Determination of Exosome Concentration in Solution Using Surface Plasmon Resonance Spectroscopy

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 toward 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 the 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 improve if the exosome deformation is negligible.

Total internal reflection fluorescence microscopy for determination of size of individual immobilized vesicles: Theory and experiment

“…8 Such systems can be fabricated via adsorption of vesicles without or with subsequent spontaneous or peptide-induced rupture 8,9 or via the "solventassisted" procedure. 10 The corresponding mass uptake can be determined optically by ellipsometry, waveguide lightmode spectroscopy and surface plasmon resonance, [11][12][13] or by quartz crystal microbalance. 12 Vesicles directly attached to a support are however deformed.…”

Bulk immunoassays for analysis of extracellular vesicles

“…The time data allow the study of kinetics of interaction between EV and the surface. Because the refractive index contrast between buffer and EV is sufficiently large, no reporter is needed for readout [15][16][17][18], which is why SPR is sometimes referred to as a label-free technique. The change in refractive index provides information on the amount of captured material [19].…”

“…The variation in definitions is most extreme in the case of SPR, where authors have determined the detection limit as number of EV/mL [16], µg EV/mL [17], or number of EV/cm 2 [18]. While each of the units for limit of detection are sensible for some of the discussed assays, we selected number of EV/mL because it is definable for all assays.…”

“…NTA does not detect the concentration of EV, but the concentration of all particles inside the detection size range (typically 80-500 nm [25]). Furthermore, a wide array of reference samples is used, including EV derived from different cell cultures [13,14,16,17,20,21,23,26] and/or plasma [21,27] were used. All these factors lead to considerable uncertainty in the limit of detection.…”

“…For example, in all surface-based immunosorbent assays the transport of EV to the surface is limited by diffusion [17]. Diffusion scales with the inverse of the diameter, so a 50 nm EV will have a 20-fold higher diffusion constant as a 1,000 nm EV.…”