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

Biochemistry. 2018 Aug 28;57(34):5117-5126. doi: 10.1021/acs.biochem.8b00523. Epub 2018 Aug 13.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aptamers, Nucleotide / chemistry
  • Aptamers, Nucleotide / metabolism*
  • Benzidines / chemistry
  • Benzidines / metabolism
  • Biosensing Techniques / methods*
  • Colorimetry / methods*
  • G-Quadruplexes*
  • Nucleic Acid Amplification Techniques / methods*
  • RNA, Catalytic / chemistry
  • RNA, Catalytic / metabolism*
  • Theophylline / analysis*

Substances

  • Aptamers, Nucleotide
  • Benzidines
  • RNA, Catalytic
  • hammerhead ribozyme
  • 3,3',5,5'-tetramethylbenzidine
  • Theophylline