Nanopore-Based Fingerprint Immunoassay Based on Rolling Circle Amplification and DNA Fragmentation

Abstract

In recent years, nanopore-based sequencers have become robust tools with unique advantages for genomics applications. However, progress toward applying nanopores as highly sensitive, quantitative diagnostic tools has been impeded by several challenges. One major limitation is the insufficient sensitivity of nanopores in detecting disease biomarkers, which are typically present at pM or lower concentrations in biological fluids, while a second limitation is the general absence of unique nanopore signals for different analytes. To bridge this gap, we have developed a strategy for nanopore-based biomarker detection that utilizes immunocapture, isothermal rolling circle amplification, and sequence-specific fragmentation of the product to release multiple DNA reporter molecules for nanopore detection. These DNA fragment reporters produce sets of nanopore signals that form distinctive fingerprints, or clusters. This fingerprint signature therefore allows the identification and quantification of biomarker analytes. As a proof of concept, we quantify human epididymis protein 4 (HE4) at low pM levels in a few hours. Future improvement of this method by integration with a nanopore array and microfluidics-based chemistry can further reduce the limit of detection, allow multiplexed biomarker detection, and further reduce the footprint and cost of existing laboratory and point-of-care devices.

Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses

“…Although all experiments in this study involve micromolar concentrations of WDR5, the detection threshold using the resistive-pulse technique and nanopores can routinely reach nanomolar ,,− and even picomolar levels of proteins. On one hand, the detection threshold can be improved through amplification of the capture rate of proteins via electrostatic interactions (e.g., lowering the salt concentration), driving force (e.g., increasing the transmembrane potential), or local electroosmotic pressure (e.g., enriching the sample buffer with osmolytes).…”

Single-Molecule RNA Sizing Enables Quantitative Analysis of Alternative Transcription Termination

“…As analytes pass through the pore, driven by electrophoretic force, ions are depleted, causing ionic current blockage associated with the analyte's volume, conformation, or size 22 . The technique enables single-molecule detection and has facilitated the study of a wide range of biological species, including DNA 23,24 , proteins 19,25,26 antibodies 19,27 , RNAs [28][29][30][31] , ribosomes 32 , and viruses 33,34 . Recently, DNA-based labelling of RNA has been performed, allowing for RNA structural mapping with nanopores 35 .…”

Template-controllable rolling circle amplification for dual protein sensitive analysis

“…Therefore, RCA technology can be designed for the analysis of various protein biomarkers, providing more accurate analysis solutions for disease diagnosis. 8–10…”

“…Therefore, RCA technology can be designed for the analysis of various protein biomarkers, providing more accurate analysis solutions for disease diagnosis. [8][9][10] Protein detection methods involving RCA are often primercontrolled. Specifically, by fixing the number of circular templates, protein targets and primers can be designed in one-to-one or oneto-more patterns.…”