Investigation of ultrafast laser-photonic material interactions: Challenges for directly written glass photonics

Currently, direct-write waveguide fabrication is probably the most widely studied application of femtosecond laser micromachining in transparent dielectrics. Devices such as buried waveguides, power splitters, couplers, gratings, and optical amplifiers have all been demonstrated. Waveguide properties depend critically on the sample material properties and writing laser characteristics. In this paper, we discuss the challenges facing researchers using the femtosecond laser direct-write technique with specific emphasis being placed on the suitability of fused silica and phosphate glass as device hosts for different applications.

Integration of a 3D hydrogel matrix within a hollow core photonic crystal fibre for DNA probe immobilization

In this paper, we demonstrate the integration of a 3D hydrogel matrix within a hollow core photonic crystal fibre (HC-PCF). In addition, we also show the fluorescence of Cy5-labelled DNA molecules immobilized within the hydrogel formed in two different types of HC-PCF. The 3D hydrogel matrix is designed to bind with the amino groups of biomolecules using an appropriate cross-linker, providing higher sensitivity and selectivity than the standard 2D coverage, enabling a greater number of probe molecules to be available per unit area. The HC-PCFs, on the other hand, can be designed to maximize the capture of fluorescence to improve sensitivity and provide longer interaction lengths. This could enable the development of fibre-based point-of-care and remote systems, where the enhanced sensitivity would relax the constraints placed on sources and detectors. In this paper, we will discuss the formation of such polyethylene glycol diacrylate (PEGDA) hydrogels within a HC-PCF, including their optical properties such as light propagation and auto-fluorescence.

Development of a quality control material for the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an in vivo marker of oxidative stress, and comparison of results from different laboratories

The measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine is an increasingly popular marker of in vivo oxidative damage to DNA. A random-sequence 21-mer oligonucleotide 5'-TCA GXC GTA CGT GAT CTC AGT-3' in which X was 8-oxo-guanine (8-oxo-G) was purified and accurate determination of the oxidised base was confirmed by a 32P-end labelling strategy. The lyophilised material was analysed for its absolute content of 8-oxo-dG by several major laboratories in Europe and one in Japan. Most laboratories using HPLC-ECD underestimated, while GC-MS-SIM overestimated the level of the lesion. HPLC-ECD measured the target value with greatest accuracy. The results also suggest that none of the procedures can accurately quantitate levels of 1 in 10(6) 8-oxo-(d)G in DNA.

Advances in femtosecond micromachining and inscription of micro and nano photonic devices

This thesis has focused on three key areas of interest for femtosecond micromachining and inscription. The first area is micromachining where the work has focused on the ability to process highly repeatable, high precision machining with often extremely complex geometrical structures with little or no damage. High aspect ratio features have been demonstrated in transparent materials, metals and ceramics. Etch depth control was demonstrated especially in the work on phase mask fabrication. Practical chemical sensing and microfluidic devices were also fabricated to demonstrate the capability of the techniques developed during this work. The second area is femtosecond inscription. Here, the work has utilised the non-linear absorption mechanisms associated with femtosecond pulse-material interactions to create highly localised refractive index changes in transparent materials to create complex 3D structures. The techniques employed were then utilised in the fabrication of Phase masks and Optical Coherence Tomography (OCT) phantom calibration artefacts both of which show the potential to fill voids in the development of the fields. This especially the case for the OCT phantoms where there exists no previous artefacts of known shape, allowing for the initial specification of parameters associated with the quality of OCT machines that are being taken up across the world in industry and research. Finally the third area of focus was the combination of all of the techniques developed through work in planar samples to create a range of artefacts in optical fibres. The development of techniques and methods for compensating for the geometrical complexities associated with working with the cylindrical samples with varying refractive indices allowed for fundamental inscription parameters to be examined, structures for use as power monitors and polarisers with the optical fibres and finally the combination of femtosecond inscription and ablation techniques to create a magnetic field sensor with an optical fibre coated in Terfenol-D with directional capability. Through the development of understanding, practical techniques and equipment the work presented here demonstrates several novel pieces of research in the field of femtosecond micromachining and inscription that has provided a broad range of related fields with practical devices that were previously unavailable or that would take great cost and time to facilitate.

LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications

Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged Lacl protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35-50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.

Purification of plasmid DNA for use in human gene therapy

Two key issues defined the focus of this research in manufacturing plasmid DNA for use In human gene therapy. First, the processing of E.coli bacterial cells to effect the separation of therapeutic plasmid DNA from cellular debris and adventitious material. Second, the affinity purification of the plasmid DNA in a Simple one-stage process. The need arises when considering the concerns that have been recently voiced by the FDA concerning the scalability and reproducibility of the current manufacturing processes in meeting the quality criteria of purity, potency, efficacy, and safety for a recombinant drug substance for use in humans. To develop a preliminary purification procedure, an EFD cross-flow micro-filtration module was assessed for its ability to effect the 20-fold concentration, 6-time diafiltration, and final clarification of the plasmid DNA from the subsequent cell lysate that is derived from a 1 liter E.coli bacterial cell culture. Historically, the employment of cross-flow filtration modules within procedures for harvesting cells from bacterial cultures have failed to reach the required standards dictated by existing continuous centrifuge technologies, frequently resulting in the rapid blinding of the membrane with bacterial cells that substantially reduces the permeate flux. By challenging the EFD module, containing six helical wound tubular membranes promoting centrifugal instabilities known as Dean vortices, with distilled water between the Dean number's of 187Dn and 818Dn,and the transmembrane pressures (TMP) of 0 to 5 psi. The data demonstrated that the fluid dynamics significantly influenced the permeation rate, displaying a maximum at 227Dn (312 Imh) and minimum at 818Dn (130 Imh) for a transmembrane pressure of 1 psi. Numerical studies indicated that the initial increase and subsequent decrease resulted from a competition between the centrifugal and viscous forces that create the Dean vortices. At Dean numbers between 187Dn and 227Dn , the forces combine constructively to increase the apparent strength and influence of the Dean vortices. However, as the Dean number in increases above 227 On the centrifugal force dominates the viscous forces, compressing the Dean vortices into the membrane walls and reducing their influence on the radial transmembrane pressure i.e. the permeate flux reduced. When investigating the action of the Dean vortices in controlling tile fouling rate of E.coli bacterial cells, it was demonstrated that the optimum cross-flow rate at which to effect the concentration of a bacterial cell culture was 579Dn and 3 psi TMP, processing in excess of 400 Imh for 20 minutes (i.e., concentrating a 1L culture to 50 ml in 10 minutes at an average of 450 Imh). The data demonstrated that there was a conflict between the Dean number at which the shear rate could control the cell fouling, and the Dean number at which tile optimum flux enhancement was found. Hence, the internal geometry of the EFD module was shown to sub-optimal for this application. At 579Dn and 3 psi TMP, the 6-fold diafiltration was shown to occupy 3.6 minutes of process time, processing at an average flux of 400 Imh. Again, at 579Dn and 3 psi TMP the clarification of the plasmid from tile resulting freeze-thaw cell lysate was achieved at 120 Iml1, passing 83% (2,5 mg) of the plasmid DNA (6,3 ng μ-1 10.8 mg of genomic DNA (∼23,00 Obp, 36 ng μ-1 ), and 7.2 mg of cellular proteins (5-100 kDa, 21.4 ngμ-1 ) into the post-EFD process stream. Hence the EFD module was shown to be effective, achieving the desired objectives in approximately 25 minutes. On the basis of its ability to intercalate into low molecular weight dsDNA present in dilute cell lysates, and be electrophoresed through agarose, the fluorophore PicoGreen was selected for the development of a suitable dsDNA assay. It was assesseel for its accuracy, and reliability, In determining the concentration and identity of DNA present in samples that were eleclrophoresed through agarose gels. The signal emitted by intercalated PicoGreen was shown to be constant and linear, and that the mobility of the PicaGreen-DNA complex was not affected by the intercalation. Concerning the secondary purification procedure, various anion-exchange membranes were assessed for their ability to capture plasmid DNA from the post-EFD process stream. For a commercially available Sartorius Sartobind Q15 membrane, the reduction in the equilibriumbinding capacity for  ctDNA in buffer of increasing ionic demonstrated that DNA was being.adsorbed by electrostatic  interactions only. However, the problems associated with fluid distribution across the membrane demonstrated that the membrane housing was the predominant cause of the .erratic breakthrough curves. Consequently, this would need to be rectified before such a membrane could be integrated into the current system, or indeed be scaled beyond laboratory scale. However, when challenged with the process material, the data showed that considerable quantities of protein (1150 μg) were adsorbed preferentially to the plasmid DNA (44 μg). This was also shown for derived Pall Gelman UltraBind US450 membranes that had been functionalised by varying molecular weight poly-L~lysine and polyethyleneimine ligands. Hence the anion-exchange membranes were shown to be ineffective in capturing plasmid DNA from the process stream. Finally, work was performed to integrate a sequence-specific DNA·binding protein into a single-stage DNA chromatography, isolating plasmid DNA from E.coli cells whilst minimising the contamination from genomic DNA and cellular protein. Preliminary work demonstrated that the fusion protein was capable of isolating pUC19 DNA into which the recognition sequence for the fusion-protein had been inserted (pTS DNA) when in the presence of the conditioned process material. Althougth the pTS recognition sequence differs from native pUC19 sequences by only 2 bp, the fusion protein was shown to act as a highly selective affinity ligand for pTS DNA alone. Subsequently, the scale of the process was scaled 25-fold and positioned directly following the EFD system. In conclusion, the integration of the EFD micro-filtration system and zinc-finger affinity purification technique resulted in the capture of approximately 1 mg of plasmid DNA was purified from 1L of E.coli  culture in a simple two stage process, resulting in the complete removal of genomic DNA and 96.7% of cellular protein in less than 1 hour of process time.

Characterisation of femtosecond laser inscribed long period gratings in photonic crystal fibre

The use of high intensity femtosecond laser sources for inscribing fibre gratings has attained significant interest. The principal advantage of high-energy pulses is their ability for grating inscription in any material type without preprocessing or special core doping - the inscription process is controlled multi-photon absorption, void generation and subsequent local refractive index changes. The formation of grating structures in photonics crystal fibre has proven difficult, as the presence of holes within the fibre that allow wave-guidance impair and scatter the femtosecond inscription beam. Here we report on the consistent manufacture of long period gratings in endlessly single mode microstructure fibre and on their characterisation to external perturbations. Long period gratings are currently the subject of considerable research interest due to their potential applications as filters and as sensing devices, responsive to strain, temperature, bending and refractive index. Compared to the more mature fibre Bragg grating sensors, LPGs have more complex spectra, usually with broader spectral features. On the other hand they are intrinsically sensitive to bending and refractive index. Perhaps more importantly, the fibre design and choice of grating period can have a considerable influence over the sensitivity to the various parameters, for example allowing the creation of a bend sensor with minimal temperature cross-sensitivity. This control is not possible with FBG sensors. Here we compare the effects of symmetric and asymmetric femtosecond laser inscription.

Comparison between femtosecond laser and fusion-arc inscribed long period gratings in photonic crystal fibre

The use of high intensity femtosecond laser sources for inscribing fibre gratings has attained significant interest. The principal advantage of high-energy pulses is their ability for grating inscription in any material type without preprocessing or special core doping. In the field of fibre optical sensing LPGs written in photonic crystal fibre have a distinct advantage of low temperature sensitivity over gratings written in conventional fibre and thus minimal temperature cross-sensitivity. Previous studies have indicated that LPGs written by a point-by-point inscription scheme using a low repetition femtosecond laser exhibit post-fabrication evolution leading to temporal instabilities at room temperatures with respect to spectral location, strength and birefringence of the attenuation bands. These spectral instabilities of LPGs are studied in photonic crystal fibres (endlessly single mode microstructure fibre) to moderately high temperatures 100°C to 200°C and their performance compared to fusion-arc fabricated LPG. Initial results suggest that the fusion-arc fabricated LPG demonstrate less spectral instability for a given constant and moderate temperature, and are similar to the results obtained when inscribed in a standard single mode fibre.

Functional carbon nanotubes for photonic applications

Carbon nanomaterials are an active frontier of research in current nanotechnology. Single wall Carbon Nanotube (SWNT) is a unique material which has already found several applications in photonics, electronics, sensors and drug delivery. This thesis presents a summary of the author’s research on functionalisation of SWNTs, a study of their optical properties, and potential for an application in laser physics. The first significant result is a breakthrough in controlling the size of SWNT bundles by varying the salt concentrations in N-methyl 2-pyrrolidone (NMP) through a salting out effect. The addition of Sodium iodide leads to self-assembly of CNTs into recognizable bundles. Furthermore, a stable dispersion can be made via addition polyvinylpyrrolidone (PVP) polymer to SWNTs-NMP dispersion, which indicates a promising direction for SWNT bundle engineering in organic solvents. The second set of experiments are concerned with enhancement of photoluminescence (PL), through the formation of novel macromolecular complexes of SWNTs with polymethine dyes with emission from enhanced nanotubes in the range of dye excitation. The effect appears to originate from exciton energy transfer within the solution. Thirdly, SWNT base-saturable absorbers (SA) were developed and applied to mode locking of fibre lasers. SWNT-based SAs were applied in both composite and liquid dispersion forms and achieved stable ultrashort generation at 1000nm, 1550nm, and 1800 nm for Ytterbium, Erbium and Thulium-doped fibre laser respectively. The work presented here demonstrates several innovative approaches for development of rapid functionalised SWNT-based dispersions and composites with potential for application in various photonic devices at low cost.