Two-photon fluorescence coincidence analysis: Rapid measurements of enzyme kinetics

Dual-color fluorescence cross-correlation analysis is a powerful tool for probing interactions of different fluorescently labeled molecules in aqueous solution. The concept is the selective observation of coordinated spontaneous fluctuations in two separate detection channels that unambiguously reflect the existence of physical or chemical linkages among the different fluorescent species. It has previously been shown that the evaluation of cross-correlation amplitudes, i.e., coincidence factors, is sufficient to extract essential information about the kinetics of formation or cleavage of chemical or physical bonds. Confocal fluorescence coincidence analysis (CFCA) (Winkler et al., Proc. Natl. Acad. Sci. U.S.A. 96:13751378, 1999) emphasizes short analysis times and simplified data evaluation and is thus particularly useful for screening applications or measurements on live cells where small illumination doses need to be applied. The recent use of two-photon fluorescence excitation has simplified dual- or multicolor measurements by enabling the simultaneous excitation of largely different dye molecules by a single infra-red laser line (Heinze et al., Proc. Natl. Acad. Sci. U.S.A. 97:10377- 10382, 2000). It is demonstrated here that a combination of CFCA with two-photon excitation allows for minimization of analysis times for multicomponent systems down to some hundreds of milliseconds, while preserving all known advantages of two- photon excitation. By introducing crucial measurement parameters, experimental limits for the reduction of sampling times are discussed for the special case of distinguishing positive from negative samples in an endonucleolytic cleavage assay

Two-photon fluorescence coincidence analysis: Rapid measurements of enzyme kinetics

Dual-color fluorescence cross-correlation analysis is a powerful tool for probing interactions of different fluorescently labeled molecules in aqueous solution. The concept is the selective observation of coordinated spontaneous fluctuations in two separate detection channels that unambiguously reflect the existence of physical or chemical linkages among the different fluorescent species. It has previously been shown that the evaluation of cross-correlation amplitudes, i.e., coincidence factors, is sufficient to extract essential information about the kinetics of formation or cleavage of chemical or physical bonds. Confocal fluorescence coincidence analysis (CFCA) (Winkler et al., Proc. Natl. Acad. Sci. U.S.A. 96:13751378, 1999) emphasizes short analysis times and simplified data evaluation and is thus particularly useful for screening applications or measurements on live cells where small illumination doses need to be applied. The recent use of two-photon fluorescence excitation has simplified dual- or multicolor measurements by enabling the simultaneous excitation of largely different dye molecules by a single infra-red laser line (Heinze et al., Proc. Natl. Acad. Sci. U.S.A. 97:10377- 10382, 2000). It is demonstrated here that a combination of CFCA with two-photon excitation allows for minimization of analysis times for multicomponent systems down to some hundreds of milliseconds, while preserving all known advantages of two- photon excitation. By introducing crucial measurement parameters, experimental limits for the reduction of sampling times are discussed for the special case of distinguishing positive from negative samples in an endonucleolytic cleavage assay

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

Hollars CW, Puls J, Bakajin O, et al. Bio-assay based on single molecule fluorescence detection in microfluidic channels. Analytical and Bioanalytical Chemistry. 2006;385(8):1384-1388.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

Tunable Blinking Kinetics of Cy5 for Precise DNA Quantification and Single-Nucleotide Difference Detection

Fluorescence correlation spectroscopy (FCS) can resolve the intrinsic fast-blinking kinetics (FBKs) of fluorescent molecules that occur on the order of microseconds. These FBKs can be heavily influenced by the microenvironments in which the fluorescent molecules are contained. In this work, FCS is used to monitor the dynamics of fluorescence emission from Cy5 labeled on DNA probes. We found that the FBKs of Cy5 can be tuned by having more or less unpaired guanines (upG) and thymines (upT) around the Cy5 dye. The observed FBKs of Cy5 are found to predominantly originate from the isomerization and back-isomerization processes of Cy5, and Cy5-nucleobase interactions are shown to slow down these processes. These findings lead to a more precise quantification of DNA hybridization using FCS analysis, in which the FBKs play a major role rather than the diffusion kinetics. We further show that the alterations of the FBKs of Cy5 on probe hybridization can be used to differentiate DNA targets with single-nucleotide differences. This discrimination relies on the design of a probe-target-probe DNA three-way-junction, whose basepairing configuration can be altered as a consequence of a single-nucleotide substitution on the target. Reconfiguration of the three-way-junction alters the Cy5-upG or Cy5-upT interactions, therefore resulting in a measurable change in Cy5 FBKs. Detection of single-nucleotide variations within a sequence selected from the Kras gene is carried out to validate the concept of this new method

Recent advances in fluorescence cross-correlation spectroscopy

10.1007/s12013-007-0042-5Cell Biochemistry and Biophysics4911-13CBBI