pH-dependent complexation of histamine H1 receptor antagonists and human serum albumin studied by UV resonance Raman spectroscopy.

UV resonance Raman spectroscopy was used to characterize the binding of three first-generation histamine H(1) receptor antagonists-tripelennamine (TRP), mepyramine (MEP), and brompheniramine (BPA)-to human serum albumin (HSA) at pH 7.2 and pH 9.0. Binding constants differ at these pH values, which can be ascribed to the different extent of protonation of the ethylamino side chain of the ligands. We have recently shown [Tardioli et al. J. Raman Spectrosc. 2011, 42, 1016-1024] that for the solution conformation of TRP and MEP the side chain plays an important role by allowing an internal hydrogen bond with the aminopyridine nitrogen in TRP and MEP. Results presented in this paper suggest that the existence of such molecular structures has serious biological significance on the binding affinity of those ligands to HSA. At pH 7.2, only the stretched conformers of protonated TRP and MEP bind in HSA binding site I. Using UV absorption data, we derived binding constants for the neutral and protonated forms of TRP to HSA. The neutral species seems to be conjugated to a positive group of the protein, affecting both the tryptophan W214 and some of the tyrosine (Y) vibrations. BPA, for which the structure with an intramolecular hydrogen bonded side chain is not possible, is H bound to the indole ring nitrogen of W214, of which the side chain rotates over a certain angle to accommodate the drug in site I. We propose that the protonated BPA is also bound in site I, where the Y150 residue stabilizes the presence of this compound in the binding pocket. No spectroscopic evidence was found for conformational changes of the protein affecting the spectroscopic properties of W and Y in this pH range.

Noninvasive detection of concealed explosives: depth profiling through opaque plastics by time-resolved Raman spectroscopy.

The detection of explosives concealed behind opaque, diffusely scattering materials is a challenge that requires noninvasive analytical techniques for identification without having to manipulate the package. In this context, this study focuses on the application of time-resolved Raman spectroscopy (TRRS) with a picosecond pulsed laser and an intensified charge-coupled device (ICCD) detector for the noninvasive identification of explosive materials through several millimeters of opaque polymers or plastic packaging materials. By means of a short (250 ps) gate which can be delayed several hundred picoseconds after the laser pulse, the ICCD detector allows for the temporal discrimination between photons from the surface of a sample and those from deeper layers. TRRS was applied for the detection of the two main isomers of dinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene as well as for various other components of explosive mixtures, including akardite II, diphenylamine, and ethyl centralite. Spectra were obtained through different diffuse scattering white polymer materials: polytetrafluoroethylene (PTFE), polyoxymethylene (POM), and polyethylene (PE). Common packaging materials of various thicknesses were also selected, including polystyrene (PS) and polyvinyl chloride (PVC). With the demonstration of the ability to detect concealed, explosives-related compounds through an opaque first layer, this study may have important applications in the security and forensic fields.

Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography.

Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-ß-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of L-MTX and its enantiomeric impurity D-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10(-7) M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of L-MTX. Furthermore, L-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples.

Sensitized phosphorescence as detection method for the enantioseparation of bupropion by capillary electrophoresis.

A new CE detection method was developed for the chiral drug bupropion (a second-generation antidepressant), based on phosphorescence both in the direct and in the sensitized mode using pulsed laser excitation at 266 nm. Electrokinetic chromatography using 5 mM sulfated-α-CD as chiral selector in 25 mM phosphate buffer at pH 3 allowed the separation of bupropion enantiomers with a high chiral resolution (Rs>3). In the sensitized phosphorescence detection mode, excitation energy is transferred from the analyte to an acceptor (1-bromo-4-napthhalenesulfonic acid or biacetyl) followed by time-resolved phosphorescence detection under deoxygenated buffer conditions. Using 2 × 10(-4) M biacetyl as the acceptor an LOD of 2 × 10(-7) M was obtained for each enantiomer, about 40 times better than in the direct mode. Under these separation conditions, no significantly different phosphorescence lifetimes (measured on-line) were obtained for the two bupropion enantiomers. The suitability of the method was demonstrated with the quantification of bupropion in a pharmaceutical formulation and its determination in a spiked urine sample.

Sensitized enantioselective laser-induced phosphorescence detection in chiral capillary electrophoresis.

The sensitivity of enantioselective cyclodextrin-induced room-temperature phosphorescence detection of camphorquinone (CQ) is enhanced using sensitization via a donor with a high extinction coefficient. The enantiomeric distinction is based on the different phosphorescence lifetimes of (+)-CQ and (-)-CQ after their complexation with α-cyclodextrin (α-CD). The collisional Dexter energy transfer from the selected donor 2,6-naphthalenedisulfonic acid (2,6-NS) to the acceptor CQ is still very efficient despite the inclusion of the acceptor into CD. For coupling to the chiral separation of (±)-CQ in cyclodextrin-based electrokinetic chromatography, the donor was added to the deoxygenated background electrolyte that consisted of 20 mM α-CD, 10 mM carboxymethyl-ß-CD, and 25 mM borate buffer at pH 9.0. Time-resolved batch studies on sensitized phosphorescence show a significant enantioselectivity for (+)- and (-)-CQ in the presence of both α-CD and CM-ß-CD although the lifetime difference is somewhat reduced with respect to direct excitation. The enantiomers were distinguished after their separation using an online time-resolved detection system. Excitation was performed at 266 nm with a pulsed, small-sized, quadrupled Nd:YAG laser. With 1 × 10(-5) M 2,6-NS, limits of detection of 4.1 × 10(-8) M and 5.2 × 10(-8) M were found for (+)-CQ and (-)-CQ, respectively. The online measured lifetimes were 238 ± 8 µs for (+)-CQ and 126 ± 10 µs for (-)-CQ. The method was used to determine the concentration of (±)-CQ leaching from a cured dental resin into water. The extracts contained 4.7 ± 0.1 × 10(-7) M of both (+)-CQ and (-)-CQ.

Time-resolved spatially offset Raman spectroscopy for depth analysis of diffusely scattering layers.

The objective of this study is to use time-resolved (TR) Raman spectroscopy, spatially offset Raman spectroscopy (SORS), and a combination of these approaches to obtain high quality Raman spectra from materials hidden underneath an opaque layer. Both TR Raman and SORS are advanced techniques that allow for an increased relative selectivity of photons from deeper layers within a sample. Time-resolved detection reduces fluorescence background, and the selectivity for the second layer is improved. By combining this with spatially offset excitation we additionally increased selectivity for deeper layers. Test samples were opaque white polymer blocks of several mm thicknesses. Excitation was carried out with a frequency-doubled Ti:sapphire laser at 460 nm, 3 ps pulse width and 76 MHz repetition rate. Detection was either with a continuous-wave CCD camera or in time-resolved mode using an intensified CCD camera with a 250 ps gate width. The Raman photons were collected in backscatter mode, with or without lateral offset. By measuring the delay of the Raman signal from the second layer (polyethylene terephthalate/PET/Arnite), the net photon migration speeds through Teflon, polythene, Delrin and Nylon were determined. Raman spectra could be obtained from a second layer of PET through Teflon layers up to 7 mm of thickness. The ability to obtain chemical information through layers of diffusely scattering materials has powerful potential for biomedical applications.

Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays.

One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC(50) values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture.

Picosecond Raman spectroscopy with a fast intensified CCD camera for depth analysis of diffusely scattering media.

A spectroscopic depth profiling approach is demonstrated for layers of non-transparent, diffusely scattering materials. The technique is based on the temporal discrimination between Raman photons emitted from the surface and Raman photons originating from a deeper layer. Excitation was carried out with a frequency-doubled, 3 ps Ti:sapphire laser system (398 nm; 76 MHz repetition rate). Time-resolved detection was carried out with an intensified CCD camera that can be gated with a 250 ps gate width. The performance of the system was assessed using 1 mm and 2 mm pathlength cuvettes with powdered PMMA and trans-stilbene (TS) crystals, respectively, or solid white polymer blocks: Arnite (polyethylene terephthalate), Delrin (polyoxymethylene), polythene (polyethylene) and Teflon (polytetrafluoroethylene). These samples were pressed together in different configurations and Raman photons were collected in backscatter mode in order to study the time difference in such media corresponding with several mm of extra net photon migration distance. We also studied the lateral contrast between two different second layers. The results demonstrate that by means of a picosecond laser system and the time discrimination of a gated intensified CCD camera, molecular spectroscopic information can be obtained through a turbid surface layer. In the case of the PMMA/TS two-layer system, time-resolved detection with a 400 ps delay improved the relative intensity of the Raman bands of the second layer with a factor of 124 in comparison with the spectrum recorded with a 100 ps delay (which is more selective for the first layer) and with a factor of 14 in comparison with a non-gated setup. Possible applications will be discussed, as well as advantages/disadvantages over other Raman techniques for diffusely scattering media.

Phosphorescence for sensitive enantioselective detection in chiral capillary electrophoresis.

Enantioselective phosphorescence lifetime detection was combined with chiral cyclodextrin-based electrokinetic chromatography for the analysis of camphorquinone (CQ). A time-gated detection system based on a pulsed light-emitting diode for excitation at 465 nm was developed for the online lifetime determination. The background electrolyte for the chiral separation consisted of 20 mM alpha-cyclodextrin (alpha-CD), 10 mM carboxymethyl-beta-CD, and 25 mM borate buffer at pH 9.0. The separation of (+)-CQ and (-)-CQ is caused by a difference in association constants of these enantiomers with alpha-CD. Under the separation conditions, different phosphorescence lifetimes were obtained for (+)-CQ and (-)-CQ (tau = 384 +/- 8 and 143 +/- 5 micros, respectively), which could be used to distinguish the enantiomers. This selectivity in detection is based on a difference in protection of the enantiomers against phosphorescence quenching after their complexation with alpha-CD. Concentration detection limits were 2 x 10(-7) and 1 x 10(-6) M for (+)-CQ and (-)-CQ, respectively. After correction for the lifetime shortening by triplet-triplet annihilation at higher CQ concentrations, a linear dynamic range was obtained from the detection limit up to 2 mM. The system was used to determine the enantiomeric impurity levels of commercial samples of (+)-CQ and (-)-CQ; 0.2% and 0.1%, respectively.

Fluorescence rejection in resonance Raman spectroscopy using a picosecond-gated intensified charge-coupled device camera.

A Raman instrument was assembled and tested that rejects typically 98-99% of background fluorescence. Use is made of short (picosecond) laser pulses and time-gated detection in order to record the Raman signals during the pulse while blocking most of the fluorescence. Our approach uses an ultrafast-gated intensified charge-coupled device (ICCD) camera as a simple and straightforward alternative to ps Kerr gating. The fluorescence rejection efficiency depends mainly on the fluorescence lifetime and on the closing speed of the gate (which is about 80 ps in our setup). A formula to calculate this rejection factor is presented. The gated intensifier can be operated at 80 MHz, so high repetition rates and low pulse energies can be used, thus minimizing photodegradation. For excitation we use a frequency-tripled or -doubled Ti : sapphire laser with a pulse width of 3 ps; it should not be shorter in view of the required spectral resolution. Other critical aspects tested include intensifier efficiency as a function of gate width, uniformity of the gate pulse across the spectrum, and spectral resolution in comparison with ungated detection. The total instrumental resolution is 7 cm(-1) in the blue and 15 cm(-1) in the ultraviolet (UV) region. The setup allows one to use resonance Raman spectroscopy (RRS) for extra sensitivity and selectivity, even in the case of strong background fluorescence. Excitation wavelengths in the visible or UV range no longer have to be avoided. The effectiveness of this setup is demonstrated on a test system: pyrene in the presence of toluene fluorescence (lambda(exc) = 257 nm). Furthermore, good time-gated RRS spectra are shown for a strongly fluorescent flavoprotein (lambda(exc) = 405 nm). Advantages and disadvantages of this approach for RRS are discussed.

Hexamerization of the bacteriophage T4 capsid protein gp23 and its W13V mutant studied by time-resolved tryptophan fluorescence.

The bacteriophage T4 capsid protein gp23 was studied using time-resolved and steady-state fluorescence of the intrinsic protein fluorophore tryptophan. In-vitro gp23 consists mostly of monomers at low temperature but forms hexamers at room temperature. To extend our knowledge of the structure and hexamerization characteristics of gp23, the temperature-dependent fluorescence properties of a tryptophan mutant (W13V) were compared to those of wild-type gp23. The W13V mutation is located in the N-terminal part of the protein, which is cleaved off after prohead formation in the live bacteriophage. Results show that W13 plays a role in the hexamerization process but is not needed to stabilize the hexamer once it is formed. Furthermore, besides the monomer-to-hexamer temperature transition (15-23 degrees C and 12-43 degrees C for wild-type and W13V gp23, respectively), we were able to observe denaturation of the N-terminus in hexameric wild-type gp23 around 40 degrees C. In addition, with the aid of a recently published homology model of gp23, the lifetimes obtained from time-resolved fluorescence measurements could tentatively be assigned to specific tryptophan residues.

High resolution slice imaging of a molecular speed distribution.

High resolution slice imaging experiments are reported measuring the speed distribution of molecular fragments, recoiling at a most probable speed v(mp), with a full-width-half-maximum (FWHM) speed resolution near the permille level: FWHM(v)/v(mp) = 1.9 x 10(-3). We implemented a high resolution single-particle slice imaging detector and used a two-colour resonance-enhanced multi-photon ionisation (REMPI) scheme to reduce broadening of the speed distribution due to the electron kick. The results on the measurement of the CD(3) speed distribution from photolysis of CD(3)I show that it is possible to image the three-dimensional speed distribution at a resolution down to FWHM(v) = 6.7 m s(-1), when the fragment has an absolute speed of v(mp) = 3473 m s(-1). The new experiments demonstrate the potential of slice imaging to measure with high resolution the three-dimensional speed distribution of a cloud of molecules.

Time-resolved fluorescence of the bacteriophage T4 capsid protein gp23.

The time-resolved fluorescence properties of the bacteriophage T4 capsid protein gp23 are investigated. The structural characteristics of this protein are largely unknown and can be probed by recording time-resolved and decay-associated fluorescence spectra and intensity decay curves using a 200 ps-gated intensified CCD-camera. Spectral and decay data are recorded simultaneously, which makes data acquisition fast compared to time-correlated single-photon counting. A red-shift of the emission maximum within the first nanosecond of decay is observed, which can be explained by the different decay-associated spectra of fluorescence lifetimes of the protein in combination with dipolar relaxation. In addition, iodide quenching experiments are performed, to study the degree of exposure of the various tryptophan residues. A model for the origin of the observed lifetimes of 0.032 +/- 0.003, 0.39 +/- 0.06, 2.1 +/- 0.1 and 6.8 +/- 0.8 ns is presented: the 32 ps lifetime can be assigned to the emission of a buried tryptophan residue, the 0.4 and 2.1 ns lifetimes to two partly buried residues, and the 6.8 ns lifetime to a single tryptophan outside the bulk of the folded gp23.

A flow injection kinase assay system based on time-resolved fluorescence resonance energy-transfer detection in the millisecond range.

A flow injection analysis (FIA) system for biochemical assays using time-resolved fluorescence resonance energy transfer (TR-FRET) in the millisecond time scale was developed. As a model system, we studied a kinase assay, measuring the phosphorylation of poly(GT)-biotin (substrate) by a receptor tyrosine kinase (epidermal growth factor receptor). A streptavidin labeled with XL665 (SA-XL665)-the acceptor-was coupled to the biotin moiety, and an antiphosphotyrosine antibody labeled with europium cryptate (Ab-EuK)-the donor-was coupled to the phosphorylated tyrosine group(s). Long-lived FRET can only occur if the substrate is successfully phosphorylated. For the time-resolved detection of such long-lived luminescence phenomena in a flow system, the repetition rate of the excitation source plays a crucial role. Good results were obtained for a small-sized commercially available quadrupled Nd:YAG laser emitting at 266 nm with a repetition rate of 7.8 kHz and a pulse width of 0.3 ns. The long-lived emissions of the donor at 625 nm and that of the acceptor at 665 nm were monitored simultaneously with two photomultipliers, using a delay time of 50 micros and a gate time of 75 micros to exclude background fluorescence interferences. In the FIA experiments, the Ab-EuK concentration was 6 nM and the substrate concentration and SA-XL665 concentrations were 7 nM. By monitoring the intensity changes at 625 and 665 nm, the inhibition of tyrosine kinase by tyrphostin AG1478 was studied and an IC(50) value of 5.1 +/- 0.4 nM obtained.

Fast-gated intensified charge-coupled device camera to record time-resolved fluorescence spectra of tryptophan.

The possibilities of a 200 ps gated intensified charge-coupled device (CCD) camera to record time-resolved fluorescence were explored using the fluorescing amino acid tryptophan and its derivative Nacetyl-tryptophan amide (NATA) as model compounds. The results were compared to complementary data from time-correlated single-photon counting (TCSPC) experiments. If a spectral resolution of 1-2 nm is desired, the fast-gated intensified CCD (ICCD) camera is the method of choice. For a 10(-5) M tryptophan solution, time-resolved emission spectra and intensity decays (measured over 12 ns at 25 ps resolution) could be obtained in typically 10 minutes, giving the well-known lifetimes of 0.5 and 3 ns. In addition, a longer lifetime of 7 ns was found at the red edge of the spectrum. The very short gate time of the ICCD camera allowed us to observe a shift in the emission maximum of tryptophan even within the first nanosecond of decay of the fluorescence emission. As expected from the tryptophan rotamer model, such a shift is not observed in NATA. Using amplitudes obtained by global analysis, decay-associated spectra of these lifetimes were constructed.