Solid-state Nanopore CRISPR-assisted Diagnostic Systems Toward Digital Nucleic Acid Testing

Download or read book Solid-state Nanopore CRISPR-assisted Diagnostic Systems Toward Digital Nucleic Acid Testing written by Reza Nouri and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Whether caring for an individual patient with an infectious disease or responding to a worldwide pandemic, the accurate diagnosis of a pathogen is fundamental to quality care. While different techniques, such as enzyme-linked immunosorbent assay and cell culture, have been introduced for diagnostics, Nucleic Acid Test (NAT) is the most sensitive and specific method. In the majority of NAT systems, an amplification step is utilized for detecting the target RNA or DNA in a sample. In the conventional amplification-based NAT quantification method, the absolute concentration of target templates remains unknown until calibrated with standard samples. However, utilizing digital assays where samples are partitioned into numerous and separated reaction chambers provides a platform for calibration-free quantification of targets in a sample. Therefore, exploring digital diagnostic platforms is strongly needed to optimize clinical care and guide infection control to limit disease spread. This thesis mainly focuses on exploring the digital quantification of target nucleic acids using solid-state nanopores and CRISPR towards rapid, label-free nucleic acid testing. We studied the experiment-relevant parameters and their effects on molecular transport dynamics through the nanopore, which would help us to design an experimental setup with higher throughput and accuracy. We further developed a calibration-free model for concentration estimation to address the pore-to-pore variations issues of nanopores. Afterward, we combined the high specificity offered by the CRISPR Cas technology and the high sensitivity offered by the nanopore sensor towards an electronic sensing platform for sequence-specific HIV-1 and SARS-COV-2 detection. Furthermore, we developed a quantitative CRISPR sensing figure of merit and explored performance improvement strategies to improve CRISPR diagnostic systems. Based on our model, which was validated by 57 existing CRISPR systems, digitalization was the most promising amplification-free method for achieving comparable performances. Therefore, we finally developed a self-digitalization through an automated membrane-based partitioning technique to digitalize the CRISPR-Cas13 assay for amplification-free and absolute quantification of HIV-1 viral RNAs. The results showed that the samples spanning 4 orders of dynamic range between 100 aM to 1 pM could be quantified using our system. We also obtain the limit of detection 100 aM within 30 min of reaction. We envision that the nanopore-based and digital CRISPR platform provided in this work will provide accurate, sensitive, and specific diagnostic systems for different types of pathogens.