CERES :: Browsing by Author "Cullen, David C."

Browsing by Author "Cullen, David C."

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Open Access
CASS•E: Cranfield astrobiological stratospheric sampling experiment
(2010-12-31T00:00:00Z) Naicker, L.; Grama, V. V.; Juanes-Vallejo, Clara M.; Katramados, Ioannis; Rato, Carla Cristina Pereira Salgueiro Catarino; Rix, Catherine S.; Sanchez, E.; Cullen, David C.
CASS•E is a life detectionexperimentthat aims to be capable of collecting microorganisms in Earth's Stratosphere. Theexperiment will be launched on astratosphericballoon in collaboration with Eurolaunch through the BEXUS (Balloon-borneExperimentsfor Universitv Students) program from Esrange Sweden in October 2010. It essentially consists of a pump which draws air from the Stratosphere through a collection filter mechanism. Due to the low number density of microbes in the Stratosphere compared to the known levels of contamination at ground level, theexperimentincorporated Planetary Protection and Contamination Control (PP&CC) protocols in its design and construction in order to confirm that any microbes detected are trulyStratosphericin origin. Space qualified cleaning and sterilisation techniques were employed throughout Assembly Integration and Testing (AIT) as well as biobarriers which were designed to open only in the stratosphere and so prevent recontamination of the instrument alter sterilisation. The material presented here covers the design and AIT of CASS•E. Copyright ©2010 by the International Astronautical Federation. All rights rese
Open Access
Correlations between life-detection techniques and implications for sampling site selection in planetary analog missions
(Mary Ann Liebert, Inc., 2017-10-01) Gentry, Diana M.; Amador, Elena S.; Cable, Morgan L.; Chaudry, Nosheen; Cullen, Thomas; Jacobsen, Malene B.; Murukesan, Gayathri; Schwieterman, Edward W.; Stevens, Adam H.; Stockton, Amanda; Tan, George; Yin, Chang; Cullen, David C.; Geppert, Wolf
We conducted an analog sampling expedition under simulated mission constraints to areas dominated by basaltic tephra of the Eldfell and Fimmvörðuháls lava fields (Iceland). Sites were selected to be “homogeneous” at a coarse remote sensing resolution (10–100 m) in apparent color, morphology, moisture, and grain size, with best-effort realism in numbers of locations and replicates. Three different biomarker assays (counting of nucleic-acid-stained cells via fluorescent microscopy, a luciferin/luciferase assay for adenosine triphosphate, and quantitative polymerase chain reaction (qPCR) to detect DNA associated with bacteria, archaea, and fungi) were characterized at four nested spatial scales (1 m, 10 m, 100 m, and >1 km) by using five common metrics for sample site representativeness (sample mean variance, group F tests, pairwise t tests, and the distribution-free rank sum H and u tests). Correlations between all assays were characterized with Spearman's rank test. The bioluminescence assay showed the most variance across the sites, followed by qPCR for bacterial and archaeal DNA; these results could not be considered representative at the finest resolution tested (1 m). Cell concentration and fungal DNA also had significant local variation, but they were homogeneous over scales of >1 km. These results show that the selection of life detection assays and the number, distribution, and location of sampling sites in a low biomass environment with limited a priori characterization can yield both contrasting and complementary results, and that their interdependence must be given due consideration to maximize science return in future biomarker sampling expeditions.
Open Access
CubeSat 3U-payload for in-situ resource utilisation demonstration at C-type near Earth asteroids
(International Astronautical Federation (IAF), 2018-10-05) Elioenai, Sitepu; Cullen, David C.
payloads capable of performing cost effective early stage in situ demonstrations of key steps for various types of ISRU. Such demonstrations would be proof-of-concept and de-risking exercises that would enable future pilot scale and eventually full-scaled implementations.This presentation will focus on a systems design for a 3U payload to demonstration at a C-type NEA the low temperature extraction of water and the subsequent electrolysis of this to dioxygen and dihydrogen. The system has the following features: sample acquisition via counter rotating brushes, extraction of volatile components via ovens with electrical resistive heating, trapping of condensable volatiles – primarily water, electrolysis of the trapped water into dioxygen and dihydrogen gas, and analysis of volatiles at various stages of the process with a miniaturised ion-trap mass-spectrometer. The baseline design allows for the collection and processing of 4 discrete samples using a carousel with 4 single use ovens. Each oven has a nominal internal volume of 7m3. Additionally the input assumptions concerning regolith properties, modelling studies and the development and implementation of a number of laboratory breadboards of various sub-systems will be presented.The design is intend to be compatible with use as part of a free-flying interplanetary 6U CubeSat, a 6U CubeSat hosted by and released by a larger parent spacecraft local to a NEA, or permanently hosted on a larger NEA surface rendezvous spacecraft.
Open Access
Defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction
(BMC (part of Springer Nature), 2015-10-24) Al Qaraghuli, Mohammed M.; Palliyil, Soumya; Broadbent, Gillian; Cullen, David C.; Charlton, Keith A.; Porter, Andrew J.
Background: Low molecular weight haptens (<1000 Da) cannot be recognized by the immune system unless conjugated to larger carrier molecules. Antibodies to these exceptionally small antigens can still be generated with exquisite sensitivity. A detailed understanding at the molecular level of this incredible ability of antibodies to recognize haptens, is still limited compared to other antigen classes. Methods: Different hapten targets with a broad range of structural flexibility and polarity were conjugated to carrier proteins, and utilized in sheep immunization. Three antibody libraries were constructed and used as potential pools to isolate specific antibodies to each target. The isolated antibodies were analysed in term of CDR length, canonical structure, and binding site shape and electrostatic potential. Results: The simple, chemically naïve structure of squalane (SQA) was recognized with micromolar sensitivity. An increase in structural rigidity of the hydrophobic and cyclic coprostane (COP) did not improve this binding sensitivity beyond the micromolar range, whilst the polar etioporphyrin (POR) was detected with nanomolar sensitivity. Homoserine lactone (HSL) molecules, which combine molecular flexibility and polarity, generated super-sensitive (picomolar) interactions. To better understand this range of antibody-hapten interactions, analyses were extended to examine the binding loop canonical structures and CDR lengths of a series of anti-hapten clones. Analyses of the pre and post- selection (panning of the phage displayed libraries) sequences revealed more conserved sites (123) within the post-selection sequences, when compared to their pre-selection counterparts (28). The strong selection pressure, generated by panning against these haptens resulted in the isolation of antibodies with significant sequence conservation in the FW regions, and suitable binding site cavities, representing only a relatively small subset of the available full repertoire sequence and structural diversity. As part of this process, the important influence of CDR H2 on antigen binding was observed through its direct interaction with individual antigens and indirect impact on the orientation and the pocket shape, when combined with CDRs H3 and L3. The binding pockets also displayed electrostatic surfaces that were complementary to the hydrophobic nature of COP, SQA, and POR, and the negatively charged HSL. Conclusions: The best binding antibodies have shown improved capacity to recognize these haptens by establishing complementary binding pockets in terms of size, shape, and electrostatic potential.
Open Access
Demonstration of a multi-technique approach to assess glacial microbial populations in the field
(Cambridge University Press, 2016-04-04) Barnett, Megan J.; Pawlett, Mark; Wadham, Jemma L.; Jackson, Miriam; Cullen, David C.
The ability to perform microbial detection and characterization in-field at extreme environments, rather than on returned samples, has the potential to improve the efficiency, relevance and quantity of data from field campaigns. To date, few examples of this approach have been reported. Therefore, we demonstrate that the approach is feasible in subglacial environments by deploying four techniques for microbial detection: real-time polymerase chain reaction; microscopic fluorescence cell counts, adenosine triphosphate bioluminescence assay and recombinant Factor C assay (to detect lipopolysaccharide). Each technique was applied to 12 subglacial ice samples, 12 meltwater samples and two snow samples from Engabreen, Northern Norway. Using this multi-technique approach, the detected biomarker levels were as expected, being highest in debris-rich subglacial ice, moderate in glacial meltwater and low in clean ice (debris-poor) and snow. Principal component analysis was applied to the resulting dataset and could be performed in-field to rapidly aid the allocation of resources for further sample analysis. We anticipate that in-field data collection will allow for multiple rounds of sampling, analysis, interpretation and refinement within a single field campaign, resulting in the collection of larger and more appropriate datasets, ultimately with more efficient science return.
Open Access
The design of beyond LEO mission scenario for a biological payload with a cubesat.
(2019-06) Pratnekar, Marko; Cullen, David C.; Kingston, Jennifer
CubeSat technology has been well established in the area of space engineering for almost two decades. Because of standardisation of components and procedures, development and launch costs of space missions are greatly reduced and space based experiments become more affordable for the broader community. Up to now, all CubeSat missions except for one have been launched in Low Earth Orbit. With recent developments and new launch opportunities, sending CubeSat missions with various on board experiments beyond Low Earth Orbit into interplanetary space becomes possible. Major space agencies have ambitious plans to send human space missions to Mars and other bodies in the Solar System. Traveling beyond Earth’s orbit, living cells in the human body will be exposed to harmful effects of space radiation. Therefore, before such interplanetary mission takes place, detailed study of effects of space radiation on human like mammalian cells should be conducted. An interplanetary mission based on the CubeSat platform would be the most affordable way of conducting such experiment. The main aim of the reported research work is to investigate if adequate space radiation protection and strict thermal environment requirement can be achieved and maintained for biological payload with higher forms of living cells, within a CubeSat spacecraft platform during interplanetary flight. This thesis is divided into a theoretical part – the literature review and methodological part – numerical simulations which are for space radiation performed by NASA developed software OLTARIS and for thermal analysis of the spacecraft and installed components with ESATAN –TMS modelling software. From the performed research work it can be concluded that adequate radiation protection can be implemented within the CubeSat payload compartment, so as not to exceed the acute dose limit set even during long duration interplanetary space flight, while at the same time leaving enough payload volume for the installation of the experimental biological payload and experimental instrumentation within the extra installed radiation protection. In maintaining the thermal environment inside the payload bay with biological material as well as in maintaining the survival temperature of some electronic components, careful heat management and active thermal control – additional electrical heating is required. There was no requirement for active cooling in the realistic mission scenarios considered.
Open Access
Detecting life on Mars and the life marker chip : antibody assays for detecting organic molecules in liquid extracts of Martian samples
(Cranfield University, 2012-01) Rix, Catherine S.; Cullen, David C.
The Life Marker Chip instrument, which has been selected to fly as part of the 2018 ExoMars rover mission payload, aims to detect up to 25 organic molecules in martian rocks and regolith, as markers of extant life, extinct life, meteoritic in-fall and spacecraft contamination. Martian samples will be extracted with a solvent and the resulting liquid extracts will be analysed using multiplexed microarray-format immunoassays. The LMC is under development by an international consortium led by the University of Leicester and the work described within this thesis was carried out at Cranfield University as part of the consortium’s broader program of work preparing the LMC instrument for flight in 2018. Within this thesis four specific areas of LMC instrument development are addressed: the investigation of immunoassay compatible liquid extraction solvents, the study of likely interactions of martian sample matrix with immunoassays, the development of antibodies for the detection of markers of extinct life and demonstration of solvent extraction and immunoassay detection in a flight representative format. Cont/d.
Open Access
Development of a fluidic sensor for the detection of herbicides using thylakoid preparations immobilised on magnetic beads to aid regenerability
(Cranfield University, 2008-12) Varsamis, Dimitrios Georgios; Cullen, David C.
Following the industrial revolution and advances in chemical science, the pollution of the environment with trace organic pollutants has been steadily increasing, which is of concern, due to their effect on the environmental and human health. Tighter legislation that has been introduced in order to minimise the release of harmful pollutants has led to the initiation of monitoring programmes. For example, drinking water suppliers are obliged to systematically monitor drinking water supplied for human consumption for a large range of pollutants. The same applies for waste water treatment facilities. The well-established standard methods of environmental waters analysis require sampling and transportation of samples to the laboratory for detailed measurements. Therefore, the timescale from sampling to reporting is not ideal, as a considerable lag occurs. There is therefore the potential for the use of in situ methods that overcome this issue. As these do not currently exist, a need to address this is identified. Biosensors are sensing devices that rely on a biologically-derived component as an integral part of their detection mechanism. Biosensors that respond to pollutants could be used for rapid, low cost, field-based pre-screening of water samples. Herbicides are considered to be the most important class of pesticides used in the E.U. Herbicides can be highly toxic for human and animal health, and increase in the application of herbicides in agriculture during recent decades has resulted in immense pollution of both soil and water. About half of the herbicides used at present in agriculture inhibit the light reactions in photosynthesis, mostly by targeting the Photosystem II (PSII) complex. A method of detecting certain classes of herbicides is therefore proposed; the photosynthesis-inhibiting herbicides act by binding to PS II, a chlorophyll– protein complex which plays a vital role in photosynthesis, located in the thylakoid membrane of algae, cyanobacteria and higher plants. The inhibition of PS II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by the HRP-mediated luminol chemiluminescence reaction. The design and development of a fluidic sensor unit for the detection of such herbicides, based upon their inhibition of the hydrogen peroxide production, will employ the use of superparamagnetic beads in order to address issues of reuse and regenerability. The illumination-dependent production of hydrogen peroxide by isolated thylakoids, and its inhibition by herbicides in a concentration-dependent manner, were achieved and measured with the HRP-mediated chemiluminescence reaction with luminol in a cuvette, batch format, allowing for the detection of herbicides down to 6.0 x 10-09.The integration of the above reactions has been achieved by designing and constructing a fluidic unit that combines the herbicide-dependent production and the detection of hydrogen peroxide in a single fluidic assay by combining all the individual steps in a compact, portable format, with both HRP and thylakoids covalently coupled on superparamagnetic beads. This addresses issues of regenerability, as the beads are introduced, used and discarded following a measurement, controlled only by magnetic and flow forces. Herbicide detection was achieved to a lower LOD of 5.5 x 10-10 M. The concept development, design and construction of the fluidic unit, as well as results of the detection of herbicides with the batch assay method has been published, in a paper by the author (Talanta, 2008, vol. 77, no. 1, pp. 42-47), Considerable progress has therefore been made towards developing a system that would be suitable for automated, field deployment applications for the detection of the most frequently used classes of herbicides; the lower LOD however is not within the stringent legislated maximum permissible limits set for herbicides measured in water, in European waters. An immediate step forward would be to achieve the required lower LOD, with the unit's development into a prototype instrument that can be field deployed being the further goal.
Open Access
Development of an automated identification system for nanocrystal encoded microspheres in flow cytometry
(Cranfield University, 2009-04) Gallagher, Clair; Cullen, David C.; Larcombe, Lee
Aim: This work sets out to use haplotype-based tagSNP selection and a systematic in silico analysis for design of multiplex-compatible PCR primer and SAT probe sets to capture maximum variation with minimum tests across candidate genes IGF1, IGFBP1 and IGFBP3. Additionally, the work aims to develop a number of robust, high-efficiency, high-specificity multiplex PCR constructs for amplification of these targets and to demonstrate the applicability of these target types to suspension array genotyping for non-insulin-dependant diabetes mellitus association facilitation. Methods: Haplotypes for predominantly European Caucasian populations were constructed and tagSNP selection performed using Haploview to capture maximum variation across candidate genes IGF1, IGFBP1 and IGFBP3. Extensive in silico analysis was performed for design, evaluation and selection of robust high-specificity primer and probe pairs, suitable for downstream multiplex PCR and SAT analysis. Singleplex endpoint and real-time PCR was performed for primer pair profile determination which informed multiplex PCR set construction and optimisation. The applicability of this complex target type to suspension array-based genotyping was investigated using a model probe pair using both quantum dot-encoded and fluorophore-encoded microspheres. Results: Haploview was used for haplotype construction and linkage disequilibriumbased tagSNP selection across candidate genes, reducing the number of SNP targets from 292 to 32 with minimal information loss. Extensive evaluation of potential tagSNPs was performed and 29 SNPs, representing 29 bins across target genes were designed for multiplex analysis. Singleplex end-point and real-time PCR was performed for primer pair profile determination which allowed four multiplex PCR sets to be constructed and optimised for simultaneous amplification of 14, six, five and two targets. The applicability of this complex target type (14-plex) to suspension array-based genotyping was demonstrated using a model probe pair. Conclusion: In silico analysis techniques have been applied for successful development of four robust multiplex PCR sets (14-plex, 6-plex, 5-plex and 2-plex) which display high-efficiency and target-specific amplification of tagSNPs, capturing maximum assaycompatible variation across candidate genes IGF1, IGFBP1 and IGFBP3 for European Caucasian populations. The applicability of these multiplex PCR constructs to suspension array-based genotyping has been demonstrated, thus paving the way for development of large multiplex suspension array-based genotyping assays using probes designed during the course of this work. This work offers the potential for comprehensive association analyses to become more accessible to the wider-scientific community by facilitating reduced genotyping burdens which allow increased accessibility for powerful association.
Open Access
Development of antibodies for life-detection experiment in extreme environments; implications for astrobiology
(Cranfield University, 2010-11) Derveni, Maria Elisavet; Cullen, David C.
The Life Marker Chip (LMC) instrument is an antibody assay-based system which will attempt to detect molecular signatures of Life in the Martian subsurface as part of the payload on board the European Space Agency (ESA) ExoMars mission rover, currently scheduled for launch in 2018. The LMC will have the ability to detect up to 25 different molecular targets of different origins that are associated with meteoritic in-fall, extinct or extant Life, prebiotic chemistry and spacecraft contamination. Regolith / crushed rock samples will be collected for the LMC by the rover and subjected to solvent extraction to extract organic molecules for analysis by the immunoassays. One of the key stages in the development of the LMC is the selection of antibodies to be used in the flight instrument. The challenge lies in the nature of the molecules or classes of molecules that are LMC targets and the need for antibodies that remain functional in the extreme conditions during a planetary exploration mission, especially the radiation environments. The work described within focuses on two main aspects of the search for LMC-relevant antibodies; the effect of space radiation on antibody performance [in the form of both ground-based and Low Earth Orbit (LEO)-set studies] and the development of ―customised‖antibodies against some of the molecules that are being investigated as potential LMC targets. The need to study the effects of space radiation on antibodies arose due to lack of any heritage of their use in interplanetary missions. For all the antibodies in the LMC, the ability to resist inactivation due to space radiation seen during a Mars mission will be a prerequisite. The objective of the ground-based radiation studies was to expose a number of LMC-relevant antibodies to simulated Mars mission radiation in the form of proton and neutron radiation which are the components of the mission radiation environment that are expected to have the dominant effect on the operation of the LMC. Cont/d.
Open Access
Development of life marker chip technology for in-situ life detection on Mars
(Cranfield University, 2007-04) Wilson, P. K.; Cullen, David C.; Magan, Naresh
The European Space Agency (ESA) is currently developing its flagship Life Detection Mission, ExoMars, which is scheduled to fly to Mars in 2013. The primary goal of this mission is to compliment the Phoenix NASA mission in confirming the presence of organic material on Mars, and, for the first time, analyse this organic material to determine the presence of organic species indicative of presence of past or present Life. One of the proposed Life detection technologies is the Life Marker Chip (LMC), which uses immunoassays with fluorescent readout to detect small organics and proteins in a microarray format within microfluidic channel structures. This PhD thesis encompasses the work done by the author on the development of the SMILE LMC during the period prior to, and during part of the first phase of, the Life Marker Chip Technology Readiness Level Upgrade Study funded by ESA from 2005 and 2007. Cont/d.
Open Access
Engineering design instrumentation for life detection planetary exploration missions
(Cranfield University, 2011-10) Juanes-Vallejo, Clara M.; Cullen, David C.; Roberts, Peter
The aim of the research documented in this thesis was to explore issues associated with the development of instrumentation for life detection and characterisation in a planetary exploration context. Within this aim, the following objectives had to be achieved: 1. To consider current and near-future single molecule detection (ultra-low lower limit of detection) analytical techniques that would be compatible with development into a Space qualifiable in situ analytical instrument for the detection of biomarkers in a planetary exploration context. 2. To practically consider the consequences of Planetary Protection and Contamination Control on the development of a sample return instrumentation in a planetary exploration context. 3. To consider the implications of flying an in situ instrument on-board a stratospheric balloon platform in order to apply them into a specific planetary exploration mission: In order to achieve the objectives described above, the following work was pursued:  A desk-based European Space Agency (ESA) study was carried out which entailed producing a literature review on single molecule detection technologies that had to be validated by the expert community. This was done by organising an International Workshop on Single Molecule Detection Technologies for Space Applications in March 2009 at Cranfield University, UK. The approved technologies then had to be analysed with standard analytical techniques (i.e., tradeoffs) in order to propose a specific technology for development and present its breadboard implementation and test plans at the end of the study.  A sample return experiment implementing PP&CC constraints and protocols was designed, built, tested and flown on-board the ESA, Swedish Space Corporation (SSC), Swedish National Space Board (SNSB) and German Space Agency (DLR) BEXUS stratospheric balloon platform. The biological and engineering results obtained from the sample return flight were then analysed and lessons learnt obtained for future flights.  Another desk-based study was performed to research future stratospheric balloon platforms for the exploration of Venus’ cloud layer. The in situ instrument previously proposed for the detection of biomarkers for planetary exploration missions was then put forward as a possible payload for a Venusian stratospheric balloon platform and approved by experts during the Venus Exploration Analysis Group (VEXAG) conference held in August 2011 in Washington D.C, USA. The first part of the research involved studying ultra-low lower limit of detection technologies as these have the potential to impact significantly on the technological and scientific requirements of future Space missions. Two systems were proposed: one based on Tandem Mass Spectrometry (with Cylindrical Ion Trap analysers) followed by Surface Enhanced Raman Scattering spectroscopy to create an MS/MS-SERS instrument for the detection of astrobiology biomarkers in Martian regolith, Europan ice and samples from Titan’s hydrocarbon lakes; and a second one as a Stand-Alone SERS system for the detection of biomarkers in Enceladean plumes, Venusian clouds and cometary coma. The second part of the research practically explored the design of instrumentation for stratospheric balloon platforms. CASS•E, the Cranfield Astrobiological Stratospheric Sampling Experiment, was a life detection experiment that aimed to be capable of detecting stratospheric microorganisms. The experiment consisted of a pump which drew air from the Stratosphere through a 0.2 μm collection filter which retained any microorganisms and >0.2 μm particulates present in the pumped air. Due to the expected rarity of microbes in the Stratosphere compared to the known levels of contamination at ground level, Planetary Protection and Contamination Control (PP&CC)constraints were introduced. Therefore PP&CC protocols were followed to implement Space qualified cleaning and sterilisation techniques; biobarrier technology was implemented to prevent re-contamination of the instrument after sterilisation; and cleanliness and contamination was monitored throughout assembly, integration and testing. The third part of the research demonstrated how an instrument from the first part of the study could be proposed as a payload on-board a stratospheric balloon platform with a focused mission context, i.e., a life detection mission for Venus. Therefore, the research concluded with the proposal of a payload for a Venus mission based on SERS technology on-board a stratospheric balloon platform to search for life above or in the mid Venusian cloud cover.
Open Access
Extraction of polar and nonpolar biomarkers from the martian soil using aqueous surfactant solutions
(Elsevier Science B.V., Amsterdam., 2012-12-31T00:00:00Z) Court, Richard W.; Rix, Catherine S.; Sims, Mark R.; Cullen, David C.; Sephton, Mark A.
The Life Marker Chip intends to use an aqueous surfactant solution to extract both polar AND nonpolar biomarkers from the martian soil for transport to an antibody-based detection system. Currently, a solution of 1.5 g l-1 polysorbate 80 in 20:80 (vol:vol) methanol:water is being considered and appears to be suitable. However, should this solution be shown to be unsuitable for the LMC or the martian environment, it will be necessary to use a different surfactant. Here, we have investigated the ability of a range of other surfactant solutions to extract a suite of eight standards spiked on the surfaces of the martian soil simulant JSC Mars-1 and tested the compatibility of the best two surfactants with a representative antibody assay for the detection of pyrene. The results show that using 20:80 (vol:vol) methanol:water as the solvent leads to greater recoveries of standards than using water alone. The poloxamer surfactants Pluronic® F-68 and F-108 are not effective at extracting the standards from JSC Mars-1 at any of the concentrations tested here. The fluorosurfactant Zonyl® FS- 300 is able to extract the standards, but not as efficiently as polysorbate 80 solutions. Most successful of the alternative surfactants was the siloxane-based surfactant poly[dimethylsiloxane-co-[3-(2-(2- hydroxyethoxy)ethoxy)propyl]methylsiloxane] (PDMSHEPMS) which is able to extract the standards from JSC Mars-1 about as efficiently as polysorbate 80 solutions. Enhanced recovery of the standards using polysorbate 80 solutions can be achieved by increasing the concentration of polysorbate 80, from 1.5 g l-1 to 10 g l-1, leading to an increase in the recovery of standards of about 50%; a similar increase in effectiveness is also apparent for PDMSHEPMS. Polysorbate 80 at concentrations of 1.5 g l-1 and 10 g l-1 and Zonyl® FS-300 and PDMSHEPMS (both at a concentration of 10 g l-1) are also compatible with the representative pyrene antibody ass
Open Access
Granular sample collection simulation via counter rotating wheels sampler for small-sized system at reduced gravity environment
(Elsevier, 2023-09-13) Sitepu, Elioenai; Cullen, David C.
The launching and planning of space missions for asteroid exploration have increased due to their scientific interest and potential resources for future use, such as composition analysis and In-Situ Resource Utilization (ISRU) demonstration. Furthermore, the design of sample acquisition systems has been limited to accommodate CubeSats or small-sized payloads, as the utilization of CubeSats for scientific missions is also on the rise. Therefore, sample collection is a critical step in preparing for these missions. The focus of this study is to analyse a conventional counter rotating wheels sample acquisition concept on a granular bed in microgravity conditions on an asteroid, as well as on the Moon and Mars with different gravitational forces. The study introduces a set of Archimedes screws into the wheels as a novel sample acquisition approach and utilizes Discrete Element Modelling (DEM) to analyse it. The simulation shows that the addition of an Archimedes screw sampler is more efficient in collecting samples in a short sampling time (<2 s) than a conventional counter rotating wheels. For implementation on planetary bodies with significant gravitational force, such as the Moon or Mars, a limitation is observed and redesigning the sample collection chamber might be necessary to capture the particles. Finally, the study highlights potential future work that could provide a more detailed and comprehensive understanding of the newly designed counter rotating wheels sampler.
Open Access
High frequency thin-film bulk acoustic wave resonators for gas- and bio-analytical applications
(2000-10) Ashley, Greg M.; Kirby, Paul B.; Cullen, David C.
Thin Film Bulk Acoustic Wave Resonators (FBAR) are mechanical micro scale devices that operate in the UHF/Microwave frequency range. This high frequency of operation potentially offers increased sensitivity to the addition of surface mass loading as implied by the famous Sauerbrey equation. FBAR was shown to be responsive to physical and chemical changes in the environment and was further adapted to act as bio-sensor. Thus indicating a universal platform from which to launch an enhanced sensing technology. This thesis follows the research and development of a prototype chemical and biological sensor based on FBAR FBAR devices were fabricated in a clean room and on die RF measurements were made to identify the units with performance characteristics of high enough quality to be useful as sensors. The FBAR design was then adapted so that it could be environmentally isolated, and microwave circuitry was devised to allow the FBAR to remain in electrical contact with the outside world during its isolation. This allowed for controllable environments in which to test FBAR responses to chemical and biological agents free from interfering signals. A software suite was written to specifically address the requirements for accurate and sensitive data processing of FBAR responses to measured analytes in real time. The isolation assembly and software was tested thoroughly, and the ultimate limits of resolution and sensitivity for the instrumentation were found using temperature change as the variable input parameter. A gas delivery apparatus was constructed and the FBAR was coated with hygroscopic polymer layers to sensitise the device to water vapour. Changes in the concentration of water vapour in a gas stream were tracked and the range of detection was established along with stability and resolution of the chemically sensitised FBAR. FBAR device gold surfaces were coated with biological antibodies, these made the devices ultra specific to measurand. Direct experimental comparisons between the FBAR and the relative performance of well established but lower frequency acoustic wave immunosensor technology systems were made and the relative increase in sensitivity was established for the FBAR based immunosensor. Optical methods were used to compliment the acoustic ones in determining the thickness and density of the protein layers adsorbed to equivalent gold surfaces. The thesis concludes with a section of speculative ideas for future work, with the experimental results for a potential rheological probe device shown. A brief demonstration of the FBAR performance when submerged in semi-infinite liquid environments is shown. Arrays of FBAR devices are software modelled in a novel way and demonstration of their possible applications are presented.
Open Access
Immunological detection of small organic molecules in the presence of perchlorates: relevance to the life marker chip and life detection on Mars.
(Mary Ann Leibert, 2011-11-17T00:00:00Z) Rix, Catherine S.; Sims, Mark R.; Cullen, David C.
The proposed ExoMars mission, due to launch in 2018, aims to look for evidence of extant and extinct life in martian rocks and regolith. Previous attempts to detect organic molecules of biological or abiotic origin on Mars have been unsuccessful, which may be attributable to destruction of these molecules by perchlorate salts during pyrolysis sample extraction techniques. Organic molecules can also be extracted and measured with solvent-based systems. The ExoMars payload includes the Life Marker Chip (LMC) instrument, capable of detecting biomarker molecules of extant and extinct Earth-like life in liquid extracts of martian samples with an antibody microarray assay. The aim of the work reported here was to investigate whether the presence of perchlorate salts, at levels similar to those at the NASA Phoenix landing site, would compromise the LMC extraction and detection method. To test this, we implemented an LMC- representative sample extraction process with an LMC-representative antibody assay and used these to extract and analyze a model sample that consisted of a Mars analog sample matrix (JSC Mars-1) spiked with a representative organic molecular target (pyrene, an example of abiotic meteoritic infall targets) in the presence of perchlorate salts. We found no significant change in immunoassay function when using pyrene standards with added perchlorate salts. When model samples spiked with perchlorate salts were subjected to an LMC-representative liquid extraction, immunoassays functioned in a liquid extract and detected extracted pyrene. For the same model sample matrix without perchlorate salts, we observed anomalous assay signals that coincided with yellow coloration of the extracts. This unexpected observation is being studied further. This initial study indicates that the presence of perchlorate salts, at levels similar to those detected at the NASA Phoenix landing site, is unlikely to prevent the LMC from extracting and detecting organic molecules from martian samples.
Open Access
Implementation of in-field life detection and characterisation techniques in icy environments
(Cranfield University, 2010-06) Barnett, Megan; Wadham, Jemma L.; Cullen, David C.
An emerging trend towards non-laboratory based biological and microbiological marker analysis is occurring in multiple sectors of science and industry. In the medical sector, these trends have demonstrated that conducting sample analyses away from centralised laboratories not only makes analyses quicker and more convenient (e.g. a home pregnancy test), but can offer services that are otherwise impractical (e.g. mobile laboratories to diagnose disease in the developing world). In the environmental sector, similar benefits, plus the ability to develop and test hypotheses, protocols and sampling strategies within a field campaign, are possible with in-field analyses. Icy environments in particular would benefit from in situ or in-field life detection as they are typically remote, and hence impart high logistical costs for repeated field campaigns and associated sample return with the implication that the efficiency of scientific return is poor. Unfortunately, most equipment and protocols developed for microbiological analyses in other sectors of science and industry are unsuitable for direct application to in-field use in icy environments because of poor compatibility with icy environment sample matrices and frequently inappropriate microbiological targets. Hence within this work, two hypotheses were tested: that (i) microbiological detection infield in icy environments is possible and through this (ii) unique and more efficient scientific studies can be conducted. Cont/d.
Open Access
Investigation of denugue virus envelope gene quasispecies variation in patient samples : implications for virus virulence and disease pathognesis
(Cranfield University, 2011-09) Love, Hannah; Cullen, David C.; Burton, Jane; Richards, Kevin
Due to the error-prone nature of RNA virus replication, each dengue virus (DV) exists as a quasispecies within the host. To investigate the hypothesis that DV quasispecies populations affect disease severity, serum samples were obtained from dengue patients hospitalised in Ragama, Sri Lanka. From the patient sera, DV envelope glycoprotein (E) genes were amplified by high-fidelity RT-PCR, cloned, and multiple clones per sample sequenced to identify mutations within the quasispecies population. A mean quasispecies diversity of 0.018% was observed, consistent with reported error rates for viral RNA polymerases (0.01%; Smith et al., 1997). However, previous studies reported 8.9 to 21.1-fold greater mean diversities (0.16% to 0.38%; Craig et al., 2003; Lin et al., 2004; Wang et al., 2002a). This discrepancy was shown to result from the lower fidelity of the RT-PCR enzymes used by these groups for viral RNA amplification. Previous studies should therefore be re-examined to account for the high number of mutations introduced by the amplification process. Nonsynonymous mutation locations were modelled to the crystal structure of DV E, identifying those with the potential to affect virulence due to their proximity to important structural features. No correlation was observed between the extent of quasispecies variation and disease severity. However, genome-defective quasispecies variants, and variants with surface accessible amino acid substitutions, or those proximal to the fusion peptide, proposed receptor binding sites, or other E oligomers, were observed predominantly in patients with severe dengue. Recombinant virus-like particles were produced for nine quasispecies variants, and the effects of the mutations on protein function assessed. Altered heparin binding abilities were demonstrated for four of the nine variants, indicative of differing cell attachment capabilities. Further work is required to assess differences in antibody binding, replication efficiency, virion and oligomer assembly, low pH-induced conformational changes required for fusion, and transmissibility of variants.
Open Access
Investigation of the effect of structured hyaluronic acid surfaces on cell proliferation and expression of HA cellular receptors:CD44 and RHAMM
(Cranfield University, 2011-03) Marques, Ana Catia Ferrao; Morgan, Sarah; Cullen, David C.
Hyaluronic acid (HA) is one of the major components of the extracellular matrix; and may exhibit different biological functions, dependent on polymer molecular weight (MW). The signalling events performed by HA are mediated through interactions with its main cell receptors: CD44 and RHAMM. However, the direct effect between the HA MW and the expression of CD44 and RHAMM remains unclear. This study aimed to investigate whether different HA polymer MW alters the proliferation of tumour-derived cell lines, and whether different HA-sized has an effect on the regulation of the expression of CD44 and RHAMM. In order to determine size-specific responses of tumour cells of defined fragment MW, this investigation was undertaken using HA-tethered culture surfaces. Four surfaces were constructed, coated with polymers of different MWs. HA (4, 234, 2590 kDa) and an oligomer mixture were tethered onto an aminosilane (AHAPTMS)-treated glass surfaces using a carbodiimide reaction. Surfaces were analysed using a toolbox of in situ characterisation techniques, including wettability measurements, QCM, AFM and confocal microscopy. Using the constructed surfaces was demonstrated that HA-polymer MW modulates cell proliferation of human bladder (RT112 and T24) and prostate (PC3 and PNT1A) cell lines, with low HA MW (HA4) increasing proliferation, whereas a decrease is seen in the presence of medium (HA234) and high MW fragments (HA2590). The proliferation stimulus performed by HA was found to be phenotype dependent, with HA4 surfaces stimulating an increased proliferation in those less invasive cell lines (T24 and PNT1A), while HA234 and HA2590 inducing a sharper decrease in the most malignant tumour cell lines (RT112 and PC3). It was also demonstrated that the regulation of CD44 and RHAMM transcripts expression appears to be phenotype dependent but not HA-MW dependent. HA down-regulates CD44 and RHAMM in the most malignant cell lines; with up-regulation of the expression of the cell receptors in the less invasive cell lines. In addition, the presence of exogenous HA was shown to be involved in the regulation of the expression of CD44 variants expression. The results obtained for the CD44 and RHAMM protein expression were also found to be correlated with the obtained transcripts expression. However, the significance of these findings in tumourigenesis remains unclear. Findings from this investigation may help in the design and development of biocompatible implants with controlled surface properties to be used in cancer therapeutics; with medium and large HA polysaccharides being potential biopolymer candidates, useful for the development of novel therapies for highly invasive cancer. In addition, implications from this work can serve as a base for future research, and can lead to ideas for drugs and methods to be used in cancer therapeutic approaches.
Open Access
The life marker chip : potential use of aptamers against small molecules and consideration of instrument planetary protection
(Cranfield University, 2013-05) Rato, Carla Cristina Pereira Salgueiro Catarino; Cullen, David C.
The Life Marker Chip (LMC) instrument was developed with the aim to detect evidence of life on Mars. The detection was based on an inhibition immunoassay. In this work aptamers were evaluated as potential alternative to antibodies for the LMC. Aptamers were synthetic oligonucleotides able to bind specifically with high affinity to a wide range of target molecules, and have been also integrated as bioreceptors in several detection instruments. The generation of new aptamers against two small molecules using the FluMag-SELEX method was tested and was verified the adaptability of pre-existing aptamers against small targets to the LMC assay type. Based on the fact that the LMC was going to be integrated into the space programme ExoMars, it was also implemented into a small scale experiment the Planetary Protection and Contamination Control requirements found on a life-search mission. In addition to that aptamers compatibility with a sterilisation procedure used in life-search missions was also tested. Furthermore because of the nature of the small molecules studied, multiple analytical chemistry techniques were assessed to verify covalent chemistry surface immobilisation. Within the project timeline it was not possible to achieve a full aptamer generation process but it was possible to understand the methodology behind the procedure and give input for future work. It was found that the direct implementation of existing aptamers against small molecules into the LMC assay was not successful. It was also seen that in the case of aptamer integration onto the LMC some assay changes would probably have to be made. This information was very useful to understand if aptamers could be an alternative to antibodies and be implemented directly into the LMC. It was found that aptamers survived the preliminary sterilisation method applied, which might open the possibility of making aptamers convenient space bioreceptors, reducing time and costs of instrument Planetary Protection implementation. In conclusion aptamers were not straightforward alternatives to antibodies for the LMC because aptamers interacted differently with their targets in comparison to antibodies, particularly with small molecules. Also the biochemical simplicity of the small molecule targets introduced difficulties in aptamers generation that more complex targets would have not. Although aptamers shown incompatibility with the LMC assay format against small targets, they presented resilience to a sterilisation procedure implemented on space missions which could lead to the development of more robust bioreceptors for space missions. This information was helpful in understanding which assay formats were better for detection of small molecules using aptamers and that might contribute for future assay choices applied in detection instruments. It was also possible to make recommendations for the LMC regarding design and validation methods used in life-search missions based on the lessons learn from the developed of a small scale experiment. The developed work was presented at conferences and mentioned in an article journal, and in that way contributed to the knowledge of the space community in general.