Article pubs.acs.org/acssensors
Improving FoRe: A New Inlet Design for Filtering Samples through Individual Microarray Spots Victoria de Lange, Marco Habegger, Marco Schmidt, and János Vörös* Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, University and ETH Zurich, Gloriastrasse 35, 8092 Zurich, Switzerland S Supporting Information *
ABSTRACT: In this publication we present an improvement to our previously introduced vertical flow microarray, the FoRe array, which capitalizes on the fusion of immunofiltration and densely packed micron test sites. Filtering samples through individual microarray spots allows us to rapidly analyze dilute samples with high-throughput and high signal-to-noise. Unlike other flowthrough microarrays, in the FoRe design samples are injected into micron channels and sequentially exposed to different targets. This arrangement makes it possible to increase the sensitivity of the microarray by simply increasing the sample volume or to rapidly reconcentrate samples after preprocessing steps dilute the analyte. Here we present a new inlet system which allows us to increase the analyzed sample volume without compromising the micron spot size and dense layout. We combined this with a model assay to demonstrate that the device is sensitive to the amount of antigen, and as a result, sample volume directly correlates to sensitivity. We introduced a simple technique for analysis of blood, which previously clogged the nanometer-sized pores, requiring only microliter volumes expected from an infant heel prick. A drop of blood is mixed with buffer to separate the plasma before reconcentrating the sample on the microarray spot. We demonstrated the success of this procedure by spiking TNF-α into blood and achieved a limit of detection of 18 pM. Compared to traditional protein microarrays, the FoRe array is still inexpensive, customizable, and simple to use, and thanks to these improvements has a broad range of applications from small animal studies to environmental monitoring. KEYWORDS: protein microarray, vertical flow assay, nitrocellulose, wax printing, affinity columns, FoRe microarray, whole blood, immunofiltration
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without compromising the amount of captured analyte. This is especially attractive for highly viscous or complex samples, e.g., whole blood, which can be diluted without loss of sensitivity. We also introduce a simple technique to analyze a finger prick of blood, by diluting the sample with buffer before briefly spinning down the blood cells. The entire supernatant then flows through the microchannels to reconcentrate the analytes on the array spots. Microfiltration devices are commercially available in a 96-well format as enzyme-linked immunofiltration (ELIFA) or dot blot systems for parallel, vertical flow analysis.4 A membrane made from, for example, nylon,2 nitrocellulose,4b or cellulose acetate5, is clamped between two plastic well plates and samples are isolated through the use of rubber gaskets. We chose nitrocellulose in our system for its high protein binding capacity and compatibility with inexpensive wax-printing.6 By patterning hydrophobic wax barriers directly on the membrane we can isolate samples without the need for gaskets.
rotein microarrays consist of spatially addressable test sites with micro- to nanodimensions for highly multiplexed sensing. Miniature, planar test sites have several advantages (e.g., they are insensitive to sample volume errors, have high signal-to-noise and high throughput), but are not well suited for analyzing dilute samples because of the long incubation times needed to reach equilibrium.1 In contrast, immunofiltration assays can rapidly detect low amounts of analyte by flowing samples vertically through membranes dense with capture probes. However, relatively large spot diameters and issues isolating samples mean that these systems lack the high signalto-noise and throughput of microarrays.2 We previously introduced a vertical flow microarray, the FoRe array, which combines micron test sites with high capture probe density for rapid and sensitive analysis of several samples in parallel.3 The 3D microarray performs multiplexed analyte detection on each sample and requires only microliter volumes. In this publication we describe an improvement in the FoRe device with an array of angled microfluidic inlets to increase the sample volume flowing through the miniaturized test sites. With this new design we have the unique ability to tune the sensitivity of a microarray, depending on the available sample volume, and to perform preprocessing or extraction steps © XXXX American Chemical Society
Received: April 20, 2016 Accepted: February 28, 2017
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DOI: 10.1021/acssensors.6b00271 ACS Sens. XXXX, XXX, XXX−XXX
Article
ACS Sensors
Figure 1. Schematic of the FoRe microarray. (A) Each layer of nitrocellulose is patterned with an array of 400 μm wax channels and functionalized with a different capture antibody. The layers are aligned with the help of four pins, creating an array of multiplexed affinity columns. (B) The nitrocellulose stack is clamped between a solid PMMA inlet and outlet to ensure contact and prevent leaking. Samples are funneled from a 1.3 mm inlet well through 500 μm angled channels in PDMS to the wax-patterned nitrocellulose channels.
Speed is the main attraction for vertical flow assays; thanks to the high surface to volume ratio of the porous membrane, binding kinetics closely resemble that of proteins in solution.1a,2,5 This dramatically reduces assay time from hours for a solid-phase immunoassay to minutes for an immunofiltration assay, and is particularly beneficial when analyzing low concentration samples.1a,4a In 1991 Poulsen and Bjerrum demonstrated that, in addition to speed, vertical flow can also increase the sensitivity of an immunoassay by concentrating dilute analytes in the membrane.7 The high capture probe density and nanometer pores make it possible to bind all the analyte flowing through the matrix, creating a system sensitive to total antigen amount instead of concentration. This discovery was not well explored, likely because the minimum sample volumes were already hundreds of microliters and millimeter-diameter spots would have poor signal-to-noise for dilute samples. For multiplexed analyte detection within the wells the membranes can be pre-spotted with capture probes.1a,8 The captured analytes are confined to a smaller test site for higher signal-to-noise (micron spots compared to millimeter wells); however, introducing several test sites within a sample well means analytes can pass through the membrane undetected in the areas surrounding the microspots. An alternative method for creating vertical flowthrough arrays is to pattern channels directly into the membranes.9 The membranes can then be irreversibly stacked10 or folded in the style of origami11 to form three-dimensional paper-based analytical devices. With this design the patterned layers serve to distribute the sample from the inlet channel to multiple detection zones. While this approach is less expensive than robotic spotting and relies only on capillary forces, it also does not take advantage of analyte concentration during vertical flow. With the FoRe microarray we bring immunofiltration from the milliscale to the microscale and combine microarray multiplexing with rapid low concentration sensing. The 3D FoRe array is formed by stacking wax-patterned nitrocellulose membranes, each functionalized with a different capture probe. The wax forms hydrophobic barriers around the array of antibody-loaded spots. This allows us to restrict the channel
diameter, reducing the minimum required volume (from hundreds of microliters for an ELIFA to
