Rapid and reagentless detection of thrombin in clinic samples via microfluidic aptasensors with multiple target-binding sites.

A reusable and straightforward aptasensor with the implementation of open-ended porous silicon (OEPSi) membranes was introduced for thrombin detection. When passing through the nanochannels of OEPSi integrated in a microfluidic cell, thrombin in sample solution could be captured by thrombin-binding aptamers (TBA) immobilized along the inner walls. The formation of thrombin-aptamer complex causes refractive index changes which can be measured by reflective interferometric Fourier transform spectroscopy (RIFTS). And this flow-through configuration with OEPSi has proven more efficient in capturing thrombin than the flow-over configuration with closed-ended PSi. For higher sensitivity, we investigated how the pore size, ionic strength, pH and aptamers affected the thrombin-aptamer interaction in nanopores. Under optimized conditions, the limits of detection (LOD) for thrombin detection in the buffer and serum were ∼6.70 nM and ∼8.21 nM respectively and a wide linear detection range (10-1000 nM) was observed. More importantly, this work reveals the sensitivity of the label-free biosensor can be significantly improved by attaching newly designed aptamers with two thrombin-binding sites. This phenomenon also indicates the potential of aptamer probes in adjusting effective pore size and enhancing the interaction between aptamers and targets through meticulous sequence design. Furthermore, the proposed strategy has been applied in thrombin detection in clinic samples successfully, which was verified by Enzyme-Linked Immunosorbent Assays (ELISA).