Biology subjects

Flax, J.

Publications and source records attributed to Flax, J..

5 recordsLinked to original sources

bioRxiv2026

Catch-and-Display Immunoassay as an Accessible Platform for Digital Biomarker Detection

Digital immunoassays provide exceptional analytical sensitivity for detecting low-abundance biomarkers, but their broad adoption is limited by practical barriers. Commercial platforms are prohibitively expensive for routine use by individual laboratories, and laboratory-scale concepts typically describe specialized biosensors and sophisticated workflows. Here, we introduce a nanomembrane-based Catch-and-Display Immunoassay (CAD-IA) as a cost-effective and laboratory-friendly digital immunoassay for common research settings. In CAD-IA, fluorescent nanoparticles are "captured" by the nanoscale pores of ultrathin silicon nitride membranes through a pipette powered filtration. The captured nanoparticles serve as optically isolated hotspots for fluorescent immunocomplex formation when target antigen is present. Co-localization of the fluorescent particles and fluorescent immunocomplexes are then "displayed" and quantified by standard confocal microscopy to generate digital signals. CAD-IA is implemented using the {micro}SiM-DX (microfluidic device featuring an ultrathin silicon membrane for diagnostics) platform, which is manually assembled from mass produced, cost-effective components. Using the traumatic brain injury (TBI) biomarker S100B as a model, we demonstrate that CAD-IA provides consistent digital outputs and linear quantification with a dynamic range of at least two orders of magnitude when digital and analog analysis are combined on the same image sets. We further demonstrate that the assay maintains linearity in serum matrices and achieves suitable sensitivity (LoD = 0.02 g/mL) for clinically relevant diagnostic with the addition of tyramide signal amplification (TSA). While further optimization of CAD-IA is possible, these results constitute a proof-of-concept demonstration of a novel digital immunoassay deployable in most research laboratories.

bioengineering↗

bioRxiv2024

Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using 'Catch and Display' on Ultrathin Nanoporous Silicon Nitride Membranes

Extracellular vesicles (EVs) are particles secreted by all cells that carry bioactive cargo and facilitate intercellular communication with roles in normal physiology and disease pathogenesis. EVs have tremendous diagnostic and therapeutic potential and accordingly, the EV field has grown exponentially in recent years. Bulk assays lack the sensitivity to detect rare EV subsets relevant to disease, and while single EV analysis techniques remedy this, they are undermined by complicated detection schemes often coupled with prohibitive instrumentation. To address these issues, we propose a microfluidic technique for EV characterization called catch and display for liquid biopsy (CAD-LB). CAD-LB rapidly captures fluorescently labeled EVs in the similarly-sized pores of an ultrathin silicon nitride membrane. Minimally processed sample is introduced via pipette injection into a simple microfluidic device which is directly imaged using fluorescence microscopy for a rapid assessment of EV number and biomarker colocalization. In this work, nanoparticles were first used to define the accuracy and dynamic range for counting and colocalization by CAD-LB. Following this, the same assessments were made for purified EVs and for unpurified EVs in plasma. Biomarker detection was validated using CD9 in which Western blot analysis confirmed that CAD-LB faithfully recapitulated differing expression levels among samples. We further verified that CAD-LB captured the known increase in EV-associated ICAM-1 following the cytokine stimulation of endothelial cells. Finally, to demonstrate CAD-LBs clinical potential, we show that EV biomarkers indicative of immunotherapy responsiveness are successfully detected in the plasma of bladder cancer patients undergoing immune checkpoint blockade.

bioengineering↗

bioRxiv2022

The Modular μSiM: a Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro

Advanced in vitro tissue chip models can reduce and replace animal experimentation and may eventually support on-chip clinical trials. To realize this potential, however, tissue chip platforms must be both mass-produced and reconfigurable to allow for customized design. To address these unmet needs, we introduce an extension of our {micro}SiM (microdevice featuring a silicon-nitride membrane) platform. The modular {micro}SiM (m-{micro}SiM) uses mass-produced components to enable rapid assembly and reconfiguration by laboratories without knowledge of microfabrication. We demonstrate the utility of the m-{micro}SiM by establishing an hiPSC-derived blood-brain barrier (BBB) in bioengineering and non-engineering, brain barriers focused laboratories. We develop and validate in situ and sampling-based assays of small molecule diffusion as a measure of barrier function. BBB properties show excellent interlaboratory agreement and match expectations from literature, validating the m-{micro}SiM as a platform for barrier models and demonstrating successful dissemination of components and protocols. We then demonstrate the ability to quickly reconfigure the m-{micro}SiM for co-culture and immune cell transmigration studies through addition of accessories and/or quick exchange of components. Because the development of modified components and accessories is easily achieved, custom designs of the m-{micro}SiM should be accessible to any laboratory desiring a barrier-style tissue chip platform.

bioengineering↗

bioRxiv2019

Tangential flow microfluidics for the capture and release of nanoparticles and extracellular vesicles on conventional and ultrathin membranes

Membranes have been used extensively for the purification and separation of biological species. A persistent challenge is the purification of species from concentrated feed solutions such as extracellular vesicles (EVs) from biological fluids. We investigated a new method to isolate micro- and nano-scale species termed tangential flow for analyte capture (TFAC), which is an extension of traditional tangential flow filtration (TFF). Initially, EV purification from plasma on ultrathin nanomembranes was compared between both normal flow filtration (NFF) and TFF. NFF resulted in rapid formation of a protein cake which completely obscured any captured EVs and also prevented further transport across the membrane. On the other hand, TFF showed capture of CD63 positive EVs with minimal contamination. We explored the use of TFF to capture target species over membrane pores, wash and then release in a physical process that does not rely upon affinity or chemical interactions. This process of TFAC was studied with model particles on both ultrathin nanomembranes and conventional thickness membranes (polycarbonate track-etch). Successful capture and release of model particles was observed using both membranes. Ultrathin nanomembranes showed higher efficiency of capture and release with significantly lower pressures indicating that ultrathin nanomembranes are well-suited for TFAC of delicate nanoscale particles such as EVs.

bioengineering↗

bioRxiv2019

Exosome labeling by lipophilic dye PKH26 results in significant increase in vesicle size

Exosomes are membrane vesicles secreted by cells and distributed widely in all biofluids. Exosomes can modulate the biological activities of cells in a paracrine or endocrine manner, in part by transferring their content, such as miRNA, following uptake in recipient cells. Fluorescent labelling of exosomes is a commonly used technique for understanding their cellular targeting and biodistribution. Lipophilic fluorescent dyes such as those in the PKH family have been widely used for exosome labelling. One concern with the use of lipophilic dyes is an increase in the exosome size due to membrane fusion or intercalation. This size shift alone may undermine the validity of exosomes tracing studies as small changes in the size of inorganic nanoparticles are known to affect their cellular uptake and biodistribution. Here, the possibility of minimizing the size shift of PKH labelled exosomes was systematically studied by changing the labelling condition. Unfortunately, the size shift towards larger particles was observed in all the PKH labelling conditions, including those where the labelled exosomes were below the fluorescent detection limit. As opposed to lipophilic dyes, no significant shift in the size of labelled exosomes was detected with protein binding dyes. Since the size shifts identified in all the PKH labelling conditions are likely to affect the cellular uptake and biodistribution, PKH may not be a reliable technique for exosomes tracking.

biochemistry↗