Multiplexed, label-free detection of biomarkers using aptamers and Tunable Resistive Pulse Sensing (AptaTRPS).

Diagnostics that are capable of detecting multiple biomarkers are improving the accuracy and efficiency of bioassays. In previous work we have demonstrated the potential of an aptamer-based sensor (aptasensor) utilising Tunable Resistive Pulse Sensing (TRPS). Here, we have advanced the technique identifying key experimental designs for potential POC assays. The assay utilised superparamagnetic beads, and using TRPS monitored their translocations through a pore. If the surfaces of the beads are modified with an aptamer, the frequency of beads (translocations/min) through the pore can be related to the concentration of specific proteins in the solution. Herein, we have demonstrated the successful use of TRPS to observe the binding of two proteins, to their specific aptamers simultaneously. We describe a series of experiments illustrating key factors which we believe are integral to bead-based assays and demonstrate a general method for a multiplexed assay. In summary, we have explored the effects of beads size, concentration, potential bias, pH and aptamer affinity to enhance the sensitivity and practically of a TRPS aptasensor. The method utilises the fact the binding of the aptamer to the protein results in a change in charge density on the bead surface, the isoelectric point of the protein then dominates the mobility of the beads, creating a multiplexed assay termed AptaTRPS. By alteration of the applied potential to the instrument it is possible to produce a positive signal in a simple multiplexed assay.