High throughput single molecule detection for monitoring biochemical reactions

Abstract

The design, performance and application of a novel optical system for high throughput single molecule detection (SMD) configured in a continuous flow format using microfluidics is reported. The system consisted of a microfabricated polymer-based multi-channel fluidic network situated within the optical path of a laser source (λex = 660 nm) with photon transduction accomplished using an electron-multiplying charge coupled device (EMCCD) operated in a frame transfer mode that allowed tracking single molecules as they passed through a large field-of-view (FoV) illumination zone. The microfluidic device consisted of 30 microchannels possessing dimensions of 30 μm (width) × 20 μm (depth) with a 25 mm pitch. Individual molecules were electrokinetically driven through the fluidic network and excited within the wide-field illumination area with the resulting fluorescence collected via an objective and imaged onto the EMCCD camera. The detection system demonstrated sufficient sensitivity to detect single DNA molecules labeled with a fluorescent tag (AlexaFluor 660) identified through their characteristic emission wavelength and the burst of photons produced during their transit through the excitation volume. In its present configuration and fluidic architecture, the sample processing throughput was ∼4.02 × 105 molecules s−1, but could be increased dramatically through the use of narrower channels and a smaller pitch. The system was further evaluated using a single molecule-based fluorescence quenching assay for measuring the population differences between duplexed and single-stranded DNA molecules as a function of temperature for determining the duplex melting temperature, Tm.

Recent Advances in Microfluidic Detection Systems

“…The system demonstrated a sample throughput of 2208 molecules per second with a theoretical capacity of 1.7 × 10 7 molecules per second, although inefficiencies in the optical set-up made the detection of single fluorophores with this system impossible. The same group later refined this system to allow detection of singly labeled DNA molecules [6]. The principle refinements of this work included use of an electron-multiplying CCD operated in frame transfer mode and a wide field epi-illumination fluorescence system.…”

Recent advances in single‐molecule detection on micro‐ and nano‐fluidic devices

“…Parallel microchannels with multiple inlets and a single outlet were shown to create a multi-laminar flow system, and each laminar flow stream could be delivered to the desired location in the jointing channel by varying the flow velocity combinations for molecular patterning [65]. Okagbare et al demonstrated that as many as thirty parallel microchannels can be fabricated within the large field-of-view under widefield illumination for the simultaneous monitoring of single-molecule biochemical reactions [66].…”

Recent and near-future advances in nucleic acid-based diagnosis of stroke

“…For 30 mm wide microfluidic channels, the multiplexing power (the number of different mRNAs that can be detected in a single run) would be 30 [80].…”