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Determination of exosome concentration in solution using surface plasmon resonance spectroscopy Deborah L. M. Rupert, Cecilia Lässer, Maria Eldh, Stephan Block, Vladimir P. Zhdanov, Jan Lotvall, Marta Bally, and Fredrik Höök Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac500931f • Publication Date (Web): 22 May 2014 Downloaded from http://pubs.acs.org on May 26, 2014
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Analytical Chemistry
Determination of exosome concentration in solution using surface plasmon resonance spectroscopy Déborah L. M. Rupert1, Cecilia Lässer2, Maria Eldh2, Stephan Block1, Vladimir P. Zhdanov1,3, Jan O. Lotvall2, Marta Bally1,4, Fredrik Höök1* 1)
2)
Department of Applied Physics, Chalmers University of Technology, Gothenburg, Sweden
Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, University of Gothenburg, Gothenburg, Sweden 3)
4)
Boreskov Institute of Catalysis, Russian Academy of Sciences, Novosibirsk, Russia
Institut Curie, Centrede Recherche, CNRS, UMR168, Physico-ChimieCurie, Paris, France-
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ABSTRACT Exosomes are cell-secreted nanometer-sized extracellular vesicles that have been reported to play an important role in intercellular communication. They are also considered potential diagnostic markers for various health disorders, and intense investigations are presently directed towards their use as carriers in drug-delivery and gene-therapy applications. This has generated a growing need for sensitive methods capable of accurately and specifically determining the concentration of exosomes in complex biological fluids. Here, we explore the use of label-free surface-based sensing with surface plasmon resonance (SPR) read-out to determine the concentration of exosomes in solution. Human mast cell secreted exosomes carrying the tetraspanin membrane protein CD63 were analyzed by measuring their diffusion-limited binding rate to an SPR sensor surface functionalized with anti-CD63 antibodies. The concentration of suspended exosomes was determined by first converting the SPR response into surface-bound mass. The increase in mass uptake over time was then related to the exosome concentration in solution using a formalism describing diffusion-limited binding under controlled flow conditions. The proposed quantification method is based on a calibration and control measurements performed with proteins and synthetic lipid vesicles and takes into account i) the influence of the broad size distribution of the exosomes on the surface coverage, ii) the fact that their size is comparable to the ~150 nm probing depth of SPR, and iii) possible deformation of exosomes upon adsorption. Under those considerations, the accuracy of the concentration determination was estimated to be better than ±50% and significantly better if the exosome deformation is negligible.
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Analytical Chemistry
INTRODUCTION Extracellular vesicles are naturally produced membrane-enclosed biomolecular carriers secreted by various types of cells. Such vesicles can be found in a variety of body fluids including blood1, urine2, saliva3 and breast milk4. First, they were considered cellular debris5, but their relevance has later been increasingly highlighted by discoveries showing that they are in fact key actors of the intercellular communication network6. With a size in the hundred nanometer range7, exosomes are a specific type of extracellular vesicles produced by the inner cell machinery as part of a complex endosomal secretory pathway8. For this reason, the composition of the exosome membrane differs from that of the plasma membrane of the cell they originate from. In particular, they are enriched in specific proteins such as tetraspanins (CD63, CD81, CD9), heat shock proteins (Hsp70, Hsp90) and membrane fusion proteins (Annexins, Rabs).9 The core of exosomes contains both soluble proteins and genetic material, in particular RNA10 such as mRNA and miRNA. Although the crucial functions of exosomes have just started to be unrevealed, it is becoming more and more evident that these extracellular vesicles play an important role in a variety of biological processes, including intercellular communication within the immune system, inflammation processes and tissue regeneration.11 They are also influenced by a range of diseases including asthma and allergic reactions12, neurodegenerative diseases13 and cancer14. For example, although exosomes are also present in body fluids of healthy individuals, their concentration has been observed to be altered in case of disease.15 Consequently, there is a growing demand for accurate means to characterize not only the molecular composition of exosomes, but also their absolute concentration in body fluids. Further refinements of already existing methods will not only contribute to broaden the understanding of
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the biological role of exosomes, but are also likely to accelerate the implementation of exosomes as biomarkers in clinical diagnostics16 and as therapeutic agents17. Among the most commonly used techniques for exosome concentration determination are colorimetric protein assays, which measure the total mass of proteins. Accurate quantification with this method is, however, possible provided that the exosome suspension is free of other types of extracellular vesicles and soluble proteins, which puts high requirements on purification procedures. In addition, since the relative protein concentration in exosomes may vary, this approach is considered a rough guide at best. More recently, alternative methods relying on direct particle counting have become popular. One of them is nanoparticle tracking analysis (NTA)18, which is based on real-time imaging of the light scattered by individual nanoscale particles freely diffusing in solution. Another one is the tunable elastomeric pore sensing method19, which measures alterations of the electrical impedance in a small aperture upon passage of single nanoscale particles. While these techniques are in principle directly applicable to vesicle quantification, they are primarily designed for accurate size distribution and/or zetapotential determinations. Their quantification accuracy may suffer from ill-defined probe volumes, uncertainties with respect to threshold settings for reliable nanoparticle counting and a risk of not counting nanoparticles below a certain size or optical density.20 An additional limitation of the above-mentioned methods is that they do not offer a possibility to selectively identify well-defined exosome subpopulations characterized by the presence of specific biomolecular markers. This lack of exosome selectivity prevents determination of relative ratios of different subpopulations of extracellular vesicles, information that may be critical in diagnostic applications.
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The approaches that have emerged to specifically quantify sub-populations of exosomes rely on a combination of nanoparticle counting and immunostaining. Exosomes are in this case typically revealed using fluorescently labeled antibodies directed against selected membranebound markers. Direct counting of individually labeled exosomes can be achieved using either NTA operating in fluorescence mode18 or high-sensitivity variants of flow cytometry21, as both are capable of detecting individual nanoscale vesicles. However, these approaches require a sufficient amount of markers on the exosome surface to make the fluorescence emission of individual exosomes detectable. In addition, the filtration steps employed to clear the solution from unbound fluorescent antibodies are likely to result in loss of exosomal material. These potential limitations are in principle circumvented by employing surface-based detection. In this case, specific molecular recognition probes, e.g. antibodies, are immobilized on a surface to specifically capture exosomes. The measured surface coverage can then be correlated to the concentration of a specific exosome sub-population in solution. For antibodies with sufficient affinity, binding of exosomes to antibody-modified surfaces should require no more than one or a few copies of a specific molecular marker per exosome, and does not require extensive purification steps. Surface-based capturing in combination with fluorescence-based read-out for quantitative concentration determination using various calibration schemes is welldocumented for proteins22 and nucleotide material23. For exosome quantification, enzyme-linked immunosorbent assay (ELISA) analysis has been proposed24, but the strategy remains relatively uncertain, since precise quantification of the amount of bound exosomes relies on external calibrations correlating the obtained signal to the bulk concentration determined using e.g. NTA or total protein content measurement.
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Certain optical methods, such as ellipsometry, optical waveguide light-mode spectroscopy and surface plasmon resonance (SPR) allow for mass-uptake determination without engaging fluorescent labels, and are usually better than fluorescence-based methods for quantifying biomolecular binding. These techniques are all based on measuring optical contrast originating from a change in interfacial refractive index upon biomolecular adsorption. SPR has become an increasingly popular tool in biotechnology25, primarily because of its i) high signal-to-noise ratio for measurements of binding events26, ii) compatibility with microfluidic liquid handling offering a precise control of flow speed27 and iii) compatibility with advanced chemical surface modifications28, which enable immobilization onto the surface of one of the binding partners under investigation, while simultaneously ensuring low non-specific biomolecule binding. SPR is based on illumination of a surface with plane polarized light which can, under certain conditions, excite a global resonance of surface-confined free electrons in the gold film, socalled surface plasmons. The conditions required to excite surface plasmons depend, among other parameters, on the effective refractive index in close proximity to the sensor (gold) surface. Biomolecular adsorption kinetics can thus be measured by monitoring of the temporal changes in interfacial refractive index converted into bound mass per surface area,
, (references 29 and
30) as detailed in the Supporting Information. The possibility to convert the temporal change in into concentration, C, of suspended biomolecules takes advantage of the fact that most biomolecules are relatively slow-diffusing objects. It therefore becomes possible to operate SPR under flow conditions at which the binding rate of the biomolecules to the sensor surface is limited by their diffusion rather than by the binding reaction itself. Using, for example, a cylindrical or rectangular flow-cell with the sensor element positioned on one of the channel
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walls, the time dependence of the initial mass-uptake can be expressed as31 (for the physics behind the derivation, see reference 32)
Γt
D 2Q
1/ 3
(1)
C t
where D is the analyte diffusion constant, Q the volumetric flow rate, and
a constant related to
the geometry of the flow cell. For a rectangular geometry with the optical measurements performed in the center33, is given by 0.98
2 2 h wl
1/ 3
(2)
where h, w and l are the height, width and length of the channel, respectively. Hence, the concentration of suspended biomolecules can be determined from their initial binding rate by converting the sensor response (which in the case of the Biacore instrumentation is given as dimensionless resonance units, RU) to a surface-bound mass30 (see Supporting Information for derivation).
C
RU d 2 1/ 3 t ( D Q) S (dn dC)[1 exp( d
)]
(3)
where RU/ t is the temporal change in resonance units, d the effective film thickness of the adsorbed biomolecular film, dn/dC is the derivative of the refractive index with respect to the biomolecule concentration in the solution, S is the sensitivity in terms of RU per change in bulk refractive index unit (RIU) and δ is the decay length of the evanescent field34, which is around 150 nm. The value of S is typically in the order of 106 RU/RIU (see reference 34). In the thin-film regime, where d
