FO‐SPR biosensor calibrated with recombinant extracellular vesicles enables specific and sensitive detection directly in complex matrices

Abstract

Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well‐characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO‐SPR) bioassay. In this context, EV binding on the FO‐SPR probes was achieved only with EV‐specific antibodies (e.g. anti‐CD9 and anti‐CD63) but not with non‐specific anti‐IgG. To increase detection sensitivity, we tested six different combinations of EV‐specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti‐CD9/ B anti‐CD81 and anti‐CD63/ B anti‐CD9), resulting in 10 3 and 10 4 times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti‐CD63/ B anti‐CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti‐EpCAM antibody on the FO‐SPR surface. The obtained results combined with FO‐SPR real‐time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis.

Innovative FO-SPR Label-free Strategy for Detecting Anti-RBD Antibodies in COVID-19 Patient Serum and Whole Blood

“…In the sandwich bioassays, the signal amplification was performed after the label-free step, with the FO probes immersed in AuNPs (optical density, OD = 1.0), functionalized with GAH IgG or GAH IgG, IgM, and IgA antibody, for 30 min, as described in the previously developed sandwich bioassays. 22 − 24 Specifically, the serum or whole blood samples were diluted 10-fold for label-free bioassays and 500-fold for sandwich bioassays as will be further elaborated in the following sections.…”

“…Starting from these clear advantages of SPR technology, we present in this work label-free fiber optic (FO)-SPR serological bioassays that enable the measuring of antibody levels as well as kinetic profiling of the polyclonal anti-SARS-CoV-2 antibody response in COVID-19 patient samples. In this context, the main benefit of the FO-SPR technology is its compatibility with complex matrices, − thereby allowing inspection of both antibody levels and their kinetic profiling directly in complex matrices (and hence in patient samples), an aspect difficult to attain with conventional chip-SPR configurations because of the potential clogging of microfluidic channels. Therefore, here we establish the FO-SPR bioassays not only in serum but also for the first time directly in the whole blood of convalescent patients (Figure ).…”

“…Compared to the more frequently reported sandwich bioassays in the context of COVID-19 serological studies, our FO-SPR label-free bioassay offers at least four apparent advantages: (1) information about kinetic binding profiles of antibody responses (not attainable with sandwich bioassays), (2) insight into the complete patient antibody response, (3) shorter time-to-result (i.e., 30 min) compared to sandwich bioassays (e.g., 4 h for a conventional ELISA), and (4) exceptional compatibility with whole blood samples, omitting the need for any sample processing. These innovative aspects, combined with other previously demonstrated FO-SPR attributes, like the low-cost sensor probes, ease of operation, low-demand in sample volume, and even flexibility to integrate with microfluidic platform toward a point-of-care (POC) test, − position the FO-SPR technology at the forefront of other (rapid) COVID-19 serological tests published to date. Moreover, because our FO-SPR technology has been proven as a highly adaptable platform for detecting >10 different targets, − ,− the same approach is easily amenable to other applications in need for testing the levels and kinetic profiling of antibodies.…”

“…These innovative aspects, combined with other previously demonstrated FO-SPR attributes, like the low-cost sensor probes, ease of operation, low-demand in sample volume, and even flexibility to integrate with microfluidic platform toward a point-of-care (POC) test, − position the FO-SPR technology at the forefront of other (rapid) COVID-19 serological tests published to date. Moreover, because our FO-SPR technology has been proven as a highly adaptable platform for detecting >10 different targets, − ,− the same approach is easily amenable to other applications in need for testing the levels and kinetic profiling of antibodies.…”

Microfluidic Approaches for Affinity-Based Exosome Separation

“…Previously considered as cellular waste [ 2 , 3 ], new research suggests that their specific composition can be used to determine the status of cancer, auto-immune or cardiovascular diseases from mainly non-invasive liquid biopsies [ 4 , 5 , 6 , 7 , 8 ]. Moreover, it is possible to turn them into targeted drug delivery systems for theranostic application with biochemical engineering methods [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. To exploit these diagnostic and therapeutic opportunities, efficient methods for separating EVs from biological solutions must be found.…”