Electrical Detection of Nucleic Acid Amplification Using an On-Chip Quasi-Reference Electrode and a PVC REFET

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.

Quantitative determination of target gene with electrical sensor

“…The sharp decline in the conductivity continues for ~110 s, suggesting a rather high efficiency of the biochemical reaction. Thereafter the decrease of output potential slows down to a gentle decline, indicating that the amplification reaction slows considerably, which may be due to the inhibition of polymerase activity by the fall of pH 16 , or the decrease of the concentration of Mg 2+ 28 . Note, the usage of more ThermoPol ® reaction buffer than the commended dosage, e.g., ≥1.2×, benefits to obtain stable curves of conductivity responses (see Supplementary Note 4 and Supplementary Fig.…”

“…The alternative, electrochemical methods, are faster, lower cost, simpler and can be more readily be miniaturized by eliminating the requirement of photoelectric converter 10 . Unfortunately, for continuously monitoring the progression of the LAMP reaction in real time with contact electrodes, either with voltammetry 11 12 13 14 or potentiometry 15 16 , there is still the issue of low reproducibility due to the fouling and passivation of working electrode, by biological species in the amplification reaction vessel. Furthermore, accompanied with this issue of electrode fouling is the increased risk of carryover contamination.…”

Real-time electrochemical LAMP: a rational comparative study of different DNA intercalating and non-intercalating redox probes

“…One exception is the real-time monitoring of LAMP reaction through a pH change, but the resulting LAMP performance suffers from a lack of sensitivity because of the need to record very small pH variations. 18 For the other approaches, two detection strategies were proposed. The first one takes advantage of a change in the electrical conductivity of the reaction solution to monitor the progress of an LAMP reaction, 16,17 while the second one is an adaptation of a detection strategy previously demonstrated for real-time electrochemical PCR.…”

“…22 However, the poor temporal resolution as well as the high data scattering in the published work hamper judgement of the LAMP performances with this particular redox probe. Though the analytical performances of real-time electrochemical LAMPs so far developed look attractive and potentially competitive with optical fluorescence-based methods, [14][15][16][17][18][19]22 it is not possible to accurately compare them, each being carried out under different conditions using different DNA targets and redox probes. It is therefore not obvious to identify which redox probes are more favorable for LAMP.…”

Rapid quantification of microRNAs in plasma using a fast real-time PCR system

“…Although many prototype rapid PCR systems have been reported with extremely fast ramp rates and miniaturization (5, 6, 31), very few proceed to commercial release. Therefore, the xxpress rapid thermal cycler, particularly given its standard block-based design, offers a unique opportunity for the wider molecular research community to adopt fast PCR.…”