Abstracts Izon Science Research Symposium 2015
Designing the ideal assay for TRPS
1Centre of Analytical Sciences, Department of Chemistry, Loughborough University, Loughborough, Leicestershire, UK
Researchers have been developing, testing and implementing the TRPS platform in a wide range experiments. From the characterisation of emulsion droplets, inorganic materials and biological analytes (see the review by Weatherall and Willmott (Analyst, 2015, 140, 3318-3334)). TRPS is being shown to rival alternative characterisation platforms and with certain samples offer a more detailed analysis of the analyte in situ. Here I focus and review the use of the TRPS platform for the characterisation of Biological analysts. I present some of our own work aimed at designing the idal assay format to improve sensitivity, dynamic range and versatility for both multiple analytes and quick analysis times.
Investigating Protein-Aptamer Interactions using Zeta Potential Measurements through TRPS
Emma L C J Blundell1,2 and Mark Platt1
1Centre for Analytical Science, Department of Chemistry, School of Science, Loughborough University, Loughborough, Leicestershire, LE11 3TU, UK
2Izon Science Limited, Magdalen Centre, The Oxford Science Park, 1 Robert Robinson Avenue, Oxford, OX4 4GA, UK
The detection and characterization of proteins and biologically relevant molecules is a challenge in nanotechnology due to the requirements for highly sensitive technologies. The use of an aptamer-based assay alongside TRPS technology has allowed for the successful detection of a range of biological targets1.
Aptamers are short, single-stranded DNA or RNA molecules that can bind to target analytes with great specificity and high affinity. Understanding the interactions between the aptamer and its relative targets is important in the development of a selective and sensitive assay platform. Successfully detecting and monitoring protein-aptamer interactions is a valuable benefit in a variety of diagnostic fields.
Here we present a particle-particle conjugation assay design for nanoparticles (70-120 nm in diameter) functionalized with a particular aptamer to those conjugated to its protein target. We are using TRPS to monitor the relative changes in signal observed when a protein-aptamer interaction is present. Although this interaction may not introduce any significant size changes to the nanoparticles, a change in particle velocity and therefore zeta potential will be incurred as the surface chemistry of the nanoparticles will have been modified. The capability of TRPS to complete size and zeta potential measurements simultaneously is advantageous for this study and assay design.
1. Billinge, E. R., Broom, M. & Platt, M. Monitoring Aptamer–Protein Interactions Using Tunable Resistive Pulse Sensing. Anal. Chem.86, 1030–1037 (2013).
Extraction and detection of copper (II) using modified silica nanoparticles and tunable resistive pulse sensing
Laura Mayne1, Steve Christie & Mark Platt
1Department of Chemistry, Loughborough University, Loughborough, Leicestershire, LE11 3TU
There are many techniques for removing metal contaminants from aqueous solutions; including chemical precipitation, ion and solvent exchange and adsorption.1–3 Nanoparticles offer new opportunities for waste treatments. They offer a large surface area, high adsorption capacity and fast adsorption rate. Silica can be used to support ligands for surface modification of the particles, due to its stability.4
Here we are modifying silica nanoparticles with (3-aminopropyl)triethoxysilane for the removal of copper (II) ions from aqueous solutions. By varying the pH and electrolyte concentration, the copper extraction and the TRPS signal could be optimised. Optimising the conditions allows for the characterisation of the sorption material using TRPS. The copper (II) extracted from solution, using the modified nanoparticles, was monitored by TRPS by studying the changes in velocities of the particles.
The changes in particle velocity allow the particles with and without copper to be distinguished and can be attributed to the change in charge on the nanoparticle when the copper binds to the ligand on the nanoparticles.
1. Duff, M. C., Coughlin, J. U. & Hunter, D. B. Uranium co-precipitation with iron oxide minerals. Geochim. Cosmochim. Acta 66, 3533–3547 (2002).
2. Praveen, A., Kumar, R., Kumar, P. & Kumar, R. Removal of heavy metals from drinking water supplies through the ion exchange membrane- A Review. J. Appl. Phys. 3, 25–39 (2013).
3. Liu, C.-H. et al. Mechanism of Arsenic Adsorption on Magnetite Nanoparticles from Water: Thermodynamic and Spectroscopic Studies. Environ. Sci. Technol. 150619062736000 (2015). doi:10.1021/acs.est.5b00381
4. Goswami, A. & Singh, A. K. Enrichment of iron(III), cobalt(II), nickel(II), and copper(II) by solid-phase extraction with 1,8-dihydroxyanthraquinone anchored to silica gel before their determination by flame atomic absorption spectrometry. Anal. Bioanal. Chem. 374, 554–60 (2002).
Unravelling the potential of Tunable Resistive Pulse Sensing
1Izon Science, 8C Homersham Place, Christchurch, New Zealand
2School of Mathematics and Physics, The University of Queensland, Brisbane, Australia
Tunable resistive pulse sensing (TRPS) provides a direct measure of particle concentration, and high resolution analysis of particle size and surface charge. The use of a tunable system increases the dynamic range, making analysis of very polydisperse samples possible. It also increases the analytical sensitivity as the pore diameter is optimised to the particulate system at hand. A unique feature of TRPS is its potential to measure individual particle charge and zeta potential, based on the analysis of the duration of the resistive pulse. The single particle nature of TRPS means that sub-populations with different zeta potential and/or size within a sample can be discriminated.
Due to its versatility the potential of TRPS for industrial, medical and research applications is vast. In this talk a range of case studies are presented which will help and guide TRPS users to tap into this potential. The case studies cover a wide range of applications and particulate systems such as extracellular vesicles, liposomes and nano/microbubbles.
Use of MES as a non-complexing buffer for TRPS determinations with variable Ca concentrations
Diego Soto-Gómez1, Marcos Paradelo-Pérez1,2, Paula Pérez-Rodríguez1; José Eugenio López-Periago1
1Área de Edafoloxía e Química Agrícola, Depto. Bioloxía Vexetal e Ciencia do Solo, Facultade de Ciencias, Universidade de Vigo, Ourense 32004, España.
2Departement of Agroecology, Faculty of Science and Technology, Aarhus University, Blichers Allé 20, P.O. Box 50, 8830 Tjele, Denmark
TRPS has promising applications to examine the aggregation and mobility of pathogenic viruses and bacteria in groundwater. But chemistry of groundwaters varies more than the biological fluids1. Groundwater samples may contain divalent cations (e.g. Ca2+) that can be complexed and co-precipitate with the phosphate buffers. In addition, with variable electrolyte composition will change the intensity of the baseline and therefore will hinder the TRPS calibration. Baseline current when measuring samples should not vary more than 3% regarding the current during calibration2.
To study these effects, we measured SKP200B and SKP400E Izon standards with different CaCl2 concentrations (0.1, 0.01 and 0.001M of Ca+2), and two electrolytes: PBS (Phosphate Buffered Saline) and MES (2-(N-morpholino) ethanesulfonic acid).
PBS co-precipitated with Ca very quickly at concentrations higher than 0.01M. With less concentrated samples (0.001M Ca2+) a small increase in the average size (6 to 17nm) occurred for the 200nm standard. MES buffer gave reproducible results even in samples with 0.01M Ca2+. With 0.1M Ca2+ measurements were less accurate. MES is non-complexing buffer suitable for aggregation studies of nanoparticles and virus in waters with varying multivalent electrolyte concentrations.
We found that the ratio of blockade/baseline current is linear. This can be used to correct the error produced in the baseline by the presence of Ca2+ in the samples.
- Harvey, R. W., Metge, D. W., Barber. L. B. & Aiken G. R. (2010). Effects of altered groundwater chemistry upon the pH-dependency and magnitude of bacterial attachment during transport within an organically contaminated sandy aquifer. Water Research, 44(4) 1062-1071 10.1016/j.watres.2009.09.008.
- Maas, S. L. N., Vrij, J. D., & Broekman, M. L. D. (2014). Quantification and size-profiling of extracellular vesicles using tunable resistive pulse sensing. Journal of Visualized Experiments, (92). 10.3791/51623
Tunable Resistive Pulse Sensing; methodological challenges of a novel method for microvesicles analysis
A. Gregorius1, S. Chlopicki1,2
1 Jagiellonian Centre of Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland
2 Department of Experimental Pharmacology, Jagiellonian University, Grzegorzecka 16, 31-531 Krakow, Poland
Microvesicles (MVs) are small (0,1 -1μm) fragments of the cell membrane released by all cells under the process of activation or apoptosis. MVs play important role in physiology and pathophysiology of cardiovascular system. In particular, the increased MVs formation appears to be linked with endothelial dysfunction representing a hallmark of various cardiovascular diseases including diabetes, metabolic syndrome and atherosclerosis.
In MVs field, flow cytometry is the method of choice for MVs analysis, but this technique requires an experienced operator due to the MVs size bordering the level of detection. Additionally it is hampered by their polydyspersity and low refractive index. In the present study Tunable Resistive Pulse Sensing (TRPS, IZON qNano, New Zealand) for MVs analysis is presented that is well suited to confirm size and fraction purity of isolated MVs. Some pitfalls of this method are discussed.
MVs were obtained from endothelial cells culture (EAhy 926) and mouse blood in different experimental conditions. After few selected isolation techniques (ultracentrifugation, filtration, gradient ultracentrifugation) the mean, mode size and mean MVs concentration in the manner of calibration particles was evaluated.
In summary, the main difficulties of TRPS involve frequent blockades of nanopore due to heterogeneity of MVs sizes and aggregates formation (requirement of additional filtration and sample purification), or non-optimal nanopore selection. Preanalitical phase of sample preparation including sample dilution is mandatory for good measurements. If successful, this technique allows to characterize MVs size and concentration with a high accuracy. Low sample volume and relatively quick information on MVs fraction are the most desired TRPS features that makes this technique a highly useful tool to analyze MVs prior to flow cytometry analysis.
Acknowledgments: This study was supported by European Union from the resources of the European Regional Development Fund under the Innovative Economy Pro- gramme (grant coordinated by JCET-UJ, No POIG.01.01.02-00-069/09).
Isolation of extracellular vesicles by size-exclusion chromatography
A.N. Böing1, N. Hajji, F.A.W Coumans, E. van der Pol, A.E. Grootemaat, A. Sturk and R. Nieuwland
1Department of Experimental Clinical Chemistry, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands
The isolation of extracellular vesicles (EVs) of body fluids is a challenge due to the presence of proteins and lipoproteins, and the viscosity of the fluids. Isolation of EVs by ultracentrifugation results in co-isolation of vesicles and HDL, in isolation of protein aggregates, and in the formation of vesicle aggregates. Recently we introduced isolation of vesicles by size-exclusion chromatography (SEC)1. Isolation of EVs of 1 mL body fluid by a SEC column of 10 mL results in a recovery of 60-80% with an 8-fold and 70-fold enrichment compared to HDL and protein. EVs isolated by SEC are still biological active, and can directly be used for transmission electron microscopy, proteomics and RNA isolation. Since the amount of body fluid can be limited, a SEC column of 3.65 mL was also tested with a sample volume of 125 µL. Isolation of EVs from 125 µL plasma results in a recovery of 60-70%, with a contamination of less than 1% of the original protein content. In conclusion, EVs can be isolated by both SEC columns with high recovery and less contamination of protein and HDL. EVs isolated by SEC can directly be used for various analyses.
1. Single-step isolation of extracellular vesicles by size-exclusion chromatography. Böing AN, van der Pol E, Grootemaat AE, Coumans FA, Sturk A, Nieuwland R. J Extracell Vesicles. 2014 Sep 8;3.
Role of microvesicles in non-targeted effects (NTE) of radiation: in vivo study
Munira Kadhim1, Scott Bright, Deborah Bowler, Fiona Lyng, Katalin Lumniczky Elizabeth Ainsbury
1 Genomic Instability Group, Oxford Brookes University, Gipsy Lane Campus, Headington, Oxford OX3 0BP, UK
Non-targeted effects (NTE) of radiation exposure, describes cellular damaging responses that can be inherited over several generations’ resulting in genomic instability (GI). Such cells also release signalling molecules that can induce damage in the genomes of neighbouring, un-irradiated cells. This process is known as the bystander effect (BE) and can also lead to long-term inherited GI in bystander cells (Kadhim et al 2013). Various pathways have been implicated in the process; these include pro-inflammatory cytokine signalling, oxidative stress and microvesicle secretion.
Recently we have shown radiation-induced NTE are mediated by a sub-class of microvesicles (MV) called exosomes. This was observed in vitro in MCF7 breast epithelial cancer cell line following radiation exposure and was seen as a persistent effect. Exosomal RNA and protein was found to work in a synergistic manner to initiate BE (Al-Mayah, 2012, 2015). However, in order to determine the role of microvesicles in NTE in vivo we used C57BL/6 male mice exposed to a range of X-ray doses. For this study, MV were isolated and analysed for a number of traits including size and concentration using the qNano (Izon ScienceTM) as well as microvesicle cargo. In parallel, isolated MV / exosomes were injected into un-irradiated recipient animals and used in an in vivo and ex-vivo bystander approach to investigate in the recipient animals, direct cellular damage (chromosomal instability, ɣH2AX); phenotypical and functional alterations as well as stress and inflammation markers. All data are currently being analysed and the available results will be discussed in this presentation.
- Kadhim, M., Salomaa, S., Wright, E., Hildebrandt, G., Belyakov, O. V., Prise, K. M., and Little, M. P. Non-targeted effects of ionising radiation-Implications for low dose risk. Mutat. Res. 752, 84-98 (2013).
- Al-Mayah, A. H., Irons, S. L., Pink, R. C., Carter, D. R., and Kadhim, M. A. Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation. Radiat. Res. 177, 539-45 (2012).
- Ammar Al-Mayaha, Scott Brighta, Kim Chapmana, Sarah Ironsb, Ping Luoc, David Carterd,Edwin Goodwine, Munira Kadhima. The non-targeted effects of radiation are perpetuated by exosomes.Mutation Research 772 (2015) 38–45
Comparative analysis of exosomes from brain tissue and CSF of Alzheimer's and healthy subjects
Maitrayee S. Sinha, Anna Ansell, Martin Hallbeck
1Department of Clinical and Experimental Medicine, Linköping University, Sweden
Introduction: Exosomes are nano-vesicles (40-100 nm) of endosomal origin, released from most cell types including neurons . They have important role in intercellular communication, cell-cell signaling or removal of toxic proteins by serving as vehicles for transferring proteins, lipids, mRNAs and miRNAs between cells As exosomes can be isolated from circulating fluids such as serum, urine, and cerebrospinal fluid (CSF), they provide a potential source of biomarkers for neurological conditions . The aim of this study is to provide a comprehensive analysis of size and distribution of exosomes isolated from CSF and brain tissue as well as their different protein content in Alzheimer’s and healthy subjects.
Methods and Results: Exosomes from brain were isolated by differential centrifugation method  and exosomes from CSF were isolated by commercially available kit and resuspended in PBS. Tunable Resistive Pulse Sensing (TRPS) analysis was performed using izon qNano system. Exosomesfrom brain andCSF samples evaluated by TRPS showed median sizes of ~100 - ~120 nm diameter vesicles. Size of exosomes were also checked by electron microscopy. Exosomal marker proteins Flotillin1 and TSG101 were detected in isolated exosomes by immunoblotting. The level of oligomeric amyloid beta protein (oAβ) which accumulates in AD brain during disease pathogenesis, was compared in exosomes isolated from brain tissue and CSF of Alzheimer’s and age matched healthy subjects.
Conclusion: Accurate, high-resolution characterization of exosomes is critical to understanding their properties, function and potential use as predictive markers or therapeutic agents. Furthermore, evaluating toxic proteins like amyloid beta in exosomes from CSF or other body fluids can act as potential biomarkers for AD and other related diseases diagnosis.
- Urbanelli L, Magini A, Buratta S, Brozzi A, Sagini K, Polchi A, Tancini B, Emiliani C. Signaling Pathways in Exosomes Biogenesis, Secretion and Fate. Genes. 20134(2), 152-170
- Record M, Subra C, Silvente-Poirot S, Poirot M. Exosomes as intercellular signalosomes and pharmacological effectors. Biochem Pharmacol. 2011, 81(10):1171-82
- Perez-Gonzalez R1, Gauthier SA, Kumar A, Levy E. The exosome secretory pathway transports amyloid precursor protein carboxyl-terminal fragments from the cell into the brain extracellular space. J Biol Chem, 2012, 287(51):43108-15.
Exosome switching: an mTORC1- dependent cellular stress response
Sumeth M Perera1*, Shih-Jung Fan1, Ben Kroeger1, Siamak Redhai1, Aaron Leiblich1,2, Mark S. Wainwright1, Laura Corrigan1, Kristie McCormick1, Helen Sheldon3, Kate E Carr1, John F. Morris1, Freddie C Hamdy2, Adrian L Harris3, Clive Wilson1, Deborah C I Goberdhan1
1Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Rd, OX1 3QX
2Nuffield Department of Surgical Sciences, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ
3The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS
In order to survive adverse conditions and to adapt to microenvironmental stress, cancer cells produce a range of secreted signals via poorly characterised mechanisms that restore homeostasis in their surroundings. Exosomes, membrane-bound vesicles classically produced by the late endosomal multivesicular bodies, represent a particularly complex signal that can reprogramme target cells via the action of multiple active cargos. Late endosomes and lysosomes (LELs) more recently have emerged as an intracellular signalling hub, responding to microenvironmental and growth factor signalling to modulate cell growth via the LEL-associated, amino acid-sensitive kinase complex, mechanistic Target of Rapamycin Complex 1 (mTORC1). We investigated whether mTORC1 activity and exosome secretion might be mechanistically linked.
By blocking different components of the PI3-kinase/mTORC1 growth regulatory pathway, including the putative amino acid-sensing intracellular transporter, Proton-Assisted Amino Acid Transporter 4 (PAT4), we demonstrate that in colorectal HCT116 cells, the rapamycin-resistant-mTORC1 regulates exosome cargo, producing a so-called ‘exosome switch’. For example, exosome numbers are generally unaffected by PAT4 knockdown, but levels of the exosome marker CD63 are drastically reduced in exosome preparations, while levels of the lipid raft protein, Caveolin-1, are upregulated. Analysis of secreted exosomes after mTORC1 inhibitor treatment suggests that rapamycin-resistant mTORC1 plays a key role in this switch, which is also induced in the MDA-231 breast cancer cell line. Interestingly, Tunable Resistive Pulse Sensing analysis of these vesicles showed subtle changes in their size indicating that PP242 like drugs behave differently to PAT4kd and may have effects via targets other than mTORC1.
Furthermore, we demonstrate that these different exosome populations have different cancer-relevant activities. Surprisingly, using confocal analysis and through collaborative studies of a new fly exosome model, we show that different endosomal compartments, marked by the monomeric Rab GTPases, Rab7 and Rab11, appear to make secreted vesicles and the exosome switch may involve a trafficking switch between these compartments.
We conclude that rapamycin-resistant mTORC1 controls exosome output, leading to functional changes in cell-cell signalling in response to microenvironmental stresses and drug treatments.
Differential detergent sensitivity of extracellular vesicle subpopulations
Xabier Osteikoetxea1, Barbara Sódar1, Andrea Németh1, Katalin Szabó-Taylor1, Krisztina Pálóczi1, Krisztina V Vukman1, Viola Tamási1, Ágnes Kittel2, Éva Pállinger1, Edit Irén Buzás1#
1 Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary
2 Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary
Extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) are currently attracting rapidly increasing attention from various fields of biology due to their ability to carry complex information and act as autocrine, paracrine and even endocrine intercellular messengers.
In the present study we investigated the sensitivity of size-based subpopulations of extracellular vesicles to different concentrations of detergents including sodium dodecyl sulphate, Triton X-100, Tween 20 and deoxycholate. We determined the required detergent concentration that lysed each of the vesicle subpopulations secreted by Jurkat, THP-1, MiaPaCa and U937 human cell lines. We characterized the vesicles by tunable resistive pulse sensing, flow cytometry and transmission electron microscopy.
Microvesicles and apoptotic bodies were found to be more sensitive to detergent lysis than exosomes. Furthermore, we found evidence that sodium dodecyl sulphate and Triton X-100 were more effective in vesicle lysis at low concentrations than deoxycholate or Tween 20.
Taken together, our data suggest that a combination of differential detergent lysis with tunable resistive pulse sensing or flow cytometry may prove useful for simple and fast differentiation between exosomes and other extracellular vesicle subpopulations as well as between vesicular and non-vesicular structures.
- Buzas EI, Gyorgy B, Nagy G, Falus A, Gay S (2014) Emerging role of extracellular vesicles in inflammatory diseases. Nat Rev Rheumatol 10:356-64.
- György B, Szabó T, Pásztói M, Pál Z, Misják P, et al. (2011) Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cellular and Molecular Life Sciences 68: 2667-2688.
- Osteikoetxea X, Balogh A, Szabó-Taylor K, Németh A, Szabó TG, Pálóczi K, Sódar B, Kittel Á, György B, Pállinger É, Matkó J, Buzás EI. Improved characterization of EV preparations based on protein to lipid ratio and lipid properties. PLoS One. 2015 Mar 23;10(3):e0121184.
Extracellular vesicles (EVs) in the oral cancer microenvironment
M. Ofield, B. Peacock, K. Alkasah, D. Lambert and S. Hunt
Unit of Oral and Maxillofacial Pathology. The School of Clinical Dentistry. The University of Sheffield. Sheffield, United Kingdom
Introduction: Worldwide, there are 560,000 new cases of head and neck cancer diagnosed and 300,000 deaths annually. Due to their ability to spread they have a poor outcome, with only ~50% of patients surviving 5 years after diagnosis. Head and neck cancer is strongly associated with lifestyle and environmental risk factors, which include tobacco, alcohol consumption and HPV infection1. Extracellular vesicles (EVs) are released by a variety of cell types in the human body and can be found in many biological fluids such as saliva2. They are believed to facilitate cell-to-cell communication as they are vehicles carrying molecular messages between cells. The cargo of EVs is altered in cancer, which has a pro-tumorigenic impact on this cell-to-cell communication and supports the development and spread of tumours3.
Methods: EVs were isolated from the conditioned medium of oral cancer cell lines and also from the saliva of healthy volunteers by size exclusion chromatography. EVs were characterized by TRPS. RNA and protein contents were quantified for subsequent transcriptomic and proteomic analysis.
Results: We have successfully isolated EVs from a panel of oral cancer cell lines and also human saliva. TRPS has allowed the comparison of EV size and concentration between samples and removed the need for indirect detection of EVs in preparations.
Conclusions: Our preliminary data shows the potential for salivary EVs to be used as a biomarker for the detection of oral cancer.
- Boyle P et al. World Cancer Report 2008. International Agency for Research on Cancer.
- Michael A et al. (2010). Exosomes from human saliva as a source of microRNA biomarkers. Oral Diseases.16: 34–38.
- Muralidharan-Chari V et al. (2010). Microvesicles: mediators of extracellular communication during cancer progression. Journal of Cell Science. 123(10): 1603-1611.
Lab-On-a-Chip biosensor approaches for real-time monitoring of antibody-EV interaction
Baharak Hosseinkhani1, Katrijn Vanschoenbeek, Nynke van den Akker, Daniel G. M. Molin , Inge Nelissen, Jef Hooyberghs, Luc Michiels
1Hasselt University, BIOMED, Martelarenlaan 42, 3590 Diepenbeek, Belgium
EV-based diagnostic tools are expected to become a breakthrough technology for early, less invasive diagnosis of various diseases and effective monitoring of disease progression at multiple time points during therapy (1). Over the past years, the major challenge in this field has been the assessment of a methodology for biomarker profiling of EVs in relation to their physiological perturbations (2). Surface Plasmon Resonance (SPR) offers a promising real-time, label-free and highly versatile sensing paradigm with enormous potential in biomarker detection (3). Therefore, we aim to develop a uniform, label-free and high-throughput method using SPR methodology for detecting the specific surface markers on intact EVs. EVs from HUVECs were isolated by different isolation procedure. The biomarker profile of EVs was first verified using ultrastructural immunogold labeling and Enzyme-Linked Immunosorbent Assay (ELISA). Then, we developed a highly sensitive methodology for biomarker profiling of EVs on a high-end SPR Biacore T200 platform.
We have previously demonstrated that the number of isolated EVs from TNF-α conditioned HUVECs-2 was 1.2- 1.4 fold higher as compared to untreated cells. Here, we discovered that ICAM-1 and CD63 have potential to discriminate between EVs derived from normal and stressed cells. Higher responses of binding interaction between immobilized anti-ICAM-1 and EVs (TNF-α conditioned and unconditioned) were also detected on SPR in comparison with anti-CD63. In addition, sandwich SPR was applied using two different biomarker antibodies to monitor behavior of these double marker containing EVs. The presented approach opened up a variety of novel opportunities for in-depth and label free investigation of EV biomarker profiling.
- Hornick NI, Huan J, Doron B, Goloviznina NA, Lapidus J, Chang BH, et al. Serum Exosome MicroRNA as a Minimally-Invasive Early Biomarker of AML. Sci Rep. 2015;5.
- Properzi F, Logozzi M, Fais S. Exosomes: the future of biomarkers in medicine. Biomarkers in Medicine. 2013;7(5):769-78.
- Im H, Shao H, Park YI, Peterson VM, Castro CM, Weissleder R, et al. Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor. Nat Biotech. 2014;32(5):490-5.
Characterisation of silica nanoparticles and adsorbed protein using complementary particle size and ζ-potential techniques.
Aneta E. Sikora a1, Vikram Kestensb, Daniel Geilerc, Alex G. Sharda, Caterina Minellia
a1National Physical Laboratory, Analytical Sciences, Teddington, UK.
bEuropean Commission, Joint Research Centre (JRC), Institute for Reference Materials and Measurements (IRMM), Geel, Belgium.
cBAM Federal Institute for Materials Research and Testing, Division 1.10 Biophotonics, Berlin, Germany.
It is recognised that the surface charge along with size of nanoparticles plays an important role in the way nanoparticles interact with biological systems. Such interactions are important for the application of nanoparticles in disease diagnosis and treatment and would play a role in the hypothesised toxicity of some nanoparticles.
We characterised silica NPs dispersed in different media, including a complex serum matrix by measuring their size, size distribution, protein shell thickness using complementary techniques. Particle size and size distributions in buffer and serum-based biological media were measured using Tunable Resistive Pulse Sensing (TRPS), as well as Differential Centrifugal Sedimentation (DCS) and Dynamic Light Scattering (DLS). Additionally, we have compared measurement strategies based on the use of ensemble (ELS) and particle-by-particle (TRPS) based techniques for the measurement of ζ-potential.
TRPS measurements showed an increase in size of the NPs upon acquisition of adsorbed proteins and the thickness of the protein corona was determined to be 5 nm, in agreement with literature 1, 2. A sample aggregation was studied by DCS, which provided high resolution size distribution measurements. DLS was unable to provide an accurate measurement of the protein corona thickness due to the presence of aggregates in serum. Despite differences between the basic measurement principles of the methods, the ζ-potential results are overall in good agreement.Particle-by-particle technique (TRPS) provided additional information in terms of distribution of ζ-potential over the whole sample and allow better understanding of the fundamental behaviour of silica NPs in biological media.
- Monopoli MP et al., Journal of the American Chemical Society, 2011, 133 (8), 2525-2534.
- Walczyk D et al., Journal of the American Chemical Society, 2010, 132 (16), 5761-5768.
Attracted to Death: Characterisation of Apoptotic Cell-derived Extracellular Vesicles
Allan M. Cameron, Lois Hawkins, Parbata Chauhan, Andrew Devitt
School of Life & Health Sciences, Aston University, Birmingham, B4 7ET, UK.
Apoptosis, an anti-inflammatory process of cell death is intrinsic to maintenance of healthy tissue and is involved in wide ranging pathologies. Recently extracellular vesicles derived from apoptotic cells (ACdEV) were recognised as a mechanism by which dying cells communicate their presence to enable safe cellular waste disposal. Failure of this communication system can lead to the inflammation and disease.
To date, comprehensive analysis of ACdEV has never been carried out and may shed light on the importance of factors such as dose, size and charge in cellular responses to apoptosis. Furthermore due to their longevity, considerable potential is seen in the field for development of biocompatible EV-matching drug delivery systems.
Here we induced apoptosis in Jurkat, Mutu, THP-1 and HeLa cell lines by administering UV irradiation at 60mJ/cm2. ACdEV production was monitored hourly over 24h after induction. Cell corpses and debris were successfully removed by 20 min centrifugation at 2000g. Size and concentration of ACdEV was determined using Tuneable Resistive Pulse Sensing technology.
Diverse concentrations of vesicles were observed in all cell lines with THP-1 (Monocyte line) producing substantially more vesicles from induction compared to Jurkat or Mutu cells. Pilot chemotaxis studies were carried out to determine the role of ACdEV in Macrophage migration.
Our data suggest that dose but not size profile is variable between ACdEV populations, which may influence the observed chemoattractive effect. Further functional studies will need to be undertaken as well as bioanalytical assessment to fully elucidate ACdEVs role.
Approaches to microvesicle extraction
Scott Bright1,Munira Kadhim1
1Genomic instability research group, Oxford Brookes University, Oxford, OX3 0BP
The field of extracellular vesicle (EV) research has undergone a massive expansion in the last few years1. However there is still much to be standardized particularly with regard to isolation and classification, there are also issues with contaminants particularly in vitro with the use of fetal bovine serum (FBS). Currently the most common techniques involve ultracentrifugation or chemical isolation both have advantages and disadvantages. However a relatively new isolation method using the IZON qEV separation column has been become available. Here we compare different EV isolation methods using the breast cancer cell line, MCF7. Varying levels of FBS will also be used to investigate its effect on EV release profile as well as cell proliferation and viability.
Having understood some of the issues associated with EV isolation we plan to present data on EV’s from human ex vivo samples. Much of the current research in this field is focused around EV’s in cancer there are far fewer studies related to EV’s in radiation biology. Recently we have shown in vitro that EV’s are involved in radiation induced non-targeted effects2. We therefore propose that they may have potential as biomarkers in radiation response. In the current study human ex vivo whole blood was placed into culture and irradiated with 0, 0.1 or 0.25 Gy X-rays. Data on the dose response will be presented for all relevant biological endpoints.
- Van der Pol, E. et al. (2012) Pharmacological reviews 64(3) 676 - 705
- Al-Mayah, A. et al. (2015) Mutation Research 772 38 - 45