Single-Step FRET-Based Detection of Femtomoles DNA

Abstract

Sensitive detection of nucleic acids and identification of single nucleotide polymorphism (SNP) is crucial in diagnosis of genetic diseases. Many strategies have been developed for detection and analysis of DNA, including fluorescence, electrical, optical, and mechanical methods. Recent advances in fluorescence resonance energy transfer (FRET)-based sensing have provided a new avenue for sensitive and quantitative detection of various types of biomolecules in simple, rapid, and recyclable platforms. Here, we report single-step FRET-based DNA sensors designed to work via a toehold-mediated strand displacement (TMSD) process, leading to a distinct change in the FRET efficiency upon target binding. Using single-molecule FRET (smFRET), we show that these sensors can be regenerated in situ, and they allow detection of femtomoles DNA without the need for target amplification while still using a dramatically small sample size (fewer than three orders of magnitude compared to the typical sample size of bulk fluorescence). In addition, these single-molecule sensors exhibit a dynamic range of approximately two orders of magnitude. Using one of the sensors, we demonstrate that the single-base mismatch sequence can be discriminated from a fully matched DNA target, showing a high specificity of the method. These sensors with simple and recyclable design, sensitive detection of DNA, and the ability to discriminate single-base mismatch sequences may find applications in quantitative analysis of nucleic acid biomarkers.

FRET-Based Aptasensor for the Selective and Sensitive Detection of Lysozyme

“…Subsequently, 1 µM H1 strand along with lysozyme at different concentrations were prepared in the imaging buffer, injected, and incubated in the flow cell for 20 min before recording the movies. Movies were recorded using Single.exe software as described [44,49]. The Cy3 fluorophore was continuously excited with a 532 nm He-Ne laser, and the resulting fluorescence emissions of both the Cy3 and Cy5 fluorophores were concurrently recorded through green and red channels (512 × 256 pixels) using an EMCCD camera (iXon 897, Andor) with 100 ms time resolution.…”

“…Briefly, a 20 pM aptasensor solution was injected into the flow cell and incubated for less than a minute. The unbound molecules were washed away by flushing an imaging buffer containing an oxygen scavenging system (OSS), which served to curtail fluorophore blinking and photobleaching upon laser illumination [49,52]. The flow cell was then irradiated with a 532 nm laser to create an evanescent field, which excites the Cy3 fluorophores of surface-tethered molecules [49].…”

“…The unbound molecules were washed away by flushing an imaging buffer containing an oxygen scavenging system (OSS), which served to curtail fluorophore blinking and photobleaching upon laser illumination [49,52]. The flow cell was then irradiated with a 532 nm laser to create an evanescent field, which excites the Cy3 fluorophores of surface-tethered molecules [49]. The excited Cy3 fluorophores transfer energy to the Cy5 fluorophore present in the same molecule via dipole-dipole coupling, a process known as fluorescence resonance energy transfer (FRET) [35].…”

“…The aptasensor can be used for up to three weeks by storing at 4 • C or annealed fresh, as only a little sample is needed for single-molecule experiments. The stem between the internal bulge and the Cy5-labeled nucleotide was optimized to be 6 bp to allow full recycling of aptasensors for multiple rounds of detection [49].…”

Scanning single-molecule counting system for Eprobe with highly simple and effective approach

“…Ultrasensitive detection has also been realized by flow channels [19][20][21][22][23] and rotating containers [24]. To increase the sensitivity, the counting of high-brightness nanoparticles [25,26] and oligonucleotide arrays [27] constructed from the target nucleic acids have been reported. These methods can be used to detect molecules modified with multiple fluorophores or large particles, but they experience difficulty attaining sensitivity beyond the femtomolar level for a single fluorescent molecule.…”