Article pubs.acs.org/ac
Electrochemical Sandwich Immunosensor for Determination of Exosomes Based on Surface Marker-Mediated Signal Amplification Ximena Doldán,† Pablo Fagúndez,† Alfonso Cayota,‡,§ Justo Laíz,† and Juan Pablo Tosar*,†,‡ †
Analytical Biochemistry Unit, Nuclear Research Center, Faculty of Sciences, Universidad de la República, Mataojo 2055, Montevideo 11400, Uruguay ‡ Functional Genomics Laboratory, Institut Pasteur de Montevideo. Mataojo 2020, Montevideo 11400, Uruguay § Department of Medicine, Faculty of Medicine, Universidad de la República, Av. Italia S/N, Montevideo 11600, Uruguay
Anal. Chem. 2016.88:10466-10473. Downloaded from pubs.acs.org by DURHAM UNIV on 08/29/18. For personal use only.
S Supporting Information *
ABSTRACT: Extracellular vesicles (EVs), namely, exosomes and microvesicles, are important mediators of intercellular communication pathways. Since EVs can be detected in a variety of biofluids and contain a specific set of biomarkers which are reminiscent of their parental cells, they show great promise in clinical diagnostics as EV analysis can be performed in minimally invasive liquid biopsies. However, reliable, fast and cost-effective methods for their determination are still needed, especially if decentralized analysis is intended. In this study, we developed an electrochemical biosensor which works with 1.5 μL sample volume and can detect as low as 200 exosomes per microliter, with a linear range spanning almost 4 orders of magnitude. The sensor is specific and readily differentiates exosomes from microvesicles in samples containing 1000-fold excess of the latter. Capability of detecting exosomes in real samples (diluted serum) was shown. This was achieved by immobilizing rabbit antihuman CD9 antibodies on gold substrates and using monoclonal antibodies against CD9 for detection of captured exosomes. Signal amplification is presumably obtained from the fact that multiple detector antibodies bind to the surface of each captured vesicle. Detection is performed based on electrochemical reduction of 3,3′,5,5′-tetramethyl benzidine (TMB) after addition of horseradish peroxidase (HRP)-conjugated anti-IgG antibodies. This amperometric biosensor can be easily incorporated into future miniaturized and semiautomatic devices for EV determination.
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and these particles also differ in their buoyant density and morphology when analyzed by transmission electron microscopy. There is also a number of well-known exosomal markers, which include the intraluminal protein TSG-101 and the transmembrane proteins CD9 and CD63.12 Purification of EVs from biofluids or cell culture conditioned media is usually performed by density gradient or differential centrifugation, the latter being considered as the “gold standard” because of its long track record.13 MVs are usually collected by mediumspeed centrifugation (>10 000g), while high-speed centrifugation (100 000g) of the supernatants is needed to pellet EXOs. While these techniques show excellent performance for research lab applications, they are not suitable for routine screening or point-of-care (POC) testing. Rapid purification from biofluids can be achieved by the use of certain commercial kits, which are based on the use of polymers to precipitate EVs. However, these methods are likely to coisolate other molecules like protein complexes or aggregates.12 Routine screening of EVs in biological fluids will benefit from the implementation of new analytical devices capable of EV
xtracellular vesicles (EVs) are submicrometer membranederived particles which are secreted to the extracellular space by most eukaryotic cells.1 It is now widely accepted that these particles contribute to intercellular communication by transporting proteins, lipids, and RNA to recipient cells.2,3 For instance, cancer-derived EVs have been recently described as the soluble factors responsible for premetastatic niche formation in specific target organs.4,5 Since EVs can reach the bloodstream and other body fluids and contain a specific set of proteins and RNA which are characteristic of their parental cells, EV-associated biomarker profiling might serve as a novel minimally invasive method to screen risk patients for the outbreak of a disease.6 Importantly, cancer cells not only release EVs with specific biomarkers which can be assayed in plasma7 but they also affect the overall plasma EV concentration.8−10 Thus, EV quantization is important for current biomedical research and future medical diagnosis. According to their biogenesis, EVs are classified in exosomes (EXOs) and microvesicles (MVs).2 Exosomes have classically been defined as originating from the endosomal compartment by fusion of multivesicular bodies (MVB) with the cell’s surface, while MVs originate from direct outward budding of the plasma membrane.11 The size distribution of EXOs (50− 150 nm) is usually narrower than that of MVs (100−1000 nm), © 2016 American Chemical Society
Received: June 23, 2016 Accepted: October 13, 2016 Published: October 13, 2016 10466
DOI: 10.1021/acs.analchem.6b02421 Anal. Chem. 2016, 88, 10466−10473
Article
Analytical Chemistry
3,3′,5,5′ tetramethylbenzidine (TMB, ≥98%) were purchased from Sigma. Dulbecco’s Phosphate-Buffered Saline (PBS) was from Gibco. MCF-7 cells were purchased from the American Tissue Type Culture Collection and used at low passage (1000-fold dilutions of human blood plasma. This is of great advantage because of the small volumes which should be required, but mainly because dilution diminishes the matrix effects of difficult real-life application of analytical devices which show promising performance in the lab. Indeed, we were able to obtain analytical signals inside the dynamic range of the method in 1/1000 (0.1%) dilutions of fetal bovine serum, which were almost completely abrogated in ultracentrifuged (EV-depleted) serum. Furthermore, matrix interference was discarded as a significant problem at such dilution ranges. We also showed near-optimal performance in mixtures of EVs containing a 1000-fold excess of MVs, the most plausible interference expected in biologically relevant samples. Altogether, we report a sensitive and specific method which, upon further optimization to reduce incubation times and steps, will aid in the determination of extracellular vesicles in clinical samples, bypassing the need of expensive equipment and laborious purification.
for future diagnostic methods based on minimally invasive liquid biopsies.26 Nanoparticle tracking analysis and flow cytometry are widely used techniques in most reports focused on EV research. However, these procedures demand access to relatively costly equipment and are therefore not suitable for decentralized or POC testing. Several companies offer ELISA kits which simplify exosome determination, but again, these techniques are hard to implement outside the lab. Electrochemical biosensors hold great promise because of their robustness, easy miniaturization, excellent sensitivity with small volumes, and ability to be used in turbid biofluids with optically absorbing compounds.27 In spite of this, only a few attempts to obtain an exosome-specific electrochemical biosensor have been described.20,21 With sample volumes as low as 1.5 μL, we have achieved very sensitive electrochemical detection of EXOs, with a limit of detection of 2 × 102 particles/μL and a linear response spanning almost 4 orders of magnitude. This outperforms previous descriptions of electrochemical aptamer-based sensors for EXO detection (LOD, 1 × 103 particles/μL; linear range, 2 orders of magnitude) and commercial immunoassays.20 The dynamic range of our biosensor is comparable to the one obtained with a recently described ultrasensitive microfluidicbased fluorogenic ELISA platform,15 but much simpler and lesscostly equipment are needed with the amperometric readout of our method (Table 1). Curiously, the cited report was also based on a sandwich-type configuration, so it is tempting to speculate that surface marker-mediated signal amplification is on the basis of the low detection limit achieved with the signalon method described in this work. NTA-based estimations of exosome concentrations showed values of ∼1 × 109 and 1−6 × 108 particles per μL for healthy blood plasma and ovarian carcinoma cell lines, respectively.25 Considering we have obtained a dynamic range in the 2 × 102 to 1 × 106 particles per μL region, our electrochemical biosensor should be able to reliably quantify exosomes in
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b02421. Optimization of electrode blocking with BSA in Au/ MUA electrodes, scanning electron micrographs, and evaluation of antibody specificity by the signal-off method (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partially funded by the Comisión Sectorial de ́ Investigación Cientifica (CSIC, Uruguay), Grant Number 10472
DOI: 10.1021/acs.analchem.6b02421 Anal. Chem. 2016, 88, 10466−10473
Article
Analytical Chemistry
Gallichotte, E. N.; et al. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 14888−14893. (26) Chi, K. R. Nature 2016, 532, 269−271. (27) Grieshaber, D.; MacKenzie, R.; Vörös, J.; Reimhult, E. Sensors 2008, 8, 1400. (28) Zong, S.; Wang, L.; Chen, C.; Lu, J.; Zhu, D.; Zhang, Y.; Wang, Z.; Cui, Y. Anal. Methods 2016, 8, 5001−5008. (29) Ko, J.; Hemphill, M. A.; Gabrieli, D.; Wu, L.; Yelleswarapu, V.; Lawrence, G.; Pennycooke, W.; Singh, A.; Meaney, D. F.; Issadore, D. Sci. Rep. 2016, 6, 31215.
PAIE2014_ID68. J.P.T. and A.C. are members of the Sistema Nacional de Investigadores (SNI, ANII, Uruguay). The authors want to thank Gabriela Casanova, Magela Rodao (Universidad de la República, Uruguay), and Kenneth Witwer (Johns Hopkins University, USA) for their support with electron microscopy and nanoparticle tracking analysis. Fabiana Gámbaro (Institut Pasteur de Montevideo) helped with Western blot analysis. Belén Fernández and Julio Berbejillo (Universidad de la República) helped with real simple analysis.
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REFERENCES
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DOI: 10.1021/acs.analchem.6b02421 Anal. Chem. 2016, 88, 10466−10473
