An integrated optics microfluidic device for detecting single DNA molecules

Abstract

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.

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

“…An integrated optics microfluidic device was introduced to avoid such expensive optical parts [42]: a plano-aspheric refractive lens for improved fluorescence excitation and a solid parabolic reflective mirror for high-efficiency fluorescence collection. Emory et al presented for the first time the ability to track single molecules simultaneously in multiple microfluidic channels by employing a charge-coupled device (CCD) camera operated in time-delayed integration mode as a means of increasing throughput [43].…”

Design and microfabrication of a miniature fiber optic probe with integrated lenses and mirrors for Raman and fluorescence measurements

“…Miniaturized Raman probes have been made by assembly of compact optical components (e.g., lenses and filters) and fiber optics [8, 9]. Such probes are commercially available, but they are typically expensive because of costs associated with material handling and precise optical alignment at small scale.…”

Fabrication and Performance of a Photonic-Microfluidic Integrated Device

“…This leads to the manipulation of volumes in the nano to atto liter range [1] and allows the manipulation and detection of single molecules [2]. By exploiting non-conventional phenomena that occurs in such small volumes of liquids, new and intriguing applications have surfaced [3].…”

“…A fully integrated device is termed a Lab-on-a-chip (LOC) or Micro-totalanaylsis-system (μTAS) [15,16], and these devices have the ability to revolutionize medical and laboratorial procedures. Currently, microchip devices can be found in practical applications from electrophoresis [4,5,17], single DNA detection [18,19], proteomics [20], immunoassays [21], and online lactate monitoring [22].…”