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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.
Quantum dots (QDs) are common as luminescing markers for imaging in biological applications because their optical properties seem to be inert against their surrounding solvent. This, together with broad and strong absorption bands and intense, sharp tuneable luminescence bands, makes them interesting candidates for methods utilizing Förster Resonance Energy Transfer (FRET), e. g. for sensitive homogeneous fluoroimmunoassays (FIA). In this work we demonstrate energy transfer from Eu<SUP>3+</SUP>-trisbipyridin (Eu-TBP) donors to CdSe-ZnS-QD acceptors in solutions with and without serum. The QDs are commercially available CdSe-ZnS core-shell particles emitting at 655 nm (QD655). The FRET system was achieved by the binding of the streptavidin conjugated donors with the biotin conjugated acceptors. After excitation of Eu-TBP and as result of the energy transfer, the luminescence of the QD655 acceptors also showed lengthened decay times like the donors. The energy transfer efficiency, as calculated from the decay times of the bound and the unbound components, amounted to 37%. The Förster-radius, estimated from the absorption and emission bands, was ca. 77 Å. The effective binding ratio, which not only depends on the ratio of binding pairs but also on unspecific binding, was obtained from the donor emission dependent on the concentration. As serum promotes unspecific binding, the overall FRET efficiency of the assay was reduced. We conclude that QDs are good substitutes for acceptors in FRET if combined with slow decay donors like Europium. The investigation of the influence of the serum provides guidance towards improving binding properties of QD assays.
In this work, ion mobility (IM) spectra of more than 50 aromatic compounds were recorded with a laser-based IM spectrometer at atmospheric pressure. IM spectra of PAH in the laser desorption experiment show a high complexity resulting from the occurrence of monomeric, dimeric, and oligomeric cluster ions. The mobilities of all compounds were determined in helium as drift gas. This allows the calculation of the diffusion cross sections (Omega(calc)) on the basis of the exact hard sphere scattering model and their comparison with the experimentally determined diffusion cross sections (Omega(exp)). Extended Omega(exp)/Omega(calc) and Omega(exp/)mass correlations were performed in order to gain insight into conformational properties of cationic alkyl benzenes and internal rotation of phenyl rings in aromatic ions. This is demonstrated with some examples, such as the evaluation of the dihedral angle of the ions of 9,10- diphenylanthracene, o- and m-terphenyl, and 1,2,3- and 1,3,5-triphenylbenzene. Furthermore, sandwich and T-structures of dimeric PAH cations are discussed. The analysis was extended to oligomeric ions with up to nine monomer units. Experimental evidence is presented suggesting the formation of pi-stacks with a transition toward modified pi-stacks with increasing cluster size. The distance between monomeric units in dimeric and oligomeric ions was obtained
A long-standing and profound problem in astronomy is the difficulty in obtaining deep near-infrared observations due to the extreme brightness and variability of the night sky at these wavelengths. A solution to this problem is crucial if we are to obtain the deepest possible observations of the early Universe, as redshifted starlight from distant galaxies appears at these wavelengths. The atmospheric emission between 1,000 and 1,800 nm arises almost entirely from a forest of extremely bright, very narrow hydroxyl emission lines that varies on timescales of minutes. The astronomical community has long envisaged the prospect of selectively removing these lines, while retaining high throughput between them. Here we demonstrate such a filter for the first time, presenting results from the first on-sky tests. Its use on current 8 m telescopes and future 30 m telescopes will open up many new research avenues in the years to come.
Laser-based ion mobility (IM) spectrometry was used for the detection of neuroleptics and PAH. A gas chromatograph was connected to the IM spectrometer in order to investigate compounds with low vapour pressure. The substances were ionized by resonant two-photon ionization at the wavelengths lambda = 213 and 266 nm and pulse energies between 50 and 300 mu J. Ion mobilities, linear ranges, limits of detection and response factors are reported. Limits of detection for the substances are in the range of 1-50 fmol. Additionally, the mechanism of laser ionization at atmospheric pressure was investigated. First, the primary product ions were determined by a laser-based time-of-flight mass spectrometer with effusive sample introduction. Then, a combination of a laser-based IM spectrometer and an ion trap mass spectrometer was developed and characterized to elucidate secondary ion-molecule reactions that can occur at atmospheric pressure. Some substances, namely naphthalene, anthracene, promazine and thioridazine, could be detected as primary ions (radical cations), while other substances, in particular acridine, phenothiazine and chlorprothixene, are detected as secondary ions (protonated molecules). The results are interpreted on the basis of quantum chemical calculations, and an ionization mechanism is proposed.
Its properties make copper one of the world’s most important functional metals. Numerous megatrends are increasing the demand for copper. This requires the prospection and exploration of new deposits, as well as the monitoring of copper quality in the various production steps. A promising technique to perform these tasks is Laser Induced Breakdown Spectroscopy (LIBS). Its unique feature, among others, is the ability to measure on site without sample collection and preparation. In this work, copper-bearing minerals from two different deposits are studied. The first set of field samples come from a volcanogenic massive sulfide (VMS) deposit, the second part from a stratiform sedimentary copper (SSC) deposit. Different approaches are used to analyze the data. First, univariate regression (UVR) is used. However, due to the strong influence of matrix effects, this is not suitable for the quantitative analysis of copper grades. Second, the multivariate method of partial least squares regression (PLSR) is used, which is more suitable for quantification. In addition, the effects of the surrounding matrices on the LIBS data are characterized by principal component analysis (PCA), alternative regression methods to PLSR are tested and the PLSR calibration is validated using field samples.
Its properties make copper one of the world’s most important functional metals. Numerous megatrends are increasing the demand for copper. This requires the prospection and exploration of new deposits, as well as the monitoring of copper quality in the various production steps. A promising technique to perform these tasks is Laser Induced Breakdown Spectroscopy (LIBS). Its unique feature, among others, is the ability to measure on site without sample collection and preparation. In this work, copper-bearing minerals from two different deposits are studied. The first set of field samples come from a volcanogenic massive sulfide (VMS) deposit, the second part from a stratiform sedimentary copper (SSC) deposit. Different approaches are used to analyze the data. First, univariate regression (UVR) is used. However, due to the strong influence of matrix effects, this is not suitable for the quantitative analysis of copper grades. Second, the multivariate method of partial least squares regression (PLSR) is used, which is more suitable for quantification. In addition, the effects of the surrounding matrices on the LIBS data are characterized by principal component analysis (PCA), alternative regression methods to PLSR are tested and the PLSR calibration is validated using field samples.
Nanobioconjugates have been synthesized using cadmium selenide quantum dots (QDs), europium complexes (EuCs), and biotin. In those conjugates, long-lived photoluminescence (PL) is provided by the europium complexes, which efficiently transfer energy via Forster resonance energy transfer (FRET) to the QDs in close spatial proximity. As a result, the conjugates have a PL emission spectrum characteristic for QDs combined with the long PL decay time characteristic for EuCs. The nanobioconjugates synthesis strategy and photo-physical properties are described as well as their performance in a time-resolved streptavidin-biotin PL assay. In order to prepare the QD-EuC-biotin conjugates, first an amphiphilic polymer has been functionalized with the EuC and biotin. Then, the polymer has been brought onto the surface of the QDs (either QD655 or QD705) to provide functionality and to make the QDs water dispersible. Due to a short distance between EuC and QD, an efficient FRET can be observed. Additionally, the QD-EuC-biotin conjugates' functionality has been demonstrated in a PL assay yielding good signal discrimination, both from autofluorescence and directly excited QDs. These newly designed QD-EuC-biotin conjugates expand the class of highly sensitive tools for bioanalytical optical detection methods for diagnostic and imaging applications. (C) 2014 Society of Photo-Optical Instrumentation Engineers (SPIE)
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 new functional luminescent lanthanide complex (LLC) has been synthesized with terbium as a central lanthanide ion and biotin as a functional moiety. Unlike in typical lanthanide complexes assembled via carboxylic moieties, in the presented complex, four phosphate groups are chelating the central lanthanide ion. This special chemical assembly enhances the complex stability in phosphate buffers conventionally used in biochemistry. The complex synthesis strategy and photophysical properties are described as well as the performance in time-resolved Forster Resonance Energy Transfer (FRET) assays. In those assays, this biotin-LLC transferred energy either to acceptor organic dyes (Cy5 or AF680) labelled on streptavidin or to quantum dots (QD655 or QD705) surfacefunctionalised with streptavidins. The permanent spatial donor-acceptor proximity is assured through strong and stable biotin-streptavidin binding. The energy transfer is evidenced from the quenching observed in donor emission and from a decrease in donor luminescence decay, both associated with simultaneous increase in acceptor intensity and in the decay time. The dye-based assays are realised in TRIS and in PBS, whereas QD-based systems are studied in borate buffer. The delayed emission analysis allows for quantifying the recognition process and for auto-fluorescence-free detection, which is particularly relevant for application in bioanalysis. In accordance with Forster theory, Forsterradii (R0) were found to be around 60 angstrom for organic dyes and around 105 angstrom for QDs. The FRET efficiency (Z) reached 80% and 25% for dye and QD acceptors, respectively. Physical donor-acceptor distances (r) have been determined in the range 45-60 angstrom for organic dye acceptors, while for acceptor QDs between 120 angstrom and 145 angstrom. This newly synthesised biotin-LLC extends the class of highly sensitive analytical tools to be applied in the bioanalytical methods such as time-resolved fluoroimmunoassays (TR-FIA), luminescent imaging and biosensing.
L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Forster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10 -500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA). (C) 2015 Elsevier B.V. All rights reserved.
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 new functional luminescent lanthanide complex (LLC) has been synthesized with terbium as a central lanthanide ion and biotin as a functional moiety. Unlike in typical lanthanide complexes assembled via carboxylic moieties, in the presented complex, four phosphate groups are chelating the central lanthanide ion. This special chemical assembly enhances the complex stability in phosphate buffers conventionally used in biochemistry. The complex synthesis strategy and photophysical properties are described as well as the performance in time-resolved Förster Resonance Energy Transfer (FRET) assays. In those assays, this biotin-LLC transferred energy either to acceptor organic dyes (Cy5 or AF680) labelled on streptavidin or to quantum dots (QD655 or QD705) surface-functionalised with streptavidins. The permanent spatial donor–acceptor proximity is assured through strong and stable biotin–streptavidin binding. The energy transfer is evidenced from the quenching observed in donor emission and from a decrease in donor luminescence decay, both associated with simultaneous increase in acceptor intensity and in the decay time. The dye-based assays are realised in TRIS and in PBS, whereas QD-based systems are studied in borate buffer. The delayed emission analysis allows for quantifying the recognition process and for auto-fluorescence-free detection, which is particularly relevant for application in bioanalysis. In accordance with Förster theory, Förster-radii (R0) were found to be around 60 Å for organic dyes and around 105 Å for QDs. The FRET efficiency (η) reached 80% and 25% for dye and QD acceptors, respectively. Physical donor–acceptor distances (r) have been determined in the range 45–60 Å for organic dye acceptors, while for acceptor QDs between 120 Å and 145 Å. This newly synthesised biotin-LLC extends the class of highly sensitive analytical tools to be applied in the bioanalytical methods such as time-resolved fluoroimmunoassays (TR-FIA), luminescent imaging and biosensing.
Deuteration effects on the vibronic structure of the emission and excitation spectra of triangular [ 4] phenylene (D-3h [4]phenylene) were studied using laser-excited Shpol'skii spectroscopy (LESS) in an octane matrix at 4.2 K. For correct assignment of the vibrational modes, the experimental results were compared with calculated frequencies (B3LYP/6-31G*). CH vibrations were identified by their characteristic isotopic shifts in the spectra of deuterated triangular [4]phenylenes. Two CC stretching modes, at 100 cm(-1) and 1176 cm(-1), suitable as probes for bond strength changes in the excited state, were identified. The isotope effect on the internal conversion rates of triangular [4] phenylene was evaluated from measurements of temperature dependent lifetime. Isotope dependency and the magnitude of the internal conversion rates indicate that internal conversion in triangular [4] phenylene is most likely induced by CH vibrations. The results obtained by LESS and lifetime measurements were compared with PM3 PECI calculations of the excited state structure. The theoretical results and the relation between ground and excited state vibration energies of the 1176 cm(-1) probe vibration indicate a reduction of bond alternation of the central cyclohexatriene ring in the excited state
In the present study, photophysical properties of [N]phenylenes were studied by means of stationary and time-resolved absorption and fluorescence spectroscopy (in THF at room temperature). For biphenylene (1) and linear [3]phenylene (2a), internal conversion (IC) with quantum yields ΦIC > 0.99 is by far the dominant mechanism of S1 state deactivation. Angular [3]phenylene (3a), the zig-zag [4]- and [5]phenylenes (3b), (3c), and the triangular [4]phenylene (4) show fluorescence emission with fluorescence quantum yieds and lifetimes between ΦF = 0.07 for (3a) and 0.21 for (3c) and τF = 20 ns for (3a) and 81 ns for (4). Also, compounds (3) and (4) exhibit triplet formation upon photoexcitation with quantum yields as high as ΦISC = 0.45 for (3c). The strong differences in the fluorescence properties and in the triplet fromation efficiencies between (1) and (2a) on one hand and (3) and (4) on the other are related to the remarkable variation of the internal conversion (IC) rate constants kIC. A tentative classification of (1) and (2a) as “fast IC compounds”, with kIC > 109 s-1, and of (3) and (4) as “slow IC compounds”, with kIC ≈ 107 s-1, is suggested. This classification cannot simply be related to Hückel’s rule-type concepts of aromaticity, because the group of “fast IC compounds” consists of “antiaromatic” (1) and “aromatic” (2a), and the group of “slow IC compounds” consists of “antiaromatic” (3b), (4) and “aromatic” (3a), (3c). The IC in the [N]phenylenes is discussed within the framework of the so-called energy gap law established for non-radiative processes in benzenoid hydrocarbons.
First studies of electron transfer in [N]phenylenes were performed in bimolecular quenching reactions of angular [3]- and triangular [4]phenylene with various electron acceptors. The relation between the quenching rate constants kq and the free energy change of the electron transfer (ΔG0CS ) could be described by the Rehm-Weller equation. From the experimental results, a reorganization energy λ of 0.7 eV was derived. Intramolecular electron transfer reactions were studied in an [N]phenylene bichomophore and a corresponding reference compound. Fluorescence lifetime and quantum yield of the bichromophor display a characteristic dependence on the solvent polarity, whereas the corresponding values of the reference compound remain constant. From the results, a nearly isoenergonic ΔG0CS can be determined. As the triplet quantum yield is nearly independent of the polarity, charge recombination leads to the population of the triplet state.
First studies of electron transfer in [N]phenylenes were performed in bimolecular quenching reactions of angular [3]- and triangular [4]phenylene with various electron acceptors. The relation between the quenching rate constants k(q) and the free energy change of the electron transfer (Delta G(CS)(0)) could be described by the Rehm- Weller equation. From the experimental results, a reorganization energy lambda of 0.7 eV was derived. Intramolecular electron transfer reactions were studied in an [N]phenylene bichomophore and a corresponding reference compound. Fluorescence lifetime and quantum yield of the bichromophor display a characteristic dependence on the solvent polarity, whereas the corresponding values of the reference compound remain constant. From the results, a nearly isoenergonic charge separation process can be determined. As the triplet quantum yield is nearly independent of the polarity, charge recombination leads to the population of the triplet state.
In many biological and environmental applications spatially resolved sensing of molecular oxygen is desirable. A powerful tool for distributed measurements is optical time domain reflectometry (OTDR) which is often used in the field of telecommunications. We combine this technique with a novel optical oxygen sensor dye, triangular-[4] phenylene (TP), immobilized in a polymer matrix. The TP luminescence decay time is 86 ns. The short decay time of the sensor dye is suitable to achieve a spatial resolution of some meters. In this paper we present the development and characterization of a reflectometer in the UV range of the electromagnetic spectrum as well as optical oxygen sensing with different fiber arrangements.
In many biological and environmental applications spatially resolved sensing of molecular oxygen is desirable. A powerful tool for distributed measurements is optical time domain reflectometry (OTDR) which is often used in the field of telecommunications. We combine this technique with a novel optical oxygen sensor dye, triangular-[4] phenylene (TP), immobilized in a polymer matrix. The TP luminescence decay time is 86 ns. The short decay time of the sensor dye is suitable to achieve a spatial resolution of some meters. In this paper we present the development and characterization of a reflectometer in the UV range of the electromagnetic spectrum as well as optical oxygen sensing with different fiber arrangements.
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 similar to 1000, and the expected increase in the signal-to-noise in the interline regions compared to GNOSIS is a factor of similar to 9 with the GNOSIS gratings and a factor of similar to 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.
Suppression of the near-infrared OH night-sky lines with fibre Bragg gratings - first results
(2012)
The background noise between 1 and 1.8 ?mu m in ground-based instruments is dominated by atmospheric emission from hydroxyl molecules. We have built and commissioned a new instrument, the Gemini Near-infrared OH Suppression Integral Field Unit (IFU) System (GNOSIS), which suppresses 103 OH doublets between 1.47 and 1.7?mu m by a factor of 1000 with a resolving power of 10?000. We present the first results from the commissioning of GNOSIS using the IRIS2 spectrograph at the Anglo-Australian Telescope. We present measurements of sensitivity, background and throughput. The combined throughput of the GNOSIS fore-optics, grating unit and relay optics is 36?per cent, but this could be improved to 46?per cent with a more optimal design. We measure strong suppression of the OH lines, confirming that OH suppression with fibre Bragg gratings will be a powerful technology for low-resolution spectroscopy. The integrated OH suppressed background between 1.5 and 1.7 mu m is reduced by a factor of 9 compared to a control spectrum using the same system without suppression. The potential of low-resolution OH-suppressed spectroscopy is illustrated with example observations of Seyfert galaxies and a low-mass star. The GNOSIS background is dominated by detector dark current below 1.67 mu m and by thermal emission above 1.67 mu m. After subtracting these, we detect an unidentified residual interline component of 860 +/- 210 photons s-1 m-2?arcsec-2?mu m-1, comparable to previous measurements. This component is equally bright in the suppressed and control spectra. We have investigated the possible source of the interline component, but were unable to discriminate between a possible instrumental artefact and intrinsic atmospheric emission. Resolving the source of this emission is crucial for the design of fully optimized OH suppression spectrographs. The next-generation OH suppression spectrograph will be focused on resolving the source of the interline component, taking advantage of better optimization for a fibre Bragg grating feed incorporating refinements of design based on our findings from GNOSIS. We quantify the necessary improvements for an optimal OH suppressing fibre spectrograph design.
Optical methods play an important role in process analytical technologies (PAT). Four examples of optical process and quality sensing (OPQS) are presented, which are based on three important experimental techniques: near-infrared absorption, luminescence quenching, and a novel method, photon density wave (PDW) spectroscopy. These are used to evaluate four process and quality parameters related to beer brewing and polyurethane (PU) foaming processes: the ethanol content and the oxygen (O2) content in beer, the biomass in a bioreactor, and the cellular structures of PU foam produced in a pilot production plant.
Optical methods play an important role in process analytical technologies (PAT). Four examples of optical process and quality sensing (OPQS) are presented, which are based on three important experimental techniques: near- infrared absorption, luminescence quenching, and a novel method, photon density wave (PDW) spectroscopy. These are used to evaluate four process and quality parameters related to beer brewing and polyurethane (PU) foaming processes: the ethanol content and the oxygen (O-2) content in beer, the biomass in a bioreactor, and the cellular structures of PU foam produced in a pilot production plant
On the basis of absorption measurements in the near-infrared (NIR) spectral range, a new method for the quantification of the ethanol content of beer is presented. Instead of the multivariate calibration models most commonly employed in NIR spectroscopic works, we use interpretive difference spectroscopy: Two wavelengths are selected according to the assignment of the absorption bands of the main substances of content of beer in the NIR region, and the difference between the absorbances at these wavelengths is used for ethanol quantification. Absorption spectra of the dominating beer ingredients are discussed and the calibration procedure with ethanol/water mixtures is shown. Robustness against the carbohydrate content of beer samples was demonstrated by analyzing solutions of ethanol and maltose in water. Validation of the method was performed with various beer samples with an ethanol concentration range between 0.5 and 7.7 vol %. The pertinent advantage of the procedure developed in this work is the indication that the results are independent from seasonal variations of the ingredients, which is of high interest for products with natural ingredients such as beer
Precision agriculture (PA) strongly relies on spatially differentiated sensor information. Handheld instruments based on laser-induced breakdown spectroscopy (LIBS) are a promising sensor technique for the in-field determination of various soil parameters. In this work, the potential of handheld LIBS for the determination of the total mass fractions of the major nutrients Ca, K, Mg, N, P and the trace nutrients Mn, Fe was evaluated. Additionally, other soil parameters, such as humus content, soil pH value and plant available P content, were determined. Since the quantification of nutrients by LIBS depends strongly on the soil matrix, various multivariate regression methods were used for calibration and prediction. These include partial least squares regression (PLSR), least absolute shrinkage and selection operator regression (Lasso), and Gaussian process regression (GPR). The best prediction results were obtained for Ca, K, Mg and Fe. The coefficients of determination obtained for other nutrients were smaller. This is due to much lower concentrations in the case of Mn, while the low number of lines and very weak intensities are the reason for the deviation of N and P. Soil parameters that are not directly related to one element, such as pH, could also be predicted. Lasso and GPR yielded slightly better results than PLSR. Additionally, several methods of data pretreatment were investigated.
Precision agriculture (PA) strongly relies on spatially differentiated sensor information. Handheld instruments based on laser-induced breakdown spectroscopy (LIBS) are a promising sensor technique for the in-field determination of various soil parameters. In this work, the potential of handheld LIBS for the determination of the total mass fractions of the major nutrients Ca, K, Mg, N, P and the trace nutrients Mn, Fe was evaluated. Additionally, other soil parameters, such as humus content, soil pH value and plant available P content, were determined. Since the quantification of nutrients by LIBS depends strongly on the soil matrix, various multivariate regression methods were used for calibration and prediction. These include partial least squares regression (PLSR), least absolute shrinkage and selection operator regression (Lasso), and Gaussian process regression (GPR). The best prediction results were obtained for Ca, K, Mg and Fe. The coefficients of determination obtained for other nutrients were smaller. This is due to much lower concentrations in the case of Mn, while the low number of lines and very weak intensities are the reason for the deviation of N and P. Soil parameters that are not directly related to one element, such as pH, could also be predicted. Lasso and GPR yielded slightly better results than PLSR. Additionally, several methods of data pretreatment were investigated.
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.
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.
In precision agriculture, the estimation of soil parameters via sensors and the creation of nutrient maps are a prerequisite for farmers to take targeted measures such as spatially resolved fertilization. In this work, 68 soil samples uniformly distributed over a field near Bonn are investigated using laser-induced breakdown spectroscopy (LIBS). These investigations include the determination of the total contents of macro- and micronutrients as well as further soil parameters such as soil pH, soil organic matter (SOM) content, and soil texture. The applied LIBS instruments are a handheld and a platform spectrometer, which potentially allows for the single-point measurement and scanning of whole fields, respectively. Their results are compared with a high-resolution lab spectrometer. The prediction of soil parameters was based on multivariate methods. Different feature selection methods and regression methods like PLS, PCR, SVM, Lasso, and Gaussian processes were tested and compared. While good predictions were obtained for Ca, Mg, P, Mn, Cu, and silt content, excellent predictions were obtained for K, Fe, and clay content. The comparison of the three different spectrometers showed that although the lab spectrometer gives the best results, measurements with both field spectrometers also yield good results. This allows for a method transfer to the in-field measurements.
Six-color time-resolved forster resonance energy transfer for ultrasensitive multiplexed biosensing
(2013)
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.
Multiplexed diagnostics and spectroscopic ruler applications with terbium to quantum dots FRET
(2008)
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 Eu3+ 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 Forster 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.
Quantum dots (QDs) are common as luminescing markers for imaging in biological applications because their optical properties seem to be inert against their surrounding solvent. This, together with broad and strong absorption bands and intense, sharp tuneable luminescence bands, makes them interesting candidates for methods utilizing Forster Resonance Energy Transfer (FRET), e. g. for sensitive homogeneous fluoroimmunoassays (FIA). In this work we demonstrate energy transfer from Eu3+-trisbipyridin (Eu-TBP) donors to CdSe-ZnS-QD acceptors in solutions with and without serum. The QDs are commercially available CdSe-ZnS core-shell particles emitting at 655 nm (QD655). The FRET system was achieved by the binding of the streptavidin conjugated donors with the biotin conjugated acceptors. After excitation of Eu-TBP and as result of the energy transfer, the luminescence of the QD655 acceptors also showed lengthened decay times like the donors. The energy transfer efficiency, as calculated from the decay times of the bound and the unbound components, amounted to 37%. The Forster-radius, estimated from the absorption and emission bands, was ca. 77Å. The effective binding ratio, which not only depends on the ratio of binding pairs but also on unspecific binding, was obtained from the donor emission dependent on the concentration. As serum promotes unspecific binding, the overall FRET efficiency of the assay was reduced. We conclude that QDs are good substitutes for acceptors in FRET if combined with slow decay donors like Europium. The investigation of the influence of the serum provides guidance towards improving binding properties of QD assays.
pH sensing in living cells represents one of the most prominent topics in biochemistry and physiology. In this study we performed one-photon and two-photon time-domain fluorescence lifetime imaging with a laser-scanning microscope using the time-correlated single-photon counting technique for imaging intracellular pH levels. The suitability of different commercial fluorescence dyes for lifetime-based pH sensing is discussed on the basis of in vitro as well of in situ measurements. Although the tested dyes are suitable for intensity-based ratiometric measurements, for lifetime- based techniques in the time-domain so far only BCECF seems to meet the requirements of reliable intracellular pH recordings in living cells.
Two-photon fluorescence lifetime imaging of intracellular chloride in cockroach salivary glands
(2009)
The performance of a home-built tunable diode laser (TDL) spectrometer, aimed at multi-line detection of carbon dioxide, has been evaluated and optimized. In the regime of the (3001)(III) <-- (000) band of (CO2)-C-12 around 1.6 mum, the dominating isotope species (CO2)-C-12, (CO2)-C-13, and (COO)-C-12-O-18-O-16 were detected simultaneously without interference by water vapor. Detection limits in the range of few ppmv were obtained for each species utilizing wavelength modulation (WM) spectroscopy with balanced detection in a long-path absorption cell set-up. High sensitivity in conjunction with high precision-typically +/-1% and +/-6% for 3% and 0.7% of CO2, respectively-renders this experimental approach a promising analytical concept for isotope-ratio determination of carbon dioxide in soil and breath gas. For a moderate (CO2)-C-12 line, the pressure dependence of the line profile was characterized in detail, to account for pressure effects on sensitive measurements
The performance of a home-built tunable diode laser (TDL) spectrometer, aimed at multi-line detection of carbon dioxide, has been evaluated and optimized. In the regime of the (30<SUP>0</SUP>1)<SUB>III</SUB> / (000) band of <SUP>12</SUP>CO<SUB>2</SUB> around 1.6 μm, the dominating isotope species <SUP>12</SUP>CO<SUB>2</SUB>, <SUP>13</SUP>CO<SUB>2</SUB>, and <SUP>12</SUP>C<SUP>18</SUP>O<SUP>16</SUP>O were detected simultaneously without interference by water vapor. Detection limits in the range of few ppmv were obtained for each species utilizing wavelength modulation (WM) spectroscopy with balanced detection in a long-path absorption cell set-up. High sensitivity in conjunction with high precision —typically ±1‰ and ±6‰ for 3% and 0.7% of CO<SUB>2</SUB>, respectively— renders this experimental approach a promising analytical concept for isotope-ratio determination of carbon dioxide in soil and breath gas. For a moderate <SUP>12</SUP>CO<SUB>2</SUB> line, the pressure dependence of the line profile was characterized in detail, to account for pressure effects on sensitive measurements.
The performance of a home-built tunable diode laser (TDL) spectrometer has been optimized regarding multi-line detection of carbon dioxide in natural gases. In the regime of the (30<SUP>0</SUP>1)<SUB>III</SUB> ← (000) band of <SUP>12</SUP>CO<SUB>2</SUB> around 1.6 μm, the dominating isotope species <SUP>12</SUP>CO<SUB>2</SUB>, <SUP>13</SUP>CO<SUB>2</SUB>, and <SUP>12</SUP>C<SUP>18</SUP>O<SUP>16</SUP>O were detected simultaneously. In contrast to most established techniques, selective measurements are performed without any sample preparation. This is possible since the CO<SUB>2</SUB> detection is free of interference from water, ubiquitous in natural gases. Detection limits in the range of a few ppmv were obtained for each species utilizing wavelength modulation (WM) spectroscopy with balanced detection in a long-path absorption cell set-up. Linear calibration plots cover a dynamic range of four orders of magnitude, allowing for quantitative CO<SUB>2</SUB> detection in various samples, like soil and breath gas. High isotopic resolution enables the excellent selectivity, sensitivity, and stability of the chosen analytical concept. The obtained isotopic resolution of typically ± 1.0 ‰ and ± 1.5 ‰ (for 3 vol. % and 0.7 vol. % of CO<SUB>2</SUB>, respectively) offers a promising analytical tool for isotope-ratio determination of carbon dioxide in soil gas. Preliminary experiments on soil respiration for the first time combine the on-line quantification of the overall carbon dioxide content with an optode sensor and isotopic determination (TDL system) of natural gas species.
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.
Near-infrared (NIR) absorption spectroscopy with tunable diode lasers allows the simultaneous detection of the three most important isotopologues of carbon dioxide (<SUP>12</SUP>CO<SUB>2</SUB>, <SUP>13</SUP>CO<SUB>2</SUB>, <SUP>12</SUP>C<SUP>18</SUP>O<SUP>16</SUP>O) and carbon monoxide (<SUP>12</SUP>CO, <SUP>13</SUP>CO, <SUP>12</SUP>C<SUP>18</SUP>O). The flexible and compact fiber-optic tunable diode laser absorption spectrometer (TDLAS) allows selective measurements of CO<SUB>2</SUB> and CO with high isotopic resolution without sample preparation since there is no interference with water vapour. For each species, linear calibration plots with a dynamic range of four orders of magnitude and detection limits (LOD) in the range of a few ppm were obtained utilizing wavelength modulation spectroscopy (WMS) with balanced detection in a Herriott-type multipass cell. The high performance of the apparatus is illustrated by fill-evacuation-refill cycles.