Nanoarrays of Individual Liposomes and Bacterial Outer Membrane Vesicles by Liftoff Nanocontact Printing

Abstract

identified and analyzed by making meas urements on a population of single enti ties, for example, single liposomes [4][5][6][7][8][9] or EVs. [10,11] Traditional assays that treat a population as an ensemble obscure the distributions of particle properties, and they are blind to the asynchronous events that are revealed at the single cell, particle or molecule level.Single entity measurements can be obtained using microscopy, [12][13][14][15] spec troscopy, [16,17] flow cytometry, [10,18] mass spectrometry, [19,20] electrical, [21] electro chemical, [22] and separation methods, [23] all of which can reveal particle heteroge neities. However, many of these methods have weaknesses that limit their appli cability. For example, electron micro scopy has the requisite spatial resolution to observe individual small particles, but because of the required sample processing and fixing, temporal dynamics among particles cannot be determined. Flow cytometry is unable to characterize nanoscale (50-500 nm) objects, such as EVs, without special instrument modifications. Additionally, flow cytometry, capillary electrophoresis, and elec trochemical cytometry have highthroughput capabilities, but they are unable to analyze a single isolated particle over long periods of time, and specific individual particles are gener ally not recoverable for further analysis. Also, some of these methods destroy the particles as part of the measurement pro cess, which prevents multiplex analysis of individual particles. To overcome these drawbacks, new approaches are necessary that leverage the strengths of optical microscopy and enable multiplex highthroughput measurements on single biological particles to reveal their physical, chemical, and physiological heterogeneities.Chemicallyspecific capture of individual particles on sur faces can enable single particle analysis. [24,25] Furthermore, this approach can be improved by patterning the capture molecules in densely packed, highly ordered arrays, resulting in densely packed, highly ordered arrays of particles. [7] Pat terning the capture molecules on a surface results in the cap ture of thousands of particles in ordered arrays within a single image frame. Patterning capture molecules into highly ordered arrays ensures that neighboring particles are optically resolv able, while at the same time particle surface density is maxi mized. Additionally, capture molecule nanodots that are similar Analytical characterization of small biological particles, such as extracellular vesicles (EVs), is complicated by their extreme heterogeneity in size, lipid, membrane protein, and cargo composition. Analysis of individual particles is essential for illuminating particle property distributions that are obscured by ensemble measurements. To enable high-throughput analysis of individual particles, liftoff nanocontact printing (LNCP) is used to define hexagonal antibody and toxin arrays that have a 425 nm dot size, on average, and 700 nm periodicity. The LNCP process is rapid, simple, and does not require access ...

Multivariate Analysis of Individual Bacterial Outer Membrane Vesicles Using Fluorescence Microscopy

“…After exposing OMVs to biotin ap-PE, which incorporates into the membrane due to its hydrophobic nature, we captured them on streptavidin- and BSA-passivated glass (Figure b). We opted to employ the biotin–streptavidin immobilization approach, as it offers a high affinity between biotin–streptavidin, guaranteeing firm immobilization of OMVs throughout the sizing and heterogeneity experiments. , As an alternate, an antibody could also be used to capture the OMVs; however, this would require the prior knowledge of the presence of certain proteins. We specifically utilized biotin Cap-PE, a membrane-specific label, to label our OMVs, which guarantees the unbiased capture of all OMVs regardless of their size or the presence of specific surface markers.…”

Outer Membrane Vesicles Released from Garlic Exosome‐like Nanoparticles (GaELNs) Train Gut Bacteria that Reverses Type 2 Diabetes via the Gut‐Brain Axis

“…[16,[52][53][54][55] This diversity within EVs stems from variances in their biogenesis, leading to classifications into different populations, further subdivided into various subsets. [53][54][55] OMVs, being extracellular nanovesicles released by bacteria, exhibit heterogeneity in size (40-300 nm diameter), [11,[56][57][58][59][60] where the size dictates both the entry into host cells and the protein composition. [61][62][63] In our investigation, we noted that a fraction of orally administered A. muciniphila OMVs evades uptake during the journey from the gut to the brain, escaping uptake by other cell types and ultimately being taken up by microglial cells.…”