The life marker chip : potential use of aptamers against small molecules and consideration of instrument planetary protection

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

Optimisation of an aptamer-biosensor to detect C-reactive protein

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2009A proteína C reactiva (PCR) é um biomarcador de doenças caracterizadas por uma resposta inflamatória aguda; tais como: doenças coronárias, diabetes mellitus ou carcinomas. A detecção desta proteína em fluidos corporais, em especial em soro humano pode ainda determinar risco de desenvolvimento futuro de doenças coronárias em indivíduos aparentemente saudáveis. Foi realizado a optimização de um novo biosensor aplicando a técnica de Voltametria de Pulso Diferencial (DPV). A detecção foi realizada em solução tampão aquosa utilizando eléctrodos de carbono e o produto enzimático electroactivo da reacção de 1- naftil fosfato com alcalina fosfatase. Neste trabalho foi optimizado, em solução tampão, o tempo de incubação e o valor de concentração da alcalina fosfatase conjugada com streptavidina. As condições finais foram testadas em solução de soro humano livre de PCR diluído 1:10. A proteína C-reactiva foi retirada do meio líquido aplicando a técnica de ensaio imunológica “sandwich”. O ensaio requereu como suporte sólido esferas magnéticas cobertas por streptavidina e como captores biológicos um aptamero de RNA e um anticorpo monoclonal, ambos biotinilados. O tempo optimizado de incubação foi de 10 min e a concentração óptima de fosfatase alcalina acoplada a streptavidina foi de 0.22U/ml. Estas condições em meio tampão demonstraram um limite de detecção de pelo menos 0.1mg/l de PCR com um coeficiente de variação igual a ±14.45%. Para as mesmas condições, mas em soro humano livre de PCR, o limite de detecção foi de pelo menos 0.5mg/l de concentração proteica e o coeficiente de variância de ±7.32%. O biosensor desenvolvido demonstrou elevada exactidão, reprodutibilidade, relativa sensibilidade e rápida concretização do ensaio quando comparado com outros métodos de detecção tradicionais. Deste modo o novo biosensor desenvolvido apresenta-se como um excelente candidato à detecção da PCR no campo do diagnóstico médico.C-reactive protein is a biomarker for diseases where an acute inflammatory response is present. Examples are coronary disease, diabetes mellitus and cancer. The presence of the CRP protein in body fluids, especially in serum may also predict risk of future coronary complications in apparently healthy people. A new aptamer biosensor based on the electrochemical differential pulse voltammetry detection method was optimised. The detection was done in solution using screenprinted carbon electrodes and the properties of the electroactive enzymatic product of 1- naphytl phosphate with alkaline phosphatase. In this work the optimisation of the time of incubation and concentration of the alkaline phosphatase enzyme conjugated with streptavidin was performed in buffer medium and the optimised conditions tested in CRP-free human serum with a total dilution of 1:10. The C-reactive protein was captured from the media, using a sandwich immunoassay technique. The solid phase used for the realisation of the sandwich technique in solution was magnetic beads covered with streptavidin. The sandwich assay was performed with an RNA aptamer and a monoclonal antibody, both biotinylated. The optimised time of incubation was 10 minutes and the optimised concentration of the streptavidin-alkaline phosphatase conjugate 0.22U/ml. These conditions in buffer medium showed a detection limit of at least 0.1mg/l of CRP concentration with a mean of coefficient of variance equal to ±14.45%. For the same conditions in CRP-free human serum, the detection limit was at least 0.5mg/l of protein concentration and the coefficient of variance ±7.32%. The developed aptamer biosensor presented elevated accuracy, reproducibility, relative sensitivity due to some matrix effects and less consumed laboratory time when compared with traditional tests. For these reasons the new aptamer biosensor developed appears to be an excellent candidate to detect CRP in the diagnostic field

CASS•E: Cranfield astrobiological stratospheric sampling experiment

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 res