Joanne V. Volponi

In vivo lipidomics using single-cell Raman spectroscopy

We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics.

Development of sensors for direct detection of organophosphates. Part I: Immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports.

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (< 1 mm2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol-gel process to create highly porous and stable silica matrices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol-gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol-gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.

Kinetics and mechanism of metal-organic framework thin film growth: Systematic investigation of HKUST-1 deposition on QCM electrodes.

We describe a systematic investigation of the factors controlling step-by-step growth of the metal–organic framework (MOF) [Cu3(btc)2(H2O)3]·xH2O (also known as HKUST-1), using quartz crystal microbalance (QCM) electrodes as an in situ probe of the reaction kinetics and mechanism. Electrodes coated with silica, alumina and gold functionalized with OH– and COOH–terminated self-assembled monolayers (SAMs) were employed to determine the effects of surface properties on nucleation. Deposition rates were measured using the high sensitivity available from QCM-D (D = dissipation) techniques to determine rate constants in the early stage of the process. Films were characterized using grazing incidence XRD, SEM, AFM, profilometry and reflection–absorption IR spectroscopy. The effects of reaction time, concentration, temperature and substrate on the deposition rates, film crystallinity and surface morphology were evaluated. The initial growth step, in which the surface is exposed to copper ions (in the form of an ethanolic solution of copper(II) acetate) is fast and independent of temperature, after which all subsequent steps are thermally activated over the temperature range 22–62 °C. Using these data, we propose a kinetic model for the Cu3(btc)2 growth on surfaces that includes rate constants for the individual steps. The magnitude of the activation energies, in particular the large entropy decrease, suggests an associative reaction with a tight transition state. The measured activation energies for the step-by-step MOF growth are an order of magnitude lower than the value previously reported for bulk Cu3(btc)2 crystals. Finally, the results of this investigation demonstrate that the QCM method is a powerful tool for quantitative, in situ monitoring of MOF growth in real time.

Development of sensors for direct detection of organophosphates. Part II : Sol-gel modified field effect transistor with immobilized organophosphate hydrolase

Abstract pH-sensitive field effect transistors (FET) were modified with organophosphate hydrolase (OPH) and used for direct detection of organophosphate compounds. OPH is the organophosphate degrading gene product isolated from Pseudomonas diminuta . OPH was selected as an alternative to acetylcholinesterase, which requires inhibition mode sensor operation, enzyme regeneration before reuse, long sample incubation times, and a constant source of acetylcholine substrate. OPH was covalently immobilized directly to the exposed silicon nitride gate insulator of the FET. Alternatively, silica microspheres of 20 or 200 nm were formed via a base catalyzed sol–gel process and were dip-coated onto the gate surface; enzyme was then covalently immobilized to this modified surface. All sensors were tested with paraoxon and displayed rapid response ( −6 molar. The 200 nm sol–gel gate modification enhanced the signal of enzyme-modified devices without effecting device pH sensitivity. Sensors were stored at 4°C in buffer and tested multiple times. Devices coated with 200 nm silica microspheres maintained significant enzymatic activity over a period of 10 weeks while uncoated devices lost all enzyme activity during the same period. The 20 nm sol–gel modification did not enhance device response or enzyme stability. Successful reuse of sensor chips was demonstrated after stripping inactive enzyme with an RF oxygen plasma system and reimmobilizing active enzyme.

The effect of allene addition on the structure of a rich C2H2/O2/Ar flame

Abstract We have studied the effects of adding allene (C 3 H 4 ) to a rich (φ = 1.67) C 2 H 2 /O 2 /Ar flame. Temperatures were measured by thermocouple and by OH laser-induced fluorescence. Stable species profiles were determined from mass spectrometer measurements using a quartz microprobe, and OH and CH concentrations were determined using LIF. The experiments were analyzed with the aid of a chemical kinetic model. The most noteworthy result of our experiments is that significant quantities of benzene appear in the flame with allene, whereas there is no detectable benzene in the pure acetylene flame. This result lends support to the theory that the reaction between two propargyl radicals is an important “cyclization step” in flames. Various aspects of the flame chemistry are discussed in depth.

The Oxidation of Allene in a Low-Pressure H2 / O2 / Ar-C3 H4 Flame

ABSTRACT We have studied the oxidation of allene in a rich ( φ = 1.5), low-pressure H2/O2/Ar flame to which one percent altene was added. Temperatures were measured by thermocouple and by OH laser-induced fluorescence. Stable species profiles were determined from mass spectrometer measurements using a quartz microprobe, and OH concentrations were determined using LIF. The experiments were analyzed with the aid of a chemical kinetic model. Significant quantities of methane and acetylene are formed early in the flame as a consequence of recombination of propargyl with hydrogen atoms, H + C2H3 (+ M) ↔ C3H4P H + C3H4P ↔CH3 + C2H2 H+CH3( + M) ↔ CH4( + M), where C3H4P is propyne. However, the main oxidation path appears to be C2H3 + H↔ C2H2 + H2 C3H2 + O2 ↔ products The underlying flame chemistry is discussed in depth, including the thermochemistry and molecular structure of various C3,H2 isomers.

Structure of endoglucanase Cel9A from the thermoacidophilic Alicyclobacillus acidocaldarius

The production of biofuels using biomass is an alternative route to support the growing global demand for energy and to also reduce the environmental problems caused by the burning of fossil fuels. Cellulases are likely to play an important role in the degradation of biomass and the production of sugars for subsequent fermentation to fuel. Here, the crystal structure of an endoglucanase, Cel9A, from Alicyclobacillus acidocaldarius (Aa_Cel9A) is reported which displays a modular architecture composed of an N-terminal Ig-like domain connected to the catalytic domain. This paper describes the overall structure and the detailed contacts between the two modules. Analysis suggests that the interaction involving the residues Gln13 (from the Ig-like module) and Phe439 (from the catalytic module) is important in maintaining the correct conformation of the catalytic module required for protein activity. Moreover, the Aa_Cel9A structure shows three metal-binding sites that are associated with the thermostability and/or substrate affinity of the enzyme.

Multiplex fluorometric assessment of nutrient limitation as a strategy for enhanced lipid enrichment and harvesting of Neochloris oleoabundans

Detailed in this study are the results of fluorometric assays used to assess the impact of gradual nutrient limitation versus punctuated nitrate limitation on the lipid content and morphology of Neochloris oleoabundans cells in batch culture. Punctuated nitrate limitation was imposed during pre‐log, log, late‐log, stationary, and senescent growth phases, and the cells were analyzed by bulk fluorescence emission, flow cytometry, and hyperspectral fluorescence imaging. In addition to intrinsic spectroscopic signatures provided by scatter and endogenous fluorescence, Nile Red staining was employed to monitor relative changes in lipid concentration. Analysis of the fluorescence images and temporal data sets was performed using multivariate curve resolution and fitting to logistic growth models to extract parameters of interest. The spectral components independently isolated from the image and temporal data sets showed close agreement with one another, especially relating to chlorophylls and Nile Red in polar and neutral lipid fractions, respectively. The fastest accumulation and highest total neutral lipid per cell and per chlorophyll were obtained with punctuated nitrate limitation during log phase growth on day 4 of culture. The presence of unbound chlorophyll in the resulting lipid bodies supports a membrane recycling TAG accumulation mechanism mediated by chloropolast–ER lipid exchange. Furthermore, an increase in cell size, indicated by forward scatter, was also found to correlate with increased neutral lipid, providing a size selection mechanism for passive harvest of algal cells at peak lipid enrichment. Biotechnol. Bioeng. 2012; 109: 2503–2512.

The structure and reaction mechanism of rich, non-sooting C2H2/O2/Ar flames

We have studied three rich, low-pressure C2H2/O2/Ar flames (O=1.03, 1.67, and 2.0, P=25 Torr). Thermocouples and OH laser-induced flourescence are used to measure the temperature. Quartz-microprobe sampling is used to measure concentrations of the stable species, LIF to measure concentrations of CH and OH, and multiphoton-excited fluorescence to measure H-atom concentrations. The measurements are compared with the predictions of a comprehensive chemical kinetic model. The agreement between model and experiment is generally good. Possible sources of the discrepancies are discussed. Analysis of the reaction mechanism clearly shows the importance of reactions of 1CH2 in rich acetylene flames, particularly in the formation of higher hydrocarbons.

Flame structure of C2H2−O2—Argon and C2H2−NO2—argon laminar premixed flames

Measurements of the profiles of gas composition and temperature and temperature are made in C2H2−O2−Ar and C2H2−NO2−Ar laminar premixed flames at a pressure of 25 torr. The composition of stable species is measured by probe sampling and mass spectrometric gas analysis, the composition of unstable species is measured by linear laser-induced fluorescence, and the temperature is measured by thermocouples on the burner surface and OH rotational spectra at positions above the burner. Measured flame structure is compared with flame modeling in order to develop a reaction mechanism for the C2H2−NO2−Ar flame. The comparison of the measured and experimental profiles for the two flames is good. Results for the O2 flame support an existing mechanism for C2H2 oxidation. The results for the previously unstudied NO2 flame support a reaction mechanism based on C2H2−O2 flame modeling, H2−NO2 flame modeling and H−C−N species interactions.