Single-molecule detection and identification of multiple species by multiparameter fluorescence detection
2006 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 78, no 6, 2039-2050 p.Article in journal (Refereed) Published
Two general strategies are introduced to identify and quantify single molecules in dilute solutions by employing a spectroscopic method for data registration and specific burst analysis, denoted multiparameter fluorescence detection (MFD). MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time and allows for selection of specific events for subsequent analysis. Using the multiple fluorescence dimensions, we demonstrate a dye labeling scheme of oligonucleotides, by which it is possible to identify and separate 16 different compounds in the mixture via their characteristic pattern by MFD. Such identification procedures and multiplex assays with single-molecule sensitivity may have a great impact on screening of species and events that do not lend themselves so easily to amplification, such as disease-specific proteins and their interactions.
Place, publisher, year, edition, pages
2006. Vol. 78, no 6, 2039-2050 p.
resonance energy-transfer, intensity distribution analysis, alternating-laser excitation, correlation spectroscopy, detection technology, 2-photon excitation, aqueous-solution, nucleic-acids, photon, microscopy
IdentifiersURN: urn:nbn:se:kth:diva-15550DOI: 10.1021/ac0522759ISI: 000236307700041ScopusID: 2-s2.0-33645241242OAI: oai:DiVA.org:kth-15550DiVA: diva2:333591
QC 201005252010-08-052010-08-05Bibliographically approved