Extracellular vesicles (EVs) are membrane-bound vesicles released from cells to the extracellular milieu, playing an important role in the biological processes, including intercellular communications (1). EVs can be generally divided into exosomes, microvesicles (MVs), and apoptotic bodies largely by their size, despite debate about their definition (2). Recently, the application of EVs as a tool for non-invasive liquid biopsy in tumor specimens has been investigated in prostate cancer (3), glioblastoma (4), and pancreatic cancer (5). Therefore, it is important to develop a better detection technique for the surface antigen of EVs, leading to the discovery of a precise biomarker for the diagnosis and prognosis of tumor. Currently, there are several techniques being used for the detection and characterization of EVs, including nanoparticle tracking analysis, dynamic light scattering analysis, zeta potential analysis, tunable resistive pulse sensing, Raman spectroscopy, electron microscopy, enzyme-linked immunosorbent assay, western blot, and “omics” such as proteomics and RNA-seq (6). In addition, surface plasmon resonance (SPR) sensing has emerged as a widespread biophysical sensing tool for the analysis of membrane molecules, including surface proteins and carbohydrates. Recently, a plethora of research on the detection of EVs by SPR technique has been carried out for various purposes (Table 1).
In the paper entitled “Surface plasmon resonance is an analytically sensitive method for antigen profiling of extracellular vesicles”, Gool EL et al. established a sensitive method to detect membrane proteins, such as Her2 and EGFR, in the EVs derived from human breast cancer cell lines, including HS578T, MCF7, and SKBR3. Importantly, the density of surface antigens in these cells and their exosomes was accurately measured via employing surface plasmon resonance imaging (SPRi) and the technique was much sensitive to detect small amount of surface antigens above the limit of detection (LOD) (16). The common Kretschmann configuration setup of the SPR for the detection of EVs has been represented in Figure 1.
Gool EL et al. established the SPRi device in which the sensor surface was precoated with a gold- and 3D hydrogel- layer, leading to the reduction of nonspecific background. An array of 48 spots functionalized with various antibodies was used to capture EVs, making the conduction of high-throughput experiments much easier. Once the chip is illuminated, light goes through the coupling crystal at a constant incident angle and the alteration of reflected light from the SPR gold surface are transformed into the altered refractive index resulting from the binding of EVs. Furthermore, this whole set of event is recorded by a charge-coupled device (CCD) camera which eventually yields plot as a component of time. The stage is quick and exceptionally subtle and requires a very low volume of samples. For high throughput multiplex estimations, a multichannel fluidic cell framework is necessary, alongside the identification framework incorporated with optics comprising of a laser diode and a CCD camera (16).
In recent studies, various exosomal proteins, including exosomal membrane antigens, have been proposed as diagnostic biomarkers for cancer, such as Glypican-1 (GPC1), Apbblip, Daf2, Foxp1, Incenp, BCO31781, Aspn, and Gng2 in pancreatic cancer, CD34 in acute myeloid leukemia (AML), phosphatase and tensin homolog (PTEN) in prostate cancer, and EDIL-3/Del1 in bladder cancer (6). However, the membrane of EVs generally contains small level of antigens associated with tumors, resulting in the challenge of the phenotyping of EVs by various biosensor tools, including flow cytometry (FCM). Gool EL et al. in their paper demonstrated that SPRi had high sensitivity to detect 31 out of 33 antibody-EV combinations above the LOD by the reduction of the background signals via using hydrogel on the sensor as well as the increase of the “back-and-forth” flow. However, only 5 among 33 antibody-EV combinations exceeded the LOD by FCM, suggesting its low sensitivity for phenotyping of EVs. Particularly, in comparison with FCM, SPRi can be efficiently used as a high-throughput screening platform for the discovery of novel exosomal biomarkers in various diseases, including tumors. However, FCM can only analyze a number of biomarkers ranging from 6 to 11 at a time on a routine basis, nonetheless various types of liquid biopsies still can be conducted by FCM via using the samples from plasma, cell culture supernatant, urine, and cerebrospinal fluid (18).
Importantly, Gool EL et al. demonstrated that the SPRi response was proportional to the antigen (Her2) density of SKBR3 cells and their EVs, which was correlated with the speciﬁc ﬂuorescence intensity (SFI) of SKBR3 cells by FCM, confirming the precise detection of exosomal Her2 by SPRi. In addition, the sensitivity of the detection by the SPRi also was determined; for example, 2×105 cells/mL were necessary on Her2 spots to surpass the LOD within 10 min of binding, whereas 2×108 SKBR3 EVs/mL were necessary to surpass the LOD.
Gool EL et al. (16) clearly demonstrated the strength of SPRi in the accurate detection of exosomal biomarkers, such as Her2, through the experimental tests of the specificity and sensitivity for various exosomal proteins. Qualitative determination of exosomal antigen could be achieved by the SPRi, however, quantitative phenotyping of EVs was not clearly demonstrated because the contribution of the concentrations and diameters of the EV particles on the SPRi response could not be determined. Finding both optimal detection methods and targets of membrane antigens of EVs might be critical for conducting better quantitative analysis of EVs by SPRi. Nonetheless, in future, quantitative analysis of exosomal biomarkers in disease progression, including tumors, can be a promising biosensing method as a liquid biopsy in the laboratories as well as the clinics.
One of the major concerns in the research of EVs is the dearth of appropriate terminology and techniques for the description of experimental results. Although the authors have referred EVs to all the vesicles in the aforementioned article, accumulated evidence has demonstrated a clear distinction between three major types of EVs, exosomes, MVs, and apoptotic bodies. Each type of EVs are synthesized and released by different biological pathways. Importantly, different type of EVs has different membrane property (2) and it contains different components, prompting us to investigate their specific role in physiological- and pathological- conditions. Therefore, the detection of specific type of EVs by SPRi might be a good diagnostic method and it also can provide a great insight into the underlying mechanism of each type of EVs in the progression of disease, such as tumor metastasis.
Funding: This work was supported by the start-up grants of City University of Hong Kong awarded to Dr. Y. Lee (Grant No. 9610340 and 7200472).
Conflicts of Interest: The authors have no conflicts of interest to declare.
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Cite this article as: Thakur A, Qiu G, NG SP, Wu CM, Lee Y. Detection of membrane antigens of extracellular vesicles by surface plasmon resonance. J Lab Precis Med 2017;2:98.