Electronic readout of DNA amplification in nanoliter chambers. Strategies towards highly parallel semiconductor-based nucleic acid amplification testing.

Polymerase chain reaction (PCR) has been the most significant driver in the field of nucleic acid testing (NAT) since its invention. Popularized as an abbreviation by the Covid-19 pandemic, PCR-based methods are the gold standard in the field of diagnostics and research. Constantly maturing on the technical front, PCR has already achieved the ultimate goal where it can detect the presence of a single nucleic acid mole-cule in the reaction mixture. However, such technological capability proved less than optimal during the outbreak of the Covid-19 pandemic. In fact, it led to the revelation that the next generation of NAT needs solutions that are low-cost, high-throughput and decentralized. The last decade and a half have already seen trends in a similar direction, only to be vindicated and accelerated by the emergence of the Covid-19 pandemic. One of the major reasons for the high cost and centralized nature of NAT is the use of optical labels as a readout method. High material costs, requiring complex optics for readout and being prone to deterioration in ambient conditions have restricted optical-NAT methods to specialized laboratory set-tings. Steps to ameliorate these shortcomings have led to the development of label-free isothermal NAT meth-ods. These readout methods sense chemical species like H+ or pyrophosphates, which are formed as by-products of the biochemical reactions used to amplify nucleic acids. Sensing accumulation of H+ ions or change in the reaction pH is one of the most popular label-free methods as its readout can be carried out electrically on relatively inexpensive CMOS chips. The principle is widely used by Ion Torrent in their next-generation sequencing solution, where they use millions of ion-sensitive field effect transistors (ISFET), fabricated by CMOS technology to sense H+ by incorporation of a nucleotide. ISFET-based NAT solutions offer the unique advantage of compact size, high parallelism, integrated readout and low-cost per chip due to highly optimized manufacturing technology. Being a general method, readout based on H+ sensing can be adapted to any amplification method, thereby reducing the complexity of the assay. The extreme-ly high sensitivity of ISFETs, when combined with ultra-low volume partitioning microfluidics has the po-tential to offer low-cost and rapid NAT platforms that offer a wide range of assay choices, all on a centi-meter-sized footprint.However, to realize such solutions technological bottlenecks, specific to ISFETs need to be addressed. This thesis aims to address these challenges and lays down strategies for the realization of highly parallel semi-conductor NAT devices. To avoid negatively affecting the signal-to-noise ratio of ISFET and minimize evaporation losses in ultra-low volumes, two molecular assays that can be operated at low temperatures have been developed. This work demonstrates a new microfluidic packaging strategy that requires no post-CMOS processing and allows for large-scale microfluidic integration on CMOS chips. The thesis con-cludes with a demonstration of DNA amplification readout via ISFETs in a nanoliter-sized volume, which can be scaled up in density to realize high-through non-optical digital-PCR-like solutions.