A Novel Inexpensive, Rapid, Simple, and Sensitive Aptamer-Based Sandwich Assay for Colorimetric Thrombin Biosensing

Abstract

In this paper, we report the development of an aptamer-based colorimetric assay for the inexpensive, rapid, simple, and sensitive detection and quantifcation of biomolecules based on the principle of sandwich ELISA. Thrombin was selected as a model analyte to validate the assay design, which involved the selective binding of one or two diferent protein epitopes with DNA aptamers. Two diferent substrates were employed: covalently modifed cellulose and glass microscope slides, with the covalently modifed cellulose immobilizing the target analyte via non-specifc divinyl sulfone (DVS) chemistry while the glass microscope slide strategy utilized a physisorbed thrombin aptamer as a capture agent. The captured biomolecules were then labeled by iron oxide nanoparticle-aptamer conjugates, which was then visualized via the reduction of Cu2+ ions onto the surface of the nanoparticles. An electrophoresis mobility shift assay and agarose gel electrophoresis were used to confrm the aptamer-thrombin binding and aptamer-nanoparticle conjugation, respectively. The formation of the copper flm on the iron oxide nanoparticles was then modeled via a nucleation-catalytic growth model in order to optimize the assay run time. Both sensor formats had a high sensitivity with the cellulose-based assay having an limit of detection (LOD) of 50 pM whereas the glass-based assay demonstrated a higher sensitivity with an LOD of 20 pM. In addition, the sensors demonstrated high specifcity to thrombin with no signifcant signal being elicited from exposure to Bovine-Serum Albumin (BSA). The cellulose-based assay had a material cost of $0.10 and took 15 minutes to run, representing a 60 times improvement in cost and a 16 times improvement in time when compared to the gold-standard for protein detection, ELISA. Thus, the developed assay represents an inexpensive, rapid, simple, sensitive, and versatile method for the detection of biomolecules for a host of applications from diagnostics to biodefense.