Improving Multicolor Colocalization in Single-Vesicle Flow Cytometry with Vesicle Transit Time.

Immunophenotyping of vesicles, such as extracellular vesicles (EVs), is essential to understanding their origin and biological role. We previously described a custom-built flow analyzer that utilizes a gravity-driven flow, high numerical aperture objective, and micrometer-sized flow channels to reach the sensitivity needed for fast multidimensional analysis of the surface proteins of EVs, even down to the smallest EVs (e.g., ∼30-40 nm). It is difficult to flow focus small EVs, and thus, the transiting EVs exhibit a distribution in particle velocities due to the laminar flow. This distribution of vesicle velocities leads to potentially incorrect results when immunophenotyping nanometer-sized vesicles using cross-correlation analysis (Xcorr), as the order of appearance of the vesicles might not be the same at different spatially offset laser excitation regions. Here, we describe an alternative cross-correlation analysis strategy (Scorr), which uses information on particle transit time across the laser excitation beam width to improve multicolor colocalization in single-vesicle immunoprofiling. We tested the performance of the algorithm for colocalization analysis of multicolor nanobeads and EVs experimentally and via simulations and found that Scorr improved both the efficiency and accuracy of colocalization versus Xcorr. As shown from Monte Carlo simulations, Scorr provided an ∼1.2-4.7-fold increase in the number of colocalized peaks and ensured negligible colocalization of peaks. In silico results were in good agreement with experimental data, which showed an increase in colocalized peaks of ∼1.3-2.5-fold and ∼1.2-2-fold for multicolor beads and EVs, respectively.

High-Throughput Counting and Superresolution Mapping of Tetraspanins on Exosomes Using a Single-Molecule Sensitive Flow Technique and Transistor-like Semiconducting Polymer Dots.

A method for high-throughput counting and superresolution mapping of surface proteins on exosomes is described. The method combines a single-molecule sensitive flow technique and an adaptive superresolution imaging method. Exosomes stained with membrane dye and dye-conjugated antibodies were analyzed using a microfluidic platform at a flow rate of 100 exosome s-1 to determine size and protein copy number. Superresolution mapping was performed with exosomes labeled with novel transistor-like, semiconducting polymer dots (Pdots), which exhibit spontaneous blinking with <5 nm localization error and a broad range of optical-adjustable duty cycles. Based on the copy numbers extracted from the flow analysis, the switch-on frequency of the Pdots were finely adjusted so that structures of hundreds of exosomes were obtained within five minutes. The high throughput and high sensitivity of this method offer clear advantages for characterization of exosomes and similar biological vesicles.

Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer.

Extracellular vesicles (EVs) are membranous particles released by most cells in our body, which are involved in many cell-to-cell signaling processes. Given the nanometer sizes and heterogeneity of EVs, highly sensitive methods with single-molecule resolution are fundamental to investigating their biophysical properties. Here, we demonstrate the sizing of EVs using a fluorescence-based flow analyzer with single-molecule sensitivity. Using a dye that selectively partitions into the vesicle's membrane, we show that the fluorescence intensity of a vesicle is proportional to its diameter. We discuss the constraints in sample preparation which are inherent to sizing nanoscale vesicles with a fluorescent membrane dye and propose several guidelines to improve data consistency. After optimizing staining conditions, we were able to measure the size of vesicles in the range ∼35-300 nm, covering the spectrum of EV sizes. Lastly, we developed a method to correct the signal intensity from each vesicle based on its traveling speed inside the microfluidic channel, by operating at a high sampling rate (10 kHz) and measuring the time required for the particle to cross the laser beam. Using this correction, we obtained a threefold greater accuracy in EV sizing, with a precision of ±15-25%.

Thermochemiluminescent semiconducting polymer dots as sensitive nanoprobes for reagentless immunoassay.

Thermochemiluminescence (TCL) is a potentially simple and sensitive detection principle, as the light emission is simply elicited by thermally-triggered decomposition of a molecule to produce a singlet excited-state product. Here we report about TCL semiconductive polymer dots (TCL-Pdots) obtained by doping fluorescent cyano-polyphenylene vinylene (CN-PPV) Pdots with an acridine 1,2-dioxetane derivative. The TCL-Pdots showed remarkable stability over time and minimum leaching of the thermo-responsive species. Furthermore, detectability of TCL-Pdots was improved by taking advantage of both the high number of 1,2-dioxetanes entrapped in each nanoparticle (about 20 molecules per Pdot) and the 5-fold enhancement of TCL emission due to energy transfer from 1,2-dioxetane to the polymer matrix, which itself acted as an energy acceptor. Indeed, upon heating the TCL-Pdots to 110 °C, 1,2-dioxetane decomposes generating an acridanone product in its electronically excited state. The latter transfers its energy to the surrounding CN-PPV chains via the Förster mechanism (φFRET about 80%), resulting in intense yellow light emission (550 nm wavelength). We next conjugated streptavidin onto the surface of these TCL-Pdots and demonstrated their suitability for use in biological studies. In particular, we used TCL-Pdots as labels in a model non-competitive immunoassay for IgG detection, which showed a LOD of 13 nM IgG and a dynamic range extending up to 230 nM. By combining the biocompatibility, brightness and tunability of Pdot fluorescence emission with the thermally-triggered reagentless light generation from TCL 1,2-dioxetanes, a broad panel of ultrabright TCL nanosystems could be designed for a variety of bioscience applications, even in multiplexed formats.

Synthesis of 1,2-Dioxetanes as Thermochemiluminescent Labels for Ultrasensitive Bioassays: Rational Prediction of Olefin Photooxygenation Outcome by Using a Chemometric Approach.

Great interest in new thermochemiluminescent (TCL) molecules, for example, in bioanalytical assays, has prompted the design and synthesis of a small library of more than 30 olefins to be subjected to photooxygenation, with the aim of obtaining new 1,2-dioxetane-based TCL labels with optimized properties. Fluorine atoms on the acridan system remarkably stabilize 1,2-dioxetanes when they are located in the 3- and/or 6-position (4 h and 4 i). On the other hand, 2,7-difluorinated acridan dioxetane (4 j) showed a significantly enhanced fluorescence quantum yield with respect to the unsubstituted dioxetane (4 a). Some of the synthesized olefins did not undergo singlet oxygen addition and a rationale was sought to ease the photooxygenation step, leading to the TCL dioxetanes. A chemometric approach has been adopted to exploit principal component analysis and linear discriminant analysis of the structural and electronic molecular descriptors obtained by DFT optimizations of olefins 3. This approach allows the steric and electronic parameters that govern dioxetane formation to be revealed.