title: Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device.
abstract: We demonstrate sensitive and multiplexed detection of DNA sequences through a surface enhanced resonance Raman spectroscopy (SERRS)-based competitive displacement assay in an integrated microsystem. The use of the competitive displacement scheme, in which the target DNA sequence displaces a Raman-labeled reporter sequence that has lower affinity for the immobilized probe, enables detection of unlabeled target DNA sequences with a simple single-step procedure. In our implementation, the displacement reaction occurs in a microporous packed column of silica beads prefunctionalized with probe-reporter pairs. The use of a functionalized packed-bead column in a microfluidic channel provides two major advantages: (i) immobilization surface chemistry can be performed as a batch process instead of on a chip-by-chip basis, and (ii) the microporous network eliminates the diffusion limitations of a typical biological assay, which increases the sensitivity. Packed silica beads are also leveraged to improve the SERRS detection of the Raman-labeled reporter. Following displacement, the reporter adsorbs onto aggregated silver nanoparticles in a microfluidic mixer; the nanoparticle-reporter conjugates are then trapped and concentrated in the silica bead matrix, which leads to a significant increase in plasmonic nanoparticles and adsorbed Raman reporters within the detection volume as compared to an open microfluidic channel. The experimental results reported here demonstrate detection down to 100 pM of the target DNA sequence, and the experiments are shown to be specific, repeatable, and quantitative. Furthermore, we illustrate the advantage of using SERRS by demonstrating multiplexed detection. The sensitivity of the assay, combined with the advantages of multiplexed detection and single-step operation with unlabeled target sequences makes this method attractive for practical applications. Importantly, while we illustrate DNA sequence detection, the SERRS-based competitive displacement assay is applicable to detection of a variety of biological macromolecules, including proteins and proteolytic enzymes.
authors.affiliation: Fischell Department of Bioengineering, University of Maryland , College Park, Maryland 20742, United States.
authors.forename: Soroush H
authors.initials: SH
authors.lastname: Yazdi
authors.forename: Kristen L
authors.initials: KL
authors.lastname: Giles
authors.forename: Ian M
authors.initials: IM
authors.lastname: White
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008856:Microscopy, Fluorescence; D013059:Spectrum Analysis, Raman
chemical_list: D017931:DNA Primers; D004247:DNA