Development of a surface-enhanced raman spectroscopy method for the detection of benzodiazepines in urine

Benzodiazepines are among the most prescribed compounds for anti-anxiety and are present in many toxicological screens. These drugs are also prominent in the commission of drug facilitated sexual assaults due their effects on the central nervous system. Due to their potency, a low dose of these compounds is often administered to victims; therefore, the target detection limit for these compounds in biological samples is 10 ng/mL. Currently these compounds are predominantly analyzed using immunoassay techniques; however more specific screening methods are needed. ^ The goal of this dissertation was to develop a rapid, specific screening technique for benzodiazepines in urine samples utilizing surface-enhanced Raman spectroscopy (SERS), which has previously been shown be capable of to detect trace quantities of pharmaceutical compounds in aqueous solutions. Surface enhanced Raman spectroscopy has the advantage of overcoming the low sensitivity and fluorescence effects seen with conventional Raman spectroscopy. The spectra are obtained by applying an analyte onto a SERS-active metal substrate such as colloidal metal particles. SERS signals can be further increased with the addition of aggregate solutions. These agents cause the nanoparticles to amass and form hot-spots which increase the signal intensity. ^ In this work, the colloidal particles are spherical gold nanoparticles in aqueous solution with an average size of approximately 30 nm. The optimum aggregating agent for the detection of benzodiazepines was determined to be 16.7 mM MgCl2, providing the highest signal intensities at the lowest drug concentrations with limits of detection between 0.5 and 127 ng/mL. A supported liquid extraction technique was utilized as a rapid clean extraction for benzodiazepines from urine at a pH of 5.0, allowing for clean extraction with limits of detection between 6 and 640 ng/mL. It was shown that at this pH other drugs that are prevalent in urine samples can be removed providing the selective detection of the benzodiazepine of interest. ^ This technique has been shown to provide rapid (less than twenty minutes), sensitive, and specific detection of benzodiazepines at low concentrations in urine. It provides the forensic community with a sensitive and specific screening technique for the detection of benzodiazepines in drug facilitated assault cases.^

Rapid quantification and validation of lipid concentrations within liposomes

Quantification of the lipid content in liposomal adjuvants for subunit vaccine formulation is of extreme importance, since this concentration impacts both efficacy and stability. In this paper, we outline a high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method that allows for the rapid and simultaneous quantification of lipid concentrations within liposomal systems prepared by three liposomal manufacturing techniques (lipid film hydration, high shear mixing, and microfluidics). The ELSD system was used to quantify four lipids: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, dimethyldioctadecylammonium (DDA) bromide, and D-(+)-trehalose 6,6′-dibehenate (TDB). The developed method offers rapidity, high sensitivity, direct linearity, and a good consistency on the responses (R2 > 0.993 for the four lipids tested). The corresponding limit of detection (LOD) and limit of quantification (LOQ) were 0.11 and 0.36 mg/mL (DMPC), 0.02 and 0.80 mg/mL (cholesterol), 0.06 and 0.20 mg/mL (DDA), and 0.05 and 0.16 mg/mL (TDB), respectively. HPLC-ELSD was shown to be a rapid and effective method for the quantification of lipids within liposome formulations without the need for lipid extraction processes.

Development of a Surface-Enhanced Raman Spectroscopy Method for the Detection of Benzodiazepines in Urine

Benzodiazepines are among the most prescribed compounds for anti-anxiety and are present in many toxicological screens. These drugs are also prominent in the commission of drug facilitated sexual assaults due their effects on the central nervous system. Due to their potency, a low dose of these compounds is often administered to victims; therefore, the target detection limit for these compounds in biological samples is 10 ng/mL. Currently these compounds are predominantly analyzed using immunoassay techniques; however more specific screening methods are needed. The goal of this dissertation was to develop a rapid, specific screening technique for benzodiazepines in urine samples utilizing surface-enhanced Raman spectroscopy (SERS), which has previously been shown be capable of to detect trace quantities of pharmaceutical compounds in aqueous solutions. Surface enhanced Raman spectroscopy has the advantage of overcoming the low sensitivity and fluorescence effects seen with conventional Raman spectroscopy. The spectra are obtained by applying an analyte onto a SERS-active metal substrate such as colloidal metal particles. SERS signals can be further increased with the addition of aggregate solutions. These agents cause the nanoparticles to amass and form hot-spots which increase the signal intensity. In this work, the colloidal particles are spherical gold nanoparticles in aqueous solution with an average size of approximately 30 nm. The optimum aggregating agent for the detection of benzodiazepines was determined to be 16.7 mM MgCl2, providing the highest signal intensities at the lowest drug concentrations with limits of detection between 0.5 and 127 ng/mL. A supported liquid extraction technique was utilized as a rapid clean extraction for benzodiazepines from urine at a pH of 5.0, allowing for clean extraction with limits of detection between 6 and 640 ng/mL. It was shown that at this pH other drugs that are prevalent in urine samples can be removed providing the selective detection of the benzodiazepine of interest. This technique has been shown to provide rapid (less than twenty minutes), sensitive, and specific detection of benzodiazepines at low concentrations in urine. It provides the forensic community with a sensitive and specific screening technique for the detection of benzodiazepines in drug facilitated assault cases.

An Environment for Rapid Derivatives Design and Experimentation

In the highly competitive world of modern finance, new derivatives are continually required to take advantage of changes in financial markets, and to hedge businesses against new risks. The research described in this paper aims to accelerate the development and pricing of new derivatives in two different ways. Firstly, new derivatives can be specified mathematically within a general framework, enabling new mathematical formulae to be specified rather than just new parameter settings. This Generic Pricing Engine (GPE) is expressively powerful enough to specify a wide range of stand¬ard pricing engines. Secondly, the associated price simulation using the Monte Carlo method is accelerated using GPU or multicore hardware. The parallel implementation (in OpenCL) is automatically derived from the mathematical description of the derivative. As a test, for a Basket Option Pricing Engine (BOPE) generated using the GPE, on the largest problem size, an NVidia GPU runs the generated pricing engine at 45 times the speed of a sequential, specific hand-coded implementation of the same BOPE. Thus a user can more rapidly devise, simulate and experiment with new derivatives without actual programming.

Growth and spectroscopic characterizations: properties of Nd:LiLaMosub(2)Osub(8) single crystal fibers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of time-resolved spectroscopy as a method to monitor carotenoids present in tomato extract obtained using ultrasound treatment

Introduction Compounds exhibiting antioxidant activity have received much interest in the food industry because of their potential health benefits. Carotenoids such as lycopene, which in the human diet mainly derives from tomatoes (Solanum lycopersicum), have attracted much attention in this aspect and the study of their extraction, processing and storage procedures is of importance. Optical techniques potentially offer advantageous non-invasive and specific methods to monitor them. Objectives To obtain both fluorescence and Raman information to ascertain if ultrasound assisted extraction from tomato pulp has a detrimental effect on lycopene. Method Use of time-resolved fluorescence spectroscopy to monitor carotenoids in a hexane extract obtained from tomato pulp with application of ultrasound treatment (583 kHz). The resultant spectra were a combination of scattering and fluorescence. Because of their different timescales, decay associated spectra could be used to separate fluorescence and Raman information. This simultaneous acquisition of two complementary techniques was coupled with a very high time-resolution fluorescence lifetime measurement of the lycopene. Results Spectroscopic data showed the presence of phytofluene and chlorophyll in addition to lycopene in the tomato extract. The time-resolved spectral measurement containing both fluorescence and Raman data, coupled with high resolution time-resolved measurements, where a lifetime of ~5 ps was attributed to lycopene, indicated lycopene appeared unaltered by ultrasound treatment. Detrimental changes were, however, observed in both chlorophyll and phytofluene contributions. Conclusion Extracted lycopene appeared unaffected by ultrasound treatment, while other constituents (chlorophyll and phytofluene) were degraded.

A deformation model of flexible, high area-to-mass ratio debris under perturbations and validation method

A new type of space debris was recently discovered by Schildknecht in near -geosynchronous orbit (GEO). These objects were later identified as exhibiting properties associated with High Area-to-Mass ratio (HAMR) objects. According to their brightness magnitudes (light curve), high rotation rates and composition properties (albedo, amount of specular and diffuse reflection, colour, etc), it is thought that these objects are multilayer insulation (MLI). Observations have shown that this debris type is very sensitive to environmental disturbances, particularly solar radiation pressure, due to the fact that their shapes are easily deformed leading to changes in the Area-to-Mass ratio (AMR) over time. This thesis proposes a simple effective flexible model of the thin, deformable membrane with two different methods. Firstly, this debris is modelled with Finite Element Analysis (FEA) by using Bernoulli-Euler theory called “Bernoulli model”. The Bernoulli model is constructed with beam elements consisting 2 nodes and each node has six degrees of freedom (DoF). The mass of membrane is distributed in beam elements. Secondly, the debris based on multibody dynamics theory call “Multibody model” is modelled as a series of lump masses, connected through flexible joints, representing the flexibility of the membrane itself. The mass of the membrane, albeit low, is taken into account with lump masses in the joints. The dynamic equations for the masses, including the constraints defined by the connecting rigid rod, are derived using fundamental Newtonian mechanics. The physical properties of both flexible models required by the models (membrane density, reflectivity, composition, etc.), are assumed to be those of multilayer insulation. Both flexible membrane models are then propagated together with classical orbital and attitude equations of motion near GEO region to predict the orbital evolution under the perturbations of solar radiation pressure, Earth’s gravity field, luni-solar gravitational fields and self-shadowing effect. These results are then compared to two rigid body models (cannonball and flat rigid plate). In this investigation, when comparing with a rigid model, the evolutions of orbital elements of the flexible models indicate the difference of inclination and secular eccentricity evolutions, rapid irregular attitude motion and unstable cross-section area due to a deformation over time. Then, the Monte Carlo simulations by varying initial attitude dynamics and deformed angle are investigated and compared with rigid models over 100 days. As the results of the simulations, the different initial conditions provide unique orbital motions, which is significantly different in term of orbital motions of both rigid models. Furthermore, this thesis presents a methodology to determine the material dynamic properties of thin membranes and validates the deformation of the multibody model with real MLI materials. Experiments are performed in a high vacuum chamber (10-4 mbar) replicating space environment. A thin membrane is hinged at one end but free at the other. The free motion experiment, the first experiment, is a free vibration test to determine the damping coefficient and natural frequency of the thin membrane. In this test, the membrane is allowed to fall freely in the chamber with the motion tracked and captured through high velocity video frames. A Kalman filter technique is implemented in the tracking algorithm to reduce noise and increase the tracking accuracy of the oscillating motion. The forced motion experiment, the last test, is performed to determine the deformation characteristics of the object. A high power spotlight (500-2000W) is used to illuminate the MLI and the displacements are measured by means of a high resolution laser sensor. Finite Element Analysis (FEA) and multibody dynamics of the experimental setups are used for the validation of the flexible model by comparing with the experimental results of displacements and natural frequencies.

Inertial dissipation method applied to derive turbulent fluxed over the ocean during the Surface of the Ocean, Fluxes and Interactions with the Atmosphere Atlantic Stratocumulus Transition Experiment (SOFIA/ASTEX) and Structure des Echanges Mer-Atmosphere, Proprietes des Heterogeneites Oceaniques: Recherche Experimentale (SEMAPHORE) experiments with low to moderate wind speed

The transfer coefficients for momentum and heat have been determined for 10 m neutral wind speeds (U-10n) between 0 and 12 m/s using data from the Surface of the Ocean, Fluxes and Interactions with the Atmosphere (SOFIA) and Structure des Echanges Mer-Atmosphere, Proprietes des Heterogeneites Oceaniques: Recherche Experimentale (SEMAPHORE) experiments. The inertial dissipation method was applied to wind and pseudo virtual temperature spectra from a sonic anemometer, mounted on a platform (ship) which was moving through the turbulence held. Under unstable conditions the assumptions concerning the turbulent kinetic energy (TKE) budget appeared incorrect. Using a bulk estimate for the stability parameter, Z/L (where Z is the height and L is the Obukhov length), this resulted in anomalously low drag coefficients compared to neutral conditions. Determining Z/L iteratively, a low rate of convergence was achieved. It was concluded that the divergence of the turbulent transport of TKE was not negligible under unstable conditions. By minimizing the dependence of the calculated neutral drag coefficient on stability, this term was estimated at about -0.65Z/L. The resulting turbulent fluxes were then in close agreement with other studies at moderate wind speed. The drag and exchange coefficients for low wind speeds were found to be C-en x 10(3) = 2.79U(10n)(-1) + 0.66 (U-10n < 5.2 m/s), C-en x 10(3) = C-hn x 10(3) = 1.2 (U-10n greater than or equal to 5.2 m/s), and C-dn x 10(3) = 11.7U(10n)(-2) + 0.668 (U-10n < 5.5 m/s), which imply a rapid increase of the coefficient values as the wind decreased within the smooth flow regime. The frozen turbulence hypothesis and the assumptions of isotropy and an inertial subrange were found to remain valid at these low wind speeds for these shipboard measurements. Incorporation of a free convection parameterization had little effect.

Biomarkers of Nitrosative Stress: Development and validation of a new analytical method for 3-Nitrotyrosine quantification

Background: The nitration of tyrosine residues in proteins is associated with nitrosative stress, resulting in the formation of 3-nitrotyrosine (3-NT). 3-NT levels in biological samples have been associated with numerous physiological and pathological conditions. For this reason, several attempts have been made in order to develop methods that accurately quantify 3-NT in biological samples. Regarding chromatographic methods, they seem to be very accurate, showing very good sensibility and specificity. However, accurate quantification of this molecule, which is present at very low concentrations both at physiological and pathological states, is always a complex task and a target of intense research. Objectives: We aimed to develop a simple, rapid, low-cost and sensitive 3-NT quantification method for use in medical laboratories as an additional tool for diagnosis and/or treatment monitoring of a wide range of pathologies. We also aimed to evaluate the performance of the HPLC-based method developed here in a wide range of biological matrices. Material and methods: All experiments were performed on a Hitachi LaChrom Elite® HPLC system and separation was carried out using a Lichrocart® 250-4 Lichrospher 100 RP-18 (5μm) column. The method was further validated according to ICH guidelines. The biological matrices tested were serum, whole blood, urine, B16 F-10 melanoma cell line, growth medium conditioned with the same cell line, bacterial and yeast suspensions. Results: From all the protocols tested, the best results were obtained using 0.5% CH3COOH:MeOH:H2O (15:15:70) as the mobile phase, with detection at wavelengths 215, 276 and 356 nm, at 25ºC, and using a flow rate of 1 mL/min. By using this protocol, it was possible to obtain a linear calibration curve (correlation coefficient = 1), limits of detection and quantification in the order of ng/mL, and a short analysis time (<15 minutes per sample). Additionally, the developed protocol allowed the successful detection and quantification of 3-NT in all biological matrices tested, with detection at 356 nm. Conclusion: The method described in this study, which was successfully developed and validated for 3-NT quantification, is simple, cheap and fast, rendering it suitable for analysis in a wide range of biological matrices.

Comparative assessment of seller’s staining test (SST) and direct fluorescent antibody test for rapid and accurate laboratory diagnosis of rabies.

Background: Rabies causes 55, 000 annual human deaths globally and about 10,000 people are exposed annually in Nigeria. Diagnosis of animal rabies in most African countries has been by direct microscopic examination. In Nigeria, the Seller’s stain test (SST) was employed until 2009. Before then, both SST and dFAT were used concurrently until the dFAT became the only standard method. Objective: This study was designed to assess the sensitivity and specificity of the SST in relation to the ‘gold standard’ dFAT in diagnosis of rabies in Nigeria. Methods: A total of 88 animal specimens submitted to the Rabies National Reference Laboratory, Nigeria were routinely tested for rabies by SST and dFAT. Results: Overall, 65.9% of the specimens were positive for rabies by SST, while 81.8% were positive by dFAT. The sensitivity of SST in relation to the gold standard dFAT was 81.0% (95% CIs; 69.7% - 88.6%), while the specificity was 100% (95% CIs; 76% - 100%). Conclusion: The relatively low sensitivity of the SST observed in this study calls for its replacement with the dFAT for accurate diagnosis of rabies and timely decisions on administration of PEP to prevent untimely deaths of exposed humans.

The quality of rapid HIV testing in South Africa: an assessment of testers’ compliance.

Background: The aim of this study was to assess the quality of rapid HIV testing in South Africa. Method: A two-stage sampling procedure was used to select HCT sites in eight provinces of South Africa. The study employed both semi-structured interviews with HIV testers and observation of testing sessions as a means of data collection. In total, 63 HCT sites (one HIV tester per site) were included in the survey assessing qualification, training, testing practices and attitudes towards rapid tests. Quantitative data was analysed using descriptive statistics and qualitative data was content analysed. Results: Of the 63 HIV testers, 20.6% had a nursing qualification, 14.3% were professional counsellors, 58.7% were lay HIV counsellors and testers and 6.4% were from other professions. Most HIV testers (87.3%) had had a formal training in testing, which ranged between 10-14 days, while 6 (9.5%) had none. Findings revealed sub-standard practices in relation to testing. These were mainly related to non-adherence to testing algorithms, poor external quality control practices, poor handling and communication of discordant results. Conclusion: Quality of HIV rapid testing may be highly compromised through poor adherence to guidelines as observed in our study.

Near-infrared Instrumentation For Rapid-response Astronomy

Ɣ-ray bursts (GRBs) are the Universe's most luminous transient events. Since the discovery of GRBs was announced in 1973, efforts have been ongoing to obtain data over a broader range of the electromagnetic spectrum at the earliest possible times following the initial detection. The discovery of the theorized ``afterglow'' emission in radio through X-ray bands in the late 1990s confirmed the cosmological nature of these events. At present, GRB afterglows are among the best probes of the early Universe (z ≳ 9). In addition to informing theories about GRBs themselves, observations of afterglows probe the circum-burst medium (CBM), properties of the host galaxies and the progress of cosmic reionization. To explore the early-time variability of afterglows, I have developed a generalized analysis framework which models near-infrared (NIR), optical, ultra-violet (UV) and X-ray light curves without assuming an underlying model. These fits are then used to construct the spectral energy distribution (SED) of afterglows at arbitrary times within the observed window. Physical models are then used to explore the evolution of the SED parameter space with time. I demonstrate that this framework produces evidence of the photodestruction of dust in the CBM of GRB 120119A, similar to the findings from a previous study of this afterglow. The framework is additionally applied to the afterglows of GRB 140419A and GRB 080607. In these cases the evolution of the SEDs appears consistent with the standard fireball model. Having introduced the scientific motivations for early-time observations, I introduce the Rapid Infrared Imager-Spectrometer (RIMAS). Once commissioned on the 4.3 meter Discovery Channel Telescope (DCT), RIMAS will be used to study the afterglows of GRBs through photometric and spectroscopic observations beginning within minutes of the initial burst. The instrument will operate in the NIR, from 0.97 μm to 2.37 μm, permitting the detection of very high redshift (z ≳ 7) afterglows which are attenuated at shorter wavelengths by Lyman-ɑ absorption in the intergalactic medium (IGM). A majority of my graduate work has been spent designing and aligning RIMAS's cryogenic (~80 K) optical systems. Design efforts have included an original camera used to image the field surrounding spectroscopic slits, tolerancing and optimizing all of the instrument's optics, thermal modeling of optomechanical systems, and modeling the diffraction efficiencies for some of the dispersive elements. To align the cryogenic optics, I developed a procedure that was successfully used for a majority of the instrument's sub-assemblies. My work on this cryogenic instrument has necessitated experimental and computational projects to design and validate designs of several subsystems. Two of these projects describe simple and effective measurements of optomechanical components in vacuum and at cryogenic temperatures using an 8-bit CCD camera. Models of heat transfer via electrical harnesses used to provide current to motors located within the cryostat are also presented.

An hr-adaptive discontinuous Galerkin method for advection-diffusion problems

We propose an adaptive mesh refinement strategy based on exploiting a combination of a pre-processing mesh re-distribution algorithm employing a harmonic mapping technique, and standard (isotropic) mesh subdivision for discontinuous Galerkin approximations of advection-diffusion problems. Numerical experiments indicate that the resulting adaptive strategy can efficiently reduce the computed discretization error by clustering the nodes in the computational mesh where the analytical solution undergoes rapid variation.

Guided Bus Rapid Transit

Electrical Bus Rapid Transit (eBRT) is a charging electrical public transport which brings a clean, high performance, and affordable cost alternative from the conventional traffic vehicles which work with combustion and hybrid technology. These buses charge the battery in every bus stop to arrive at the next station. But, this charging system needs an appropriate infrastructure called pantograph, and it requires a high precision bus location to maintain battery lifetime, energy saving and charging time. To overcome this issue Vicomtech and Datik has planned a project based on computer vision to help to the driver to locate the vehicle in the correct place. In this document, we present a mono camera bus driver guided fast algorithm because these vehicles embedded computers do not support high computation and precision operations. In addition to the frequent lane sign, there are more accurate geometric beacons painted on the road to bring metric information to the vision system. This method uses segmentation to binarize the image discriminating the background space. Besides it detects, tracks and counts different lane mark contours in addition to classify each special painted mark. Besides it does not need any calibration task to calculate longitudinal and cross distances because we know the lane mark sizes.

Measuring sperm backflow following female orgasm: a new method

Background: Human female orgasm is a vexed question in the field while there is credible evidence of cryptic female choice that has many hallmarks of orgasm in other species. Our initial goal was to produce a proof of concept for allowing females to study an aspect of infertility in a home setting, specifically by aligning the study of human infertility and increased fertility with the study of other mammalian fertility. In the latter case - the realm of oxytocin-mediated sperm retention mechanisms seems to be at work in terms of ultimate function (differential sperm retention) while the proximate function (rapid transport or cervical tenting) remains unresolved. Method: A repeated measures design using an easily taught technique in a natural setting was used. Participants were a small (n=6), non-representative sample of females. The introduction of a sperm-simulant combined with an orgasm-producing technique using a vibrator/home massager and other easily supplied materials. Results: The sperm flowback (simulated) was measured using a technique that can be used in a home setting. There was a significant difference in simulant retention between the orgasm (M=4.08, SD=0.17) and non-orgasm (M=3.30, SD=0.22) conditions; t (5)=7.02, p=0.001. Cohen’s d=3.97, effect size r=0.89. This indicates a medium to small effect size. Conclusions: This method could allow females to test an aspect of sexual response that has been linked to lowered fertility in a home setting with minimal training. It needs to be replicated with a larger sample size.