A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. Highresolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.cell signaling ͉ immunoassay ͉ phosphorylation ͉ Western blot ͉ microfluidic
The use of arrays of immobilized DNA “probes” for high-throughput analysis of genomic
samples is expanding rapidly. The detection sensitivity of these arrays depends on the
quantity and density of immobilized probe molecules as well as on the thermodynamics and
kinetics of nucleic acid hybridization. We have prepared and investigated substrates with a
porous, “three-dimensional” surface layer as a means of increasing the surface area available
for the synthesis or immobilization of oligonucleotide probes, thereby increasing the number
of available probes and the amount of detectable bound target per unit area. Surfaces with
pores 5 nm and larger were created by spin-coating colloidal suspensions of silica particles,
followed by thermal curing. DNA arrays were synthesized on the resulting surfaces by
photolithographic patterning, and the performance on the high-capacity substrates was
compared to that on standard flat glass surfaces. The colloidal silica films created via this
route show equivalent performance to flat glass substrates in terms of the efficiency of
chemical synthesis and resolution of photolithographic patterning. DNA targets are able to
penetrate the porous layers, and under saturating conditions, the quantity of bound target
is proportional to the layer thickness. The result is an enhanced hybridization signal that
is 20 times higher than flat glass for a colloidal particle layer that is 0.5 μm thick. The
thermodynamic stability of probe/target duplexes in the matrix is the same as that for their
counterparts on flat surfaces, although the colloidal silica films reach saturation more slowly
than flat surfaces.
Colloidal silica particles were deposited on a glass substrate to produce high-capacity porous supports for high-density DNA probe arrays. Porous surfaces were used to increase the addressable surface area and number of probes available for hybridization. Surfaces derived from 70-100 nm size particles deposited in films from 0.15 to 2 microns thick exhibited excellent performance in light-directed oligonucleotide synthesis. Evaluation of these substrates in a genotyping assay is reported.
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