Robotic arm material characterisation using LIBS and Raman in a nuclear hot cell decommissioning environment.

Material characterisation in nuclear environments is an essential part of decommissioning processes. This paper explores the feasibility of deploying commercial off the shelf (COTS) laser induced breakdown spectroscopy (LIBS) and Raman spectroscopy, for use in a decommissioning hot cell environment, to inform waste operation decision making. To operate these techniques, adapters and probes were designed and constructed, for each instrument, to form tools that a robotic arm could pick up and operate remotely from an isolated control room. The developed instrumentation successfully returned live measurement data to a control room for saving and further analysis (e.g. material classification/identification). Successful testing of the solutions was performed for contact LIBS, contact Raman and stand-off Raman on a PaR M3000 robotic arm, in a simulated hot cell environment and the limitations identified. Data obtained by the techniques are analysed, classified and presented in a 3D virtual environment. The spectral data collected by a basic COTS LIBS showed potential for use in contamination identification (beryllium is used as example). Potential for COTS, LIBS and Raman in decommissioning is established and improvements to the hardware, the measurement processes and how the data is stored and used, are identified.

Optical extinction combined with phase measurements for probing DNA-small-molecule interactions using an evanescent waveguide biosensor.

We demonstrate the use of both optical extinction and phase measurements for probing the interactions between DNA and small molecules by dual polarization interferometry. On binding to DNA at the interface, mitoxantrone (MTX) and methylene blue (MB) induced reversible concentration-dependent optical extinction due to light absorption, which clearly revealed the association and dissociation of small molecules with DNA in real time. The binding constants of MTX-DNA and MB-DNA determined from the masses derived from optical extinction are 1.8 x 10(5) and 4.2 x 10(4) M(-1), respectively, and shown to be buffer salt concentration-dependent. Apart from optical extinction, phase measurements reflected the overall change of the interaction; namely, a combined result of the binding of small molecules and any changes in DNA structure. The masses derived from phase could be very different from those derived from optical extinction. The structural changes detected by phase measurements showed a contraction and densification of DNA upon intercalation by MTX or MB. The combination of optical extinction and phase measurements allows a detailed understanding of the interaction process.

Thermoresponsive copolymer nanofilms for controlling cell adhesion, growth, and detachment.

This study reports the development and use of a novel thermoresponsive polymeric nanofilm for controlling cell adhesion and growth at 37 °C, and then cell detachment for cell recovery by subsequent temperature drop to the ambient temperature, without enzymatic cleavage or mechanical scraping. A copolymer, poly(N-isopropylacrylamide-co-hydroxypropyl methacrylate-co-3-(trimethoxysilyl)propyl methacrylate) (abbreviated PNIPAAm copolymer), was synthesized by free radical polymerization. The thermoresponses of the copolymer in aqueous solution were demonstrated by dynamic light scattering (DLS) through detecting the sensitive changes of copolymer aggregation against temperature. The DLS measurements revealed the lower critical solution temperature (LCST) at approximately 30 °C. The PNIPAAm film stability and robustness was provided through silyl cross-linking within the film and with the hydroxyl groups on the substrate surface. Film thickness, stability, and reversibility with respect to temperature switches were examined by spectroscopic ellipsometry (SE), atomic force microscopy (AFM), and contact angle measurements. The results confirmed the high extent of thermosensitivity and structural restoration based on the alterations of film thickness and surface wettability. The effective control of adhesion, growth, and detachment of HeLa and HEK293 cells demonstrated the physical controllability and cellular compatibility of the copolymer nanofilms. These PNIPAAm copolymer nanofilms could open up a convenient interfacial mediation for cell film production and cell expansion by nonenzymatic and nonmechanical cell recovery.

Multiple path length dual polarization interferometry.

An optical sensor for quantitative analysis of ultrathin films and adsorbed layers is described. Quantification of both layer thickness and refractive index (density) can be made for in situ and ex-situ coated films. With the use of two polarizations, in situ measurements are made via one path length in a young's interferometer arrangement while ex-situ measurements use multiple path lengths. The multiple path length young's interferometer arrangement is embodied in a solid state waveguide configuration called the multiple path length dual polarization interferometer (MPL-DPI). The technique is demonstrated with ultrathin layers of poly(methylmethacrylate) and human serum albumin.

A new analysis workflow for discrimination of nuclear grade graphite using laser-induced breakdown spectroscopy.

Stand-off, in-situ, laser induced breakdown spectroscopy (LIBS) offers a rapid, safe, and cost-effective method for discrimination of radioactive waste materials arising during the operation of nuclear plants and from decommissioning activities. Characterisation of waste materials is a critical activity in understanding the nature of the waste, ensuring hazardous material is managed safely and that waste can be segregated for reuse, recycle or sentenced for appropriate disposal. Characterisation of materials, often in hostile environments, requires the ability to remotely differentiate between materials in terms of their chemical composition and radioactivity. This proposition was tested using a case study on nuclear grade graphite. Graphite has been used extensively as a moderator material in many nuclear reactors. Internationally, over 250,000 tons of various nuclear-grade graphite, and graphite-bearing, materials exist. These are a major issue for nuclear decommissioning and radioactive waste management, due to the long half-lives of the associated 14C and 36Cl isotopes. LIBS offers a method for discrimination of nuclear grade graphites and other carbon and non-carbon-bearing wastes. This paper describes the development of a workflow method, including LIBS measurement analysis, for the discrimination of pre-irradiated nuclear 'Pile Grade A' (PGA) graphite moderator rod and domestic lumpwood charcoal, which act as surrogates for nuclear grade graphite and other carbon-bearing wastes. A new analysis workflow comprising the examination of spectral characteristics, multivariate analysis and molecular isotopic spectroscopy is proposed to enable rapid segregation of graphite from a heterogeneous waste stream. Enhanced characterisation techniques have the potential to significantly reduce the cost of decommissioning large parts of legacy nuclear generation plants.

Analysis of contaminated nuclear plant steel by laser-induced breakdown spectroscopy.

Laser Induced Breakdown Spectroscopy (LIBS) has the potential to allow direct, standoff measurement of contaminants on nuclear plant. Here, LIBS is evaluated as an analytical tool for measurement of Sr and Cs contamination on type 304 stainless steel surfaces. Samples were reacted in model acidic (PUREX reprocessing) and alkaline (spent fuel ponds) Sr and Cs bearing liquors, with LIBS multi-pulse ablation also explored to measure contaminant penetration. The Sr II (407.77nm) and Cs I (894.35nm) emission lines could be separated from the bulk emission spectra, though only Sr could be reliably detected at surface loadings >0.5mgcm-2. Depth profiling showed decay of the Sr signal with time, but importantly, elemental analysis indicated that material expelled from LIBS craters is redistributed and may interfere in later laser shot analyses.

Gelatin modified ultrathin silk fibroin films for enhanced proliferation of cells.

Silk fibroin (SF) films were modified with gelatin (G) to explore if such SF/G films could enhance the surface biocompatibility of silk as cell growth biomaterials. Ultrathin films were coated from aqueous SF solutions pre-mixed with different amounts of G. It was found that the SF/G blended films after methanol treatment were highly stable in physiological conditions. The incorporation of G smoothed the surface morphology of the SF/G films formed. Surface-exposed RGD sequences were successfully identified on the SF/G films through specific recognition of an integrin-mimicking peptide (bearing the sequence of CWDDGWLC). Cell culture experiments with 3T3 fibroblasts demonstrated that SF/G films with 1.2-20% (w/w) G gave clear improvement in promoting cell attachment and proliferation over pure SF films. Films containing 10-20% (w/w) of G showed cell attachment and growth even superior to the pure G films. The differences as observed from this study suggest that due to the lack of mechanical strength associated with its high solubility, G could not work alone as a cell growth scaffold. The enhanced cellular responses from the blended SF/G films must result from improvement in film stability arising from SF and in cytocompatibility arising from G. The results thus indicate the potential of the SF/G blends in tissue engineering and biomedical engineering where physical and biological properties could be manipulated via mixing either as bulk biomaterials or for coating purposes.

Serum protein layers on parylene-C and silicon oxide: effect on cell adhesion.

Among the range of materials used in bioengineering, parylene-C has been used in combination with silicon oxide and in presence of the serum proteins, in cell patterning. However, the structural properties of adsorbed serum proteins on these substrates still remain elusive. In this study, we use an optical biosensing technique to decipher the properties of fibronectin (Fn) and serum albumin adsorbed on parylene-C and silicon oxide substrates. Our results show the formation of layers with distinct structural and adhesive properties. Thin, dense layers are formed on parylene-C, whereas thicker, more diffuse layers are formed on silicon oxide. These results suggest that Fn acquires a compact structure on parylene-C and a more extended structure on silicon oxide. Nonetheless, parylene-C and silicon oxide substrates coated with Fn host cell populations that exhibit focal adhesion complexes and good cell attachment. Albumin adopts a deformed structure on parylene-C and a globular structure on silicon oxide, and does not support significant cell attachment on either surface. Interestingly, the co-incubation of Fn and albumin at the ratio found in serum, results in the preferential adsorption of albumin on parylene-C and Fn on silicon oxide. This finding is supported by the exclusive formation of focal adhesion complexes in differentiated mouse embryonic stem cells (CGR8), cultured on Fn/albumin coated silicon oxide, but not on parylene-C. The detailed information provided in this study on the distinct properties of layers of serum proteins on substrates such as parylene-C and silicon oxide is highly significant in developing methods for cell patterning.

Cationic copolymer-mediated DNA immobilization: interfacial structure and composition as determined by ellipsometry, dual polarization interferometry, and neutron reflection.

DNA immobilization onto support surfaces is required in biotechnological applications such as microarrays and gene delivery. This important interfacial molecular process can be mediated from a preadsobred cationic polymer. There is, however, a lack of understanding over the control of the interfacial composition and structural distribution of the DNA immobilized. We have used a combined approach of spectroscopic ellipsometry (SE), dual polarization interferometry (DPI) and neutron reflection (NR) to determine the interfacial polymer adsorption and the subsequent DNA binding. Cationic diblock copolymers incorporating 30 phosphorylcholine (PC) groups and different diethylaminoethyl groups, referred to as MPC30-DEAn, were chosen because of their well-defined molecular architecture. While our studies revealed different effects of surface charge and hydrophobicity, the amount of copolymers adsorbed on both model surfaces showed a broad trend of increase with solution pH, indicating a strong effect arising from pH-dependent charge density on the copolymers. In contrast, the copolymer structure and solution concentration showed a weak effect under the conditions studied. The subsequent DNA binding at pH 7 showed that on both surfaces the amount of DNA immobilized followed an approximate 1:1 charge interaction for all different DNA samples studied, irrespective of single or double strand, or different DNA size, indicating the dominant effect of electrostatic interaction between the two species. Both DPI and NR revealed consistent thickness increase upon DNA binding. Furthermore, with increasing DNA size, the interfacial layer became much thicker, and charge interaction drove more extensive interfacial mixing between the two species. Our results show that the amount of DNA immobilized is controlled by the amount of cationic copolymer preadsorbed that is in turn controlled by the solution pH and surface chemistry but that is barely affected by the type and concentration of DNA or cationic copolymer.

Structural and functional characterization of recombinant matrilin-3 A-domain and implications for human genetic bone diseases.

Mutations in matrilin-3 result in multiple epiphyseal dysplasia, which is characterized by delayed and irregular bone growth and early onset osteoarthritis. The majority of disease-causing mutations are located within the beta-sheet of the single A-domain of matrilin-3, suggesting that they disrupt the structure and/or function of this important domain. Indeed, the expression of mutant matrilin-3 results in its intracellular retention within the rough endoplasmic reticulum of cells, where it elicits an unfolded protein response. To understand the folding characteristics of the matrilin-3 A-domain we determined its structure using CD, analytical ultracentrifugation, and dual polarization interferometry. This study defined novel structural features of the matrilin-3 A-domain and identified a conformational change induced by the presence or the absence of Zn(2+). In the presence of Zn(2+) the A-domain adopts a more stable "tighter" conformation. However, after the removal of Zn(2+) a potential structural rearrangement of the metal ion-dependent adhesion site motif occurs, which leads to a more "relaxed" conformation. Finally, to characterize the interactions of the matrilin-3 A-domain we performed binding studies on a BIAcore using type II and IX collagen and cartilage oligomeric matrix protein. We were able to demonstrate that it binds to type II and IX collagen and cartilage oligomeric matrix protein in a Zn(2+)-dependent manner. Furthermore, we have also determined that the matrilin-3 A-domain appears to bind exclusively to the COL3 domain of type IX collagen and that this binding is abolished in the presence of a disease causing mutation in type IX collagen.