| AVS 54th International Symposium | |
| Biomaterial Interfaces | Friday Sessions |
| Session BI-FrM |
| Session: | Microbioanalytical Systems |
| Presenter: | S.P. Mulvaney, Naval Research Laboratory |
| Authors: | S.P. Mulvaney, Naval Research Laboratory K.M. Myers, Naval Research Laboratory P.E. Sheehan, Naval Research Laboratory L.J. Whitman, Naval Research Laboratory |
| Correspondent: | Click to Email |
Assays for biomolecular detection are ideally both multiplexed and sensitive, metrics that often require conflicting solutions. For example, microarrays use spatial location for multiplexing, but target capture on the surface will ultimately be diffusion-limited. Alternatively, homogeneous assays offer very efficient target capture, but typically require multiple label types to multiplex. In our semi-homogeneous immunoassays we use microbeads for both target capture and labeling to leverage the advantages of both approaches. The sample is first mixed with secondary antibodies and microbeads that are functionalized against the secondary host. Target molecules are thereby captured onto the beads via the secondary antibodies. The target-loaded beads are then captured onto an antibody microarray, and controlled fluidic forces are applied to preferentially remove nonspecifically bound beads.1 Finally, the remaining beads are counted to determine the target(s) concentration. Utilizing such assays, we have achieved multiplexed toxin detection, including aM detection of SEB, in <20 min in a variety of complex matrices. Micrometer-scale sensors and beads are optimal for detecting nanoscale biomolecules with practical sensitivity and speed.2 However, the blending of micro- and nano-scales in such assays leads to some interesting relationships. In diffusion-limited, solid-phase assays, it takes hours-to-days for fM targets to accumulate on a nanosensor, but only seconds-to-minutes on a microsensor. In addition, microscale labels enable fluidic forces to be applied to achieve greater sensitivity and fewer false positives than possible with nanoscale labels. Finally, in contrast to nanoscale labels, individual microbeads can be easily detected. We believe the size mismatch between label and target contributes to the extreme sensitivity of our assays. Each microbead-label confines a very small volume beneath the contact area, thereby creating a high local concentration of target molecules and capture/label antibodies. This confinement greatly increases the effective binding constant, suppressing dissociation and detachment of the label. The relatively large bead size also contributes to the unusual log-linear dose response curves we obtain, that span up to nine orders of magnitude.
1 Mulvaney, et al., Biosens. Bioelectron., in press.
2 Sheehan and Whitman, Nano Lett. 5, 803 (2005).