Development of a multiplexing biosensor platform using SERS particle immunoassay technology

A thesis submitted to the University of Bedfordshire in partial fulfilment of the requirements of the degree of Doctor of PhilosophyThe purpose of this study is to demonstrate the ability of surface enhanced Raman scattering (SERS) active particles to enable multiplexed immunoassays in a lateral flow format for point of care (POC) testing. The SERS particles used for this study are chemically active glass coated gold particles, containing tracer molecules which in principle can be chosen to provide Raman Spectra with unique features allowing multiple tracers to be simultaneously measured and distinguished without interference between each other. Lateral flow immunoassay technology is the important part of this study and can be conveniently packaged for the use of other than highly skilled technicians outside of the laboratory. A well-known (single channel - simplex) device for the pregnancy test is a typical example of the lateral flow assay. Similar formats have been/are being developed by others for a range of POC applications – but most diagnostic applications require simultaneous determination of a range of biomarkers and multiplexed assays are difficult to achieve without significant interference between the individual assays. This is where SERS particles may provide some advantages over existing techniques. Cardiac markers are the growing market for point of care technology therefore biomarkers of cardiac injury (Troponin, myoglobin and CRP) have been chosen as a model. The object of the study is to establish the proof of concept multiplexing assay using these chosen biomarkers. Thus, initially all different particles were characterised in single and mixture form. Also development of conjugate chemistry between antibodies for each analyte that have been purchased from commercial sources and SERS particles were analysed using different conditions like buffer, pH and antibody loading concentration to get the optimum intensity. The selected SERS particles and their conjugates were tested for size, aggregation and immune quality using a range of techniques: ultraviolet-visible (UV/Vis) absorption spectroscopy, dynamic light scattering (DLS) and lateral flow assay. These characterisations methodologies gave the understanding of optimum conditions of the each conjugates and individual’s behaviour in mixture conditions as well. After the characterisation all conjugates were tested singularly on the lateral flow assay using buffers and serum. The results of this single analyte immunoassay explained the individual’s bioactivity on the lateral flow strip. Further in study, multiplex assay have been demonstrated in serum. These outcomes have described each candidate characteristic in a mixture form on the lateral flow strip. In order to get the optimum Raman intensity from multiplex assay, the detection and capture antibodies loading concentrations were tuned in the assay. Later on different combinations (high, medium and low concentrations) of all three analytes were analysed and has found some interferences in multiplex assay. To investigate these issues various aspect were considered. First of all, different possibilities of non-specific interactions between the co-analytes and antibodies were tested. In addition, steric hindrance and optical interference investigations were performed via several assays and analysis using Scanning electron microscopy. The outcomes have confirmed related optical interferences. Therefore other assay (wound biomarkers) established to eliminate the interferences. In summary, the works reported here have built and test the equipment and necessary reagents for individual assays before moving on the more complicated task. In addition, the entire study has given a deep knowledge of multiplex assay on a single test line including the investigation of the issues for selected cardiac biomarkers and their applications in the future

Importance of clinical and radiological assessment of patient of osteoarthritis knee foroptimum outcome in total knee replacement

Background: Total knee replacement (TKR) provides symptomatic relief in patients with severe arthritis by removing pathologic joint tissue and restoration of the joint anatomy and function by the replacing with metal or plastic components leading to more stable biomechanics. Inspite of all advances in the surgical techniques 1 out of 5 people who undergo TKA remain unsatisfied. Clinical and radiological grading of osteoarthritis of knee may be an important prognostic marker to assess the outcome following TKR. Methods: Study conducted was retrospective study. Pre-op routine and clinical assessment data noted from departmental records. Correlation between ‘initial knee society score’ (KSSi) and ‘radiological severity’ in terms of (Kellgren-Lawrence) grade and varus angle with ‘change in knee society score’ (ΔKSS) assessed to reach at conclusion. Results: Lower KSS values, higher KL grading, higher femorotibial angle and were associated significantly with higher change in KSS values, i.e. better outcome. Conclusions: Severe OA knee with high FTA and higher KL grade has shown better outcome following TKA. In early stages it is better to manage the patient conservatively with medications and physiotherapy

Spring inter monsoon algal blooms in the Eastern Arabian sea: Shallow marine encounter off Karwar and Kumbla Coast using a Hyperspectral radiometer

We encountered the presence of algal blooms in the shallow coastal waters; typical of case II conditions off Karwar (Karnataka) and Kumbla (Kerala), eastern Arabian Sea during the Inter Monsoon. The algal blooms are Trichodesmium species and appear in shades of brown strands and tufts representing T. Erythraeum. Sites of the algal blooms depict a high spectrophotometric surface chlorophyll-a value ranging from 32- 39 mgm-3 and sea surface area surrounding the bloom sites depict lower values of chlorophyll-a (1-3 mgm-3). In-situ Hyperspectral radiometer measurements depict the existence of Chlorophyll Maxima at various depths (3.0-37.31 ug/l) representing the blooms along with their surface appearance. Contrary to the Karwar blooms that are dense, the blooms at Kumbla were dispersed and scantly distributed on the surface waters. Our sea-truth data on mean Sea Surface Temperature of Karwar (30.61 °C) that simulates the Kumbla (30.34 °C) scenario and the mean salinity of Kumbla was 35.86psu, and that of Karwar that is slightly elevated (35.40psu), suggests that SST and salinity probably do not either directly enhance or provide an environment for the eterioration of algal blooms

Dental students’ ability to locate emergency equipment‐lessons learned from aviation

PurposeThe purpose of this study was to evaluate the dental student’s ability to locate medical emergency equipment/items at the University of Michigan School of Dentistry clinic.MethodsA total of 138 second‐year dental students (traditional group) participated in this study as part of a simulation‐based medical emergencies rotation course held during the winter term of 2014 and 2015. Without prior training, students were tested on their ability to locate nine predetermined items on the clinic floor using a self‐reported checklist. Six months later, a convenience sample of 18 students (novel group) from the same cohort were later trained on their location and retested individually.ResultsOf the 138 students tested, only 10.14% students could locate seven of the nine items when compared to 100% in the novel group. Only 5.07% of students in the traditional group could locate all items initially, compared with 72.22% students in the novel group.ConclusionWhilst our students have lecture‐based knowledge about medical emergencies, the results of our study identified a gap of knowledge of emergency equipment/item location amongst students. Therefore, an intervention performed with a similar group of second‐year dental students supported that proper training may be used to achieve retention of knowledge. Based on our “novel group” results, we have incorporated targeted training in the dental curriculum that leads to students being better prepared in locating emergency equipment/items. This study suggests that other populations, such as faculty or staff, may also benefit from hands‐on training.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142451/1/eje12251.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142451/2/eje12251_am.pd

Process Parameter Selection for Resistance Spot Welding through Thermal Analysis of 2mm CRCA Sheets

AbstractAn attempt is made to arrive at suitable process parameters for resistance spot welding through thermal analysis. 2mm Closed Rolled Closed Anealing (CRCA) sheet metal is selected as work piece. Nugget and electrode diameter are evaluated from emperical relations. Full factorial Design Of Experiment (DOE) is applied for weld current and time. Thermal analysis is carried out for selected DOE values using SYSweld. Temperature distribution, nugget and Heat Affected Zone dimensions are obtained from the analysis. Weld strenght is analysed through experimental trails. Optimum process parameter for spot welding of 2mm CRCA sheet is arrived by comparing simulation and experimental results

Optical Scattering Artifacts Observed in the Development of Multiplexed Surface Enhanced Raman Spectroscopy Nanotag Immunoassays

Here we describe scattering based signal suppression artifacts encountered while developing multiplex lateral flow (LF) immunoassay using surface enhanced Raman spectroscopy (SERS) “nanotags” as analyte labels. Using these SERS nanotags, we have produced a quantitative test for inflammation biomarkers that is transferable to the point of care (POC). The SERS assay shows similar performance when compared with a fluorescent nanoparticle POC test. Here, using cardiac and inflammation biomarkers, we highlight the need to carefully optimize the concentration of assay components when using SERS nanotags and a single-line multiplexing approach. We show that in certain circumstances the SERS signal may be suppressed, leading to a significant underestimation of the analyte concentrations. Using electron microscopy and optical spectroscopy, we demonstrate that the error in the measurement is associated with the light scattering properties of the nanotags. These findings will be applicable to other nanoparticle labels with high light scattering coefficients. Through careful modification of the assay to reduce the impact of light scattering, it is possible to produce quantitative assays, but potentially at the expense of multiplexing capability and assay sensitivity