Multiplex fluorescence detection of single-cell droplet microfluidics and its application in quantifying protein expression levels.

This study presented a platform of multiplex fluorescence detection of single-cell droplet microfluidics with demonstrative applications in quantifying protein expression levels. The platform of multiplex fluorescence detection mainly included optical paths adopted from conventional microscopy enabling the generation of three optical spots from three laser sources for multiple fluorescence excitation and capture of multiple fluorescence signals by four photomultiplier tubes. As to platform characterization, microscopic images of three optical spots were obtained where clear Gaussian distributions of intensities without skewness confirmed the functionality of the scanning lens, while the controllable distances among three optical spots validated the functionality of fiber collimators and the reflector lens. As to demonstration, this platform was used to quantify single-cell protein expression within droplets where four-type protein expression of α-tubulin, Ras, c-Myc, and ß-tubulin of CAL 27 (Ncell = 1921) vs WSU-HN6 (Ncell = 1881) were quantitatively estimated, which were (2.85 ± 0.72) × 105 vs (4.83 ± 1.58) × 105, (3.69 ± 1.41) × 104 vs (5.07 ± 2.13) × 104, (5.90 ± 1.45) × 104 vs (9.57 ± 2.85) × 104, and (3.84 ± 1.28) × 105 vs (3.30 ± 1.10) × 105, respectively. Neural pattern recognition was utilized for the classification of cell types, achieving successful rates of 69.0% (α-tubulin), 75.4% (Ras), 89.1% (c-Myc), 65.8% (ß-tubulin), and 99.1% in combination, validating the capability of this platform of multiplex fluorescence detection to quantify various types of single-cell proteins, which could provide comprehensive evaluations on cell status.

Electrodeposited magnetic nanoporous membrane for high-yield and high-throughput immunocapture of extracellular vesicles and lipoproteins.

Superparamagnetic nanobeads offer several advantages over microbeads for immunocapture of nanocarriers (extracellular vesicles, lipoproteins, and viruses) in a bioassay: high-yield capture, reduction in incubation time, and higher capture capacity. However, nanobeads are difficult to "pull-down" because their superparamagnetic feature requires high nanoscale magnetic field gradients. Here, an electrodeposited track-etched membrane is shown to produce a unique superparamagnetic nano-edge ring with multiple edges around nanopores. With a uniform external magnetic field, the induced monopole and dipole of this nano edge junction combine to produce a 10× higher nanobead trapping force. A dense nanobead suspension can be filtered through the magnetic nanoporous membrane (MNM) at high throughput with a 99% bead capture rate. The yield of specific nanocarriers in heterogeneous media by nanobeads/MNM exceeds 80%. Reproducibility, low loss, and concentration-independent capture rates are also demonstrated. This MNM material hence expands the application of nanobead immunocapture to physiological samples.

Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research.

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.

Elliptical Pipette Generated Large Microdroplets for POC Visual ddPCR Quantification of Low Viral Load.

Rapid point-of-care (POC) quantification of low virus RNA load would significantly reduce the turn-around time for the PCR test and help contain a fast-spreading epidemic. Herein, we report a droplet digital PCR (ddPCR) platform that can achieve this sensitivity and rapidity without bulky lab-bound equipment. The key technology is a flattened pipette tip with an elliptical cross-section, which extends a high aspect-ratio microfluidic chip design to pipette scale, for rapid (<5 min) generation of several thousand monodispersed droplets ∼150 to 350 µm in size with a CV of ∼2.3%. A block copolymer surfactant (polyoxyalkylene F127) is used to stabilize these large droplets in oil during thermal cycling. At this droplet size and number, positive droplets can be counted by eye or imaged by a smartphone with appropriate illumination/filtering to accurately quantify up to 100 target copies. We demonstrate with 2019 nCoV-PCR assay LODs of 3.8 copies per 20 µL of sample and a dynamic range of 4-100 copies. The ddPCR platform is shown to be inhibitor resistant with spiked saliva samples, suggesting RNA extraction may not be necessary. It represents a rapid 1.5-h POC quantitative PCR test that requires just a pipette equipped with elliptical pipette tip, a commercial portable thermal cycler, a smartphone, and a portable trans-illuminator, without bulky and expensive micropumps and optical detectors that prevent POC application.

Analysis of Cargo Loading Modes and Capacity of an Electrically-Powered Active Carrier.

The use of active colloids for cargo transport offers unique potential for applications ranging from targeted drug delivery to lab-on-a-chip systems. Previously, Janus particles (JPs), acting as mobile microelectrodes, have been shown to transport cargo which is trapped at the JP surface by a dielectrophoretic mechanism. Herein, we aim to characterize the cargo loading properties of mobile Janus carriers, across a broad range of frequencies and voltages. In expanding the frequency range of the carrier, we are able to compare the influences of different modes of carrier transport on the loading capacity as well as highlight the differences between cargo trapped by positive and negative dielectrophoresis. Specifically, it is shown that cargo trapping results in a reduction in carrier velocities with this effect more pronounced at low frequencies where cargo is trapped close to the substrate. Interestingly, we observe the existence of a maximum cargo loading capacity which decreases at large voltages suggesting a strong interplay between trapping and hydrodynamic shear. Finally, we demonstrate that the control of the frequency can enable different assemblies of binary colloidal solutions on the JP. The resultant findings enable the optimization of electrokinetic cargo transport and its selective application to a broad range of targets.

Thermal sensing in fluid at the micro-nano-scales.

Temperature is one of the most fundamental parameters for the characterization of a physical system. With rapid development of lab-on-a-chip and biology at single cell level, a great demand has risen for the temperature sensors with high spatial, temporal, and thermal resolution. Nevertheless, measuring temperature in liquid environment is always a technical challenge. Various factors may affect the sensing results, such as the fabrication parameters of built-in sensors, thermal property of electrical insulating layer, and stability of fluorescent thermometers in liquid environment. In this review, we focused on different kinds of micro/nano-thermometers applied in the thermal sensing for microfluidic systems and cultured cells. We discussed the advantages and limitations of these thermometers in specific applications and the challenges and possible solutions for more accurate temperature measurements in further studies.

Geometric Shape Induced Small Change of Seebeck Coefficient in Bulky Metallic Wires.

In this paper, we report the results of slight changes in the thermopower of long W, Mo, Zn, Cu, brass, and Ti wires, that resulted from changes in the wire's diameter or cross-sectional area. The samples used in the tests had a round shape with a diameter that ranged from tens of micron to 2 mm, which was much larger than the corresponding mean free paths of these materials. Nevertheless, a small change in thermopower, at the order of 1-10 nV/K, was repeatedly observed when the wire diameter was changed, or when the cross-sectional area of the wire was altered by mechanical methods, such as grinding or splitting. The results are consistent with previous observations showing that the thermopower in metallic thin film stripes changes with their width, from 100 µm to as little as 70 nm, implying a universal, geometric-boundary-related size effect of thermopower in metal materials, that occurs at the nanometer scale and continuously decreases all the way to the millimeter scale. This effect could be applied in the manufacturing of high-temperature sensors with simple structures.

Performance of Nano-Submicron-Stripe Pd Thin-Film Temperature Sensors.

Dozens of small dual-beam thin-film temperature sensors with a total width down to 430 nm were fabricated and tested. The sensors were all made from 90-nm-thick Pd thin films, where the width of the narrow stripes was 70-100 nm and that of the wide ones was 210-800 nm. Two different calibration methods showed consistent and repeatable sensitivities of 0.7-1.2 µV/K for the sensors, confirming that the sensitivity mainly depended on the width configuration of each sensor. By integrating arrays of such sensors on a practical testing platform using hybrid e-beam lithography and photolithography techniques, we demonstrated that these sensors were capable of detecting a weak surface temperature difference of 0.1-0.2 K at microscale, and they could be scaled up as built-in temperature sensors in many practical devices.

Real-Time Two-Dimensional Mapping of Relative Local Surface Temperatures with a Thin-Film Sensor Array.

Dynamic mapping of an object's local temperature distribution may offer valuable information for failure analysis, system control and improvement. In this letter we present a computerized measurement system which is equipped with a hybrid, low-noise mechanical-electrical multiplexer for real-time two-dimensional (2D) mapping of surface temperatures. We demonstrate the performance of the system on a device embedded with 32 pieces of built-in Cr-Pt thin-film thermocouples arranged in a 4 × 8 matrix. The system can display a continuous 2D mapping movie of relative temperatures with a time interval around 1 s. This technique may find applications in a variety of practical devices and systems.

High mobility flexible graphene field-effect transistors and ambipolar radio-frequency circuits.

Field-effect transistors (GFETs) were fabricated on mechanically flexible substrates using chemical vapor deposition grown graphene. High current density (nearly 200 µA µm(-1)) with saturation, almost perfect ambipolar electron-hole behavior, high transconductance (120 µS µm(-1)) and good stability over 381 days were obtained. The average carrier mobility for holes (electrons) is 13,540 cm(2) V(-1) s(-1) (12,300 cm(2) V(-1) s(-1)) with the highest value over 24,000 cm(2) V(-1) s(-1) (20,000 cm(2) V(-1) s(-1)) obtained in flexible GFETs. Ambipolar radio-frequency circuits, frequency doubler, were constructed based on the high performed flexible GFET, which show record high output power spectra purity (∼97%) and high conversion gain of -13.6 dB. Bending measurements show the flexible GFETs are able to work under modest strain. These results show that flexible GFETs are a very promising option for future flexible radio-frequency electronics.

A nano-stripe based sensor for temperature measurement at the submicrometer and nano scales.

Submicrometer dual-stripe temperature sensors made from a single piece of metal thin film (e.g., Pd) are developed. With the narrowest sensor being 900 nm in width, they show sensitivity of 1-2 µV/K for the full range of 10-300 K. The results confirm the size effect in Seebeck coefficient previously observed in microstripe sensors of the same device configuration.