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Steffen Jockusch

Publications and source records attributed to Steffen Jockusch.

At least 19 recordsLinked to original sources
PubMed2006

Combinatorial fluorescence energy transfer molecular beacons for probing nucleic acid sequences.

We report the design, synthesis, and characterization of molecular beacons (MB) consisting of three distinct fluorophores, 6-carboxyfluorescein (Fam), N,N,N',N'-tetramethyl-6-carboxyrhodamine (Tam), and Cyanine-5 (Cy5). The primary light absorber/energy donor (Fam) is located on one terminus of the MB, whereas the primary energy acceptor/secondary donor (Tam) and secondary acceptor (Cy5) are located at the other terminus of the MB. In the absence of target DNA or RNA, the MB exists in the stem-closed form. Excitation of Fam initiates an energy transfer cascade from Fam to Tam and further to Cy5 generating unique fluorescence signatures defined as the ratio of the emission from each of the three fluorophores. This energy transfer cascade was investigated in detail by steady-state and time-resolved fluorescence spectroscopy, as well as fluorescence depolarization studies. In the presence of the complementary target DNA, the MB opened efficiently and hybridized with the target separating Fam and Tam by a large distance, so that energy transfer from Fam to Tam was blocked in the stem-open form. This opening of the MB generates a "bar code" fluorescence signature, which is different from the signature of the stem-closed MB. The fluorescence signature of this combinatorial fluorescence energy transfer MB can be tuned by variation of the spacer length between the individual fluorophores.

Base Sequence↗
PubMed2006

Phosphorylation state-responsive lanthanide peptide conjugates: a luminescence switch based on reversible complex reorganization.

[reaction: see text] A luminogenic probe for peptide dephosphorylation has been developed. It consists of a serine-/tyrosine-containing peptide modified on the N-terminus with a tryptophan residue and a DTPA chelate capable of binding Tb(3+). We propose a mechanistic model for the luminescence enhancement based on the interconversion of monomeric and dimeric lanthanide species, which is affected by the phosphorylation state of the serine or tyrosine residue. The optical switch reports effectively on phosphatase-catalyzed dephosphorylation in vitro.

Alkaline Phosphatase↗
PubMed2006

Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy.

We report here the design, synthesis and application of pyrene binary oligonucleotide probes for selective detection of cellular mRNA. The detection strategy is based on the formation of a fluorescent excimer when two pyrene groups are brought into close proximity upon hybridization of the probes with the target mRNA. The pyrene excimer has a long fluorescence lifetime (>40 ns) compared with that of cellular extracts (approximately 7 ns), allowing selective detection of the excimer using time-resolved emission spectra (TRES). Optimized probes were used to target a specific region of sensorin mRNA yielding a strong excimer emission peak at 485 nm in the presence of the target and no excimer emission in the absence of the target in buffer solution. While direct fluorescence measurement of neuronal extracts showed a strong fluorescent background, obscuring the detection of the excimer signal, time-resolved emission measurements indicated that the emission decay of the cellular extracts is approximately 8 times faster than that of the pyrene excimer probes. Thus, using TRES of the pyrene probes, we are able to selectively detect mRNA in the presence of cellular extracts, demonstrating the potential for application of pyrene excimer probes for imaging mRNAs in cellular environments that have background fluorescence.

Animals↗
PubMed2006

Oximetry of oxygen supersaturated solutions using nitroxides as EPR probe.

The broadening of the three hyperfine EPR nitroxide lines in oxygen supersaturated solutions was examined. The solutions were supersaturated with oxygen either by thermal decomposition of 1,4-peroxy-1,4-dimethylnaphthalene or by pressurizing molecular oxygen above the sample solution. The linear relationship between the Lorentzian component of the line width and the O(2) concentration was proven to hold even when the hyperfine splitting is unresolved. In this region of line broadening, the requirement for spectral simulation is discussed.

Journal Article↗
PubMed2006

Molecular beacons with intrinsically fluorescent nucleotides.

We report the design, synthesis and characterization of a novel molecular beacon (MB-FB) which uses the fluorescent bases (FB) 2-aminopurine (AP) and pyrrolo-dC (P-dC) as fluorophores. Because the quantum yield of these FB depend on hybridization with complementary target, the fluorescent properties of MB-FB were tuned by placing the FB site specifically within the MB such that hybridization with complementary sequence switches from single strand to double strand for AP and vice versa for P-dC. The MB-FB produces a ratiometric fluorescence increase (the fluorescence emission of P-dC over that of AP in the presence and absence of complementary sequence) of 8.5 when excited at 310 nm, the maximum absorption of AP. This ratiometric fluorescence is increased to 14 by further optimizing excitation (325 nm). The fluorescence lifetime is also affected by the addition of target, producing a change in the long-lived component from 6.5 to 8.7 ns (Exc. 310 nm, Em. 450 nm). Thermal denaturation profiles monitored at 450 nm (P-dC emission) show a cooperative denaturation of the MB-FB with a melting temperature of 53 degrees C. The thermal denaturation profile of MB-FB hybridized with its target shows a marked fluorescence reduction at 53 degrees C, consistent with a transition from double stranded helix to random coil DNA.

2-Aminopurine↗
PubMed2006

Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences.

We report the design, synthesis, and characterization of a molecular beacon (MB) consisting of two fluorescent dyes (Alexa 488 and RedX) for DNA and RNA analysis. In the absence of the target DNA or RNA the MB is in its stem-closed form and shows efficient energy transfer from the donor (Alexa) to the acceptor (RedX), generating mostly fluorescence from RedX. In the presence of the complementary target DNA the MB opened efficiently, hybridizes with the target DNA, and energy transfer is blocked in the stem-open form. This attachment to the target generates a fluorescence signature, which is clearly distinguishable from the fluorescence signature of the stem-closed form, allowing for ratiometric analysis of the fluorescence signal. In addition to steady-state fluorescence analysis, time resolved fluorescence (ps time range) and fluorescence depolarization studies were performed. We show that fluorescence lifetime and fluorescence depolarization measurements are useful analytical tools to optimize the MB design.

Base Sequence↗
PubMed2005

Light-switching excimer probes for rapid protein monitoring in complex biological fluids.

Quantitative protein bioanalysis in complex biological fluids presents considerable challenges in biological studies and disease diagnosis. The major obstacles are the background signals from both the probe and the biological fluids where the proteins reside. We have molecularly engineered light-switching excimer aptamer probes for rapid and sensitive detection of a biomarker protein, platelet-derived growth factor (PDGF). Labeled with one pyrene at each end, the aptamer switches its fluorescence emission from approximately 400 nm (pyrene monomer) to 485 nm (pyrene excimer) upon PDGF binding. This fluorescence wavelength change from monomer to excimer emission is a result of aptamer conformation rearrangement induced by target binding. The excimer probe is able to effectively detect picomolar PDGF in homogeneous solutions. Because the excimer has a much longer fluorescence lifetime (approximately 40 ns) than that of the background (approximately 5 ns), time-resolved measurements were used to eliminate the biological background. We thus were able to detect PDGF in a cell sample quantitatively without any sample pretreatment. This molecular engineering strategy can be used to develop other aptamer probes for protein monitoring. Combined with lifetime-based measurements and molecular engineering, light-switching excimer aptamer probes hold great potential in protein analysis for biomedical studies.

Body Fluids↗
PubMed2005

Chemically induced dynamic electron polarization generated through the interaction between singlet molecular oxygen and nitroxide radicals.

An absorptive chemically induced dynamic electron polarization (CIDEP) was generated by the quenching of singlet oxygen by nitroxide radicals (TEMPO derivatives). The spin polarization decay time of the nitroxide (measured by time-resolved EPR) correlates with the lifetime of singlet oxygen (measured by singlet oxygen phosphorescence spectroscopy). In addition, a deuterium isotope effect on the spin polarization decay time was observed, a signature of singlet oxygen involvement. With use of isotope labeled nitroxides (15N, 14N), the relative spin polarization efficiencies of TEMPO, 4-oxo-TEMPO, and 4-hydroxy-TEMPO by singlet oxygen were determined. The relative spin polarization efficiencies (per quenching event) decrease in the order 4-hydroxy-TEMPO > TEMPO > 4-oxo-TEMPO, whereas an opposite trend was observed for the total quenching rate constants of singlet oxygen by the nitroxides where the order is 4-hydroxy-TEMPO < TEMPO < 4-oxo-TEMPO.

Journal Article↗
PubMed2005

Conformational changes of pyrene-labeled polyelectrolytes with pH: effect of hydrophobic modifications.

Structural changes of pyrene-labeled and unlabeled poly(maleic acid/octyl vinyl ether) (PMAOVE) and poly(maleic acid/methyl vinyl ether) (PMAMVE) with changes in pH have been investigated in this study. The changes in the photophysical properties of pyrene are interpreted to investigate uncoiling or swelling of the polymeric chains with pH. The vibrational fine structure of the pyrene fluorescence (I(3)/I(1)) and the ratio between excimer and monomer fluorescence (I(e)/I(m)) of both pyrene-labeled and unlabeled PMAMVE and PMAOVE suggest that, at pH 4, the polymers are in the coiled form and PMAOVE forms hydrophobic nanodomains. An increase in pH ionizes a number of COOH groups on both PMAMVE and PMAOVE, which leads to the stretching or swelling of the polymers.

Hydrogen-Ion Concentration↗
PubMed2005

157 nm pellicles (thin films) for photolithography: mechanistic investigation of the VUV and UV-C photolysis of fluorocarbons.

The use of 157 nm as the next lower wavelength for photolithography for the production of semiconductors has created a need for transparent and radiation-durable polymers for use in soft pellicles, the polymer films which protect the chip from particle deposition. The most promising materials for pellicles are fluorinated polymers, but currently available fluorinated polymers undergo photodegradation and/or photodarkening upon long term exposure to 157 nm irradiation. To understand the mechanism of the photodegradation and photodarkening of fluorinated polymers, mechanistic studies on the photolysis of liquid model fluorocarbons, including perfluorobutylethyl ether and perfluoro-2H-3-oxa-heptane, were performed employing UV, NMR, FTIR, GC, and GC/MS analyses. All hydrogen-containing compounds showed decreased photostability compared to the fully perfluorinated compounds. Irradiation in the presence of atmospheric oxygen showed reduced photostability compared to deoxygenated samples. Photolysis of the samples was performed at 157, 172, 185, and 254 nm and showed only minor wavelength dependence. Mechanisms for photodegradation of the fluorocarbons are proposed, which involve Rydberg excited states. Time-dependent density functional theory has been used to predict the excitation spectra of model compounds.

Journal Article↗
PubMed2005

Stereoselective photooxidation of enecarbamates: reactivity of ozone vs singlet oxygen.

[reaction: see text]. Oxazolidinone-functionalized enecarbamates show contrasting behavior upon oxidation by singlet oxygen and by ozone. The observed stereoselectivity difference indicates that the oxidation with ozone is subject to classic steric effects, whereas the very high selectivity in the photooxidation with singlet oxygen is derived from vibrational deactivation.

Journal Article↗
PubMed2005

Investigation of the mobility of amphiphilic polymer--AOT reverse microemulsion systems using electron spin resonance.

Amphiphilic polymers can be used as tools to manipulate the behavior of reverse microemulsions. EPR spectroscopy employing the spin probe 5-doxyl stearic acid was used to study the adsorption of a comb type polymer (polymaleic anhydride octyl vinyl ether) and a diblock polymer (polybutadiene-ethylene oxide) onto reverse microemulsion droplets formed from Aerosol-OT/heptane/water. The findings indicate that the comb type polymer was adsorbed by the reverse microemulsion drops at low polymer concentrations causing a structural change of the micelle.

Journal Article↗
PubMed2005

Two-photon induced uncaging of a reactive intermediate. Multiphoton in situ detection of a potentially valuable label for biological applications.

[reaction: see text] Two-photon induced Wolff rearrangement of a terphenyl diazoketone 1 was achieved by using focused laser pulses of 532 nm from a Q-switched Nd:YAG laser. The nonfluorescent terphenyl diazoketone 1 was transformed into a fluorescent ester derivative 4, which can be detected in situ using the focused laser pulses at 532 nm. Laser power dependence studies show that the Wolff rearrangement is induced by two-photon absorption of the terphenyl diazoketone 1, but suggests that more than two photons of 532 nm are involved (a multiphoton process) in excitation of the ester derivative 4.

Diazomethane↗
PubMed2004

Reversible surface oxidation and efficient luminescence quenching in semiconductor single-wall carbon nanotubes.

We have investigated reversible single-wall carbon nanotube (SWNT) oxidation by quantitative analysis of the oxide-induced absorption bleaching and luminescence quenching at low pH. These data, in combination with DFT structure calculations, suggest that the nanotube oxide is a 1,4-endoperoxide. At low pH, the endoperoxide protonates to create a hydroperoxide carbocation, introducing a hole in the SWNT valence band. Nanotube luminescence is extremely sensitive to quenching by hole-doping, while the absorption is relatively robust.

Journal Article↗