A Simple Colorimetric System for Detecting Target Antigens by a Three-Stage Signal Transformation-Amplification Strategy.

Abstract

Inexpensive, straightforward, and rapid medical diagnostics are becoming increasingly important for disease identification in time- and resource-limited settings. Previous attempts to link oligonucleotide-based aptamers and hammerhead ribozymes to form ligand-induced ribozymes have been successful in identifying a variety of small molecule and protein targets. Isothermal exponential amplification reactions (EXPAR) amplify minute amounts of nucleic acid templates without requiring special instrumentation. We introduce a colorimetric assay that we engineered using an aptamer, hammerhead ribozyme, EXPAR, and peroxidase activity in conjunction with a 3,3',5,5'-tetramethylbenzidine (TMB) substrate. This is a modular signal enhancer system that can be easily modified to detect virtually any chosen analyte target within 5-10 min with minimal technical requirements. Ligand-aptamer binding causes the ribozyme to change conformation and self-cleave. The cleaved ribozyme triggers exponential amplification of a reporter sequence during EXPAR. The amplification products fold into single-stranded DNA guanine quadruplexes that exhibit peroxidase-like activity and can oxidize a colorless TMB substrate into a colored reaction product for visual detection. As a proof of concept, we examined the bronchodilator theophylline versus its chemical analogue, caffeine. We demonstrate linear changes in absorption readout across a wide range of target concentrations (0.5-1000 μM) and the ability to visually detect theophylline at 0.5 μM with an approximately 35-fold increased specificity versus that of caffeine. This three-stage detection system is a versatile platform that has the potential to improve the rapid identification of target analytes.