Loop mediated isothermal amplification synergism with lateral flow assay based point-of- care biosensor design for diagnosis of viral infections

A biosensor is an analytical device that is used to detect and measure bio-chemical interactions and reactions by generating optical, electrical or visually detectable colorimetric signals proportional to the concentration of an analyte within a short period of time (usually some minutes). The analytical methods involving nucleic acid amplification, mainly the Polymerase Chain Reaction (PCR), the gold-standard detection method for nucleic acid targets, is still not accessible in a point-of-care format. This thesis work describes a biosensor platform combining nucleic acid amplification via isothermal method called as loop mediated isothermal amplification (LAMP) and the systematic labelling of the LAMP amplicon by using different labelling techniques, for the readout on a lateral-flow assay (LFA) platform. The chosen viral examples for LAMP based nucleic acid detection are SARS-CoV-2 virus and Dengue virus. LAMP method is a technique to isothermally amplify the nucleic acids within 30 minutes amplification time. In contrast to PCR, LAMP does not require thermal cycling or complex instrumentation. This work shows the reduced amplification times required for all nucleic acid types, e.g., for DNA amplification in 10 minutes, RNA amplification in 15 minutes and also with crude samples (swab samples) in 20 minutes. Various properties of LAMP have been explored during the investigation of this thesis e.g., LAMP amplification with untreated samples (no-purification of nucleic acids) and LAMP amplicon readout via LFA, but there are some major challenges that have been tried to overcome as well, e.g., reduction of false positive signals that can occur due to primer dimerization, using a DNA-unwinding enzyme called Helicase. This work further describes different labelling strategies used to label the LAMP amplified product for the compatibility in readout on the LFA i.e., in one-step (incorporation of two labels, FITC and biotin simultaneously during amplification), in two-steps (FITC incorporation during amplification and biotin via targeted DNA probes) or in a modified one-step approach (by immobilizing DNA probes on the LFA an reading out FITC labelled amplicon). The potential of the modified one-step-approach was demonstrated by the differential diagnostics of the two different viral infections via LFA. Lastly, the LAMP-LFA synergism has been semi-quantitated using an in-vitro-diagnostic (IVD) device for the purpose of improving the reach of the biosensor in POC-setting and improve surveillance.