Biology subjects

Shen, M. L.

Publications and source records attributed to Shen, M. L..

8 recordsLinked to original sources

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

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↗

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↗