Physisorption of Affinity Ligands Facilitates Extracellular Vesicle Detection with Low Non-Specific Binding to Plasmonic Gold Substrates

Abstract

Plasmonic biosensors are increasingly being used for the analysis of extracellular vesicles (EVs) originating from disease areas. However, the high non-specific binding of EVs to a gold-sensing surface has been a critical problem and hindered the true translational potential. Here, we report that direct antibody immobilization on the plasmonic gold surface via physisorption shows excellent capture of cancer-derived EVs with ultralow non-specific binding even at very high concentrations. Contrary to commonly used methods that involve thiol-based linker attachment and an EDC/sulfo-NHS reaction, we show a higher specific capture rate and >50-fold lower non-specific on citrate-capped plain and nanopatterned gold surfaces. The method provides a simple, fast, and reproducible means to functionalize plasmonic gold surfaces with antibodies for robust EV biosensing.

Keywords: antibody immobilization; extracellular vesicles; physisorption; plasmonic sensing; surface chemistry.

Figure 1.

(A) Schematic illustration of physisorption and chemisorption procedures for antibody immobilization on gold substrates. (B) Representative fluorescence images of captured EVs on plain gold substrates coated with anti-CD63 and IgG control antibodies using physisorption and chemisorption methods. For chemisorption methods, mercaptoundecanoic acid (MUA) and 1kDa thiol polyethylene glycol (PEG) was used as linkers. EVs from OV90 cells were fluorescently labeled with AZDye 555. Scale bars = 50 μm. (C) The number of captured EVs on plain gold substrates prepared by different methods. (D) Specific capture efficiencies, defined by the ratio between specifically and nonspecifically captured EVs, are compared between the three methods tested.

Figure 2.

Schematic illustration of interactions (A) between antibodies and gold film via physisorption and (B) between antibodies and linker/gold film in a chemisorption method. (C) Fluorescence images before and after immobilization of AF647 dye-labeled antibodies on gold substrates prepared by physisorption and chemisorption methods. The images show negligible background signals before immobilization and more uniform antibody coating on the substrate with the physisorption method than with chemisorption methods. Scale bars = 50 μm.

Figure 3.

(A) A scanning electron micrograph of a gold nanowell substrate. (B-C) Representative fluorescence images showing (B) specifically and (C) nonspecifically bound OV90 EVs on anti CD63 antibody- and IgG control antibody-immobilized nanowell substrates, respectively, which were prepared by physisorption and chemisorption methods (scale bars = 25 μm). (D-F) The numbers of captured EVs with titrating concentrations of OV90 EVs on gold nanowell substrates coated with (D) anti-CD63, (E) IgG control antibodies. (F) Net EV counts after subtracting the numbers of nonspecifically bound EVs from those from anti-CD63 coated substrates.

Figure 4.

Specificity tests of nanowell substrates prepared by the physisorption method using EVs from ovarian cancer cells mixed with EVs from benign cells or normal human plasma. (A) Representative fluorescence images of nanowell substrates functionalized with anti-CD63, anti-EpCAM, and IgG control antibodies. EVs from OV90 ovarian cancer cells were fluorescently labeled by AZDye 555 (shown green in the images). EVs from TIOSE4 benign cells were fluorescently labeled by AZDye 647 (shown red in the images). Scale bars = 50 μm. (B) A mixture of OV90 and TIOSE4-derived EVs were applied on anti-CD63, anti-EPCAM, and IgG coated nanowell substrates, and the captured EV counts were detected by fluorescence imaging. (C) Specific capture efficiencies defined by the ratio between specifically and nonspecifically captured EVs for OV90 and TIOSE4-derived EVs. (D) The number of EVs captured on anti-CD63, anti-EpCAM, and IgG antibody-coated nanowell substrates. Significantly higher numbers of cancer-derived EVs (OV90 and CaOV3) were captured on anti-EpCAM coated surfaces than those from normal plasma, while their levels on IgG-coated surfaces are comparable. (E) Specific capture efficiencies for EVs from OV90 and CaOV3 ovarian cancer cells and healthy human plasma samples.