Electrical detection of nucleic acid amplification using an on-chip quasi-reference electrode and a PVC REFET

Anal Chem. 2014 Jul 15;86(14):6968-75. doi: 10.1021/ac500897t. Epub 2014 Jun 27.

Abstract

Electrical detection of nucleic acid amplification through pH changes associated with nucleotide addition enables miniaturization, greater portability of testing apparatus, and reduced costs. However, current ion-sensitive field effect transistor methods for sensing nucleic acid amplification rely on establishing the fluid gate potential with a bulky, difficult to microfabricate reference electrode that limits the potential for massively parallel reaction detection. Here we demonstrate a novel method of utilizing a microfabricated solid-state quasi-reference electrode (QRE) paired with a pH-insensitive reference field effect transistor (REFET) for detection of real-time pH changes. The end result is a 0.18 μm, silicon-on-insulator, foundry-fabricated sensor that utilizes a platinum QRE to establish a pH-sensitive fluid gate potential and a PVC membrane REFET to enable pH detection of loop mediated isothermal amplification (LAMP). This technique is highly amendable to commercial scale-up, reduces the packaging and fabrication requirements for ISFET pH detection, and enables massively parallel droplet interrogation for applications, such as monitoring reaction progression in digital PCR.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Electrodes*
  • Equipment Design
  • Hydrogen-Ion Concentration
  • Microtechnology
  • Nucleic Acid Amplification Techniques / instrumentation*
  • Nucleic Acid Amplification Techniques / methods*
  • Oligonucleotide Array Sequence Analysis / instrumentation
  • Platinum
  • Polymerase Chain Reaction / methods
  • Polyvinyl Chloride

Substances

  • Platinum
  • Polyvinyl Chloride