Hollow organosilica beads as reference particles for optical detection of extracellular vesicles.

Essentials Standardization of extracellular vesicle (EV) measurements by flow cytometry needs improvement. Hollow organosilica beads were prepared, characterized, and tested as reference particles. Light scattering properties of hollow beads resemble that of platelet-derived EVs. Hollow beads are ideal reference particles to standardize scatter flow cytometry research on EVs. SUMMARY: Background The concentration of extracellular vesicles (EVs) in body fluids is a promising biomarker for disease, and flow cytometry remains the clinically most applicable method to identify the cellular origin of single EVs in suspension. To compare concentration measurements of EVs between flow cytometers, solid polystyrene reference beads and EVs were distributed in the first ISTH-organized interlaboratory comparison studies. The beads were used to set size gates based on light scatter, and the concentration of EVs was measured within the size gates. However, polystyrene beads lead to false size determination of EVs, owing to the mismatch in refractive index between beads and EVs. Moreover, polystyrene beads gate different EV sizes on different flow cytometers. Objective To prepare, characterize and test hollow organosilica beads (HOBs) as reference beads to set EV size gates in flow cytometry investigations. Methods HOBs were prepared with a hard template sol-gel method, and extensively characterized for morphology, size, and colloidal stability. The applicability of HOBs as reference particles was investigated by flow cytometry with HOBs and platelet-derived EVs. Results HOBs proved to be monodisperse with a homogeneous shell thickness. Two-angle light-scattering measurements by flow cytometry confirmed that HOBs have light-scattering properties similar to those of platelet-derived EVs. Conclusions Because the structure and light-scattering properties HOBs resemble those of EVs, HOBs with a given size will gate EVs of the same size. Therefore, HOBs are ideal reference beads with which to standardize optical measurements of the EV concentration within a predefined size range.

Nanoscale radiofrequency impedance sensors with unconditionally stable tuning.

Impedance sensors perform an important role in a number of biosensing applications, including particle counting, sizing, and velocimetry. Detection of nanoparticles, or changes in, e.g., the interfacial Debye-Hückel layer, can also be performed using nanoscale impedance sensors. One method for monitoring changes in the local impedance is to use radiofrequency reflectometry, which when combined with an impedance-matched sensor can afford very high sensitivity with very large detection bandwidth. Maintaining sensitivity and dynamic range, however, requires continuous tuning of the impedance matching network. Here we demonstrate a dual feedback tuning circuit, which allows us to maintain near-perfect impedance matching, even in the presence of long-term drifts in sensor impedance. We apply this tuning technique to a nanoscale interdigitated impedance sensor, designed to allow the direct detection of nanoparticles or real-time monitoring of molecular surface binding. We demonstrate optimal performance of the nanoscale sensor and tuned impedance network both when modulating the concentration of saline to which the sensor is exposed and when electronically switching between sensors configured in a two-element differential array, achieving a stabilization response time of <20 ms.

Probing the debye layer: capacitance and potential of zero charge measured using a debye-layer transistor.

We present a unique method for probing the properties of the electrolytic Debye layer, incorporating it as the active element in a novel radio frequency (rf) field-effect transistor. The capacitance of the Debye layer depends nonlinearly on the voltage applied across it, and we exploit this dependence to directly modulate the rf conductance between two nanofabricated interdigitated electrodes. We make quantitative measurements of the Debye-layer capacitance, allowing us to determine the potential of zero charge, a quantity of importance for electrochemistry and impedance-based biosensing.

A feasible approach to all-electronic digital labeling and readout for cell identification.

We present two critical innovations that enable a unique, purely electronic approach to microfluidic whole-cell analysis, focusing on the problem of cell identification and sorting. We used fully-scalable lithographic techniques to microfabricate digital barcodes, providing a means for low-cost, large volume production. We have demonstrated molecular functionalization of the barcodes, using biotin-streptavidin, as well as human CD4 antibody, and we have successfully linked the barcodes to polystyrene beads using the biotin-streptavidin complex. This functionalization allows unique barcodes to be attached to specific cell types, based on phenotype. We have also implemented an electronic barcode readout scheme, using a radio frequency microsensor integrated in an elastomeric microfluidic channel, that can read individual barcodes at rates in excess of 1000 labels s(-1). The barcodes are biologically compatible, and coupled with the electronic sensing technology, provide a route to compact, inexpensive, disposable cell identification, sorting and purification.