Mechanistic studies of reactions at the single-molecule level using microfluidics with applications in molecular diagnostics

Motivated by needs in molecular diagnostics and advances in microfabrication, researchers started to seek help from microfluidic technology, as it provides approaches to achieve high throughput, high sensitivity, and high resolution. One strategy applied in microfluidics to fulfill such requirements is to convert continuous analog signal into digitalized signal. One most commonly used example for this conversion is digital PCR, where by counting the number of reacted compartments (triggered by the presence of the target entity) out of the total number of compartments, one could use Poisson statistics to calculate the amount of input target.

However, there are still problems to be solved and assumptions to be validated before the technology is widely employed. In this dissertation, the digital quantification strategy has been examined from two angles: efficiency and robustness. The former is a critical factor for ensuring the accuracy of absolute quantification methods, and the latter is the premise for such technology to be practically implemented in diagnosis beyond the laboratory. The two angles are further framed into a “fate” and “rate” determination scheme, where the influence of different parameters is attributed to fate determination step or rate determination step. In this discussion, microfluidic platforms have been used to understand reaction mechanism at single molecule level. Although the discussion raises more challenges for digital assay development, it brings the problem to the attention of the scientific community for the first time.

This dissertation also contributes towards developing POC test in limited resource settings. On one hand, it adds ease of access to the tests by incorporating massively producible, low cost plastic material and by integrating new features that allow instant result acquisition and result feedback. On the other hand, it explores new isothermal chemistry and new strategies to address important global health concerns such as cyctatin C quantification, HIV/HCV detection and treatment monitoring as well as HCV genotyping.

Orientation-controllable self-organized microgratings induced in the bulk SrTiO3 crystal by a single femtosecond laser beam

Self-organized microgratings were induced in the bulk SrTiO3 crystal by readily scanning the laser focus in the direction perpendicular to the laser propagation axis. The groove orientations of those gratings could be controlled by changing the irradiation pulse number per unit scanning length, which could be implemented either through adjusting the scanning velocity at a fixed pulse repetition rate or through varying the pulse repetition rate at a fixed scanning velocity. This high-speed method for fabrication of microgratings will have many potential applications in the integration of micro-optical elements. The possible formation mechanism of the self-organized microgratings is also discussed. (C) 2007 Optical Society of America.

Reference-growth rate – a simple and handy parameter summarizing the influence of environmental conditions

Abstract Environmental changes may have an impact on life conditions of the fish, e.g. food supply for the fish. The prevailing environmental conditions apply evenly to all age groups of one stock. Small fish have high growth rates, whereas large fish grow with low rates. But, it can be shown on the basis of the von Bertalanffy-growth model that it is sufficient to know only the growth rate of one single age group to compute the growth rates of all other age groups. The growth rate of a reference fish GRF (e.g. a fish with a body mass of 1 kg) was introduced as a reference growth describing the current food condition of all age groups of the stock. As an example a time series of the reference-growth rate of the northern cod stock (NAFO, 3K) was computed for the time span 1979 to 1999. For the northern cod stock it can be observed that environmental conditions caused growth rates below the long-term mean for seven years in a row. After a prolonged hunger period the fish stock collapsed in 1992 also by the impact of fisheries - and this was probably not a coincidence. Now, with the reference-growth rate GRF a simple and handy parameter was found to summarize the influence of the environmental conditions on growth and other derived models and therefore makes it easier to compute the influence of environmental changes within stock assessment. Zusammenfassung Veränderungen der Umwelt können Auswirkungen auf die Lebensbedingungen der Fische haben, z. B. auf das Nahrungsangebot der Fische. Die vorherrschenden Umgebungsbedingungen wirken gleichmäßig auf alle Altersgruppen eines Bestandes, wobei typischer Weise kleineFische hohe Wachstumsraten haben, während die großen Fische mit niedrigen Raten wachsen. Auf der Grundlage des von Bertalanffy-Wachstumsmodells kann gezeigt werden, dass es ausreicht, nur die Wachstumsrate von einer einzigen Altersgruppe zu kennen, um die Wachstumsraten von allen anderen Altersgruppen berechnen zu können. Die Wachstumsrate eines Referenz-Fisches (z.B. eines Fisches mit einer Körpermasse von 1 kg) wurde als Referenz-Wachstum GRF eingeführt, die den aktuellen Zustand des Nahrungsangebots füralle Altersgruppen des Bestandes beschreibt. Als Beispiel wurde einer Zeitreihe der Referenz-Wachstumsraten des nördlichen Kabeljaubestandes (NAFO, 3K) für die Zeitsraum 1979 bis 1999 berechnet. Für diesen Kabeljaubestand war zu beobachten, dass Umgebungsbedingungen für sieben Jahre in Folge Wachstumsraten unter dem langjährigen Mittelwert verursachten. Nach einer längeren Hungerperiode kollabierte dieser Fischbestand im Jahr 1992 auch durch den Einfluß der Fischerei - und dies war sicher kein Zufall. Jetzt, mit der Referenz-Wachstumsrate GRF, ist ein einfacher und handlicher Parameter gefunden, der es gestattet den Einfluss der Umweltbedingungen auf die Wachstumsbedingungen und andere davon abgeleitete Modelle zusammenzufassen. Dies macht es einfach, den Einfluss von Umweltveränderungen innerhalb der Bestandsabschätzungen zu berechnen.

Technology for Single Cell Protein Analysis in Immunology and Cancer Prognostics

The first chapter of this thesis deals with automating data gathering for single cell microfluidic tests. The programs developed saved significant amounts of time with no loss in accuracy. The technology from this chapter was applied to experiments in both Chapters 4 and 5.

The second chapter describes the use of statistical learning to prognose if an anti-angiogenic drug (Bevacizumab) would successfully treat a glioblastoma multiforme tumor. This was conducted by first measuring protein levels from 92 blood samples using the DNA-encoded antibody library platform. This allowed the measure of 35 different proteins per sample, with comparable sensitivity to ELISA. Two statistical learning models were developed in order to predict whether the treatment would succeed. The first, logistic regression, predicted with 85% accuracy and an AUC of 0.901 using a five protein panel. These five proteins were statistically significant predictors and gave insight into the mechanism behind anti-angiogenic success/failure. The second model, an ensemble model of logistic regression, kNN, and random forest, predicted with a slightly higher accuracy of 87%.

The third chapter details the development of a photocleavable conjugate that multiplexed cell surface detection in microfluidic devices. The method successfully detected streptavidin on coated beads with 92% positive predictive rate. Furthermore, chambers with 0, 1, 2, and 3+ beads were statistically distinguishable. The method was then used to detect CD3 on Jurkat T cells, yielding a positive predictive rate of 49% and false positive rate of 0%.

The fourth chapter talks about the use of measuring T cell polyfunctionality in order to predict whether a patient will succeed an adoptive T cells transfer therapy. In 15 patients, we measured 10 proteins from individual T cells (~300 cells per patient). The polyfunctional strength index was calculated, which was then correlated with the patient's progress free survival (PFS) time. 52 other parameters measured in the single cell test were correlated with the PFS. No statistical correlator has been determined, however, and more data is necessary to reach a conclusion.

Finally, the fifth chapter talks about the interactions between T cells and how that affects their protein secretion. It was observed that T cells in direct contact selectively enhance their protein secretion, in some cases by over 5 fold. This occurred for Granzyme B, Perforin, CCL4, TNFa, and IFNg. IL- 10 was shown to decrease slightly upon contact. This phenomenon held true for T cells from all patients tested (n=8). Using single cell data, the theoretical protein secretion frequency was calculated for two cells and then compared to the observed rate of secretion for both two cells not in contact, and two cells in contact. In over 90% of cases, the theoretical protein secretion rate matched that of two cells not in contact.

Effect of size, morphology and crystallinity of seed crystal on the nucleation and growth of single grain Y-Ba-Cu-O

The effect of size, morphology and crystallinity of seed crystals on the nucleation and growth of large grain Y-Ba-Cu-O (YBCO) bulk superconductors fabricated by top seeded melt growth (TSMG) has been investigated. Seeding bulk samples with small, square shaped seed crystals leads to point nucleation and growth of the superconducting YBa2Cu3O7-y (Y-123) phase that exhibits the usual square habitual growth symmetry. The use of triangular and circular shaped seed crystals, however, modifies significantly the growth habit geometry of the grain. The use of large area seeds both increases the rate of epitaxial nucleation of the Y-123 phase and produces relatively large crystals in the incongruent melt, which decreases significantly the processing times of large grain samples. The present study is relevant to decrease processing times of samples with both preferred or no growth sectors and for multiple seeding of large grain samples which contain clean grain boundaries. © 2005 Published by Elsevier Ltd.

Large single grain (RE)-Ba-Cu-O superconductors with nano-phase inclusions

Nano-phase (5-20 nm) particles of YBa2(Cu0.5M 0.6)O6 [where M = Nb, Ta, Mo, W, Zr and Hf] have been introduced successfully into RE-Ba-Cu-O single grain superconductors. A study to enlarge the size of a single grain containing these particles has been carried out involving measurement of the growth rate as a function of YBa 2(Cu0.5M0.6)O6 phase concentration and degree of un-dercooling. The influence of the change in YBa2 (Cu0.8M0.5)O6 concentration on microstructural features is also investigated and the superconducting properties of these large grain superconductors are presented. © 2005 IEEE.

Growth window and possible mechanism of millimeter-thick single-walled carbon nanotube forests.

Our group recently reproduced the water-assisted growth method, so-called "SuperGrowth", of millimeter-thick single-walled carbon nanotube (SWNT) forests by using C2H4/H2/H2O/Ar reactant gas and Fe/Al2O3, catalyst. In this current work, a parametric study was carried out on both reaction and catalyst conditions. Results revealed that a thin Fe catalyst layer (about 0.5 nm) yielded rapid growth of SWNTs only when supported on Al2O3, and that Al2O3 support enhanced the activity of Fe, Co, and Ni catalysts. The growth window for the rapid SWNT growth was narrow, however. Optimum amount of added H2O increased the SWNT growth rate but further addition of H2O degraded both the SWNT growth rate and quality. Addition of H2 was also essential for rapid SWNT growth, but again, further addition decreased both the SWNT growth rate and quality. Because Al2O3 catalyzes hydrocarbon reforming, Al2O3 support possibly enhances the SWNT growth rate by supplying the carbon source to the catalyst nanoparticles. The origin of the narrow window for rapid SWNT growth is also discussed.

Coexistence of high-bit-rate quantum key distribution and data on optical fiber

Quantum key distribution (QKD) uniquely allows distribution of cryptographic keys with security verified by quantum mechanical limits. Both protocol execution and subsequent applications require the assistance of classical data communication channels. While using separate fibers is one option, it is economically more viable if data and quantum signals are simultaneously transmitted through a single fiber. However, noise-photon contamination arising from the intense data signal has severely restricted both the QKD distances and secure key rates. Here, we exploit a novel temporal-filtering effect for noise-photon rejection. This allows high-bit-rate QKD over fibers up to 90 km in length and populated with error-free bidirectional Gb/s data communications. With high-bit rate and range sufficient for important information infrastructures, such as smart cities and 10 Gbit Ethernet, QKD is a significant step closer towards wide-scale deployment in fiber networks.

A self-regulating valve for single-phase liquid cooling of microelectronics

This paper reports on the design, optimization and testing of a self-regulating valve for single-phase liquid cooling of microelectronics. Its purpose is to maintain the integrated circuit (IC) at constant temperature and to reduce power consumption by diminishing flow generated by the pump as a function of the cooling requirements. It uses a thermopneumatic actuation principle that combines the advantages of zero power consumption and small size in combination with a high flow rate and low manufacturing costs. The valve actuation is provided by the thermal expansion of a liquid (actuation fluid) which, at the same time, actuates the valve and provides feed-back sensing. A maximum flow rate of 38 kg h-1 passes through the valve for a heat load up to 500 W. The valve is able to reduce the pumping power by up to 60% and it has the capability to maintain the IC at a more uniform temperature. © 2011 IOP Publishing Ltd.

Nonlinear phenomena in thermoacoustics: A comparison between single-mode and multi-mode methods

Nonlinear analysis of thermoacoustic instability is essential for prediction of frequencies and amplitudes of limit cycles. In frequency domain analyses, a quasi-linear transfer function between acoustic velocity and heat release rate perturbations, called the flame describing function (FDF), is obtained from a flame model or experiments. The FDF is a function of the frequency and amplitude of velocity perturbations but only contains the heat release response at the forcing frequency. While the gain and phase of the FDF provide insight into the nonlinear dynamics of the system, the accuracy of its predictions remains to be verified for different types of nonlinearity. In time domain analyses, the governing equations of the fully coupled problem are solved to find the time evolution of the system. One method is to discretize the governing equations using a suitable basis, such as the natural acoustic modes of the system. The number of modes used in the discretization alters the accuracy of the solution. In our previous work we have shown that predictions using the FDF are almost exactly the same as those obtained from the time-domain using only one mode for the discretization. We call this the single-mode method. In this paper we compare results from the single-mode and multi-mode methods, applied to a thermoacoustic system of a premixed flame in a tube. For some cases, the results differ greatly in both amplitude as well as frequency content. This study shows that the contribution from higher and subharmonics to the nonlinear dynamics can be significant and must be considered for an accurate and comprehensive analysis of thermoacoustic systems. Hence multi-mode simulations are necessary, and the single-mode method or the FDF may be insufficient to capture some of the complex nonlinear behaviour in fhermoacoustics.

The high strain rate response of Ultra High Molecular-weight Polyethylene: From fibre to laminate

The effect of strain rate upon the uniaxial response of Ultra High Molecular-weight Polyethylene (UHMWPE) fibres, yarns and laminates of lay-up [0/90]48 has been measured in both the 0/90 and ±45 configurations. The tensile strength of the matrix-dominated ±45 laminate is two orders of magnitude less than that of the fibre-dominated 0/90 laminate, and is more sensitive to strain rate. A piezoelectric force sensor device was developed to obtain the high strain rate data, and this achieved a rise time of less than 1 μs. It is found that the failure strength (and failure strain) of the yarn is almost insensitive to strain rate within the range (10 -1-103 s-1). At low strain rates (below 10 -1 s-1), creep of the yarn dominates and the failure strain increases with diminishing strain rate. The tensile strength of the dry yarn exceeds that of the laminate by about 20%. Tests on single fibres exceed the strength of the yarn by 20%. © 2013 Elsevier Ltd. All rights reserved.

Resonant excitation and imaging of nonequilibrium exciton spins in single core-shell GaAs-AlGaAs nanowires.

Nonequilibrium spin distributions in single GaAs/AlGaAs core-shell nanowires are excited using resonant polarized excitation at 10 K. At all excitation energies, we observe strong photoluminescence polarization due to suppressed radiative recombination of excitons with dipoles aligned perpendicular to the nanowire. Excitation resonances are observed at 1- or 2-LO phonon energies above the exciton ground states. Using rate equation modeling, we show that, at the lowest energies, strongly nonequilibrium spin distributions are present and we estimate their spin relaxation rate.

Effects of tetrabrominated diphenyl ether and hexabromocyclododecanes in single and complex exposure to hepatoma HepG2 cells

This study was designed to determine cytotoxic effects of PBDE-47 and HBCDs individually or with a mixture of both compounds exposure to Hep G2 cells. The results showed PBDE-47 and HBCDs induced increase of nitric oxide synthase (NOS) activity, release of NO. dissipation of mitochondria membrane potential and cell apoptosis. Exposure to HBCDs induced ROS formation. Moreover, preincubation with PTIO (NO scavanger) and N-acetylcysteine (ROS scavanger) partially reversed cytotoxic effects of these compounds. The possible mechanism is that PBDE-47 and HBCDs could boost generation of NO and/or ROS, impact mitochondria, and result in start-ups of apoptosis program. Cells exposed to mixture of both compounds and each of them showed non-apoptotic rate significant difference, but the combination of them caused more adverse effects on cells. These results Suggest that PBDE-47 and HBCDs in single and complex exposure have the cytotoxic activity of anti-proliferation and induction of apoptosis in tumor cells in vitro. (C) 2008 Elsevier B.V. All rights reserved.

Single event molecular signalling for estimation and control

Cell biology is characterised by low molecule numbers and coupled stochastic chemical reactions with intrinsic noise permeating and dominating the interactions between molecules. Recent work [9] has shown that in such environments there are hard limits on the accuracy with which molecular populations can be controlled and estimated. These limits are predicated on a continuous diffusion approximation of the target molecule (although the remainder of the system is non-linear and discrete). The principal result of [9] assumes that the birth rate of the signalling species is linearly dependent on the target molecule population size. In this paper, we investigate the situation when the entire system is kept discrete, and arbitrary non-linear coupling is allowed between the target molecule and downstream signalling molecules. In this case it is possible, by relying solely on the event triggered nature of control and signalling reactions, to define non-linear reaction rate modulation schemes that achieve improved performance in certain parameter regimes. These schemes would not appear to be biologically relevant, raising the question of what are an appropriate set of assumptions for obtaining biologically meaningful results. © 2013 EUCA.

The toxicities of single-celled Microcystis aeruginosa PCC7820 and liberated M-aeruginosa to Daphnia carinata in the absence and presence of the green alga Scenedesmus obliquus

We evaluated the toxic effect of Microcystis aeruginosa on Daphnia carinata King using survival rate, population growth rate, and body length. When fed Microcystis aerugionsa PCC7820 and liberated colonial Microcystis spp., all D. carinata died within five days. When fed a mixture of M. aeruginosa PCC7820 and the green alga Scenedesmus obliquus, the survival rate, population growth rate, and body length of D. carinata generally increased. The survival rates were all above 80% after ten days. However, with liberated colonial M. aeruginosa, the toxic effect on D. carinata was more pronounced, and only at higher concentration of S. obliquus did that toxic effect abate. Our results indicated that green algae could greatly weaken the toxic effect of cyanobacteria.