Tentacle probe sandwich assay in porous polymer monolith improves specificity, sensitivity and kinetics

Nucleic acid sandwich assays improve low-density array analysis through the addition of a capture probe and a specific label, increasing specificity and sensitivity. Here, we employ photo-initiated porous polymer monolith (PPM) as a high-surface area substrate for sandwich assay analysis. PPMs are shown to enhance extraction efficiency by 20-fold from 2 μl of sample. We further compare the performance of labeled linear probes, quantum dot labeled probes, molecular beacons (MBs) and tentacle probes (TPs). Each probe technology was compared and contrasted with traditional hybridization methods using labeled sample. All probes demonstrated similar sensitivity and greater specificity than traditional hybridization techniques. MBs and TPs were able to bypass a wash step due to their 'on-off' signaling mechanism. TPs demonstrated reaction kinetics 37.6 times faster than MBs, resulting in the fastest assay time of 5 min. Our data further indicate TPs had the most sensitive detection limit (< 1 nM) as well as the highest specificity (> 1 × 10⁴ improvement) among all tested probes in these experiments. By matching the enhanced extraction efficiencies of PPM with the selectivity of TPs, we have created a format for improved sandwich assays.