Population Health Research Institute, the Canadian Institutes of Health Research, Heart and Stroke Foundation of Ontario, Canadian Institutes of Health Research Strategy for Patient Oriented Research through the Ontario SPOR Support Unit, the Ontario Ministry of Health and Long-Term Care, pharmaceutical companies (with major contributions from AstraZeneca [Canada], Sanofi Aventis [France and Canada], Boehringer Ingelheim [Germany amd Canada], Servier, and GlaxoSmithKline), Novartis and King Pharma, and national or local organisations in participating countries.

Full funding sources listed at the end of the paper (see Acknowledgments).

Nucleic acid detection methods are crucial for many fields such as pathogen detection and genotyping. Solid-state nanopore sensors represent a promising platform for nucleic acid detection due to its unique single molecule sensitivity and label-free electronic sensing. Here, we demonstrated the use of the glass nanopore for highly sensitive quantification of single-stranded circular DNAs (reporters), which could be degraded under the trans-cleavage activity of the target-specific CRISPR-Cas12a. We developed and optimized the Cas12a assay for HIV-1 analysis. We validated the concept of the solid-state CRISPR-Cas12a-assisted nanopores (SCAN) to specifically detect the HIV-1 DNAs. We showed that the glass nanopore sensor is effective in monitoring the cleavage activity of the target DNA-activated Cas12a. We developed a model to predict the total experimental time needed for making a statistically confident positive/negative call in a qualitative test. The SCAN concept combines the much-needed specificity and sensitivity into a single platform, and we anticipate that the SCAN would provide a compact, rapid, and low-cost method for nucleic acid detection at the point of care.

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Label-free nanopore sensors have emerged as a new generation technology of DNA sequencing and have been widely used for single molecule analysis. Since the first α-hemolysin biological nanopore, various types of nanopores made of different materials have been under extensive development. Noise represents a common challenge among all types of nanopore sensors. The nanopore noise can be decomposed into four components in the frequency domain (1/f noise, white noise, dielectric noise, and amplifier noise). In this work, we reviewed and summarized the physical models, origins, and reduction methods for each of these noise components. For the first time, we quantitatively benchmarked the root mean square (RMS) noise levels for different types of nanopores, demonstrating a clear material-dependent RMS noise. We anticipate this review article will enhance the understanding of nanopore sensor noises and provide an informative tutorial for developing future nanopore sensors with a high signal-to-noise ratio.

Solid-state nanopores have shown great promise and achieved tremendous success in label-free single-molecule analysis. However, there are three common challenges in solid-state nanopore sensors, including the nanopore size variations from batch to batch that makes the interpretation of the sensing results difficult, the incorporation of sensor specificity, and the impractical analysis time at low analyte concentration due to diffusion-limited mass transport. Here, we demonstrate a novel loop-mediated isothermal amplification (LAMP)-coupled glass nanopore counting strategy that could effectively address these challenges. By using the glass nanopore in the counting mode (versus the sizing mode), the device fabrication challenge is considerably eased since it allows a certain degree of pore size variations and no surface functionalization is needed. The specific molecule replication effectively breaks the diffusion-limited mass transport thanks to the exponential growth of the target molecules. We show the LAMP-coupled glass nanopore counting has the potential to be used in a qualitative test as well as in a quantitative nucleic acid test. This approach lends itself to most amplification strategies as long as the target template is specifically replicated in numbers. The highly sensitive and specific sensing strategy would open a new avenue for solid-state nanopore sensors toward a new form of compact, rapid, low-cost nucleic acid testing at the point of care.

The outbreak of the SARS-CoV-2 caused the disease COVID-19 to spread globally. Specific and sensitive detection of SARS-CoV-2 facilitates early intervention and prevents the disease from spreading. Here, we present a solid-state CRISPR-Cas12a-assisted nanopore (SCAN) sensing strategy for the specific detection of SARS-CoV-2. We introduced a nanopore-sized counting method to measure the cleavage ratio of reporters, which is used as a criterion for positive/negative classification. A kinetic cleavage model was developed and validated to predict the reporter size distributions. The model revealed the trade-offs between sensitivity, turnaround time, and false-positive rate of the SARS-CoV-2 SCAN. With preamplification and a 30 min CRISPR Cas12a assay, we achieved excellent specificity against other common human coronaviruses and a limit of detection of 13.5 copies/μL (22.5 aM) of viral RNA at a confidence level of 95%. These results suggested that the SCAN could provide a rapid, sensitive, and specific analysis of SARS-CoV-2.

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