On-chip immunoprecipitation for protein purification

Immunoprecipitation (IP) is one of the most widely used and selective techniques for protein purification. Here, a miniaturised, polymer-supported immunoprecipitation (µIP) method for the on-chip purification of proteins from complex mixtures is described. A 4 µl PDMS column functionalised with covalently bound antibodies was created and all critical aspects of the µIP protocol (antibody immobilisation, blocking of potential non-specific adsorption sites, sample incubation and washing conditions) were assessed and optimised. The optimised µIP method was used to obtain purified fractions of affinity-tagged protein from a bacterial lysate.

Nanotechnology and microfluidics:formulation design and on-chip manufacture of nanoparticles

Nanoparticles offer an ideal platform for the delivery of small molecule drugs, subunit vaccines and genetic constructs. Besides the necessity of a homogenous size distribution, defined loading efficiencies and reasonable production and development costs, one of the major bottlenecks in translating nanoparticles into clinical application is the need for rapid, robust and reproducible development techniques. Within this thesis, microfluidic methods were investigated for the manufacturing, drug or protein loading and purification of pharmaceutically relevant nanoparticles. Initially, methods to prepare small liposomes were evaluated and compared to a microfluidics-directed nanoprecipitation method. To support the implementation of statistical process control, design of experiment models aided the process robustness and validation for the methods investigated and gave an initial overview of the size ranges obtainable in each method whilst evaluating advantages and disadvantages of each method. The lab-on-a-chip system resulted in a high-throughput vesicle manufacturing, enabling a rapid process and a high degree of process control. To further investigate this method, cationic low transition temperature lipids, cationic bola-amphiphiles with delocalized charge centers, neutral lipids and polymers were used in the microfluidics-directed nanoprecipitation method to formulate vesicles. Whereas the total flow rate (TFR) and the ratio of solvent to aqueous stream (flow rate ratio, FRR) was shown to be influential for controlling the vesicle size in high transition temperature lipids, the factor FRR was found the most influential factor controlling the size of vesicles consisting of low transition temperature lipids and polymer-based nanoparticles. The biological activity of the resulting constructs was confirmed by an invitro transfection of pDNA constructs using cationic nanoprecipitated vesicles. Design of experiments and multivariate data analysis revealed the mathematical relationship and significance of the factors TFR and FRR in the microfluidics process to the liposome size, polydispersity and transfection efficiency. Multivariate tools were used to cluster and predict specific in-vivo immune responses dependent on key liposome adjuvant characteristics upon delivery a tuberculosis antigen in a vaccine candidate. The addition of a low solubility model drug (propofol) in the nanoprecipitation method resulted in a significantly higher solubilisation of the drug within the liposomal bilayer, compared to the control method. The microfluidics method underwent scale-up work by increasing the channel diameter and parallelisation of the mixers in a planar way, resulting in an overall 40-fold increase in throughput. Furthermore, microfluidic tools were developed based on a microfluidics-directed tangential flow filtration, which allowed for a continuous manufacturing, purification and concentration of liposomal drug products.

The application of 3D printing to study microfluidic architecture for 'on-chip' mixing systems for SRCD and UV spectroscopy

This paper details methodologies that have been explored for the fast proofing of on-chip architectures for Circular Dichroism techniques. Flow-cell devices fabricated from UV transparent Quartz are used for these experiments. The complexity of flow-cell production typically results in lead times of six months from order to delivery. Only at that point can the on-chip architecture be tested empirically and any required modifications determined ready for the next six month iteration phase. By using the proposed 3D printing and PDMS moulding techniques for fast proofing on-chip architectures the optimum design can be determined within a matter of hours prior to commitment to quartz chip production.

Distributed processing, reconfigurable processes and active network

The fast spread of the Internet and the increasing demands of the service are leading to radical changes in the structure and management of underlying telecommunications systems. Active networks (ANs) offer the ability to program the network on a per-router, per-user, or even per-packet basis, thus promise greater flexibility than current networks. To make this new network paradigm of active network being widely accepted, a lot of issues need to be solved. Management of the active network is one of the challenges. This thesis investigates an adaptive management solution based on genetic algorithm (GA). The solution uses a distributed GA inspired by bacterium on the active nodes within an active network, to provide adaptive management for the network, especially the service provision problems associated with future network. The thesis also reviews the concepts, theories and technologies associated with the management solution. By exploring the implementation of these active nodes in hardware, this thesis demonstrates the possibility of implementing a GA based adaptive management in the real network that being used today. The concurrent programming language, Handel-C, is used for the description of the design system and a re-configurable computer platform based on a FPGA process element is used for the hardware implementation. The experiment results demonstrate both the availability of the hardware implementation and the efficiency of the proposed management solution.

Network effects, network structure and consumer interaction in mobile telecommunications in Europe and Asia

This paper estimates the importance of (tariff-mediated) network effects and the impact of a consumer's social network on her choice of mobile phone provider. The study uses network data obtained from surveys of students in several European and Asian countries. We use the Quadratic Assignment Procedure, a non-parametric permutation test, to adjust for the particular error structure of network data. We find that respondents strongly coordinate their choice of mobile phone providers, but only if their provider induces network effects. This suggests that this coordination depends on network effects rather than on information contagion or pressure to conform to the social environment.

City branding:can goods and services branding models be used to brand cities?

A city's branding is investigated using generic product and services branding models. Two generic branding models and tourism segmentation models guide an investigation into city branding 'as it should be' and 'as it is' using Birmingham, England as a case study. The unique characteristics of city brands are identified and Keller's Brand Report Card provides a theoretical framework for building a picture of the brand-building activity taking place in the city. Four themes emerge and are discussed: 1) the impact of a network on brand models developed for organisations; 2) segmentation of brand elements; 3) corporate branding; and 4) the political dimension. A conclusion is that city branding would be more effective if the systems and structures of generic branding models were adopted.

Intracellular protein determination using droplet-based immunoassays

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 µM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells. © 2011 American Chemical Society.

H264 data partitioned video streaming

Motivated by the increasing demand and challenges of video streaming in this thesis, we investigate methods by which the quality of the video can be improved. We utilise overlay networks that have been created by implemented relay nodes to produce path diversity, and show through analytical and simulation models for which environments path diversity can improve the packet loss probability. We take the simulation and analytical models further by implementing a real overlay network on top of Planetlab, and show that when the network conditions remain constant the video quality received by the client can be improved. In addition, we show that in the environments where path diversity improves the video quality forward error correction can be used to further enhance the quality. We then investigate the effect of IEEE 802.11e Wireless LAN standard with quality of service enabled on the video quality received by a wireless client. We find that assigning all the video to a single class outperforms a cross class assignment scheme proposed by other researchers. The issue of virtual contention at the access point is also examined. We increase the intelligence of our relay nodes and enable them to cache video, in order to maximise the usefulness of these caches. For this purpose, we introduce a measure, called the PSNR profit, and present an optimal caching method for achieving the maximum PSNR profit at the relay nodes where partitioned video contents are stored and provide an enhanced quality for the client. We also show that the optimised cache the degradation in the video quality received by the client becomes more graceful than the non-optimised system when the network experiences packet loss or is congested.

Adaptive congestion control of DSRC vehicle networks for collaborative road safety applications

Congestion control is critical for the provisioning of quality of services (QoS) over dedicated short range communications (DSRC) vehicle networks for road safety applications. In this paper we propose a congestion control method for DSRC vehicle networks at road intersection, with the aims of providing high availability and low latency channels for high priority emergency safety applications while maximizing channel utilization for low priority routine safety applications. In this method a offline simulation based approach is used to find out the best possible configurations of message rate and MAC layer backoff exponent (BE) for a given number of vehicles equipped with DSRC radios. The identified best configurations are then used online by an roadside access point (AP) for system operation. Simulation results demonstrated that this adaptive method significantly outperforms the fixed control method under varying number of vehicles. The impact of estimation error on the number of vehicles in the network on system level performance is also investigated.

Recent advances in spin crossover research

Thermal spin transition (spin crossover), one of the most fascinating dynamic electronic structure phenomena occurring in coordination compounds of third row transition metal ions, mostly of iron(II), iron(III) and cobalt(II) with critical ligand field strengths competing with the spin pairing energy, has attracted increasing attention by many research groups. One of the reasons is the promising potential for practical applications. In this chapter we intend to cover essential recent work, primarily accomplished within the European research network on “Thermal and Optical Switching of Molecular Spin States (TOSS)”. New spin crossover compounds and their thermal spin transition behaviour, also under applied pressure, novel effects observed by irradiation and magnetic field, will be discussed. Progress in theoretical treatments of spin crossover phenomena, particularly cooperativity, will be briefly outlined. The chapter concludes with a summary of research highlights published by the partner laboratories of the TMR network TOSS.

Bessel beams from semiconductor light sources

We report on recent progress in the generation of non-diffracting (Bessel) beams from semiconductor light sources including both edge-emitting and surface-emitting semiconductor lasers as well as light-emitting diodes (LEDs). Bessel beams at the power level of Watts with central lobe diameters of a few to tens of micrometers were achieved from compact and highly efficient lasers. The practicality of reducing the central lobe size of the Bessel beam generated with high-power broad-stripe semiconductor lasers and LEDs to a level unachievable by means of traditional focusing has been demonstrated. We also discuss an approach to exceed the limit of power density for the focusing of radiation with high beam propagation parameter M2. Finally, we consider the potential of the semiconductor lasers for applications in optical trapping/tweezing and the perspectives to replace their gas and solid-state laser counterparts for a range of implementations in optical manipulation towards lab-on-chip configurations. © 2014 Elsevier Ltd.

Optical trapping with superfocused high-M2 laser diode beam

Many applications of high-power laser diodes demand tight focusing. This is often not possible due to the multimode nature of semiconductor laser radiation possessing beam propagation parameter M2 values in double-digits. We propose a method of 'interference' superfocusing of high-M2 diode laser beams with a technique developed for the generation of Bessel beams based on the employment of an axicon fabricated on the tip of a 100 μm diameter optical fiber with highprecision direct laser writing. Using axicons with apex angle 140º and rounded tip area as small as 10 μm diameter, we demonstrate 2-4 μm diameter focused laser 'needle' beams with approximately 20 μm propagation length generated from multimode diode laser with beam propagation parameter M2=18 and emission wavelength of 960 nm. This is a few-fold reduction compared to the minimal focal spot size of 11 μm that could be achieved if focused by an 'ideal' lens of unity numerical aperture. The same technique using a 160º axicon allowed us to demonstrate few-μm-wide laser 'needle' beams with nearly 100 μm propagation length with which to demonstrate optical trapping of 5-6 μm rat blood red cells in a water-heparin solution. Our results indicate the good potential of superfocused diode laser beams for applications relating to optical trapping and manipulation of microscopic objects including living biological objects with aspirations towards subsequent novel lab-on-chip configurations.

Resting state morphology predicts the effect of theta burst stimulation in false belief reasoning:ventrolateral prefrontal cortex in false belief reasoning

When required to represent a perspective that conflicts with one's own, functional magnetic resonance imaging (fMRI) suggests that the right ventrolateral prefrontal cortex (rvlPFC) supports the inhibition of that conflicting self-perspective. The present task dissociated inhibition of self-perspective from other executive control processes by contrasting belief reasoning-a cognitive state where the presence of conflicting perspectives was manipulated-with a conative desire state wherein no systematic conflict existed. Linear modeling was used to examine the effect of continuous theta burst stimulation (cTBS) to rvlPFC on participants' reaction times in belief and desire reasoning. It was anticipated that cTBS applied to rvlPFC would affect belief but not desire reasoning, by modulating activity in the Ventral Attention System (VAS). We further anticipated that this effect would be mediated by functional connectivity within this network, which was identified using resting state fMRI and an unbiased model-free approach. Simple reaction-time analysis failed to detect an effect of cTBS. However, by additionally modeling individual measures from within the stimulated network, the hypothesized effect of cTBS to belief (but, importantly, not desire) reasoning was demonstrated. Structural morphology within the stimulated region, rvlPFC, and right temporoparietal junction were demonstrated to underlie this effect. These data provide evidence that inconsistencies found with cTBS can be mediated by the composition of the functional network that is being stimulated. We suggest that the common claim that this network constitutes the VAS explains the effect of cTBS to this network on false belief reasoning. Hum Brain Mapp, 2016. © 2016 Wiley Periodicals, Inc.