Bio-assay based on single molecule fluorescence detection in microfluidic channels

A rapid bioassay is described based on the detection of colocalized fluorescent DNA probes bound to DNA targets in a pressure-driven solution flowing through a planar microfluidic channel. By employing total internal reflection excitation of the fluorescent probes and illumination of almost the entire flow channel, single fluorescent molecules can be efficiently detected leading to the rapid analysis of nearly the entire solution flowed through the device. Cross-correlation between images obtained from two spectrally distinct probes is used to determine the target concentration and efficiently reduces the number of false positives. The rapid analysis of DNA targets in the low pM range in less than a minute is demonstrated.

[1]  H Schindler,et al.  Detection of individual oligonucleotide pairing by single-molecule microscopy. , 1999, Analytical chemistry.

[2]  J. A. Hubbell,et al.  Poly(ethylene oxide)-graft-poly(L-lysine) copolymers to enhance the biocompatibility of poly(L-lysine)-alginate microcapsule membranes. , 1992, Biomaterials.

[3]  John P Nolan,et al.  A simple quenching method for fluorescence background reduction and its application to the direct, quantitative detection of specific mRNA. , 2003, Analytical chemistry.

[4]  M. Eigen,et al.  Dual-color fluorescence cross-correlation spectroscopy for monitoring the kinetics of enzyme-catalyzed reactions. , 2001, Methods.

[5]  W. Moerner,et al.  Illuminating single molecules in condensed matter. , 1999, Science.

[6]  D. Axelrod.  Cell-substrate contacts illuminated by total internal reflection fluorescence , 1981, The Journal of cell biology.

[7]  Wayne F. Patton,et al.  Detection technologies in proteome analysis. , 2002, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[8]  D. P. Fromm,et al.  Methods of single-molecule fluorescence spectroscopy and microscopy , 2003 .

[9]  S. Lane,et al.  Controlled non-classical photon emission from single conjugated polymer molecules , 2003 .

[10]  J. Fitch,et al.  Technology Challenges in Responding to Biological or Chemical Attacks in the Civilian Sector , 2003, Science.

[11]  Watt W Webb,et al.  Biological and chemical applications of fluorescence correlation spectroscopy: a review. , 2002, Biochemistry.

[12]  J. Hubbell,et al.  Electrochemical optical waveguide lightmode spectroscopy (EC-OWLS): a pilot study using evanescent-field optical sensing under voltage control to monitor polycationic polymer adsorption onto indium tin oxide (ITO)-coated waveguide chips. , 2003, Biotechnology and bioengineering.

[13]  Robert J. Chichester,et al.  Single Molecules Observed by Near-Field Scanning Optical Microscopy , 1993, Science.

[14]  M. Eigen,et al.  Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[15]  P M Goodwin,et al.  Single-molecule detection with total internal reflection excitation: comparing signal-to-background and total signals in different geometries. , 1999, Cytometry.

[16]  Shimon Weiss,et al.  Photon Arrival-Time Interval Distribution (PAID): A Novel Tool for Analyzing Molecular Interactions , 2004 .

[17]  J. Hubbell,et al.  Reduction of fibrous adhesion formation by a copolymer possessing an affinity for anionic surfaces. , 1998, Journal of biomedical materials research.

[18]  J. Shendure,et al.  Advanced sequencing technologies: methods and goals , 2004, Nature Reviews Genetics.

[19]  P. Schwille,et al.  Fluorescence correlation spectroscopy for the detection and study of single molecules in biology. , 2002, BioEssays : news and reviews in molecular, cellular and developmental biology.

[20]  W. Moerner,et al.  Optical detection and spectroscopy of single molecules in a solid. , 1989, Physical review letters.

[21]  J. Hubbell,et al.  Poly(l-lysine)-g-Poly(ethylene glycol) Layers on Metal Oxide Surfaces: Attachment Mechanism and Effects of Polymer Architecture on Resistance to Protein Adsorption† , 2000 .

[22]  Joanna S Albala,et al.  Comparison of multiplexed techniques for detection of bacterial and viral proteins. , 2004, Journal of proteome research.

[23]  H Schindler,et al.  Statistical analysis of single-molecule colocalization assays. , 2001, Analytical chemistry.

[24]  E. Yeung,et al.  High-throughput single-molecule spectroscopy in free solution. , 2000, Analytical chemistry.