A method is described for an aptamer-based affinity assay using a combination of two nonconventional techniques, temperature gradient focusing (TGF) and field-amplified continuous sample injection TGF (FACSI-TGF), with fluorescence detection. Iquman 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 CE. A range of starting aptamer concentrations are used to determine the optimal LOD for human immunodeficiency virus reverse transcriptase (HIVRT) using each approach. The results indicate that the LODs for HIVRT obtained with TGF and FACSITGF are comparable to or even lower than the LODs 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 versus 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 LOD achieved with the new aptamer assay approach is more than an order of magnitude lower than that reported for a similar CE-based aptamer assay