Konfokale Mehrfarben Einzelmolekülspektroskopie in Mikrokanälen zur Charakterisierung biologischer Proben
In this work confocal single-molecule fluorescence detection is extended to twocolor detection in microfluidic channels. The so-called two-color coincidence analysis can be applied to detect and to analyze molecular binding events, as demonstrated in this thesis. The great advantage of this method in comparison to the widely used FCS-method is its sensitivity which lies in the sub-pM regime, as successfully demonstrated in this thesis. I performed measurements with diagnostic relevance which show, due to the sensitivity, the potential adaptability to early-stage detection of viral or bacterial infections. The first part of this work shows by a number of microfluidic experiments that on the one hand, one of the basic equations, the Hagen-Poiseuille equation, holds and on the other hand it demonstrates how to describe quantitatively the increase in sensitivity. With the aid of this model it it possible to approximate the width of the microchannel with an error of about 10% and to calculate flow velocities as a function of applied pressure. Making use of this microstructure enables to achieve a (at least) fourfold increase in Signal-to-Noise (SNR). In the next part of this work a novel statistical method, the Correlation-Matrix Method (CMM), is introduced, which enables to judge the qualitiy of a twocolor coincidence measurement. Thus a measure for the eciency of molecular interaction of two (or more) species can be calculated. The characteristic parameter, the so-called Gamma-Norm, l-Gamma, yields a value of two in case of a perfect coincidence sample. The value of this Euclidian norm is smaller than or equal to one for pure statistical coincidences (for which the probability is around 7%). Values between two and one (practically between two and 1:4) indicate a mixed sample of pure coincidences and single emitters. To my knowledge in this work i report for the first time how to use the intrinsic autouorescence of M280-Beads to calibrate coincidence measurements. Experimentally the fraction of coincident events could be estimated to be approx. 85%. The corresponding Gamma-Norm is l-Gamma approx. 1.9. Both in this work developed methods, the sensitivity increase by using microfluidics and its description and the CMM, are used to quantitatively detect single DNA target sequences of the organism Streptococcus Pneumoniae with the help of two spectrally separated and efficiently quenched (QYrel = 4%) short hairpin-shaped oligonucleotides (Molecular Beacons). These smart-probes yield dissociation constants of kd = 2x 10E-10M. In this
thesis a so far unmached and unpublished aquisition time of 30 s is reported to detect a concentration of the target sequence of 10E-13M. The corresponding coincidence rate is approx 0.23Hz. The linear behavior (double-logarithmic scale) of the coincidence rate as a function of target concentration indicates that one can expect a coincidence rate of 0:04Hz at a target concentration of 1 fM. This rate corresponds to approx. 1.2 coincident events in 30 seconds. In comparison with a measurement in an open volume we achieve a 100-fold improvement in sensitivity in one-tenth of the measurement time.
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Universität Bielefeld
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