Rapid Fluorescence-Free Detection of DNA Loop-Mediated Isothermal Amplification on Bare ITO Surface Under EG-FET Scheme

Abstract

An extended gate field effect transistor (EG-FET)-based chip with a precision temperature control system is developed for real-time and fluorescence-free loop-mediated isothermal amplification (LAMP) detection. A label-free, non-optical instant method was used to detect the hydrogen ions released during nucleic acid amplification, rather than indirect measurements. The system comprises an EG-FET sensor, a temperature sensor, a heating block, and a signal processing system. Industrial grade bare indium tin oxide (ITO) is firstly demonstrated as the sensing material for nucleic acid amplification detection, such that stable and high hydrogen ion detection performance of -80.1±0.03 mV/pH with R <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> of 0.998 is achieved with the developed sensor. Moreover, a printed circuit board (PCB) with high thermal conductivity was used as the sensor base to provide a uniform temperature distribution during the reaction. The results show that the system maintains a stable LAMP assay temperature of 63°C with a variation of just 0.25°C. Moreover, in a pH-sensitive LAMP assay, the developed sensor achieves a limit-of-detection down to 10 genomic copies per reaction of lambda phage DNA within 17 minutes. The novel and performance iso-thermal EG-FET system is capable of real-time detecting the LAMP DNA amplification, and thus has significant potential for future biomedical applications.