A method is described for an aptamer-based affinity assay using a combination of two non-conventional techniques, temperature gradient focusing (TGF) and field amplified continuous sample injection TGF (FACSI-TGF), with fluorescence detection. Human immunodeficiency virus reverse transcriptase (HIVRT) is used as the protein target for the assay. The TGF and FACSI-TGF assays are compared to similar results obtained with conventional capillary electrophoresis (CE). A range of starting aptamer concentrations are used to determine the optimal limit of detection (LOD) for HIVRT using each approach. The results indicate that the detection limits for HIVRT obtained with TGF and FACSI-TGF are comparable to or even lower than the detection limits obtained with conventional CE in spite of the inferior detector used for the TGF and FACSI-TGF assays (arc lamp and low cost CCD for TGF vs. LIF with PMT for CE). It is hypothesized that this is due to the greater reproducibility of the TGF and FACSI-TGF techniques since they do not employ a defined sample injection. The lowest detection limit achieved with the new aptamer assay approach is more than two orders of magnitude lower than that reported for a similar CE-based aptamer assay for the same target.
Pub Type: Journals