A Graphene-Based Biosensing Platform Based on the Release of DNA Probes and Rolling Circle Amplification

Abstract

We report a versatile biosensing platform capable of achieving ultrasensitive detection of both small-molecule and macromolecular targets. The system features three components: reduced graphene oxide for its ability to adsorb single-stranded DNA molecules nonspecifically, DNA aptamers for their ability to bind reduced graphene oxide but undergo target-induced conformational changes that facilitate their release from the reduced graphene oxide surface, and rolling circle amplification (RCA) for its ability to amplify a primer-template recognition event into repetitive sequence units that can be easily detected. The key to the design is the tagging of a short primer to an aptamer sequence, which results in a small DNA probe that allows for both effective probe adsorption onto the reduced graphene oxide surface to mask the primer domain in the absence of the target, as well as efficient probe release in the presence of the target to make the primer available for template binding and RCA. We also made an observation that the circular template, which on its own does not cause a detectable level of probe release from the reduced graphene oxide, augments target-induced probe release. The synergistic release of DNA probes is interpreted to be a contributing factor for the high detection sensitivity. The broad utility of the platform is illustrated though engineering three different sensors that are capable of achieving ultrasensitive detection of a protein target, a DNA sequence and a small-molecule analyte. We envision that the approach described herein will find useful applications in the biological, medical, and environmental fields.

Aptamer-based nanobiosensors

“…As a result, 250-fold enhancement of sensitivity was achieved by this method. As a similar strategy, rolling circle amplification was applied in GO-based aptasensors (Liu et al, 2014b). In this method, target induced displacement of aptamers from GO was amplified and detected.…”

Nucleic acid quantification using nicking–displacement, rolling circle amplification and bio-bar-code mediated triple-amplification

“…Recently, multiple amplification approaches have developed to further improve the sensitivity [16,17]. Among the methods developed for the sensitive detection of DNAs, the fluorescence [18], electrochemistry [19] and surfaceenhanced Raman scattering (SERS) [20] are the most widely used approach.…”

Amplified electrochemical detection of mecA gene in methicillin-resistant Staphylococcus aureus based on target recycling amplification and isothermal strand-displacement polymerization reaction

“…The released target DNA opens another hairpin probe to initiate next polymerization reaction, thereby resulting in target DNA recycling and achieving signal amplification. Compared with other signal amplification techniques such as PCR [27], exonuclease-based methods [28,29], and rolling circle amplification [30,31], ISDPR does not need repeated thermal cycling, a specific recognition site, and a specially designed circular template. Thus, ISDPR can be conveniently used to fabricate DNA biosensors.…”