Gerhard M. Kresbach

Planar waveguides for ultra-high sensitivity of the analysis of nucleic acids

Abstract In the first part of this paper, the need for analytical techniques capable of highly parallel and sensitive nucleic acid analysis, with the capability of achieving very low limits of detection (LODs) and of resolving small differences in concentration, is described. Whereas the requirement for performing simultaneously multi-analyte detection is solved by the approach of nucleic acid microarrays, requirements on sensitivity can often not be satisfied by classical detection technologies. Inherent limitations of conventional fluorescence excitation and detection schemes are identified, and the implementation of planar waveguides as analytical platforms for nucleic acid microarrays, with fluorescence excitation in the evanescent field associated with the guided excitation light, is proposed. The relevant parameters for an optimization of sensitivity are discussed. In the second part of this paper, the specific formats of our planar waveguide platforms, which are compatible with established industrial standard formats allowing for integration into industrial high throughput environments, are presented, as well as the dedicated optical system for fluorescence excitation and detection that we developed. In a direct comparison with a state-of-the-art scanner, it is demonstrated that the implementation of genomic microarrays on planar waveguide platforms allows for unprecedented, direct detection of low-abundant genes in limited amounts of sample. Otherwise, when using conventional fluorescence excitation and detection configurations, the detection of such low amounts of nucleic acids requires massive sample preparation and signal or target amplification steps.

Trace detection of modified dna bases via moving-belt liquid chromatography—mass spectrometry using electrophoric derivatization and negative chemica

Electrophoric derivatives of 5-methylcytosine and 5-hydroxymethyluracil nucleobases are determined using high-performance liquid chromatography-mass spectrometry coupled via a moving-belt interface. Standards as well as samples derived from DNA are analysed. As little as 9.9 pg (signal-to-noise ratio 5) and 180 fg (signal-to-noise ratio 10) of the respective nucleobases are detected in the electron-capture negative chemical ionization mode, and linear responses are observed over a moderate dynamic range. In a comparison study, liquid chromatography-electron-capture negative chemical ionization mass spectrometry is found to have a sensitivity comparable to gas chromatography-electron-capture negative chemical ionization mass spectrometry for 5-hydroxymethyluracil. A detection limit of 60 fg (signal-to-noise ratio 5) by gas chromatography-mass spectrometry is only three-fold better than the amount detected by liquid chromatography-mass spectrometry using the same mass spectrometer.

Direct electrically heated spray device for a moving belt liquid chromatography-mass spectrometry interface

Abstract A direct electrically heated capillary spray device for the deposition of liquid chromatographic eluents onto a moving belt interface for liquid chromatographymass spectrometry (LC—MS) has been constructed. This system shows significant advantages over hot propellant gas driven or cartridge heated sprayers, especially when eluents with high water content and high flow-rates are used. Various parameters have been examined to achieve optimum conditions with respect to capillary wall temperature, peak variance and analyte yield during deposition. Reversed-phase high-performance liquid chromatographic separations with standard bore columns (4.6 mm I.D.) demonstrate the enhanced possibilities of the moving belt interface for on-line high-performance LC—MS.

Ester and related derivatives of ring N-pentafluorobenzylated 5-hydroxymethyluracil: hydrolytic stability, mass spectral properties, and trace detection by gas chromatography-electron-capture detection, gas chromatography-electron-capture negative ion mass spectrometry, and moving-belt liquid chromatography-electron-capture negative ion mass spectrometry

One consequence of radiation damage to DNA is the conversion of thymine to 5-hydroxymethyluracil (HMU). In order to sensitively detect this DNA adduct by gas chromatography (GC) or high-performance liquid chromatography (HPLC) with electron-capture detection techniques, it is necessary to derivatize it. This study was designed to select an optimum ester derivative of the aliphatic hydroxyl group on HMU. N1, N3-Bis(pentafluorobenzyl)-HMU was formed as a parent derivative, and from this a series of esters. Also O-pentafluorobenzyl and O-tetrafluorobenzyl ether derivatives were prepared. Of the esters the pivalyl derivative was the best choice because it formed easily, was relatively stable to aqueous hydrolysis (t 1/2 = 9.8 days at pH 11.5, 24 degrees C) and gave a response at fmol levels by GC and LC comparable to that of the ethers. Unanticipated was a good response as well for the parent derivative, a free hydroxyl compound, by GC and LC at this level. The work also demonstrates a high performance by LC-electron-capture negative ion mass spectrometry with a belt interface for the trace detection of derivatives of this type.

Apparatus for analytical scale hydrogenation reactions at elevated temperatures and pressures

Abstract An inexpensive, analytical-scale hydrogenation apparatus is constructed. Nearly all parts are commercially avaialble. The reaction takes place in a glass vial of ≤10 ml size with magnetic stirring up to medium temperatures (≤ 140°C) and pressures (≤ 250 p.s.i. of hydrogen). The vai is housed in a modified PTFE Digestion Vessel.