Direct detection of genomic DNA with fluidic force discrimination assays.

Herein, we describe the direct detection of genomic DNA using fluidic force discrimination (FFD) assays. Starting with extracted bacterial DNA, samples are fragmented by restriction enzymes or sonication, then thermocycled in the presence of blocking and labeling sequences, and finally detected with microbead-based FFD assays. Both strain and species identification of extracted Bacillus DNA have been demonstrated in <30 min, without amplification (e.g., PCR). Femtomolar assays can be achieved with this rapid and simple procedure.

Attomolar protein detection in complex sample matrices with semi-homogeneous fluidic force discrimination assays.

We describe a semi-homogenous (SH) implementation of a fluidic force discrimination (FFD) assay using only two reagent mixtures and three assay steps that can be performed in as little as 10min. Previously microbead labels and FFD have been combined to achieve multiplexed, femtomolar nucleic acid hybridization and immunoassays in a microarray format [Mulvaney, S.P., Cole, C.L., Kniller, M.D., Malito, M., Tamanaha, C.R., Rife, J.C., Stanton, M.W., Whitman, L.J., 2007. Biosen. Bioelectron. 23, 191-200.]. In SH FFD assays, the microbeads and any required intermediate receptors (e.g., secondary antibodies) are first mixed directly with a sample, allowing target analytes to be efficiently captured onto the beads. The target-loaded beads are then specifically captured onto a microarray surface, with nonspecifically bound beads removed by controlled, laminar fluidic forces. The remaining beads on each microarray capture spot are counted to determine the targets' identities and concentrations. SH target collection provides a 1000-fold improvement in the assay sensitivity, down to attomolar concentrations, as demonstrated by our detection of staphylococcal enterotoxin B (SEB) at 35 aM (1 fg/ml). We also show that SH assays are adaptable for extraction, preconcentration, and identification of analytes in complex sample matrices, including assays for SEB and ricin toxoid in serum and whole blood. Finally, we present a detailed model of the reaction kinetics that reveals how capturing the targets onto the beads in solution provides a significant kinetic advantage at low target concentrations where mass transport to a microarray surface is most limited.

Magnetic labeling, detection, and system integration.

Among the plethora of affinity biosensor systems based on biomolecular recognition and labeling assays, magnetic labeling and detection is emerging as a promising new approach. Magnetic labels can be non-invasively detected by a wide range of methods, are physically and chemically stable, relatively inexpensive to produce, and can be easily made biocompatible. Here we provide an overview of the various approaches developed for magnetic labeling and detection as applied to biosensing. We illustrate the challenges to integrating one such approach into a complete sensing system with a more detailed discussion of the compact Bead Array Sensor System developed at the U.S. Naval Research Laboratory, the first system to use magnetic labels and microchip-based detection.

Rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics.

A significant challenge for all biosensor systems is to achieve high assay sensitivity and specificity while minimizing sample preparation requirements, operational complexity, and sample-to-answer time. We have achieved multiplexed, unamplified, femtomolar detection of both DNA and proteins in complex matrices (including whole blood, serum, plasma, and milk) in minutes using as few as two reagents by labeling conventional assay schemes with micrometer-scale magnetic beads, and applying fluidic force discrimination (FFD). In FFD assays, analytes captured onto a microarray surface are labeled with microbeads, and a controlled laminar flow is then used to apply microfluidic forces sufficient to preferentially remove only nonspecifically bound bead labels. The density of beads that remain bound is proportional to the analyte concentration and can be determined with either optical counting or magnetoelectronic detection of the magnetic labels. Combining FFD assays with chip-based magnetoelectronic detection enables a simple, potentially handheld, platform capable of both nucleic acid hybridization assays and immunoassays, including orthogonal detection and identification of bacterial and viral pathogens, and therefore suitable for a wide range of biosensing applications.