Rupert Herrmann

Complement hybridization from solution to surface-attached probe-oligonucleotides observed by surface-plasmon-field-enhanced fluorescence spectroscopy

Abstract Surface-plasmon field-enhanced fluorescence spectroscopy is applied for the detection of hybridization reactions of fluorophore-labeled 15mer target oligonucleotides from solution to surface-attached probe DNA. The attachment of the catcher strand via a biotin-group at the 5′ end of an additional 15mer of thymines used as spacers, strongly binding to a monolayer of streptavidin at the sensor surface ensures a separation of the chromophores beyond 2 Forster radii, thus preventing any sifnificant loss of fluorescence signal due to energy transfer to the (acceptor states of the) metal (quenching). The high sensitivity thus obtainable is used to quantify the kinetics of binding (hybridization) and dissociation of different oligonucleotides exhibiting full complementarity to the catcher probes or having one or two mismatches in the base sequence, respectively. It is shown that the hybridization process follows a simple Langmuir model with a single mismatch reducing the equilibrium constant by two orders of magnitude, and a second mismatch causing another three orders in reduction.

Sample preparation for liquid chromatography-tandem mass spectrometry using functionalized ferromagnetic micro-particles

OBJECTIVES To study the applicability of functionalized ferromagnetic micro-particles in the sample preparation for quantification of small molecules by LC-MS/MS. DESIGN AND METHODS A representative analyte (itraconazole) was extracted from plasma samples using small quantities of C18-modified ferromagnetic micro-particles. RESULTS 125 microg of functionalized micro-particles was sufficient to extract the target analyte from 10 microL of plasma with a recovery rate of approximately 100%; linearity (r>0.99) and reproducibility (inter-assay CV< or =7%) of quantitative results was found. CONCLUSIONS C18-functionalized ferromagnetic particles can be used to efficiently extract small molecule analytes from complex biological matrices for quantitative analyses using LC-MS/MS.

Preparation of plasma samples for chromatographic analyses using functionalized ferromagnetic micro-particles manipulated in a high pressure liquid system

OBJECTIVES To study if functionalized ferromagnetic micro-particles used for extraction procedures prior to chromatographic analyses can be handled within an HPLC system. METHOD We used C18-functionalized ferromagnetic micro-particles, manipulated within a high-performance liquid chromatography system for extraction of plasma samples, with a novel extractor device enabling reversible immobilization of the particles within HPLC tubing. A pilot analytical system for the quantification of itraconazole in plasma--as a representative small molecule analyte--by ultraviolet detection was configured and tested. RESULTS The system allowed linear and reproducible quantification of the representative target analyte. CONCLUSIONS Sample preparation of biological samples can be achieved by handling of functionalized ferromagnetic micro-particles which are handled within a high pressure liquid chromatography configuration; this offers interesting perspectives for the development of automated sample preparation devices.