Point of Care Biosensor System for Nucleic Acid Based Ion-Mediated Real-Time Analysis of Hepatitis C Virus

Abstract

Hepatitis C virus (HCV) is one the major threat to public health being leading cause of chronic liver disease with 92 to 149 million people affected worldwide. A rapid and accurate diagnosis is the key to prevent viral transmission and management of disease progression. A point-of-care (POC) system was designed for rapid detection of HCV infection called as STALLION (Single Tube Analysis using LAMP, LED and ION-sensing). Viral RNA was initially isolated using conventional spin-column and magnetic bead based methods, however, for POC setup, two modified methods were described. The first method uses a millifluidic channel with nucleic-acid-binding-silica-membrane made with the help of a novel approach called grid sandwich method (GSM). This method uses plastic canvas grid to make fluidic channels and then canvas grid is sandwiched between two sheets of polymeth-methacrylate (PMMA) sheets and fixed with the help of suitable adhesive. The second method employs transparent soft tube with reagents separated by surface tension valves and isolation is performed by nucleic-acid-bindingmagnetic-beads. The later approach was used with the currently designed STALLION system for isolation of RNA. A custom detection probe was made to dip into PCR tube comprising an ion sensitive field effect transistor (ISFET), a micro-capillary based Ag/AgCl reference electrode and a temperature sensor. In-vitro replication of HCV genomic RNA was performed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detected in real-time by ISFET bio-sensing of released H+ ions. Heat incubation at 60°C was provided by a very low cost novel concept using dissipated heat from power LED. With this system, the HCV positive samples with 101, 102, 103 copies/ml were detected in as less as 30, 18 and 13 minutes respectively against no-template control (NTC). Detection limit was comparable to available methods such as nested PCR. No significant change was observed for HCV negative samples. The results can be viewed on associated LCD screen or alternatively can be transferred to computer or smart phone. STALLION system provides analysis in conventional PCR tube format avoiding any complex fluidics or instrumentation requirements. Such a system will be particularly useful for POC, rapid and reliable clinical diagnosis of HCV RNA with no prior expertise in limited resource settings especially for developing countries. Further, STALLION system was tested for detection Malarial parasite which is a DNA containing infectious agent. The testing was also performed using fluorescent dye and custom designed real-time detection setup. These experiments showed that STALLION system can be effectively used for the detection of any organism whose nucleic acid can be amplified with LAMP in POC settings making it suitable and convenient for testing especially in remote and under-developed areas. In conclusion, the STALLION setup provides a complete system to perform RT-LAMP (from RNA source) or LAMP (from DNA source), and easy scalability for performing other isothermal nucleic acid amplification tests using specific primer sets and standardization accordingly.