobilization applications, such as lateral
• FMIA flow-through membrane immunoassay
• chromatography paper for low-cost analytical devices.
• the first three-dimensional microfluidic paper analytical devices (3D ⁇ ) distributed samples into multiple detection zones[23, 26]. To achieve this, they used double-sided tape resulting in an irreversible stack of patterned paper. Biorecognition in these systems is not technically multiplexed[4],
• Vella et al. showed that the 3D assembly can be used to assess liver function by detecting enzymes and total protein content from a fmgerstick of blood[9].
• Origami 3D microarrays are an alternative design that does not require laser- patterned tape and allows the user to access the layers by unfolding the paper after performing the assay[27, 28].
• Ge et al. incorporated several immunoassay steps into their origami ⁇ by
• origami 3D ⁇ for sensing enzymes [29 ⁇ 31]. Even though origami exposes the layers, nitrocellulose membranes are too brittle to fold. Like in the previous design, the samples are distributed among the test sites and therefore this system is not multiplexed.
• the hydrophobic spacing material between the multiplexed affinity channels is 165 realized by wax barriers around each antibody-loaded spot or microchannel that extend through the thickness of the porous capture layer material; this allows liquid to pass through vertically while isolating samples from each other laterally.
• the preferred diameter of the samples is between 10 ⁇ and 5000 ⁇ , most preferably between 100 ⁇ ⁇ ⁇ and 1000 ⁇ .
• the pore size of the porous membranes are preferably in the order of some (1-10) tenths 170 of ⁇ -rn.
• the patterning of each layer can be realized by a solid ink printer, as proposed, for example in
• porous material could for instance be realized by nitrocellulose.
• the wax After printing alone, the wax is only on the surface; liquid would initially be confined to the spots, but would quickly diffuse out laterally as it passed through the membrane. Melting the wax pulls the pattern though the thickness of the nitrocellulose, isolating the liquid channels. 175
• the melting can for instance be carried out by placing the membranes into a pre-heated oven to the melting point of the wax for some minutes.
• the channels will shrink due to lateral spread of the wax.
• the holes should thus be printed 30-50% larger than they have to be in the final design, when the process according to [14] is used for printing.
• each square can be customized with a different target.
• the wax can be used to carry a label to keep track on which layer was exposed to which target.
• Unused capture layers may be functionalized with blocking agents, such as, for example, bovine serum albuminum, to prevent non-specific adsorption to the porous material.
• blocking agents such as, for example, bovine serum albuminum
• the slices can be rinsed in tris-buffered saline.
• the arrays could be stored dry by rinsing in ultrapure water briefly before drying with a stream of nitrogen.
• Protran B85 nitrocellulose membranes were used, which have a pore size of 0.45 ⁇ and 80 ⁇ / ⁇ 2 protein binding capacity [34] - h this embodiment, a Xerox X8560 printer was used to deposit multiple copies of the array on each sheet of nitrocellulose, to prepare 190 patterned layers for several experiments iri parallel. The print quality was set to the maximum
• the membrane functionalisation is by passively adsorbing antibodies. This could be achieved using a simple well formed from a microscopy slide, an elastomeric ring and a metallic washer. The membrane is pressed between the slide and the washer. The elastomeric ring is 195 placed between the membrane and the washer to create the walls of the well and prevent leakage.
• the elastomer ring can for instance be cut from a ⁇ 2.5 mm thick slab of PDMS using the metal washer as a guide.
• the chamber reduces the amount of liquid needed to submerge the arrays and the
• hydrophobic wax prevents protein adhesion outside of the channels. Protein adhesion could be improved by leaving the arrays in an oven pre-heated to the optimal temperature (e.g. 37°C).
• the stack is used for a direct-labelled immunoassay.
• labelled e.g. with a fluorescent dye, quantum dots, enzymes, or metallic nanoparticle
• Another option is to use the stack for a sandwich assay. In this case the sample would not be labelled.
• the layers are separated and each is incubated in the labelled antibody specific to the protein captured on that layer.
• the layers of biofunctionalised nitrocellulose are aligned with four corner pins.
• a hole (1 mm in diameter) is punched from a marked position in the corner of each layer.
• the stack is assembled by inserting the four pins into four holes in a micromachined piece of solid plastic (e.g. PMMA).
• the membranes are slide onto the pins, followed by an micromoulded elastomer (e.g. PDMS).
• the l-nim thick elastomer contains an
• this sheet has tapered inlet holes to facilitate pipetting samples into the channels.
• the sample could be pulled through the channels with a centrifuge (129 x g). After sample incubation, the stack is disassembled and the layers can be handled with tweezers.
• the detection antibodies are imaged with a fluorescence microscope.
• the membrane layers could be clamped between two microscopy slides, keeping them flat for automated imaging (e.g. with a microarray reader). Description of the Drawings
• FIG. 1 A schematic of one embodiment of the device, the 3D microarray.
• Capture antibodies are passively adsorbed by irreversible electrostatic attraction to the nitrocellulose membranes. The nitrocellulose is patterned with wax, creating hydrophobic barriers between the antibody loaded spots.
• B) Layers of patterned and biofunctionalised nitrocellulose are stacked to form an array of multiplexed affinity columns.
• C) The 3D microarray is assembled within a device to aid alignment and sample
• Capture layers are exemplarily clamped between two micromachined pieces of PMMA.
• Spacer layers prevent leakage between the PMMA and the nitrocellulose and act as a reservoir for the sample.
• Samples are pipetted into the channels using the tapered inlets.
• the device as exemplarily shown can analyse 25 samples simultaneously; the cut away side view shows one row of 5 channels.
• Figure 2 Images of wax printing on nitrocellulose for a particular embodiment of the invention.
• A) A 230 section of the Adobe Illustrator file for preparing patterned nitrocellulose layers in parallel.
• B) Images of the front and back of the nitrocellulose membrane before and after melting the wax. Initially the wax is only on the surface of the membrane and as it melts the hydrophobic barriers extend through to the backside.
• C) Microscopy images of one spot before (left) and after (right) melting. Lateral diffusion causes the spots to shrink as the wax melts. Scale bar 200 ⁇ .
• Figure 4 Stack-and-Separate immunoassay.
• A) A schematic of the four-layer stack for evaluating the design. The third layer is functionalised with rabbit IgG and the other three only contain BSA. The sample, 1 ⁇ of anti-rabbit IgG Alexa Fluor 488, is injected into the channels.
• the 3D microarray is disassembled and rinsed before imaging each layer separately.
• the anti-rabbit IgG bound to the third layer in a checkerboard pattern is expected.
• Fluorescent rings appear around the spots on the first layer of the 3D microarray stack and to a lesser extent on the subsequent layers. Reflection images indicate that this binding is on the wax edge surrounding the exposed nitrocellulose. The same ring-effect is observed in a sandwich assay when the
• Figure 5 Fluorescence images of individual spots from a sandwich assay experiment. The two rows represent two repeats. In both cases, the second layer in the stack was functionalized with anti-mouse IgG (pictured) and the other three blocked with BSA (not shown). In the first column (A and C), 0.3 ⁇ g/mL of mouse IgG was injected into the channels. The second column (C and D) is the negative
• mice IgG concentrations of mouse IgG (ranging from 1781 pM to 10 pM, spiked into BSA) were injected sequentially three times. The stack was then disassembled, rinsed and incubated in fluorescent anti- mouse IgG. The wax background was removed from the image to visualise the low concentration
• the black dotted line was used to determine the limit of detection and was calculated from the average intensity of 0 pM of mouse IgG added to 3x its standard deviation. Each point is the average signal-to-background of nine spots, taken from three different experiments. The error bars are the standard deviation between experiments.
• Figure 9 Images of another embodiment of the device: A) Materials used to make the microarray stack. B) A stack of paper arrays, aligned and held together with a temporary adhesive, pressed between a KimwipeTM and a PDMS seal and supported by a microscope slide. C) The assembled device including an inlet extension to support larger volumes (e.g for rinsing). D) Side view of the 285 assembled device.
• FIG. 10 Concentration series of fluorescent IgG passively adsorbed to patterned cellulose-based filter paper.
• A) A typical fluorescence image of one slice from the stack (slice #1).
• B) Concentration series for three stacked slices.
• the antibody in this example is non-specifically adsorbed to the fibres, which causes the intensity to drop as the antibody passes through the layers. Each point represents the 290 average of five spots. Error bars are the standard deviation.

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