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The solution of a Mannich reaction was electrosprayed to study their conversion ratio at various ES parameter. Implementing new methods demonstrated low influence of the gas phase and a high effect of the life time and the charge of the μ-droplets.

The visible-light photocatalytic E/Z isomerization of olefins can be mediated by a wide spectrum of triplet sensitizers (photocatalysts). However, the search for the most efficient photocatalysts through screenings in photo batch reactors is material and time consuming. Capillary and microchip flow reactors can accelerate this screening process. Combined with a fast analytical technique for isomer differentiation, these reactors can enable high-throughput analyses. Ion mobility (IM) spectrometry is a cost-effective technique that allows simple isomer separation and detection on the millisecond timescale. This work introduces a hyphenation method consisting of a microchip reactor and an infrared matrix-assisted laser desorption ionization (IR-MALDI) ion mobility spectrometer that has the potential for high-throughput analysis. The photocatalyzed E/Z isomerization of ethyl-3-(pyridine-3-yl)but-2-enoate (E-1) as a model substrate was chosen to demonstrate the capability of this device. Classic organic triplet sensitizers as well as Ru-, Ir-, and Cu-based complexes were tested as catalysts. The ionization efficiency of the Z-isomer is much higher at atmospheric pressure which is due to a higher proton affinity. In order to suppress proton transfer reactions by limiting the number of collisions, an IM spectrometer working at reduced pressure (max. 100 mbar) was employed. This design reduced charge transfer reactions and allowed the quantitative determination of the reaction yield in real time. Among 14 catalysts tested, four catalysts could be determined as efficient sensitizers for the E/Z isomerization of ethyl cinnamate derivative E-1. Conversion rates of up to 80% were achieved in irradiation time sequences of 10 up to 180 s. With respect to current studies found in the literature, this reduces the acquisition times from several hours to only a few minutes per scan.

Infrared matrix-assisted desorption and ionization (IR-MALDI) enables the transfer of sub-micron particles (sMP) directly from suspensions into the gas phase and their characterization with differential mobility (DM) analysis. A nanosecond laser pulse at 2940 nm induces a phase explosion of the aqueous phase, dispersing the sample into nano- and microdroplets. The particles are ejected from the aqueous phase and become charged. Using IR-MALDI on sMP of up to 500 nm in diameter made it possible to surpass the 100 nm size barrier often encountered when using nano-electrospray for ionizing supramolecular structures. Thus, the charge distribution produced by IR-MALDI could be characterized systematically in the 50-500 nm size range. Well-resolved signals for up to octuply charged particles were obtained in both polarities for different particle sizes, materials, and surface modifications spanning over four orders of magnitude in concentrations. The physicochemical characterization of the IR-MALDI process was done via a detailed analysis of the charge distribution of the emerging particles, qualitatively as well as quantitatively. The Wiedensohler charge distribution, which describes the evolution of particle charging events in the gas phase, and a Poisson-derived charge distribution, which describes the evolution of charging events in the liquid phase, were compared with one another with respect to how well they describe the experimental data. Although deviations were found in both models, the IR-MALDI charging process seems to resemble a Poisson-like charge distribution mechanism, rather than a bipolar gas phase charging one.

Acoustically levitated droplets have been suggested as compartmentalized, yet wall-less microreactors for high-throughput reaction optimization purposes. The absence of walls is envisioned to simplify up-scaling of the optimized reaction conditions found in the microliter volumes. A consequent pursuance of high-throughput chemistry calls for a fast, robust and sensitive analysis suited for online interrogation. For reaction optimization, targeted analysis with relatively low sensitivity suffices, while a fast, robust and automated sampling is paramount. To follow this approach, in this contribution, a direct coupling of levitated droplets to a homebuilt ion mobility spectrometer (IMS) is presented. The sampling, transfer to the gas phase, as well as the ionization are all performed by a single exposure of the sampling volume to the resonant output of a mid-IR laser. Once formed, the nascent spatially and temporally evolving analyte ion cloud needs to be guided out of the acoustically confined trap into the inlet of the ion mobility spectrometer. Since the IMS is operated at ambient pressure, no fluid dynamic along a pressure gradient can be employed. Instead, the transfer is achieved by the electrostatic potential gradient inside a dual ring electrode ion optics, guiding the analyte ion cloud into the first stage of the IMS linear drift tube accelerator. The design of the appropriate atmospheric pressure ion optics is based on the original vacuum ion optics design of Wiley and McLaren. The obtained experimental results nicely coincide with ion trajectory calculations based on a collisional model. Graphical Abstract.

Electrospray ionization-ion mobility spectrometry was employed for the determination of collision cross sections (CCS) of 25 synthetically produced peptides in the mass range between 540-3310 Da. The experimental measurement of the CCS is complemented by their calculation applying two different methods. One prediction method is the intrinsic size parameter (ISP) method developed by the Clemmer group. The second new method is based on the evaluation of molecular dynamics (MD) simulation trajectories as a whole, resulting in a single, averaged collision cross-section value for a given peptide in the gas phase. A high temperature MD simulation is run in order to scan through the whole conformational space. The lower temperature conformational distribution is obtained through thermodynamic reweighting. In the first part, various correlations, e.g. CCS vs. mass and inverse mobility vs. m/z correlations, are presented. Differences in CCS between peptides are also discussed in terms of their respective mass and m/z differences, as well as their respective structures. In the second part, measured and calculated CCS are compared. The agreement between the prediction results and the experimental values is in the same range for both calculation methods. While the calculation effort of the ISP method is much lower, the MD method comprises several tools providing deeper insights into the conformations of peptides. Advantages and limitations of both methods are discussed. Based on the separation of two pairs of linear and cyclic peptides of virtually the same mass, the influence of the structure on the cross sections is discussed. The shift in cross section differences and peak shape after transition from the linear to the cyclic peptide can be well understood by applying different MD tools, e.g. the root-mean-square deviation (RMSD) and the root mean square fluctuation (RMSF). (C) 2018 Elsevier B.V. All rights reserved.

The increasing development of antibiotic resistance in bacteria has been a major problem for years, both in human and veterinary medicine. Prophylactic measures, such as the use of vaccines, are of great importance in reducing the use of antibiotics in livestock. These vaccines are mainly produced based on formaldehyde inactivation. However, the latter damages the recognition elements of the bacterial proteins and thus could reduce the immune response in the animal. An alternative inactivation method developed in this work is based on gentle photodynamic inactivation using carbon nanodots (CNDs) at excitation wavelengths λ

Samples of agricultural soil were analyzed by LIBS and evaluated by multivariate methods. The multivariate models provided reliable predictions of total and plant-available nutrient contents and the soil parameters humus content, pH and texture.

A new ion mobility (IM) spectrometer, enabling mobility measurements in the pressure range between 5 and 500 mbar and in the reduced field strength range E/N of 5-90 Td, was developed and characterized. Reduced mobility (K-0) values were studied under low E/N (constant value) as well as high E/N (deviation from low field K-0) for a series of molecular ions in nitrogen. Infrared matrix-assisted laser desorption ionization (IR-MALDI) was used in two configurations: a source working at atmospheric pressure (AP) and, for the first time, an IR-MALDI source working with a liquid (aqueous) matrix at sub-ambient/reduced pressure (RP). The influence of RP on IR-MALDI was examined and new insights into the dispersion process were gained. This enabled the optimization of the IM spectrometer for best analytical performance. While ion desolvation is less efficient at RP, the transport of ions is more efficient, leading to intensity enhancement and an increased number of oligomer ions. When deciding between AP and RP IR-MALDI, a trade-off between intensity and resolving power has to be considered. Here, the low field mobility of peptide ions was first measured and compared with reference values from ESI-IM spectrometry (at AP) as well as collision cross sections obtained from molecular dynamics simulations. The second application was the determination of the reduced mobility of various substituted ammonium ions as a function of E/N in nitrogen. The mobility is constant up to a threshold at high E/N. Beyond this threshold, mobility increases were observed. This behavior can be explained by the loss of hydrated water molecules.

The applications of quantum dots (QDs) in two-photon (2P) excitation applications demand reliable data about their 2P absorption (2PA) cross sections (σ2PA). In the present study, σ2PA values have been determined for a series of commercial colloidal CdSe/ZnS QDs and CdSeTe/ZnS QDs in aqueous media. For the first time for these QDs, the σ2PA values have been determined over a wide spectral range, that is, between 720 and 900 nm, and are compared to the extinction coefficient (e) values obtained under one-photon (1P) excitation. Furthermore, we present a QD in combination with an organic dye in a biotin-streptavidin Forster resonance energy transfer bioassay under 1P and 2P excitation. The results for the bioassay under 2P excitation are compared to those obtained under 1P excitation. The results demonstrate that in the case of the 2P excitation, higher sensitivity can be achieved because of an improved signal-to-noise ratio.

The capability of electrospray ionization (ESI)-ion mobility (IM) spectrometry for reaction monitoring is assessed both as a stand-alone real-time technique and in combination with HPLC. A three-step chemical reaction, consisting of a Williamson ether synthesis followed by a hydrogenation and an N-alkylation step, is chosen for demonstration. Intermediates and products are determined with a drift time to mass-per-charge correlation. Addition of an HPLC column to the setup increases the separation power and allows the determination of further species. Monitoring of the intensities of the various species over the reaction time allows the detection of the end of reaction, determination of the rate-limiting step, observation of the system response in discontinuous processes, and optimization of the mass ratios of the starting materials. However, charge competition in ESI influences the quantitative detection of substances in the reaction mixture. Therefore, two different methods are investigated, which allow the quantification and investigation of reaction kinetics. The first method is based on the pre-separation of the compounds on an HPLC column and their subsequent individual detection in the ESI-IM spectrometer. The second method involves an extended calibration procedure, which considers charge competition effects and facilitates nearly real-time quantification.

In electrospray ionization (ESI)-ion mobility spectrometry, continuously generated ions must be desolvated in a first tube before short ion pulses are introduced into a second (drift) tube. Both tubes are separated by an ion-gate. The resolving power of the resulting drift time spectrum is strongly influenced by the design of the ion gate. In the case of the Bradbury-Nielsen gates typically used, an orthogonal field between oppositely charged, parallel wires blocks ions from entering the drift tube. However, the blocking field also distorts the entering ion cloud. One alternative, which eliminates these effects and therefore enables a potentially higher resolving power, is already known for spectrometers with small ionization volumes, where ions are formed between two electrodes and subsequently transferred into the drift tube by a high voltage pulse. Based on this setup, we introduce an alternative ion gate design for liquid samples, named field switching ion gate (FSIG). The continuous flow of ions generated by ESI is desolvated in the first tube and introduced into the space between two electrodes (repeller and transfer electrodes). A third (blocking) electrode prevents the movement of ions into the drift tube in the closed state. Ions are transferred during the open state by pulsing the voltages of the repeller and blocking electrodes. First results demonstrate an increase of the resolving power by 100% without intensity losses and further changes in the spectrometer setup. The parameters of the FSIG, such as electrode voltages and pulse width, are characterized allowing the optimization of the spectrometer’s resolving power.

The pressure dependence of sheath gas assisted electrospray ionization (ESI) was investigated based on two complementary experimental setups, namely an ESI-ion mobility (IM) spectrometer and an ESI capillary - Faraday plate setup housed in an optically accessible vacuum chamber. The ESI-IM spectrometer is capable of working in the pressure range between 300 and 1000 mbar. Another aim was the assessment of the analytical capabilities of a subambient pressure ESI-IM spectrometer. The pressure dependence of ESI was characterized by imaging the electrospray and recording current-voltage (I-U) curves. Qualitatively different behavior was observed in both setups. While the current rises continuously with the voltage in the capillary-plate setup, a sharp increase of the current was measured in the IM spectrometer above a pressure-dependent threshold voltage. The different character can be attributed to the detection of different species in both experiments. In the capillary-plate experiment, a multitude of charged species are detected while only desolvated ions attribute to the IM spectrometer signal. This finding demonstrates the utility of IM spectrometry for the characterization of ESI, since in contrast to the capillary-plate setup, the release of ions from the electrospray droplets can be observed. The I-U curves change significantly with pressure. An important result is the reduction of the maximum current with decreasing pressure. The connected loss of ionization efficiency can be compensated by a more efficient transfer of ions in the IM spectrometer at increased E/N. Thus, similar limits of detection could be obtained at 500 mbar and 1 bar.

The contamination of barley by molds on the field or in storage leads to the spoilage of grain and the production of mycotoxins, which causes major economic losses in malting facilities and breweries. Therefore, on‐site detection of hidden fungus contaminations in grain storages based on the detection of volatile marker compounds is of high interest. In this work, the volatile metabolites of 10 different fungus species are identified by gas chromatography (GC) combined with two complementary mass spectrometric methods, namely, electron impact (EI) and chemical ionization at atmospheric pressure (APCI)‐mass spectrometry (MS). The APCI source utilizes soft X‐radiation, which enables the selective protonation of the volatile metabolites largely without side reactions. Nearly 80 volatile or semivolatile compounds from different substance classes, namely, alcohols, aldehydes, ketones, carboxylic acids, esters, substituted aromatic compounds, alkenes, terpenes, oxidized terpenes, sesquiterpenes, and oxidized sesquiterpenes, could be identified. The profiles of volatile and semivolatile metabolites of the different fungus species are characteristic of them and allow their safe differentiation. The application of the same GC parameters and APCI source allows a simple method transfer from MS to ion mobility spectrometry (IMS), which permits on‐site analyses of grain stores. Characterization of IMS yields limits of detection very similar to those of APCI‐MS. Accordingly, more than 90% of the volatile metabolites found by APCI‐MS were also detected in IMS. In addition to different fungus genera, different species of one fungus genus could also be differentiated by GC‐IMS.

Contactless pressure monitoring based on Forster resonance energy transfer between donor/acceptor pairs immobilized within elastomers is demonstrated. The donor/acceptor energy transfer is employed by dispersing terbium(III) tris[(2-hydroxybenzoyl)-2-aminoethyl] amine complex (LLC, donor) and CdSe/ZnS quantum dots (QD655, acceptor) in styrene-ethylene/buthylene-styrene (SEBS) and poly(dimethylsiloxane) (PDMS). The continuous monitoring of QD luminescence showed a reversible intensity change as the pressure signal is alternated between two stable states indicating a pressure sensitivity of 6350 cps kPa(-1). Time-resolved measurements show the pressure impact on the FRET signal due to an increase of decay time (270 ms up to 420 ms) for the donor signal and parallel drop of decay time (170 mu s to 155 mu s) for the acceptor signal as the net pressure applied. The LLC/QD655 sensors enable a contactless readout as well as space resolved monitoring to enable miniaturization towards smaller integrated stretchable opto-electronics. Elastic FRET sensors can potentially lead to developing profitable analysis systems capable to outdo conventional wired electronic systems (inductive, capacitive, ultrasonic and photoelectric sensors) especially for point-of-care diagnostics, biological monitoring required for wearable electronics.

In many laser based ionization techniques with a subsequent drift time separation, the laser pulse generating the ions is considered as the start time to. Therefore, an accurate temporal definition of this event is crucial for the resolution of the experiments. In this contribution, the laser induced plume dynamics of liquids evaporating into atmospheric pressure are visualized for two distinctively different laser pulse widths, Delta t = 6 nanoseconds and Delta tau = 280 microseconds. For ns-pulses the expansion of the generated vapour against atmospheric pressure is found to lead to turbulences inside the gas phase. This results in spatial and temporal broadening of the nascent clouds. A more equilibrated expansion, without artificial smearing of the temporal resolution can, in contrast, be observed to follow mu s-pulse excitation. This leads to the counterintuitive finding that longer laser pulses results in an increased temporal vapour formation definition. To examine if this fume expansion also eventually results in a better definition of ion formation, the nascent vapour plumes were expanded into a linear drift tube ion mobility spectrometer (IMS). This time resolved detection of ion formation corroborates the temporal broadening caused by collisional impeding of the supersonic expansion at atmospheric pressure and the overall better defined ion formation by evaporation with long laser pulses. A direct comparison of the observed results strongly suggests the coexistence of two individual ion formation mechanisms that can be specifically addressed by the use of appropriate laser sources.

The combination of high-performance liquid chromatography and electrospray ionization ion mobility spectrometry facilitates the two-dimensional separation of complex mixtures in the retention and drift time plane. The ion mobility spectrometer presented here was optimized for flow rates customarily used in High-performance liquid chromatography between 100–1500 μL min−1. The characterization of the system with respect to such parameters as the peak capacity of each time dimension and of the 2-D spectrum was carried out based on a separation of a pesticide mixture containing 24 substances. While the total ion current chromatogram is coarsely resolved, exhibiting coelutions for a number of compounds, all substances can be separately detected in the 2-D plane due to the orthogonality of the separations in retention and drift dimensions. Another major advantage of the ion mobility detector is the identification of substances based on their characteristic mobilities. Electrospray ionization allows the detection of substances lacking a chromophore. As an example, the separation of a mixture of 18 amino acids is presented. A software built upon the free mass spectrometry package OpenMS was developed for processing the extensive 2-D data. The different processing steps are implemented as separate modules which can be arranged in a graphic workflow facilitating automated processing of data. This article is protected by copyright. All rights reserved

A promising replacement for the radioactive sources commonly encountered in ion mobility spectrometers is a miniaturized, energy-efficient photoionization source that produce the reactant ions via soft X-radiation (2.8 keV). In order to successfully apply the photoionization source, it is imperative to know the spectrum of reactant ions and the subsequent ionization reactions leading to the detection of analytes. To that end, an ionization chamber based on the photoionization source that reproduces the ionization processes in the ion mobility spectrometer and facilitates efficient transfer of the product ions into a mass spectrometer was developed. Photoionization of pure gasses and gas mixtures containing air, N-2, CO2 and N2O and the dopant CH2Cl2 is discussed. The main product ions of photoionization are identified and compared with the spectrum of reactant ions formed by radioactive and corona discharge sources on the basis of literature data. The results suggest that photoionization by soft X-radiation in the negative mode is more selective than the other sources. In air, adduct ions of O-2 - with H2O and CO2 were exclusively detected. Traces of CO2 impact the formation of adduct ions of O-2 - and Cl -(upon addition of dopant) and are capable of suppressing them almost completely at high CO2 concentrations. Additionally, the ionization products of four alkyl nitrates (ethylene glycol dinitrate, nitroglycerin, erythritol tetranitrate and pentaerythritol tetranitrate) formed by atmospheric pressure chemical ionization induced by X-ray photoionization in different gasses (air, N-2 and N2O) and dopants (CH2Cl2, C2H5Br and CH3I) are investigated. The experimental studies are complemented by density functional theory calculations of the most important adduct ions of the alkyl nitrates (M) used for their spectrometric identification. In addition to the adduct ions [M + NO3](-) and [M + Cl](-), adduct ions such as [M + N2O2](-), [M + Br](-) and [M+ I](-) were detected, and their gas-phase structures and energetics are investigated by density functional theory calculations. Copyright (C) 2016 John Wiley & Sons, Ltd.

The problem of atmospheric emission from OH molecules is a long standing problem for near-infrared astronomy. PRAXIS is a unique spectrograph which is fed by fibres that remove the OH background and is optimised specifically to benefit from OH-Suppression. The OH suppression is achieved with fibre Bragg gratings, which were tested successfully on the GNOSIS instrument. PRAXIS uses the same fibre Bragg gratings as GNOSIS in its first implementation, and will exploit new, cheaper and more efficient, multicore fibre Bragg gratings in the second implementation. The OH lines are suppressed by a factor of ∼ 1000, and the expected increase in the signal-to-noise in the interline regions compared to GNOSIS is a factor of ∼ 9 with the GNOSIS gratings and a factor of ∼ 17 with the new gratings. PRAXIS will enable the full exploitation of OH suppression for the first time, which was not achieved by GNOSIS (a retrofit to an existing instrument that was not OH-Suppression optimised) due to high thermal emission, low spectrograph transmission and detector noise. PRAXIS has extremely low thermal emission, through the cooling of all significantly emitting parts, including the fore-optics, the fibre Bragg gratings, a long length of fibre, and the fibre slit, and an optical design that minimises leaks of thermal emission from outside the spectrograph. PRAXIS has low detector noise through the use of a Hawaii-2RG detector, and a high throughput through a efficient VPH based spectrograph. PRAXIS will determine the absolute level of the interline continuum and enable observations of individual objects via an IFU. In this paper we give a status update and report on acceptance tests.

Mold fungi on malting barley grains cause major economic loss in malting and brewery facilities. Possible proxies for their detection are volatile and semivolatile metabolites. Among those substances, characteristic marker compounds have to be identified for a confident detection of mold fungi in varying surroundings. The analytical determination is usually performed through passive sampling with solid phase microextraction, gas chromatographic separation, and detection by electron ionization mass spectrometry (EI-MS), which often does not allow a confident determination due to the absence of molecular ions. An alternative is GC-APCI-MS, generally, allowing the determination of protonated molecular ions. Commercial atmospheric pressure chemical ionization (APCI) sources are based on corona discharges, which are often unspecific due to the occurrence of several side reactions and produce complex product ion spectra. To overcome this issue, an APCI source based on soft X-radiation is used here. This source facilitates a more specific ionization by proton transfer reactions only. In the first part, the APCI source is characterized with representative volatile fungus metabolites. Depending on the proton affinity of the metabolites, the limits of detection are up to 2 orders of magnitude below those of EI-MS. In the second part, the volatile metabolites of the mold fungus species Aspergillus, Alternaria, Fusarium, and Penicillium are investigated. In total, 86 compounds were found with GC-EI/APCI-MS. The metabolites identified belong to the substance classes of alcohols, aldehydes, ketones, carboxylic acids, esters, substituted aromatic compounds, terpenes, and sesquiterpenes. In addition to substances unspecific for the individual fungus species, characteristic patterns of metabolites, allowing their confident discrimination, were found for each of the 4 fungus species. Sixty-seven of the 86 metabolites are detected by X-ray-based APCI-MS alone. The discrimination of the fungus species based on these metabolites alone was possible. Therefore, APCI-MS in combination with collision induced dissociation alone could be used as a supervision method for the detection of mold fungi.

Total protein concentration (TPC) is a key parameter in many biochemical experiments and its quantification is often necessary for isolation, separation, and analysis of proteins. A sensitive and rapid nanobead-based TPC quantification assay based on Forster Resonance Energy Transfer (FRET) has been developed. A new, highly luminescent Tb(III) complex has been synthesised and applied as donor in this FRET assay with an organic dye (Cy5) as acceptor. FRET-induced changes in luminescence have been investigated both at donor and acceptor emission wavelength using time-resolved luminescence spectroscopy with time-gated detection. In the assay, the Tb(III) complex and fine-tuned polyglycidyl methacrylate (PGMA) nanobeads ensure that an improvement in sensitivity and background reduction is achieved. Using 40 nm large PGMA nanobeads loaded with the Tb(III) complex, it is possible to determine TPC down to 50 ng mL−1 in just 10 minutes. Through specific assay components the sensitivity has been improved when compared to existing nanobead-based assays and to currently known commercial methods. Additionally, the assay is relatively insensitive to the presence of contaminants, such as non-ionic detergents commonly found in biological samples. Due to no need for any centrifugal steps, this mix-and-measure bioassay can easily be implemented into routine TPC quantification protocols in biochemical laboratories.

A laser-based ionization source for the direct analysis of liquid samples in ion mobility (IM) spectrometry is presented and characterized. Ionization of aromatic substances in liquids is achieved, analogous to atmospheric pressure laser ionization (APLI) in mass spectrometry, by vaporizing the liquid and subsequently ionizing the aromatic substances by resonance-enhanced multiphoton ionization (REMPI). The effects of parameters, such as composition and flow rate of the solvent as well as laser wavelength and pulse energy, are systematically investigated. The characterization of the IM spectrometer is carried out by means of selected substances from diverse fields of applications, e.g., polycyclic aromatic hydrocarbons (PAH), pesticides, wood preservatives and drug compounds. Limits of detection (LOD) down to 10 fmol and linear ranges up to three orders of magnitude are established. In addition to direct laser ionization, indirect laser ionization via dopants (toluene) for substances with low ionization efficiencies is investigated. Ionization occurs as a result of proton transfer from toluene radical cations to substances of sufficiently high proton affinities. As a result of indirect laser ionization, LOD could be decreased by up to two orders of magnitude. Ionization products are investigated by means of a combination of IM and mass spectrometer. Depending on the substance investigated primary ions (radical cations) and secondary ions (protonated molecules) resulting from ion molecule reactions are formed.

Spot variation fluorescence correlation spectroscopy (SV-FCS) is a variant of the FCS techniques which may give useful information about the structural organisation of the medium in which the diffusion takes place. We show that the same results can be obtained by post-processing the photon count data from ordinary FCS measurements. By using this method, one obtains the fluorescence autocorrelation functions for sizes of confocal volume, which are effectively smaller than that of the initial FCS measurement. The photon counts of the initial experiment are first transformed into smooth intensity trace using kernel smoothing method or to a piecewise-continuous intensity trace using binning and then a non-linear transformation is applied to this trace. The result of this transformation mimics the photon count rate in an experiment performed with a smaller confocal volume. The applicability of the method is established in extensive numerical simulations and directly supported in in-vitro experiments. The procedure is then applied to the diffusion of AlexaFluor647-labeled streptavidin in living cells.

A homogeneous time-resolved luminescence resonance energy transfer (TR-LRET) assay has been developed to quantify proteins. The competitive assay is based on resonance energy transfer (RET) between two luminescent nanosized particles. Polystyrene nanoparticles loaded with Eu(3+) chelates (EuNPs) act as donors, while protein-coated quantum dots (QDs), either CdSe/ZnS emitting at 655 nm (QD655-strep) or CdSeTe/ZnS with emission wavelength at 705 nm (QD705-strep), are acceptors. In the absence of analyte protein, in our case bovine serum albumin (BSA), the protein-coated QDs bind nonspecifically to the EuNPs, leading to RET. In the presence of analyte proteins, the binding of the QDs to the EuNPs is prevented and the RET signal decreases. RET from the EuNPs to the QDs was confirmed and characterized with steady-state and time-resolved luminescence spectroscopy. In accordance with the Förster theory, the approximate average donor-acceptor distance is around 15 nm at RET efficiencies, equal to 15% for QD655 and 13% for QD705 acceptor, respectively. The limits of detection are below 10 ng of BSA with less than a 10% average coefficient of variation. The assay sensitivity is improved, when compared to the most sensitive commercial methods. The presented mix-and-measure method has potential to be implemented into routine protein quantification in biological laboratories.

Simultaneous monitoring of multiple molecular interactions and multiplexed detection of several diagnostic biomarkers at very low concentrations have become important issues in advanced biological and chemical sensing. Here we present an optically multiplexed six-color Forster resonance energy transfer (FRET) biosensor for simultaneous monitoring of five different individual binding events. We combined simultaneous FRET from one Tb complex to five different organic dyes measured in a filter-based time-resolved detection format with a sophisticated spectral crosstalk correction, which results in very efficient background suppression. The advantages and robustness of the multiplexed FRET sensor were exemplified by analyzing a 15-component lung cancer immunoassay involving 10 different antibodies and five different tumor markers in a single 50 mu L human serum sample. The multiplexed biosensor offers clinically relevant detection limits in the low picomolar (ng/mL) concentration range for all five markers, thus providing an effective early screening tool for lung cancer with the possibility of distinguishing small-cell from non-small-cell lung carcinoma. This novel technology will open new doors for multiple biomarker diagnostics as well as multiplexed real-time imaging and spectroscopy.

We discuss the application of fluorescence correlation spectroscopy (FCS) for characterization of anomalous diffusion of tracer particles in crowded environments. While the fact of anomaly may be detected by the standard fitting procedure, the value of the exponent $\ensuremath{\alpha}$ of anomalous diffusion may be not reproduced correctly for non-Gaussian anomalous diffusion processes. The important information is however contained in the asymptotic behavior of the fluorescence autocorrelation function at long and at short times. Thus, analysis of the short-time behavior gives reliable values of $\ensuremath{\alpha}$ and of lower moments of the distribution of particles' displacement, which allows us to confirm or reject its Gaussian nature. The method proposed was tested on the FCS data obtained in artificial crowded fluids and in living cells.

The combination of high-performance liquid chromatography and electrospray ionization ion mobility spectrometry facilitates the two-dimensional separation of complex mixtures in the retention and drift time plane. The ion mobility spectrometer presented here was optimized for flow rates customarily used in high-performance liquid chromatography between 100 and 1500 μL/min. The characterization of the system with respect to such parameters as the peak capacity of each time dimension and of the 2D spectrum was carried out based on a separation of a pesticide mixture containing 24 substances. While the total ion current chromatogram is coarsely resolved, exhibiting coelutions for a number of compounds, all substances can be separately detected in the 2D plane due to the orthogonality of the separations in retention and drift dimensions. Another major advantage of the ion mobility detector is the identification of substances based on their characteristic mobilities. Electrospray ionization allows the detection of substances lacking a chromophore. As an example, the separation of a mixture of 18 amino acids is presented. A software built upon the free mass spectrometry package OpenMS was developed for processing the extensive 2D data. The different processing steps are implemented as separate modules which can be arranged in a graphic workflow facilitating automated processing of data.

Pioneered by Clark's microelectrode more than half a century ago, there has been substantial interest in developing new, miniaturized optical methods to detect molecular oxygen inside cells. While extensively used for animal tissue measurements, applications of intracellular optical oxygen biosensors are still scarce in plant science. A critical aspect is the strong autofluorescence of the green plant tissue that interferes with optical signals of commonly used oxygen probes. A recently developed dual-frequency phase modulation technique can overcome this limitation, offering new perspectives for plant research. This review gives an overview on the latest optical sensing techniques and methods based on phosphorescence quenching in diverse tissues and discusses the potential pitfalls for applications in plants. The most promising oxygen sensitive probes are reviewed plus different oxygen sensing structures ranging from micro-optodes to soluble nanoparticles. Moreover, the applicability of using heterologously expressed oxygen binding proteins and fluorescent proteins to determine changes in the cellular oxygen concentration are discussed as potential non-invasive cellular oxygen reporters.

In the present work, the density distribution of rhodamine 6G ions (R6G) in the gas phase and the droplets of an electrospray plume was studied by spatial and spectral imaging. The intention is to contribute to the fundamental understanding of the release mechanism of gaseous R6G in the electrospray ionization (ESI) process. Furthermore, the influence of ESI-parameters on the release efficiency of R6G, e.g. solvent flow, R6G and salt concentration were examined via direct fluorescence imaging of R6G. A solvent-shift of the fluorescence maximum, λ max = 555 nm in methanolic solution and λ max = 505 nm in gas phase, allows the discrimination between solvated and gaseous R6G. Two experimental setups were used for our measurements. In the first experiment, the R6G fluorescence and the light scattered from the spray plume were imaged in two spatial dimensions using a tunable wavelength fil ter. The second experiment was designed for obtaining 1-dimensional spatially resolved emission spectra of the spray. Here, the intensity distribution of solvated and gaseous R6G as well as scattered light (λ=355 nm) were measured simultaneously. The results show the distribution of gaseous R6G in the plane, orthogonal to the ESI capillary, decreasing slightly towards the spray center and showing maxima at the cone margins. The distribution of gaseous R6G confirms the preferred release of gaseous ions from nano-droplets, indicating the ion evaporation model (IEM) to be the dominating release mechanism. Up to now, only a few fluorescence spectra of ionic compounds in the gas phase were published because the measurement of emission spectra of mass-selected ions in an ion trap is experimentally challenging. The fluorescence spectrum of gaseous lucigenin at atmospheric pressure is reported for the first time. This spectrum of lucigenin in the gas phase exhibits a blue shift of about Δλ=10 nm in comparison to the corresponding spectrum in methanol.

Novel fluorescent nanosensors, based on a naphthyridine receptor, have been developed for the detection of guanosine nucleotides, and both their sensitivity and selectivity to various nucleotides were evaluated. The nanosensors were constructed from polystyrene nanoparticles functionalized by (N-(7-((3-aminophenyl) ethynyl)-1,8-naphthyridin- 2-yl) acetamide) via carbodiimide ester activation. We show that this naphthyridine nanosensor binds guanosine nucleotides preferentially over adenine, cytosine, and thymidine nucleotides. Upon interaction with nucleotides, the fluorescence of the nanosensor is gradually quenched yielding Stern-Volmer constants in the range of 2.1 to 35.9mM(-1). For all the studied quenchers, limits of detection (LOD) and tolerance levels for the nanosensors were also determined. The lowest (3 sigma) LOD was found for guanosine 3',5'-cyclic monophosphate (cGMP) and it was as low as 150 ng/ml. In addition, we demonstrated that the spatial arrangement of bound analytes on the nanosensors' surfaces is what is responsible for their selectivity to different guanosine nucleotides. We found a correlation between the changes of the fluorescence signal and the number of phosphate groups of a nucleotide. Results of molecular modeling and zeta-potential measurements confirm that the arrangement of analytes on the surface provides for the selectivity of the nanosensors. These fluorescent nanosensors have the potential to be applied in multi-analyte, array-based detection platforms, as well as in multiplexed microfluidic systems.

A large variety of microporous polymer films can be prepared by the breath figure technique. Here, we report on its use for the formation of microporous zinc oxide films. Zinc acetylacetonate, a zinc oxide precursor, is either dissolved in a polymer solution that is cast at high humidity to form microporous films or is vacuum evaporated onto a preformed microporous polymer film. Annealing leads to the pyrolysis of the organic material and the formation of zinc oxide films, which show increased photocatalytic activity as compared to unstructured films.

Due to the growing demand for rapid, sensitive and inexpensive detection of contaminations in food and feed, both in the field and after harvest, non-invasive and non-destructive spectroscopic methods are reviewed for the direct identification of fungi and mycotoxins on grain and in grain products. Different model samples containing defined contaminations of fungi and/or mycotoxins as well as real-world samples were investigated using spectroscopic methods (steady-state and time-resolved fluorescence spectroscopy, ultraviolet/visible/near infrared (NIR) spectroscopy, two-photon-induced fluorescence (TPIF)). To evaluate the capability of TPIF for on-line and in-line mycotoxin analysis in real-world samples, fundamental parameters of different mycotoxins, such as two-photon cross-sections, were determined in beer and wine. A comparison showed that the disturbance due to background emission originating from matrix constituents is significantly reduced under two-photon excitation conditions, and TPIF can be applied for a qualitative and quantitative analysis of mycotoxins in wine and beer. The combination of data obtained from different spectroscopic methods (such as optimised excitation and emission wavelengths, fluorescence decay times and fluorescence quantum efficiency on the one hand side and NIR spectroscopy on the other side) is promising for the qualitative as well as quantitative identification of fungi and mycotoxins. Moreover, NIR reflection spectra yield additional information on ingredients, moisture content and the presence (or absence) of fungi in the sample. The spectroscopic techniques are complemented by chemometric tools to extract the desired chemical information, e.g. with respect to the presence of contaminations.

Time- and color-resolved detection of Foerster resonance energy transfer (FRET) from luminescent terbium complexes to different semiconductor quantum dots results in a fivefold multiplexed bioassay with sub-picomolar detection limits for all five bioanalytes (see picture). The detection of up to five biomarkers occurs with a sensitivity that is 40-240-fold higher than one of the best-established single-analyte reference assays.

We prepared honeycomb films of a crosslinkable poly(maleic anhydride-co-styrene). It was found that the surface modification of the polymer by ethanolamine had a profound effect on the adsorption of TiO2 nanoparticles from alcoholic suspensions. The other important parameter to control adsorption density of the TiO2 film on the honeycomb substrate was the water content of the alcohol. Furthermore, we found that other organic solvents can also be used to form adsorbed films with various morphologies. After pyrolysis, the microscopic honeycomb morphology was retained. [DOI: 10.1380/ejssnt.2010.309]

Infrared matrix-assisted laser dispersion and ionization (IR-MALDI) in combination with ion mobility (IM) spectrometry enables the direct analysis of biomolecules in aqueous solution. The release of ions directly from an aqueous solution is based on a phase explosion, induced by the absorption of an IR laser pulse, which disperses the liquid as vapor, nano-and micro-droplets. The ionization process is characterized initially by a broad spatial distribution of the ions, which is a result of complex fluid dynamics and desolvation kinetics. These processes have a profound effect on the shape and width of the peaks in the IM spectra. In this work, the transport of ions by the phase explosion-induced shockwave could be studied independently from the transport by the electric field. The shockwave-induced mean velocities of the ions at different time scales were determined through IM spectrometry and shadowgraphy. The results show a deceleration of the ions from 118 m.s(-1) at a distance of 400 mu m from the liquid surface to 7.1 m.s(-1) at a distance of 10 mm, which is caused by a pile-up effect. Furthermore, the desolvation kinetics were investigated and a first-order desolvation constant of 325 +/- 50 s(-1) was obtained. In the second part, the IR-MALDI-IM spectrometer is used as an HPLC detector for the two-dimensional separation of a pesticide mixture.

For the past forty years, optical fibres have found widespread use in ground-based and space-based instruments. In most applications, these fibres are used in conjunction with conventional optics to transport light. But photonics offers a huge range of optical manipulations beyond light transport that were rarely exploited before 2001. The fundamental obstacle to the broader use of photonics is the difficulty of achieving photonic action in a multimode fibre. The first step towards a general solution was the invention of the photonic lantern (Leon-Saval, Birks & Bland-Hawthorn 2005) and the delivery of high-efficiency devices (< 1 dB loss) five years on (Noordegraaf et al 2009). Multicore fibres (MCF), used in conjunction with lanterns, are now enabling an even bigger leap towards multimode photonics. Until recently, the single-moded cores in MCFs were not sufficiently uniform to achieve telecom (SMF-28) performance. Now that high-quality MCFs have been realized, we turn our attention to printing complex functions (e.g. Bragg gratings for OH suppression) into their N cores. Our first work in this direction used a Mach-Zehnder interferometer (near-field phase mask) but this approach was only adequate for N=7 MCFs as measured by the grating uniformity (Lindley et al 2014). We have now built a Sagnac interferometer that gives a three-fold increase in the depth of field sufficient to print across N > 127 cores. We achieved first light this year with our 500mW Sabre FRED laser. These are sophisticated and complex interferometers. We report on our progress to date and summarize our first-year goals which include multimode OH suppression fibres for the Anglo-Australian Telescope/PRAXIS instrument and the Discovery Channel Telescope/MOHSIS instrument under development at the University of Maryland., 14 pages

The novel combination of infrared matrix-assisted laser dispersion and ionization (IR-MALDI) with ion mobility (IM) spectrometry makes it possible to investigate biomolecules in their natural environment, liquid water. As an alternative to an ESI source, the IR-MALDI source was implemented in an in-house-developed ion mobility (IM) spectrometer. The release of ions directly from an aqueous solution is based on a phase explosion, induced by the absorption of an IR laser pulse (lambda = 2.94 mu m, 6 ns pulse width), which disperses the liquid as nano- and micro-droplets. The prerequisites for the application of IR-MALDI-IM spectrometry as an analytical method are narrow analyte ion signal peaks for a high spectrometer resolution. This can only be achieved by improving the desolvation of ions. One way to full desolvation is to give the cluster ions sufficient time to desolvate. Two methods for achieving this are studied: the implementation of an additional drift tube, as in ESI-IM-spectrometry, and the delayed extraction of the ions. As a result of this optimization procedure, limits of detection between 5 nM and 2.5 mu M as well as linear dynamic ranges of 2-3 orders of magnitude were obtained for a number of substances. The ability of this method to analyze simple mixtures is illustrated by the separation of two different surfactant mixtures.