Loop-Mediated Isothermal Amplification-Coupled Glass Nanopore Counting Toward Sensitive and Specific Nucleic Acid Testing

Abstract

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.

Rapid detection of novel coronavirus SARS-CoV-2 by RT-LAMP coupled solid-state nanopores

“…Afterward, the nanopore counting analysis was performed to examine the concentration of the resulting amplicons. This is based on the fact that the nanopore event rate has a linear relation with the analyte concentration in the diffusion-limited region ( Nouri et al, 2019 ; Tang et al, 2019 ). Fig.…”

“…Existing works on nanopores have demonstrated that nanopores can easily capture the analyte at the pM range ( Albrecht, 2019 ; Nouri et al, 2019 ). Our previous work has demonstrated a LAMP-coupled nanopore sensor for malaria nucleic acid test ( Tang et al, 2019 ). The integration and miniaturization potential of the label-free, electronics-based nanopore sensors could open a new avenue for enhancing the accessibility of the molecular testing at the point of care.…”

LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community

“…Imai et al used a MinION TM nanopore device to perform genome sequencing of LAMP amplicons to specifically identify five Plasmodium species [92]. Tang et al demonstrated qualitative and quantitative detection using a LAMP-coupled glass nanopore sensor that counts target LAMP amplicons without added probes or dyes [93]. Although direct counting of amplicons is not target-specific, sensing specific probe molecules can be easily adopted for the detection mechanism.…”

Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring

“…Unlike typical thermal cycling amplification, isothermal techniques are carried out at a constant temperature, which reduces the power needed and makes them more compatible for integration into miniaturised systems [4]. These techniques have provided biosensors with the relevant sensitivity and specificity for the detection of different analytes such as microalgae [6,7] viruses [8,9], fungi [10,11], protozoa [12,13] and bacteria, being the latter the focus of this review. Therefore, the objective of this work is to provide a comprehensive overview of the biosensors based on isothermal DNA amplification methods for bacterial detection with application in the fields of food safety and environmental monitoring, ultimately affecting human, animal and plant health.…”