Stand-off Hyperspectral Raman Imaging and Random Decision Forest Classification: A Potent Duo for the Fast, Remote Identification of Explosives.

In this study, we present a stand-off hyperspectral Raman imager (HSRI) for the fast detection and classification of different explosives at a distance of 15 m. The hyperspectral image cube is created by using a liquid crystal tunable filter (LCTF) to select a specific Raman shift and sequentially imaging spectral images onto an intensified CCD camera. The laser beam is expanded to illuminate the field of view of the HSRI and thereby improves large area scanning of suspicious surfaces. The collected hyperspectral image cube (HSI) is evaluated and classified using a random decision forest (RDF) algorithm. The RDF is trained with a training set of mg-amounts of different explosives, i.e., TNT, RDX, PETN, NaClO3, and NH4NO3, on an artificial aluminum substrate. The resulting classification is validated, and variable importance is used to optimize the RDF using spectral descriptors, effectively reducing the dimensionality of the data set. Using the gained information, a faster acquisition and calculation mode can be designed, giving improved results in classification at a much higher repetition rate.

In-Line Ultrasound-Enhanced Raman Spectroscopy Allows for Highly Sensitive Analysis with Improved Selectivity in Suspensions.

Raman spectroscopy is a nondestructive characterization method offering chemical-specific information. However, the cross-section of inelastically (Raman) scattered light is very low compared to elastically (Rayleigh) scattered light, resulting in weak signal intensities in Raman spectroscopy. Despite providing crucial information in off-line measurements, it usually is not sensitive enough for efficient, in-line process control in conjunction with low particle concentrations. To overcome this limitation, two custom-made 1.4404 stainless-steel prototype add-ons were developed for in-line Raman probes that enable ultrasound particle manipulation and thus concentration of particles in suspensions in the focus of the Raman excitation laser. Depending on size and density differences between particles and the carrier medium, particles are typically caught in the nodal planes of a quasi-standing wave field formed in an acoustic resonator in front of the sensor. Two arrangements were realized with regard to the propagation direction of the ultrasonic wave relative to the propagation direction of the laser. The parallel arrangement improved the limit of detection (LOD) by a factor of ≈30. In addition to increased sensitivity, the perpendicular arrangement offers increased selectivity: modifying the frequency of the ultrasonic wave field allows the liquid or solid phase to be moved into the focus of the Raman laser. The combination of in-line Raman spectroscopy with ultrasound particle manipulation holds promise to push the limits of conventional Raman spectroscopy, hence broadening its field of application to areas where previously Raman spectroscopy has not had sufficient sensitivity for accurate, in-line detection.

An Acoustic Trap for Bead Injection Attenuated Total Reflection Infrared Spectroscopy.

In this work, we introduce a system combining an acoustic trap for bead injection with attenuated total reflection (ATR) infrared (IR) spectroscopy. By mounting an acoustofluidic cell hosting an ultrasound source on top of a custom-built ATR fixture we were able to trap beads labeled with the enzyme alkaline phosphatase without requiring any mechanical retention elements. Sequential injection analysis was employed for reproducible sample handling and bead injection into the acoustic trap. To showcase potential applications of the presented setup for kinetic studies, we monitored the conversion of p-nitrophenylphosphate into p-nitrophenol and phosphate via beads carrying the immobilized enzyme using ATR-IR spectroscopy. Retaining the labeled beads via ultrasound particle manipulation resulted in excellent experimental reproducibility (relative standard deviation, 3.91%). It was demonstrated that trapped beads remained stably restrained with up to eight cell volumes of liquid passing through the acoustofluidic cell. Beads could be discarded in a straightforward manner by switching off the ultrasound, in contrast to systems containing mechanical retention elements, which require backflushing. Multiple experiments were performed by employing different substrate concentrations with the same batch of trapped beads as well as varying the amount of enzyme present in the cell, enabling enzyme kinetic studies and emphasizing the application of the proposed setup in studies where enzymatic reuse is desired. This proves the potential of the acoustic trap combined with ATR-IR spectroscopy to monitor the activity of immobilized enzymes and its ability to perform complex bead-based assays.

Enhanced mid-infrared multi-bounce ATR spectroscopy for online detection of hydrogen peroxide using a supercontinuum laser.

A compact multi-bounce attenuated total reflection (ATR) probe combined with a Fabry-Pérot filter spectrometer (FPFS) has been developed for detection of hydrogen peroxide used for oxidative gas scrubbing operating in the mid-infrared (MIR) spectral region. A novel MIR supercontinuum light source is employed to enhance the quantification capabilities of the sensor and is compared to a classical thermal emitter. An improvement of a factor of 4 in noise and approximately a factor of 3 in limit of detection is shown in this study allowing fast inline detection of aqueous hydrogen peroxide solutions around 0.1%.

On-line monitoring of methanol and methyl formate in the exhaust gas of an industrial formaldehyde production plant by a mid-IR gas sensor based on tunable Fabry-Pérot filter technology.

On-line monitoring of key chemicals in an industrial production plant ensures economic operation, guarantees the desired product quality, and provides additional in-depth information on the involved chemical processes. For that purpose, rapid, rugged, and flexible measurement systems at reasonable cost are required. Here, we present the application of a flexible mid-IR filtometer for industrial gas sensing. The developed prototype consists of a modulated thermal infrared source, a temperature-controlled gas cell for absorption measurement and an integrated device consisting of a Fabry-Pérot interferometer and a pyroelectric mid-IR detector. The prototype was calibrated in the research laboratory at TU Wien for measuring methanol and methyl formate in the concentration ranges from 660 to 4390 and 747 to 4610 ppmV. Subsequently, the prototype was transferred and installed at the project partner Metadynea Austria GmbH and linked to their Process Control System via a dedicated micro-controller and used for on-line monitoring of the process off-gas. Up to five process streams were sequentially monitored in a fully automated manner. The obtained readings for methanol and methyl formate concentrations provided useful information on the efficiency and correct functioning of the process plant. Of special interest for industry is the now added capability to monitor the start-up phase and process irregularities with high time resolution (5 s).

Sequential lignin depolymerization by combination of biocatalytic and formic acid/formate treatment steps.

Lignin, a complex three-dimensional amorphous polymer, is considered to be a potential natural renewable resource for the production of low-molecular-weight aromatic compounds. In the present study, a novel sequential lignin treatment method consisting of a biocatalytic oxidation step followed by a formic acid-induced lignin depolymerization step was developed and optimized using response surface methodology. The biocatalytic step employed a laccase mediator system using the redox mediator 1-hydroxybenzotriazole. Laccases were immobilized on superparamagnetic nanoparticles using a sorption-assisted surface conjugation method allowing easy separation and reuse of the biocatalysts after treatment. Under optimized conditions, as much as 45 wt% of lignin could be solubilized either in aqueous solution after the first treatment or in ethyl acetate after the second (chemical) treatment. The solubilized products were found to be mainly low-molecular-weight aromatic monomers and oligomers. The process might be used for the production of low-molecular-weight soluble aromatic products that can be purified and/or upgraded applying further downstream processes.

Production of superparamagnetic nanobiocatalysts for green chemistry applications.

Immobilization of enzymes on solid supports is a convenient method for increasing enzymatic stability and enabling enzyme reuse. In the present work, a sorption-assisted surface conjugation method was developed and optimized to immobilize enzymes on the surface of superparamagnetic nanoparticles. An oxidative enzyme, i.e., laccase from Trametes versicolor was used as model enzyme. The immobilization method consists of the production of superparamagnetic nanoparticles by co-precipitation of FeCl2 and FeCl3. Subsequently, the particle surface is modified with an organosilane containing an amino group. Next, the enzymes are adsorbed on the particle surface before a cross-linking agent, i.e., glutaraldehyde is added which links the amino groups on the particle surface with the amino groups of the enzymes and leads to internal cross-linking of the enzymes as well. The method was optimized using response surface methodology regarding optimal enzyme and glutaraldehyde amounts, pH, and reaction times. Results allowed formulation of biocatalysts having high specific enzymatic activity and improved stability. The biocatalysts showed considerably higher stability compared with the dissolved enzymes over a pH range from 3 to 9 and in the presence of several chemical denaturants. To demonstrate the reusability of the immobilized enzymes, they were applied as catalysts for the production of a phenoxazinone dye. Virtually, 100 % of the precursor was transformed to the dye in each of the ten conducted reaction cycles while on average 84.5 % of the enzymatic activity present at the beginning of a reaction cycle was retained after each cycle highlighting the considerable potential of superparamagnetic biocatalysts for application in industrial processes.

Laccases to take on the challenge of emerging organic contaminants in wastewater.

The removal of emerging organic contaminants from municipal wastewater poses a major challenge unsatisfactorily addressed by present wastewater treatment processes. Enzyme-catalyzed transformation of emerging organic contaminants (EOC) has been proposed as a possible solution to this major environmental issue more than a decade ago. Especially, laccases gained interest in this context in recent years due to their broad substrate range and since they only need molecular oxygen as a cosubstrate. In order to ensure the stability of the enzymes and allow their retention and reuse, either immobilization or insolubilization of the biocatalysts seems to be the prerequisite for continuous wastewater treatment applications. The present review summarizes the research conducted on EOC transformation with laccases and presents an overview of the possible immobilization techniques. The goal is to assess the state of the art and identify the next necessary steps that have to be undertaken in order to implement laccases as a tertiary wastewater treatment process in sewage treatment plants.

Immobilization of defined laccase combinations for enhanced oxidation of phenolic contaminants.

Immobilization is an important method to increase enzyme stability and allow enzyme reuse. One interesting application in the field of environmental biotechnology is the immobilization of laccase to eliminate phenolic contaminants via oxidation. Fumed silica nanoparticles have interesting potential as support material for laccase immobilization via sorption-assisted immobilization in the perspective of applications such as the elimination of micropollutants in aqueous phases. Based on these facts, the present work aimed to formulate laccase-nanoparticle conjugates with defined laccase combinations in order to obtain nanobiocatalysts, which are active over a broad range of pH values and possess a large substrate spectrum to suitably address pollution by multiple contaminants. A multi-enzymatic approach was investigated by immobilizing five different types of laccases originating from a Thielavia genus, Coriolopsis polyzona, Cerrena unicolor, Pleurotus ostreatus, and Trametes versicolor onto fumed silica nanoparticles, separately and in combinations. The laccases differed concerning their pH optima and substrate affinity. Exploiting their differences allowed the formulation of tailor-made nanobiocatalysts. In particular, the production of a nanobiocatalyst could be achieved that retained a higher percentage of its relative activity over the tested pH range (3-7) compared to the dissolved or separately immobilized enzymes. Furthermore, a nanobiocatalyst could be formulated able to oxidize a broader substrate range than the dissolved or separately immobilized enzymes. Thereby, the potential of the nanobiocatalyst for application in biochemical oxidation applications such as the elimination of multiple target pollutants in biologically treated wastewater has been illustrated.

Advanced enzymatic elimination of phenolic contaminants in wastewater: a nano approach at field scale.

The removal of recalcitrant chemicals in wastewater treatment systems is an increasingly relevant issue in industrialized countries. The elimination of persistent xenobiotics such as endocrine-disrupting chemicals (EDCs) emitted by municipal and industrial sewage treatment plants remains an unsolved challenge. The existing efficacious physico-chemical methods, such as advanced oxidation processes, are resource-intensive technologies. In this work, we investigated the possibility to remove phenolic EDCs [i.e., bisphenol A (BPA)] by means of a less energy and chemical consuming technology. To that end, cheap and resistant oxidative enzymes, i.e., laccases, were immobilized onto silica nanoparticles. The resulting nanobiocatalyst produced at kilogram scale was demonstrated to possess a broad substrate spectrum regarding the degradation of recalcitrant pollutants. This nanobiocatalyst was applied in a membrane reactor at technical scale for tertiary wastewater treatment. The system efficiently removed BPA and the results of long-term field tests illustrated the potential of fumed silica nanoparticles/laccase composites for advanced biological wastewater treatment.

Determination of oxidoreductase activity using a high-throughput microplate respiratory measurement.

High-throughput multiparallel activity profiling for oxygen consuming cell layers has been recently developed for extracellular flux analysis. This technology has great potential for determining the enzymatic activity of oxidoreductases (i.e., laccase) both in vivo and in vitro, which is usually measured using photometrical tests monitoring the colored oxidation products. Improvements in terms of sample throughput, comparability, and gain of information (i.e., stoichiometry, electron transfer rate) can be achieved by means of a multiwell plate-based fluorimetric oxygen sensor. In the present study, various laccases have been applied to develop protocols that allow the multiparallel measurement of O(2)-consumption by enzymatic reactions. The developed and validated method enables the comparative quantitation of laccase characteristics (i.e., profiles of activity at various pH values) and minimizes the time it usually takes to collect respiratory data of oxygen-consuming enzymes. Furthermore, the possibility to assess differences between single and multisubstrate kinetics of laccases has been demonstrated.

Multi-catalysis reactions: new prospects and challenges of biotechnology to valorize lignin.

Considerable effort has been dedicated to the chemical depolymerization of lignin, a biopolymer constituting a possible renewable source for aromatic value-added chemicals. However, these efforts yielded limited success up until now. Efficient lignin conversion might necessitate novel catalysts enabling new types of reactions. The use of multiple catalysts, including a combination of biocatalysts, might be necessary. New perspectives for the combination of bio- and inorganic catalysts in one-pot reactions are emerging, thanks to green chemistry-driven advances in enzyme engineering and immobilization and new chemical catalyst design. Such combinations could offer several advantages, especially by reducing time and yield losses associated with the isolation and purification of the reaction products, but also represent a big challenge since the optimal reaction conditions of bio- and chemical catalysis reactions are often different. This mini-review gives an overview of bio- and inorganic catalysts having the potential to be used in combination for lignin depolymerization. We also discuss key aspects to consider when combining these catalysts in one-pot reactions.

Microbeam bending of hydrated human cortical bone lamellae from the central region of the body of femur shows viscoelastic behaviour.

Bone is a biological tissue with unique mechanical properties, owing to a complex hierarchical structure ranging from the nanoscale up to the macroscale. To better understand bone mechanics, investigation of mechanical properties of all structural elements at every hierarchical level and how they interact is necessary. Testing of bone structures at the lower microscale, e.g. bone lamellae, has been least performed and remains a challenge. Focused ion beam (FIB) milling is an attractive technique for machining microscopic samples from bone material and performing mechanical testing at the microscale using atomic force microscopy (AFM) and nanoindentation setups. So far, reported studies at this length scale have been performed on bone samples of animal origin, mostly in a dehydrated state, except for one study. Here we present an AFM-based microbeam bending method for performing bending measurements in both dehydrated and rehydrated conditions at the microscale. Single lamella bone microbeams of four human donors, aged 65-94 yrs, were machined via FIB and tested both in air and fully submerged in Hank's Balanced Salt Solution (HBSS) to investigate the effect of (de)hydration and to a certain extent, of age, on bone mechanics. Bending moduli were found to reduce up to 5 times after 2 h of rehydration and no trend of change in bending moduli with respect to age could be observed. Mechanical behavior changed from almost purely elastic to viscoelastic upon rehydration and a trend of lower dissipated energy in samples from older donors could be observed in the rehydrated state. These results confirm directly the importance of water for the mechanical properties of bone tissue at the microscale. Moreover, the trend of lowered capability of energy dissipation in older donors may contribute to a decrease of fracture toughness and thus an increase in bone fragility with age.

Stand-off spatial offset Raman spectroscopy for the detection of concealed content in distant objects.

A pulsed (4.4 ns pulse length) frequency-doubled Nd:YAG laser operated at 10 Hz was used to generate Raman scattering of samples at a distance of 12 m. The scattered light was collected by a 6 in. telescope, and the Raman spectrum was recorded using an Acton SP-2750 spectrograph coupled to a gated intensified charge-coupled device (ICCD) detector. Applying a spatial offset between the point where the laser hit the sample and the focus of the telescope on the sample enabled collection of Raman photons that were predominantly generated inside the sample and not from its surface. This is especially effective when the content of concealed objects should be analyzed. High-quality Raman spectra could be recorded, within 10 s of data acquisition, from a solid (NaClO(3)) as well as a liquid (isopropyl alcohol) placed inside a 1.5 mm thick opaque low-density polyethylene (LDPE) plastic bottle. The applied spatial offset was also advantageous in cases where the surface of the container was highly fluorescent. In such a situation, Raman spectra of the sample could not be recorded when the sampling volume (telescope observation field) coincided with the focus of the excitation laser. However, with the use of a spatial offset of some millimeters, a clear Raman spectrum of the content (isopropyl alcohol) in a strongly fluorescent plastic container was obtained.

Adsorption of transgenic insecticidal Cry1Ab protein to silica particles. Effects on transport and bioactivity.

Bt crops are genetically modified to be resistant against insect pests by expressing insecticidal Cry proteins. The processes governing the fate and bioavailability of the expressed transgenic Cry proteins in soils are poorly understood. We studied adsorption of Cry1Ab to negatively charged silica (SiO(2)) particles, a major soil constituent and a model for negatively charged mineral surfaces, at pH 5 to 10 and ionic strengths I = 10 mM to 250 mM, both in solution depletion and saturated column transport experiments. Cry1Ab-SiO(2) interactions were dominated by patch-controlled electrostatic attraction (PCEA), as evident from increasing Cry1Ab attraction to SiO(2) with decreasing I at pH at which both Cry1Ab and SiO(2) were net negatively charged. Experimental and modeling evidence is provided that the surface heterogeneity of SiO(2) particles modulated PCEA, leading to a fraction of adsorption sites with slow Cry1Ab desorption kinetics. Desorption rates from these sites increased upon increasing the solution pH. In toxicity bioassays, we demonstrated that Cry1Ab retained insecticidal activity when adsorbed to SiO(2), suggesting high protein conformational stability during adsorption-desorption cycles. Models predicting Cry1A protein adsorption in soils therefore need to account for combined effects of the nonuniform protein surface charge distribution and of sorbent surface heterogeneity.

In Situ Quantification of Polyhydroxybutyrate in Photobioreactor Cultivations of Synechocystis sp. Using an Ultrasound-Enhanced ATR-FTIR Spectroscopy Probe.

Polyhydroxybutyrate (PHB) is a very promising alternative to most petroleum-based plastics with the huge advantage of biodegradability. Biotechnological production processes utilizing cyanobacteria as sustainable source of PHB require fast in situ process analytical technology (PAT) tools for sophisticated process monitoring. Spectroscopic probes supported by ultrasound particle traps provide a powerful technology for in-line, nondestructive, and real-time process analytics in photobioreactors. This work shows the great potential of using ultrasound particle manipulation to improve spectroscopic attenuated total reflection Fourier-transformed mid-infrared (ATR-FTIR) spectra as a monitoring tool for PHB production processes in photobioreactors.

Comparing mapping and direct hyperspectral imaging in stand-off Raman spectroscopy for remote material identification.

Stand-off Raman spectroscopy offers a highly selective technique to probe unknown substances from a safe distance. Often, it is necessary to scan large areas of interest. This can be done by pointwise imaging (PI), that is, spectra are sequentially acquired from an array of points over the region of interest (point-by-point mapping). Alternatively, in this paper a direct hyperspectral Raman imager is presented, where a defocused laser beam illuminates a wide area of the sample and the Raman scattered light is collected from the whole field of view (FOV) at once as a spectral snapshot filtered by a liquid crystal tunable filter to select a specific Raman shift. Both techniques are compared in terms of achievable FOV, spectral resolution, signal-to-noise performance, and time consumption during a measurement at stand-off distance of 15 m. The HSRI showed superior spectral resolution and signal-to-noise ratio, while more than doubling the FOV of the PI at laser power densities reduced by a factor of 277 at the target. Further, the output hyperspectral image data cube can be processed with state of the art chemometric algorithms like vertex component analysis in order to get a simple deterministic false color image showing the chemical composition of the target. This is shown for an artificial polymer sample, measured at a distance of 15 m.

Depth profiling for the identification of unknown substances and concealed content at remote distances using time-resolved stand-off Raman spectroscopy.

Time-resolved stand-off Raman spectroscopy was used to determine both the position and identity of substances relative to each other at remote distances (up to tens of meters). Spectral information of three xylene isomers, toluene, and sodium chlorate was obtained at a distance of 12 m from the setup. Pairs and triplets of these samples were placed at varying distances (10-60 cm) relative to each other. Via the photon time of flight the distance between the individual samples was determined to an accuracy of 7% (corresponding to a few cm) of the physically measured distance. Furthermore, at a distance of 40 m, time-resolved Raman depth profiling was used to detect sodium chlorate in a white plastic container that was non-transparent to the human eye. The combination of the ranging capabilities of Raman LIDAR (sample location usually determined using prior knowledge of the analyte of interest) with stand-off Raman spectroscopy (analyte detection at remote distances) provides the capability for depth profile identification of unknown substances and analysis of concealed content in distant objects. To achieve these results, a 532 nm laser with a pulse length of 4.4 ns was synchronized to an intensified charge-coupled device camera with a minimum gate width of 500 ps. For automated data analysis a multivariate curve resolution algorithm was employed.

Production of a robust nanobiocatalyst for municipal wastewater treatment.

Immobilization is a fundamental method to improve both enzyme activity and stability. In the present work, the process previously described for immobilizing laccase - an enzyme oxidizing phenolic compounds - onto fumed silica was optimized, in order to efficiently produce industrially relevant amounts of a nanobiocatalyst for biological micropollutant elimination, whilst saving 80% of surface modification agent (3-aminopropyl triethoxy silane) and 90% of cross-linker (glutaraldehyde). Minimized losses during preparation and favorable effects of immobilization yielded conjugates with drastically increased enzymatic activity (164% of invested activity). Long-term stability and activity regarding bisphenol A (2,2-bis(4-hydroxyphenyl)propane) removal of the synthesized biocatalyst were assessed under application-relevant conditions. With 81.1±0.4% residual activity after 7 days, stability of conjugates was drastically higher than of free laccase, which showed virtually no activity after 1.5 days. These results illustrate the huge potential of fumed silica nanoparticles/laccase-composites for innovative biological wastewater treatment.

[Lameness detection in cows by accelerometric measurement of motion at walk]. / Lahmheitserkennung bei Kühen durch Messung der Bewegung im Schritt mittels Accelerometer.

Locomotion scoring (Sprecher et al., 1997) and measurements with a tri-axial accelerometric device were performed in 15 cows before and after digit amputation (Group 1_AMP) as well as in 26 lame cows with different lameness scores (Group 1_LAHM). The evaluated data of Group 1_AMP, documented directly before and on days 2, 5, 10 and 14 after amputation were compared with reference values of 16 non-lame cows (Group 2). For this purpose the Root Mean Square (RMS) as well as the mean minimum (gMin) and maximum (gMax) acceleration were calculated for each measurement (n = 117). Basing on this data, forecast models were performed to predict lameness scores as well as to differentiate between lame and non-lame cows in order to verify the suitability of accelerometry as a technical tool for automated lameness detection in cows. The preoperative locomotion score in Group 1_AMP averaged 4.2. During the period of convalescence, lameness improved significantly (p < 0.01) to a mean score of 1.9 on day 14 after amputation. Statistically, score 1 can be expected on day 20 after surgery (95% CI). On day 35 an amputated cow can be assumed to be non-lame with aprobability of 95%. Acceleration values showed different approximation to those recorded for the reference group. Forecast models enabled prediction of determined scores of lameness with each category of measurement (RMS, gMin, gMax) as well as considering the entire data set with an accuracy of up to 61.7%. Differentiation between lame and non-lame cows was successful with a percentage of up to 91.7%, depending on the applied data. The results of this study showed that accelerometry is a suitable technical tool for automated lameness detection in cows, especially using the acceleration values (RMS, gMin, gMax) and forecast models employed in this study.