Improving FoRe: A New Inlet Design for Filtering Samples through Individual Microarray Spots.

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.

Twist on protein microarrays: layering wax-patterned nitrocellulose to create customizable and separable arrays of multiplexed affinity columns.

We developed the simple and inexpensive FoRe microarray to simultaneously test several 1 µL samples for multiple proteins. By combining forward and reverse phase microarrays into an innovative three-dimensional format, the FoRe array exploits the advantages and eliminates several drawbacks of the traditional approaches (i.e., large sample volumes, protein loss, and cross-reactivity between detection antibodies). Samples are pipetted into an array of separable, multiplexed affinity columns. Several nitrocellulose membranes, each functionalized with a different capture antibody, are stacked to create a customizable affinity column. The nitrocellulose is patterned with wax to form 25 isolated microspots on each layer, allowing us to analyze multiple samples in parallel. After running the immunoassay, the stacks are quickly disassembled, revealing 2D microarrays of different fractions from multiple samples. By combining the stack-and-separate technique with wax patterning, we keep the arrays low cost and easily tailored to a variety of applications. We successfully performed 3D multiplexing using a model system with mouse and rabbit IgG. Binding proved to be independent of the position in the stack, and the limit of detection for a mouse IgG sandwich assay was 7.3 pM in BSA and 15 pM in human plasma. The FoRe microarray makes it possible to identify protein expression patterns across several minute volume samples; for example, it could be used to analyze cell lysate in drug response studies or pricks of blood from small animal studies.

Microarrays made easy: biofunctionalized hydrogel channels for rapid protein microarray production.

We present a simple, inexpensive, and sensitive technique for producing multiple copies of a hydrogel-based protein microarray. An agarose block containing 25 biofunctionalized channels is sliced perpendicularly to produce many identical biochips. Each microarray consists of 500 µm spots, which contain protein-coated microparticles physically trapped in porous SeaPrep agarose. Proteins diffuse readily through SeaPrep agarose, while the larger microparticles are immobilized in the hydrogel matrix. Without major assay optimization, the limit of detection is 12 pM for a sandwich assay detecting human IgG. These highly flexible, multiplexed arrays can be produced rapidly without any special instrumentation and are compatible with standard fluorescence-based read-out.

Vesicles for Signal Amplification in a Biosensor for the Detection of Low Antigen Concentrations.

The sensitivity of biosensors is often not sufficient to detect diagnostically relevant biomarker concentrations. In this paper we have utilized a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) to detect dissipative losses induced by the attachment of intact vesicles. We modified a sandwich assay by coupling the secondary antibodies to vesicles. This resulted in an increase of detection sensitivity, achieving a diagnostically relevant detection limit of 5 ng/ml or 30 pM antigens. In addition, we could combine the individual assay steps to decrease the total time to result in about 30 minutes.

On-chip surface-based detection with nanohole arrays.

A microfluidic device with integrated surface plasmon resonance (SPR) chemical and biological sensors based on arrays of nanoholes in gold films is demonstrated. Widespread use of SPR for surface analysis in laboratories has not translated to microfluidic analytical chip platforms, in part due to challenges associated with scaling down the optics and the surface area required for common reflection mode operation. The resonant enhancement of light transmission through subwavelength apertures in a metallic film suggests the use of nanohole arrays as miniaturized SPR-based sensing elements. The device presented here takes advantage of the unique properties of nanohole arrays: surface-based sensitivity; transmission mode operation; a relatively small footprint; and repeatability. Proof-of-concept measurements performed on-chip indicated a response to small changes in refractive index at the array surfaces. A sensitivity of 333 nm per refractive index unit was demonstrated with the integrated device. The device was also applied to detect spatial microfluidic concentration gradients and to monitor a biochemical affinity process involving the biotin-streptavidin system. Results indicate the efficacy of nanohole arrays as surface plasmon-based sensing elements in a microfluidic platform, adding unique surface-sensitive diagnostic capabilities to the existing suite of microfluidic-based analytical tools.