Design of a single particle-interferometric reflectance imaging sensor adipo-chip for obesity biomarker screening

Abstract

In light of the obesity pandemic, there is an increasing imperative to identify obesity phenotypes that encompass factors such as adipose tissue distribution, inflammation, and insulin sensitivity, to devise tailored therapeutic strategies. Within this context, extracellular vesicles (EVs) have emerged as promising reservoirs of biomarkers. However, the inherent technical challenges associated with their isolation and analysis necessitate the development of precise, high-throughput technologies to facilitate their integration into clinical settings. The single-particle interferometric reflectance imaging sensor (SP-IRIS) has emerged as a valuable tool that enables the analysis of biomarkers in individual EVs without the need for prior purification. The fundamental principle of SP-IRIS involves the capture of EVs using functionalized chips with capture antibodies targeting standard exosomal tetraspanins, with the option of employing custom antibodies to capture cell- or tissue-specific EVs. Herein, we describe, for the first time, the design and validation of an SP-IRIS chip functionalized with two capture antibodies to assess adipose-specific (adipo-chip) secreted vesicles for biomarker assessment. Using this approach, we demonstrate that the designed adipo-chip captures EVs directly secreted by the whole adipose tissue of patients with obesity undergoing bariatric surgery, allowing the quantification of up to four previously described adipose EV-biomarkers. Thus, we demonstrate for the first time the capacity of the adipo-chip to enrich the capture of adipose-secreted EVs, enabling the detection of changes in described biomarkers with potential applications in clinical settings through liquid biopsy at the circulating level.

Keywords: Adipose tissue; Biomarkers; Extracellular vesicles; Obesity; SP-IRIS.

Fig. 1

Design of an SP-IRIS chip for adipose tissue-specific EV capture [adipo-chip] and biomarker analysis. Schematic representation of the adipo-chip design, including the capture of functionalized spots with anti-tetraspanins and custom antibodies against CAVN-1 and PLIN1. Secondary fluorescent labelled antibodies were used to detect biomarkers of interest, including representative images (created at https://BioRender.com).

Fig. 2

Development and validation of an adipose tissue-specific SP-IRIS chip [adipo-chip]. The concentrations (particles/mL) of EVs captured by each antibody arrayed on the chip, as measured by interferometry, are shown. These include the standard anti-tetraspanin-based spots: CD63 (A), CD81 (B), and CD9 (C), as well as spots functionalized with anti-adipose-specific proteins: caveolin-1 (D) and perilipin-1 (E) (created at https://BioRender.com). SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue.

Fig. 3

Evaluation of biomarkers in extracellular vesicles (EVs) derived from human obese subcutaneous and visceral adipose tissue explants using the Adipo-chip. The concentrations of biomarkers detected in the captured vesicles are depicted in histograms, indicating particles/mL for those captured by the three tetraspanins and the custom adipo-chip: TFGBI (A, E), mimecan (B, F), ceruloplasmin (C, G), and FABP4 (D, H). Statistical differences were assessed using the Mann–Whitney U test, with p ≤ 0.05 considered statistically significant (*p < 0.05, **p < 0.01) (Created in https://BioRender.com). SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue.

Fig. 4

Fluorescent biomarker colocalising in single particles captured in tetras and adipo-chip. The proportion of single vesicle fluorescent detection of individual or colocalized biomarkers for each tissue and capture spot is depicted in pie charts (A–I). Representative immunofluorescence images of captured extracellular vesicles (EVs) incubated with anti-TGFBI (red), FABP4 (far red), ceruloplasmin (green), and mimecan (blue), as well as their colocalization, captured by CD63, CD81, CD9, caveolin-1, or perilipin-1, are also presented (created at https://BioRender.com).