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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.

We report a method to detect proteins via suppression of rolling circle amplification (RCA) by using an appropriate aptamer as the linear primer (denoted as an aptaprimer) to initiate RCA. In the absence of a protein target, the aptaprimer is free to initiate RCA, which can produce long DNA products that are detected via binding of a fluorescent intercalating dye. Introduction of a target causes the primer region within the aptamer to become unavailable for binding to the circular template, inhibiting RCA. Using SYBR Gold or QuantiFluor dyes as fluorescent probes to bind to the RCA reaction product, it is possible to produce a generic protein-modulated RCA assay system that does not require fluorophore- or biotin-modified DNA species, substantially reducing complexity and cost of reagents. Based on this modulation of RCA, we demonstrate the ability to produce both solution and paper-based assays for rapid and quantitative detection of proteins including platelet derived growth factor and thrombin.

We report a new method for biosensing based on the target-mediated resistance of DNA aptamers against 5'-exonuclease digestion, allowing them to act as primers for rolling circle amplification (RCA). A target-bound DNA strand containing an aptamer region on the 5'-end and a primer region on the 3'-end is protected from 5'-exonuclease digestion by RecJ exonuclease in a target-dependent manner. As the protected aptamer is at the 5'-end, the exposed primer on the 3'-end can participate in RCA in the presence of a circular template to generate a turn-on sensor. Without target, RecJ digests the primer and prevents RCA from occurring, allowing quantitative fluorescence detection of both thrombin, a protein, and ochratoxin A (OTA), a small molecule, at picomolar concentrations.

We report a generalizable strategy for biosensing that takes advantage of the resistance of DNA aptamers against nuclease digestion when bound with their targets, coupled with toehold mediated strand displacement (TMSD) and rolling circle amplification (RCA). A DNA aptamer containing a toehold extension at its 5'-end protects it from 3'-exonuclease digestion by phi29 DNA polymerase (phi29 DP) in a concentration-dependent manner. The protected aptamer can participate in RCA in the presence of a circular template that is designed to free the aptamer from its target via TMSD. The absence of the target leads to aptamer digestion, and thus no RCA product is produced, resulting in a turn-on sensor. Using two different DNA aptamers, we demonstrate rapid and quantitative real-time fluorescence detection of two human proteins: platelet-derived growth factor (PDGF) and thrombin. Sensitive detection of PDGF was also achieved in human serum and human plasma, demonstrating the selectivity of the assay.