Squeezable Hydrogel Microparticles for Single Extracellular Vesicle Protein Profiling

Introduction: : Circulating extracellular vesicles (EVs) hold great potential for molecular diagnostics, but current analyses are challenged by the low abundance and compositional heterogeneity of EV protein expression [1-3]. To overcome these limitations, single-EV profiling requires high sensitivity, multiplexing capacity, and throughput [4]. Here, we present a single-EV analysis technique that leverages squeezable methacrylated hyaluronic acid hydrogel microparticles (MHPs) as a scaffold for EV immobilization and integrated rolling circle amplification (RCA) to enable ultra-sensitive, multiplexed detection of EV surface proteins. EVs are encapsulated into MHPs using droplet microfluidics in a high-throughput manner (Fig. 1A) and are efficiently labeled with antibody–oligonucleotide conjugates, free from steric hindrance. By engineering MHPs with high compressibility, RCA products from individual EVs can be aligned onto a single imaging plane through physical squeezing, allowing visualization with low-magnification microscopy (Fig. 1B-C). This approach establishes a streamlined, scalable imaging pipeline for multiplexed single-EV protein profiling (Fig. 1D). As a proof of concept, the method is applied to characterize the heterogeneity of cancer-associated markers on EVs derived from different cancer cell lines. These results highlight the utility of squeezable MHPs as a powerful platform for sensitive, multiplexed, and scalable single-EV analysis, with broad applications in molecular diagnostics.

Materials and

Methods: : High-throughput production of EV-loaded MHPs was achieved using a flow-focusing microfluidic droplet generator in combination with a 50 µm-tall UV polymerization chamber. Uniform MHPs were generated by mixing bulk EVs with MeHA prepolymer and subsequently photo-crosslinking the droplets to physically encapsulate EVs for downstream immuno-RCA analysis.

The integrated immuno-RCA workflow consists of four main steps: immunolabeling, ligation, amplification, and fluorescent labeling.
1. Immunolabeling: Antibody–DNA conjugates (0.125 µg/mL in 1× PBS with 5% BSA) were mixed with MHPs and incubated at 25 °C for 30 min.
2. Ligation: A ligation mix containing 20 nM padlock probe and 400 mU/µL T4 DNA ligase in 1× ligase buffer was added to MHPs and incubated at 37 °C for 30 min.
3. Amplification: Phi29 polymerase mixture (800 mU/µL phi29, 250 µM dNTPs in 1× phi29 buffer) was added and incubated at 37 °C for 3 hr.
4. Fluorescent labeling: A 250 nM fluorescent probe in 1× hybridization buffer was incubated with MHPs at 37 °C for 20 min.
All incubation steps were performed using a thermomixer set to 1500 rpm. Three sequential wash steps were carried out between each stage and post-labeling to prepare samples for imaging.

For imaging and analysis, MHPs were physically squeezed with a glass coverslip and visualized using an IX83 inverted fluorescence microscope at 20× magnification. Dot counts and fluorescence colocalization across channels were quantified using customized pipelines in CellProfiler.

Results, Conclusions, and Discussions:: This study presents a squeezable hydrogel microparticle-based platform for an ultra-sensitive, multiplexed protein analysis of single EVs. MHPs allow for physical compression, aligning RCA products into a single imaging plane and eliminating the need for z-stack imaging. This enables high-throughput, cost-effective, and simplified EV imaging with >10³ EVs analyzed per frame using an automated pipeline to assess protein marker expression and co-expression. Validation was carried out using A431-derived EVs known to express EGFR, achieving a detection limit of 28 EVs/hydrogel (Fig. 2A) The platform’s multiplexing capacity was expanded using four antibody-DNA barcode/PLP/fluorophore probe sets. Specificity was confirmed by testing distinct barcode-labeled anti-EGFR antibodies against matched and mismatched probes (Fig. 2B-C), as well as negative controls using isotype antibodies and no-EV conditions (Fig. 2D) for target multiplexing (Fig. 2E). To calibrate and validate the MHP assay, we investigated the expression of cancer markers EGFR, EpCAM, and MUC1, as well as EV marker CD9, between different parent cancer cell lines (A431, A549, MCF7, and PANC1) and EVs. The correlation analysis of cancer markers between parent cells and EVs demonstrates good concordance among protein expression profiles (R2 = 0.815) (Fig. 3A-B). Furthermore, a 4-plex analysis across EVs from different cancer cell lines revealed distinct protein expression patterns (Fig. 3C). CD9 was highly abundant (>35%), while EGFR was similarly expressed (~12–18%) across lines. Notably, co-expression patterns aligned with known cell profiles: A431 and PANC1 showed higher EGFR-CD9 co-expression, MCF7 had prominent EpCAM expression, and PANC1 EVs expressed MUC1 with or without CD9. These findings underscore single-EV heterogeneity and the value of ultrasensitive detection. The MHP-based approach addresses key limitations of current methods, including Poisson distribution constraints, limited multiplexing of digital ELISAs, and sensitivity issues, by enhancing epitope interactions and protein signals. Overall, this simple, scalable approach enables comprehensive EV profiling and holds potential for identifying rare EV subpopulations from liquid biopsies, advancing precision medicine efforts for early disease detection.

Acknowledgements and/or References (Optional)::

References:
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