A new camera for high-resolution infrared imaging of works of art

A new camera – SIRIS (scanning infrared imaging system) – developed at the National Gallery in London allows high-resolution images to be made in the near infrared region (900–1700 nm). The camera is based on a commercially available 320 × 256 pixel indium gallium arsenide area array sensor. This relatively small sensor is moved across the focal plane of the camera using two orthogonal translation stages to give images of c. 5000 × 5000 pixels. The main advantages of the SIRIS camera over scanning infrared devices or sequential image capture and mosaic assembly are its comparative portability and rapid image acquisition – making a 5000 × 5000 pixel image takes less than 20 minutes. The SIRIS camera can operate at a range of resolutions; from around 2.5 pixels per millimetre over an area of up to 2 × 2 m to 10 pixels per millimetre when examining an area measuring 0.5 × 0.5 m. The development of the mechanical, optical and electronic components of the camera, including the design of a new lens, is described. The software used to control image capture and to assemble the individual frames into a seamless mosaic image is mentioned. The camera was designed primarily to examine underdrawings in paintings; preliminary results from test targets and paintings imaged in situ are presented and the quality of the images compared with those from other cameras currently used for this application

Aptasensors using tunable resistive pulse sensing

In recent years there has been an increased drive towards point of care testing (POCT), in which assays are performed at the site of the patient. This has many benefits, critically; the time for a result to be obtained will be significantly reduced, allowing for greater and more effective decision making. Many currently used bioassay methods are not affordable in resource poor areas where infectious disease is most prevalent, in order to combat this issue many research groups are attempting to miniaturise equipment for portability and make assays more affordable and therefore more accessible. With the aims of generating a new assay platform which is highly portable and affordable, the work in this thesis presents the development of several generic methods utilising nano- and micro-scale beads coated with aptamer which are then monitored interacting with target proteins with Tunable Resistive Pulse Sensing (TRPS). Aptamers are short oligonucleotide sequences which are capable of binding to a wide range of targets with high selectivity and comparable affinity to antibodies while possessing greater stability and have begun to challenge the role of antibodies. When aptamers bind a target, they often undergo a conformational change. In the assays described herein, this conformational change is key to the observed signal changes. TRPS is a pore-based system in which beads moving through a pore cause a measurable increase in resistance which can be used to derive particle size, concentration, and mobility. During the course of this thesis several template TRPS aptasensors have been developed. TRPS was successfully used to confirm the successful coating of nano- and micro-scale beads with DNA aptamers by monitoring an increase in electrophoretic mobility when the negatively charged DNA is added to the surface. Following on from this, TRPS was used to monitor the interaction of aptamer tagged beads with thrombin protein enabling thrombin detection down to 1.4 nM and the comparison of several thrombin-aptamers with results comparable to previously published SPR data. Thrombin was postulated to shield the negative DNA, resulting in a decrease in mobility, and the magnitude of this charge shielding was found to depend upon the binding mechanism of the aptamer used. This effect is not thought to be specific to our system nor to thrombin, the principles outlined here may be applied to other RPS technologies, or by interchanging of the aptamer, different proteins. In later chapters, this method is expanded to include multiplexed detection of growth factors and a significant improvement in signal. vi Following on from this, the controlled aggregation of avidin coated beads in the presence of biotinylated-BSA was explored. Factors impacting upon this assay were discussed including magnetic separation, particle size and particle concentration, and different methods of data interpretation were presented. This aggregation study identified several key parameters in the use of TRPS in aggregation assays. Using the methods outlined by the study of aggregates, a dispersion assay was then designed in which the interaction of thrombin proteins with clusters of particles brought about the release of many small particles by the disruption of double stranded DNA linkages. This dispersion assay incorporated magnetic separation to simplify the read-out and relied on measuring particle concentration rather than mobility, enabling the use of additional pressure to increase speed and ease of use. Using this method, thrombin was able to be detected down to 100 fM, a significant advancement in TRPS aptasensors

Multiplexed, label-free detection of biomarkers using aptamers and Tunable Resistive Pulse Sensing (AptaTRPS).

Diagnostics that are capable of detecting multiple biomarkers are improving the accuracy and efficiency of bioassays. In previous work we have demonstrated the potential of an aptamer-based sensor (aptasensor) utilising Tunable Resistive Pulse Sensing (TRPS). Here, we have advanced the technique identifying key experimental designs for potential POC assays. The assay utilised superparamagnetic beads, and using TRPS monitored their translocations through a pore. If the surfaces of the beads are modified with an aptamer, the frequency of beads (translocations/min) through the pore can be related to the concentration of specific proteins in the solution. Herein, we have demonstrated the successful use of TRPS to observe the binding of two proteins, to their specific aptamers simultaneously. We describe a series of experiments illustrating key factors which we believe are integral to bead-based assays and demonstrate a general method for a multiplexed assay. In summary, we have explored the effects of beads size, concentration, potential bias, pH and aptamer affinity to enhance the sensitivity and practically of a TRPS aptasensor. The method utilises the fact the binding of the aptamer to the protein results in a change in charge density on the bead surface, the isoelectric point of the protein then dominates the mobility of the beads, creating a multiplexed assay termed AptaTRPS. By alteration of the applied potential to the instrument it is possible to produce a positive signal in a simple multiplexed assay

Aptamer based dispersion assay using tunable resistive pulse sensing (TRPS)

Aggregates of micron sized beads were formed by the binding of anti-thrombin aptamer to its complement. The addition of the thrombin protein target caused a concentration-dependant dispersion of these aggregates, and their number was measured by tunable resistive pulse sensing. The technique allowed the detection of thrombin down to sub picomolar concentrations, and an increase in sensitivity over previous assays on the same platform. The sensitivity of the assay is attributed to each thrombin protein disrupting multiple aggregates resulting in a signal amplification

Monitoring aptamer-protein interactions using Tunable Resistive Pulse Sensing

Aptamers are short single-stranded pieces of DNA or RNA capable of binding to analytes with specificity and high affinity. Due to their comparable selectivity, stability, and cost, over the last two decades, aptamers have started to challenge antibodies in their use on many technology platforms. The binding event often leads to changes in the aptamer’s secondary and tertiary structure; monitoring such changes has led to the creation of many new analytical sensors. Here, we demonstrate the use of a tunable resistive pulse sensing (TRPS) technology to monitor the interaction between several DNA aptamers and their target, thrombin. We immobilized the aptamers onto the surface of superparamagnetic beads, prior to their incubation with the thrombin protein. The protein binding to the aptamer caused a conformational change resulting in the shielding of the polyanion backbone; this was monitored by a change in the translocation time and pulse frequency of the particles traversing the pore. This signal was sensitive enough to allow the tagless detection of thrombin down to nanomolar levels. We further demonstrate the power of TRPS by performing real time detection and characterization of the aptamer–target interaction and measuring the association rates of the thrombin protein to the aptamer sequences

Neutron diffraction evidence of microscopic charge inhomogeneities in the CuO2 plane of superconducting La2-xSrxCuO4 (0 < x <0.30)

We present local structural evidence supporting the presence of charge inhomogeneities in the CuO2 planes of underdoped La2-xSrxCuO4. High-resolution atomic pair distribution functions have been obtained from neutron powder diffraction data over the range of doping 0 < x < 0.30 at 10 K. Despite the average structure getting less orthorhombic we see a broadening of the in-plane Cu-O bond distribution as a function of doping up to optimal doping. Thereafter the peak abruptly sharpens. Complementary evidence is also evident from the observation of octahedral tilt disorder in the PDF at higher atomic separation. This suggests a crossover from a charge inhomogeneous state at and below optimal doping to a homogeneous charge state above optimal doping. The strong response of the local structure to the charge-state implies a strong electron-lattice coupling in these materials.Comment: 4 pages, 3 figures, submitted to Physical Review Letters (27-th of June 1999) resubmitted to Phys. Rev. Lett. (8th of March 2000