Functional nucleic acids (FNAs), including DNA aptamers and DNAzymes, are finding increasing use as molecular recognition elements for point-of-care (POC) assays and sensors. An ongoing challenge in ...the development of FNA-based POC sensors is the ability to achieve detection of low levels of analyte without compromising assay time and ease of use. Rolling circle amplification (RCA) is a leading nucleic acid (NA) isothermal amplification method which can be coupled with FNAs for the ultrasensitive detection of non-NA targets. Herein we examine the key considerations required when designing FNA-coupled biosensors utilizing RCA. Specifically, we describe methods for using FNAs as inputs to regulate RCA, various modes of RCA amplification, and methods to detect the output of the RCA reaction, along with how these can be combined to allow detection of non-NA targets. Recent progress on development of portable optical and electrochemical POC devices that incorporate RCA is then described, followed by a summary of key challenges and opportunities in the field.
Functional nucleic acids regulate rolling circle amplification to produce multiple detection outputs suitable for the development of point-of-care diagnostic devices.
The deposition of micro- and nanolitre volumes is crucial in sessile droplet microfluidic systems. Several techniques exist for the fabrication of surfaces with patterned wettabilities; however, many ...of these fabrication techniques are time-consuming and complex. Here, we present a device that allows for deposition of multiple droplets within seconds followed by directed evaporative preconcentration. Hydrophobic-coated glass substrates are fashioned with hydrophilic surface energy traps (SETs) using picosecond laser micromachining. SETs can capture nanolitre volumed droplets of both aqueous and organic liquids through discontinuous dewetting. Modification of the machined hydrophilic shape yields a passive mechanism that preconcentrates analyte through evaporation. Studies and optimizations of SET parameters/dimensions (laser power, laser passes, ring/patch diameter) and their effect on patch wettability and degree of preconcentration are presented. As a demonstration, the optimized platform was used to improve the colourimetric detection of cadmium-containing aqueous samples. The optimized SET design demonstrated an 18-fold increase in colourimetric sensitivity compared to conventional milled SETs, suggesting the design would be well-suited for trace analysis. The evaporative preconcentration was also applied to MALDI-IMS analysis of peptides where it resulted in improved uniformity of deposited analyte and decreased analysis times. The rapid droplet deposition and directed evaporative approach can be tailored to provide different concentration factors and is compatible with a wide variety of detection schemes.
We present a microfluidic platform that rapidly deposits many samples and preconcentrates them, making it suitable for a wide range of high-throughput detection schemes.
RNA-cleaving DNAzymes (RCDs) are a class of functional nucleic acids that can bind various targets ranging in size from small molecules to large proteins, which results in activation of cleavage ...activity. The activation of RCDs results in the cleavage of a ribonucleotide site in an otherwise all-DNA substrate, leading to two cleavage fragments. In this work, a previously identified DNAzyme that binds to a protein biomarker endogenous to Helicobacter pylori (J99) crude extracellular matrix was evaluated for coupling to an isothermal amplification method termed rolling circle amplification (RCA) as a way to improve the originally reported detection limit. Three RCD constructs were designed with the goal of generating a cleavage fragment that could act as a primer to initiate RCA. The first method used the original HP DNAzyme, which liberated a short cleavage fragment that could be used as a primer. However, the primer fragment was rapidly digested by the bacterial matrix, preventing RCA. A second method evaluated use of a circularized substrate and separate RCD to generate a primer, however this system was not capable of generating a cleavage fragment. A final method redesigned the original RCD to move the substrate region from the 3’ to the 5’ end of the RCD, causing the longer RCD-containing fragment to be the primer for RCA. In this case, target-triggered cleavage was observed and the resulting primer was sufficiently resistant to digestion to allow its use as a primer for RCA. Preliminary characterization of the rearranged RCD showed that it retained selectivity similar to the original RCD, but that the cleavage rate was slower. In addition, the RCA based reaction, while successful, did not produce improved detection sensitivity relative to unamplified assays. Methods to further improve RCA performance are discussed for future work.
Thesis
Master of Science (MSc)
