Biology subjects

Juncker, D.

Publications and source records attributed to Juncker, D..

17 recordsLinked to original sources

bioRxiv2026

Bottom-up engineering of EV-mimetic vesicles reveals selective protein loading and modulates cellular uptake and biodistribution

Biological systems such as extracellular vesicles (EVs) are highly sophisticated and functionally diverse. Despite growing recognition of EVs' roles in health and disease through mediating intercellular communication, their inherent heterogeneity and challenges in purity and yield hinder their mechanistic research and clinical translation. To address this gap, we sought to translate essential EV properties into a controllable and simplified platform by engineering biomimetic synthetic vesicles (SVs) incorporating defined proteins and lipids. Using microfluidics, we generated SVs loaded with bulk membrane and cytosolic proteins from cancer cells, as well as individual organotropism-associated integrins. Bulk membrane protein incorporation into SVs reduced surface charge, modified lipid profiles, and enhanced cellular uptake, while integrin incorporation altered biodistribution. To investigate protein loading, we profiled input materials and SVs by mass spectrometry, identifying features linked to high incorporation, such as low molecular weight, positive charge, high isoelectric point, and predicted stability, globularity, lipidation, and amphipathic helices. This work presents a synthetic platform for dissecting the roles of EV-associated features and cargo, while providing insights into protein properties relevant to the optimization of delivery systems.

bioengineering↗

bioRxiv2026

Multiphase capillarics for structurally pre-programmed sequential and parallel operations of high and low cohesion liquids

Capillaric circuits (CCs) enable pre-programmed liquid handling through self-filling and passive valving governed by capillary forces, eliminating the need for external pumps and actuators. However, CCs optimized for high cohesion liquids (HCL) such as water are intrinsically unsuitable for programmed flow control of low cohesion liquids (LCL) such as oils and solvents because the high surface free energy needed for capillary flow of HCL results in uncontrolled, complete wetting by LCL. Here, we introduce a library of multiphase components including phase-to-phase valves (P2PV), hydro-pneumatic relays (HPR) and multiphase domino valves (MDVs) that collectively enable CCs to concomitantly process HCLs and LCLs. P2PVs use a pre-filled HCL to valve immiscible LCLs (e.g. oil) by confining the LCL, and upon triggering, hydraulically entrain it. To prevent uncontrolled mixing between miscible LCLs (e.g. ethanol) and the HCL, HPRs with an air gap and air waste are added to the P2PV. MDVs are further added and enable preprogrammed, sequential delivery of LCL and HCL by multiphase microfluidic chain reactions. Multiphase liquid processing is applied to automated, on-chip lipid nanoparticle (LNP) manufacturing, illustrating the potential of multiphase CCs.

bioengineering↗

bioRxiv2026

Iterative Extracellular Vesicle Protein Co-Expression Biomarker Refinement for Preoperative Classification of Histopathological Growth Patterns in Colorectal Liver Metastasis Patients

Preoperative triage of colorectal liver metastases (CRCLM) by histopathological growth pattern (HGP)--angiogenesis-dependent desmoplastic (dHGP) and vessel co-opting replacement (rHGP)--could guide anti-angiogenic therapy, yet HGP scoring requires resected tissue. We present an extracellular vesicle (EV) inner and outer protein (EVPio) co-expression assay and iterative biomarker refinement for plasma-based HGP classification. We established a minimal, high-throughput plasma pre-processing workflow (low-speed centrifugation and 0.45 m filtration) with comparable EVPio assay performance to size-exclusion chromatography. We established an EV biomarker selection template with growing cohorts--feasibility (n = 3), pilot (n = 9), discovery (n = 67)--ranking candidate protein pairs by signal quality (SNR, CV), redundancy (inter-correlation/orthogonality), and HGP separation (effect size, significance, ROC). This process reduced an initial 19x18 capture/detection set to a focused 9x9 panel (81 co-expression pairs). In a 58-patient CRCLM subset (22 dHGP, 14 rHGP, 22 mixed), three pairs achieved high signal quality with significant differential expression across HGPs. A three-feature linear discriminant model yielded 75.9% cross-validated accuracy (AUC 0.77) for classifying pure dHGP vs. non-dHGP. The results show that co-expression signatures capture defining features of HGP biology while revealing heterogeneity. The proposed EV biomarker refinement template is generalizable and our results show that co-expression signatures capture defining features of HGP biology supporting efforts towards clinically actionable, HGP-driven therapeutic guidance.

bioengineering↗

bioRxiv2025

Microfluidic sieve-detector for rapid capture and detection of single molecules

The advent of ultrasensitive assays enabled many new research and clinical applications, but the time-to-result is hours and has not significantly improved over time, limiting their potential. We introduce the microfluidic sieve and detector (MSD) that can sieve 200 microlitres in just one minute, and quantify captured analytes in four minutes, down to zeptomolar (10-19M) concentration. The MSD comprises half a million 8-m-diameter pores that bind analytes upon Brownian motion-induced wall collision. Digital sandwich assays are completed by sequentially flowing the sample, reagents, partitioning the pores, and digitally revealing single trapped analytes by enzymatic amplification. The MSD tests are specific and reproducible, exhibit a large dynamic range, and are easy-to-use, affordable, tuneable and versatile, enabling measurement of different proteins including influenza A nucleoprotein in clinical samples, and even nucleic acids while being both faster and more sensitive than PCR. As such, the MSD could open a new chapter for analysis and diagnostics.

bioengineering↗

bioRxiv2025

PiP-plex: A Particle-in-Particle System for Multiplexed Quantification of Secreted Proteins by Single Cells

Cell signaling is modulated by the secretion of various proteins, which can be used to infer a cells phenotype. However, these proteins cannot be readily detected in multiplex by commonly used methods at the single cell level. Here, we present PiP-plex, a particles-in-particle (PiPs) system comprising (i) fluorescence intensity barcoded microparticles (BMPs) co-entrapped with (ii) a single cell inside an alginate hydrogel particle for multiplex protein secretion analysis by confocal microscopy. We show that developed PiPs maintained >90 % cellular viability and allowed live cells retrieval. A seven-plex fluorescent barcoding and concomitant sandwich immunoassay in PiPs were implemented with limits of detection ranging from 0.8 pg mL-1 to 2 ng mL-1 depending on the protein. PiP-plex assays were benchmarked with bulk immunoassays and found to rival or outperform them. We applied PiP-plex to analyze protein secreted by THP-1 cells upon exposure to lipopolysaccharide and detected varying cell responses, with a significant increase in MIP-1, TNF- and IL-17A. Multivariate analysis revealed that the majority of stimulated cells secreted either MIP-1 or IL-17A, while other cytokines were typically co-secreted. Using PiP-plex, we analyzed [~]750 THP-1 cells, showcasing its potential for characterizing cells and cell-based therapeutics for cancer immunotherapies.

bioengineering↗

bioRxiv2024

High-resolution low-cost LCD 3D printing of microfluidics

The fabrication of microfluidic devices has progressed from cleanroom manufacturing to replica molding in polymers, and more recently to direct manufacturing by subtractive (e.g., laser machining) and additive (e.g., 3D printing) techniques, notably digital light processing (DLP) photopolymerization. However, many methods require technical expertise and while DLP 3D printers remain expensive at a cost [~]15-30K USD with [~]8M pixels that are 25-40 {micro}m in size. Here, we introduce (i) the use of low-cost ([~]150-600 USD) liquid crystal display (LCD) photopolymerization 3D printing with [~]8M-58M pixels that are 18-35 {micro}m in size for direct microfluidic device fabrication and (ii) a poly(ethylene glycol) diacrylate-based ink developed for LCD 3D printing (PLInk). We optimized PLInk for high resolution, fast 3D printing and biocompatibility while considering the illumination inhomogeneity and low power density of LCD 3D printers. We made lateral features as small as 75 {micro}m, 22-{micro}m-thick embedded membranes, and circular channels with a 110 {micro}m radius. We 3D printed microfluidic devices previously manufactured by other methods, including an embedded 3D micromixer, a membrane microvalve, and an autonomous capillaric circuit (CC) deployed for interferon-{gamma} detection with excellent performance (limit of detection: 12 pg mL-1, CV: 6.8%), and we demonstrated compatibility with cell culture. Finally, large area manufacturing was illustrated by printing 42 CCs with embedded microchannels in <45 min. LCD 3D printing together with tailored inks pave the way for democratizing access to high-resolution manufacturing of ready-to-use microfluidic devices by anyone, anywhere.

bioengineering↗

bioRxiv2023

Size photometry and fluorescence imaging of immobilized immersed extracellular vesicles

Immunofluorescence analysis of individual extracellular vesicles (EVs) in common fluorescence microscopes is gaining popularity due to its accessibility and high fluorescence sensitivity, however, EV number and size are only measurable using fluorescent stains requiring extensive sample manipulations. Here we introduce highly sensitive label-free EV size photometry (SP) based on interferometric scattering (iSCAT) imaging of immersed EVs immobilized on a glass coverslip. We implement SP on a common inverted epifluorescence microscope with LED illumination and a simple 50:50 beamsplitter, permitting seamless integration of SP with fluorescence imaging (SPFI). We present a high-throughput SPFI workflow recording >10,000 EVs in 7 min over multiple fields of view, pre- and post-incubation imaging to suppress background, along with automated image alignment, aberration correction, spot detection, and EV sizing. We identify the upper sizing limit of SP with 440 nm illumination and extend EVs sizing from [~]37 nm in diameter to >200 nm with dual 440 nm and 740 nm illumination. We benchmark SP to flow cytometry using calibrated silica nanoparticles and demonstrate superior, label-free sensitivity. We showcase SPFIs potential for EV analysis by experimentally distinguishing surface and volumetric EV dyes, observing the deformation of EVs adsorbed to a surface, and by uncovering distinct subpopulations in <100 nm-in-diameter EVs with fluorescently tagged membrane proteins.

bioengineering↗

bioRxiv2023

Nanoporous PEGDA ink for High-Resolution Additive Manufacturing of Scaffolds for Organ-on-a-Chip

Polydimethylsiloxane (PDMS), commonly used in organ-on-a-chip (OoC) systems, faces limitations in replicating complex geometries, hindering its effectiveness in creating 3D OoC models. In contrast, poly(ethylene glycol)diacrylate (PEGDA-250), favored for its fabrication ease and resistance to small molecule absorption, is increasingly used for 3D printing microfluidic devices. However, applications in cell culture have been limited due to poor cell adhesion. Here, we introduce a nanoporous PEGDA ink (P-PEGDA) designed to enhance cell adhesion. P-PEGDA is formulated with a porogen, photopolymerized, followed by the porogen removal. Utilizing P-PEGDA, complex microstructures and membranes as thin as 27 {micro}m were 3D-printed. Porogen concentrations from 10-30% were tested yielding constructs with increasing porosity and oxygen permeability surpassing PDMS, without compromising printing resolution. Tests across four cell lines showed >80% cell viability, with a notable 77-fold increase in MDA-MB-231 cell coverage on the porous scaffolds. Finally, we introduce an OoC model comprising a gyroid scaffold with a central opening filled with a cancer spheroid. This setup, after a 14-day co-culture, demonstrated significant endothelial sprouting and integration within the spheroid. The P-PEGDA formulation is suitable for high-resolution 3D printing of constructs for 3D cell culture and OoC owing to its printability, gas permeability, biocompatibility, and cell adhesion.

bioengineering↗

bioRxiv2023

Effect of sample preprocessing and extraction methods on the physical and molecular profiles of extracellular vesicles

Extracellular vesicles (EVs) are nanometric lipid vesicles that shuttle cargo between cells. Their analysis could shed light on health and disease conditions, but EVs must first be preserved, extracted and often pre-concentrated. Here we firstly compare plasma preservation agents, and secondly, using both plasma and cell supernatant, four EV-extraction methods including (i) ultracen-trifugation (UC), (ii) size exclusion chromatography (SEC), (iii) centrifugal filtration (LoDF), and (iv) accousto-sorting (AcS). We benchmarked them by characterizing integrity, size-distribution, concentration, purity and the expression profiles for nine proteins of EVs, as well as overall throughput, time-to-result and cost. We found that the difference between EDTA and citrate anticoagulants vary with the extraction method. In our hands, ultracentrifugation produced a high yield of EVs with low contamination; SEC is low-cost, fast, and easy to implement, but the purity of EVs is lower; LoDF and AcS are both compatible with process automation, small volume requirement, and rapid processing times. When using plasma, the LoDF was susceptible to clogging and sample contamination, while the AcS featured high purity but a lower yield of extraction. Analysis of protein profiles suggest that extraction methods extract different sub-population of EVs. Our study highlights the strength and weakness of sample preprocessing methods, and the variability in concentration, purity, and EV expression profiles of the extracted EVs. Pre-analytical parameters such as collection or pre-processing protocols must be considered as part of the entire process in order to address EV diversity and their use as clinically actionable indicators.

bioengineering↗

bioRxiv2023

High-Resolution Additive Manufacturing of a Biodegradable Elastomer with a Low-Cost LCD 3D Printer

Artificial organs and organs-on-a-chip are of great clinical and scientific interest and have recently been made by additive manufacturing, but depend on, and benefit from, biocompatible, biodegradable, and soft materials. Poly(octamethylene maleate (anhydride) citrate (POMaC) meets these criteria and has gained popularity, and as in principle, it can be photocured and is amenable to vat-photopolymerization (VP) 3D printing, but only low-resolution structures have been produced so fa. Here, we introduce a VP-POMaC ink and demonstrate 3D printing of high resolution (80 {micro}m) and complex 3D structures using low-cost ([~]US$300) liquid-crystal display (LCD) printers. The ink includes POMaC, a diluent and porogen additive to reduce viscosity within the range of VP, and a crosslinker to speed up reaction kinetics. The mechanical properties of the cured ink were tuned to match the elastic moduli of different tissues simply by varying the porogen concentration. The biocompatibility was assessed by cell culture which yielded 80% viability and the potential for tissue engineering illustrated with a 3D printed gyroid seeded with cells. VP-POMaC and low-cost LCD printers make the additive manufacturing of high resolution, elastomeric, and biodegradable constructs widely accessible, paving the way for a myriad of applications in tissue engineering, implants, organ-on-a-chip, wearables, and soft robotics.

bioengineering↗

bioRxiv2023

Digital Manufacturing of Functional Ready-to-Use Microfluidic Systems

Digital manufacturing (DM) strives for the seamless manufacture of a functional device from a digital file. DM holds great potential for microfluidics, but requirements for embedded conduits and high resolution beyond the capability of common manufacturing equipment, and microfluidic systems dependence on peripherals (e.g. connections, power supply, computer), have limited its adoption. Microfluidic capillaric circuits (CCs) are structurally-encoded, self-contained microfluidic systems that operate and self-fill thanks to precisely tailored hydrophilicity. CCs were heretofore hydrophilized in a plasma chamber, but which only produces transient hydrophilicity, lacks reproducibility, and limits CC design to open surface channels sealed with a tape. Here we introduce the additive DM of monolithic, fully functional and intrinsically hydrophilic CCs. CCs were 3D printed with commonly available light engine-based 3D printers using polyethylene(glycol)diacrylate-based ink co-polymerized with hydrophilic acrylic acid crosslinkers and optimized for hydrophilicity and printability. A new, robust capillary valve design and embedded conduits with circular cross-sections that prevent bubble trapping are presented, and complex interwoven circuit architectures created, and their use illustrated with an immunoassay. Finally, the need for external paper capillary pumps is eliminated by directly embedding the capillary pump in the chip as a porous gyroid structure, realizing fully functional, monolithic CCs. Thence, a computer-aided design file can be made into a CC by commonly available 3D printers in less than 30 minutes enabling low-cost, distributed, DM of fully functional ready-to-use microfluidic systems.

bioengineering↗

bioRxiv2023

nELISA: A high-throughput, high-plex platform enables quantitative profiling of the secretome

We present the nELISA, a high-throughput, high-fidelity, and high-plex protein profiling platform. DNA oligonucleotides are used to pre-assemble antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection. Spatial separation between non-cognate antibodies prevents the rise of reagent-driven cross-reactivity, while read-out is performed cost-efficiently and at high-throughput using flow cytometry. nELISA can measure both protein concentration and their post-translational modifications. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity nor impact on performance vs 1-plex signals, with sensitivities as low as 0.1 pg/mL and measurements spanning 7 orders of magnitude. We then performed a large-scale inflammatory-secretome perturbation screen of peripheral blood mononuclear cells (PBMCs), with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating [~]1.4M protein data points in under a week; a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel ones, that were conserved across donors and stimulation conditions. We validate nELISA for phenotypic screening, where its capacity to faithfully report hundreds of proteins make it a powerful tool across multiple stages of drug discovery.

bioengineering↗

bioRxiv2023

Microfluidics on Stretchable Strings

This paper introduces string microfluidics (SM), which consists of stretchable strings trapping discrete microdroplets within a porous matrix, and the realization of elementary microfluidic operations such as droplet formation, transport, splitting, merging, and mixing by moving and stretching the strings. While performing these operations, SM was shown to be compatible with colorimetric biological assays. SM represents a new form of microfluidics that integrates the concepts of thread microfluidics and digital microfluidics, along with mobile or reconfigurable microfluidics where liquid transport is realized by manipulating the substrate.

bioengineering↗

bioRxiv2022

3D-printed capillaric ELISA-on-a-chip with aliquoting

Sandwich immunoassays such as the enzyme-linked immunosorbent assay (ELISA) have been miniaturized and performed in a lab-on-a-chip format, but the execution of the multiple assay steps typically requires a computer or complex peripherals. Recently, an ELISA for detecting antibodies was encoded structurally in a chip thanks to the microfluidic chain reaction but the need for precise pipetting and intolerance to commonly used surfactant concentrations limited the potential for broader adoption. Here, we introduce the ELISA-on-a-chip with aliquoting functionality that obviates the need for precise pipetting, accommodates higher surfactant concentrations, includes barrier channels that delay the contact between solutions and prevent undesired mixing, and that executed a quantitative, high sensitivity assay for the SARS-CoV-2 nucleocapsid protein in 4x-diluted saliva. Upon loading the chip using disposable pipettes, capillary flow draws each reagent and the sample into a separate volumetric measuring reservoir for detection antibody (70 {micro}L), enzyme conjugate (50 {micro}L), substrate (80 {micro}L), and sample (210 {micro}L), and splits washing buffer into 4 different reservoirs of 40, 40, 60, and 20 {micro}L. The excess volume is autonomously drained via a structurally encoded capillaric aliquoting circuit, creating aliquots with an accuracy of >93%. Next, the user click-connects the assay module, comprising a nitrocellulose membrane with immobilized capture antibodies and a capillary pump, to the chip which triggers the step-by-step, timed flow of all aliquoted solutions. A colored precipitate forming a line on a nitrocellulose strip serves as an assay readout, and upon digitization, yielded a binding curve with a limit of detection of 54 and 91 pg/mL for buffer and diluted saliva respectively, vastly outperforming rapid tests. The ELISA chip is 3D-printed, modular, adaptable to other targets and assays, and could be used to automate ELISA in the lab; or as a diagnostic test at the point of care with the convenience and form factor of rapid tests while preserving the protocol and performance of central laboratory ELISA.

bioengineering↗

bioRxiv2022

Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis

Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of extracellular vesicle inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed and high-throughput assays. We captured 4 subpopulations of EVs from colorectal cancer cell lines HT29 and SW403 based on EpCAM, CD9, CD63 and CD81 expression, and quantified the co-expression of 8 outer (integrins and tetraspanins) and 4 inner (heat shock, endosomal and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

bioengineering↗

bioRxiv2020

Mechanically Matched Silicone Brain Implants Reduce Brain Foreign Body Response

Brain implants are increasingly used to treat neurological disorders and diseases. However, the brain foreign body response (FBR) elicited by implants affects neuro-electrical transduction and long-term reliability limiting their clinical adoption. The mismatch in Youngs modulus between silicon implants ([~]180 GPa) and brain tissue ([~]1-30 kPa) exacerbates the FBR resulting in the development of flexible implants from polymers such as polyimide ([~]1.5-2.5 GPa). However, a stiffness mismatch of at least two orders of magnitude remains. Here, we introduce (i) the first mechanically matched brain implant (MMBI) made from silicone ([~]20 kPa), (ii) new microfabrication methods, and (iii) a novel dissolvable sugar shuttle to reliably implant MMBIs. MMBIs were fabricated via vacuum-assisted molding using sacrificial sugar molds and were then encased in sugar shuttles that dissolved within 2 min after insertion into rat brains. Sections of rat neocortex implanted with MMBIs, PDMS implants, and silicon implants were analyzed by immunohistochemistry 3 and 9-weeks post-implantation. MMBIs resulted in significantly higher neuronal density and lower FBR within 50 {micro}m of the tissue-implant interface compared to PDMS and silicon implants suggesting that materials mechanically matched to brain further minimize the FBR and could contribute to better implant functionality and long-term reliability.

bioengineering↗

bioRxiv2019

Gravity-based microfiltration reveals unexpected prevalence of circulating tumor cell clusters in ovarian cancer

Circulating tumor cells (CTCs) are rare (few cells per milliliter of blood) and mostly isolated as single cell CTCs (scCTCs). CTC clusters (cCTCs), even rarer, are of growing interest, notably because of their higher metastatic potential, but very difficult to isolate. Here, we introduce gravity-based microfiltration (G{micro}F) for facile isolation of cCTCs while minimizing unwanted cluster disaggregation, with [~]85% capture efficiency. G{micro}F from orthotopic ovarian cancer mouse models, from 17 epithelial ovarian cancer (EOC) with either localized or metastatic disease, and from 13 metastatic colorectal cancer liver metastasis (CRCLM) patients uncovered cCTCs in every case, with between 2-100+ cells. cCTCs represented between 5-30% of all CTC capture events, and 10-80% of CTCs were clustered; remarkably, in 10 patients, most CTCs were circulating not as scCTCs, but as cCTCs. G{micro}F uncovered the unexpected prevalence and frequency of cCTCs including sometimes very large ones in EOC patients, and motivates additional studies to uncover their properties and role in disease progression.

bioengineering↗