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The second affiliation of one of the authors (Pavel Neuzil) is missing in this article. Their second affiliation should read: The Faculty of Electrical Engineering and Communication, Brno University of Technology, Technická 3058/10, 616 00 Brno-Královo Pole, Czech Republic. The corrected list of authors' affiliations for this paper is as shown below. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.

Extracellular vesicles (EVs) are cell-derived particles that exhibit diverse sizes, molecular contents, and clinical implications for various diseases depending on their specific subpopulations. However, fractionation of EV subpopulations with high resolution, efficiency, purity, and yield remains an elusive goal due to their diminutive sizes. In this study, we introduce a novel strategy that effectively separates EV subpopulations in a gel-free and label-free manner, using two-dimensional (2D) electrophoresis in a microfluidic artificial sieve. The microfabricated artificial sieve consists of periodically arranged micro-slit-well structures in a 2D array and generates an anisotropic electric field pattern to size fractionate EVs into discrete streams and steer the subpopulations into designated outlets for collection within a minute. Along with fractionating EV subpopulations, contaminants such as free proteins and short nucleic acids can be simultaneously directed to waste outlets, thus accomplishing both size fractionation and purification of EVs with high performance. Our platform offers a simple, rapid, and versatile solution for EV subpopulation isolation, which can potentially facilitate the discovery of biomarkers for specific EV subtypes and the development of EV-based therapeutics.

Synchronous coefficient of drag alteration refers to a multidimensional transport mechanism where a net drift of molecules is achieved under a zero-time-average alternating motive force by perturbing their drag coefficient synchronously with the applied force. An electrophoretic form of the method is often applied to focus and purify nucleic acids in a gel under rotating electric fields. However, this method requires lengthy operation due to the use of limited field strengths. Here, using DNA as target molecules, we demonstrate that the operation time can be reduced from hours to minutes by replacing polymer gel with a microfabricated artificial sieve. We also describe an electrophoretic protocol that facilitates the collection of purified DNA from the sieve, which is shown to yield amplifiable DNA from crude samples including the lysates of cultured cells and whole blood. The sieve can be further equipped with nucleic acid amplification and detection functions for a point-of-care diagnostic application.

Differential scanning calorimetry (DSC) is a popular thermal analysis technique. The miniaturization of DSC on chip as thin-film DSC (tfDSC) has been pioneered for the analysis of ultrathin polymer films at temperature scan rates and sensitivities far superior to those attainable with DSC instruments. The adoption of tfDSC chips for the analysis of liquid samples is, however, confronted with various issues including sample evaporation due to the lack of sealed enclosures. Although the subsequent integration of enclosures has been demonstrated in various designs, rarely did those designs exceed the scan rates of DSC instruments mainly because of their bulky features and requirement for exterior heating. Here, we present a tfDSC chip featuring sub-nL thin-film enclosures integrated with resistance temperature detectors (RTDs) and heaters. The chip attains an unprecedented sensitivity of 11 V W-1 and a rapid time constant of 600 ms owing to its low-addenda design and residual heat conduction (similar to 6 mu W K-1). We present results on the phase transition of common liquid crystals which we leverage to calibrate the RTDs and characterize the thermal lag with scan rates up to 900 degrees C min(-1). We then present results on the heat denaturation of lysozyme at various pH values, concentrations, and scan rates. The chip can provide excess heat capacity peaks and enthalpy change steps without much alteration induced by the thermal lag at elevated scan rates up to 100 degrees C min(-1), which is an order of magnitude faster than those of many chip counterparts.

Extracellular vesicles (EVs) are cell-derived bioparticles that play significant roles in various biological processes including cell-to-cell communication and intercellular delivery. Additionally, they hold great potential as liquid biopsy biomarkers for pre-diagnostic applications. However, the isolation of EV subpopulations, especially exosomes from a biological fluid remains a challenge due to their submicron range. Here, we demonstrate continuous-flow label-free size fractionation of EVs for the first time through a synergistic combination of electrothermal fluid rolls and dielectrophoresis in a microfluidic device. The device features three dimensional microelectrodes with unique sidewall contours that give rise to effective electrothermal fluid rolls in cooperation with dielectrophoretic forces for the electrokinetic manipulation and size separation of submicron particles. We first validate the device functionality by separating submicron polystyrene particles from binary mixtures with a cut-off size of ∼200 nm and then isolate intact exosomes from cell culture medium or blood serum with a high recovery rate and purity (∼80%). The device operation in a high-conductivity medium renders the method ideal for the purification of target bioparticles directly from physiological fluids, and may offer a robust and versatile platform for EV related diagnostic applications.

A novel and cost-effective method for creating ordered submicrometer-wide surface patterns using PDMS cracks is presented in this study. Cracks are first formed on a flat PDMS substrate by stretching an oxidized PDMS slab, and then the cracks are imprinted onto a target substrate to create uniform and ordered surface patterns. The resulting submicrometer-wide patterns are difficult to achieve using conventional fabrication methods limited by photolithography's resolution. Applications of the imprinted patterns, such as selective particle capture and DNA combing, are demonstrated. This approach offers a new way to utilize PDMS cracks and provides a simple and low-cost method for creating submicrometer-wide surface patterns with potential applications in various fields.

Here, we demonstrate continuous-flow separation of submicron particles under dielectrophoretic (DEP) force through a microfluidic device with 3D microelectrodes. Under optimal activation, the device is capable of effectively separating submicron particles at a high resolution of 200 nm. © 2022 MicroTAS 2022 - 26th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

This paper presents a sessile droplet differential scanning calorimetry (DSC) chip for liquid crystal phase transition characterization. The chip achieves higher sensitivity and less signal distortion compared to commercial DSC, while the chip's sample consumption is two orders of magnitude less than that of commercial DSC. The improved sessile sample droplet configuration greatly facilitates the sample handling and paves the way for the calorimetric measurements of living cells and tissues. © 2022 MicroTAS 2022 - 26th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

For studying protein–protein interactions (PPIs) in general, a powerful and commonly used technique is conventional coimmunoprecipitation (co-IP/pulldown) followed by western blotting. However, the technique does not provide precise information regarding the kinetics and stoichiometry of PPIs. Another drawback is that the sensitivity of conventional co-IP is not suitable for examining PPIs in rare cells such as sensory hair cells, circulating tumor cells, embryonic stem cells, and subsets of immune cells. The current single-molecule pulldown (SiMPull) assay can potentially be used for studying PPIs in rare cells but its wide application is hindered by the high technical barrier and time consumption. We report an innovative, agarose microbead-based approach for SiMPull. We used commercially available, pre-surface-functionalized agarose microbeads to capture the protein of interest together with its binding partners specifically from cell extracts and observed these interactions under a microscope at the single-molecule level. Relative to the original method, microbead-based SiMPull is considerably faster, easier to use, and more reproducible and yet provides similar sensitivity and signal-to-background ratio; specifically, with the new method, sample-preparation time is substantially decreased (from ∼10 to ∼3 h). These crucial features should facilitate wide application of the powerful and versatile SiMPull method in common biological and clinical laboratories. Notably, by exploiting the simplicity and ultrahigh sensitivity of microbead-based SiMPull, we used the method in the study of rare auditory hair cells and γδ T cells for the first time.

Continuous-flow electrokinetic enrichment/separation of nanoparticles is demonstrated in a device based on target nanoparticles railing along 3D microelectrode tracks. The device utilizes dielectrophoresis and electrohydrodynamic drag including electrothermal flow in high-conductive media and AC electroosmosis in low-conductive media. An enrichment efficiency of 100% is noted for 500 nm and 1 μm polystyrene spheres in media at a conductivity of 0.2 and 0.01 S/m. Size-based separation of the two is also shown efficient in deionized water using a modified design. © 2021 IEEE.

Here, we introduce a microchip for differential measurements of aqueous samples in twin calorimetric arms with a sub-nL enclosed space. Each arm features a fluidic microchannel located in close proximity to a thin-film resistance thermometer and heater patterned on a low-stress SiN membrane held over a Si cavity with suspension beams. The chip exhibits a rapid response with a thermal time constant of less than 10 ms in atmosphere and a residual thermal conductance below 6 μW·K⁻¹, one of the lowest reported and accounts for the excellent sensitivity registered in vacuum up to 11 V·W⁻¹. These values are in good agreement with those ones predicted by modeling using finite element method, lumped-capacitance analysis, and calorimeter electrothermal circuit simulation together with the measurement system. Long suspension beams in each arm ensure uniform temperature maintained across its membrane as revealed by the fluorescence-based melting curve analysis. We share the specific heat capacity values of water, glycerin, and mineral oil accurately measured by the chip. Lastly, we demonstrate the chip calorimeter utility on the measurement of the photothermal transduction efficiency of plasmonic nanoparticles, which makes our study further unique in expanding the applications of chip calorimeters. [2021-0044] IEEE

We demonstrate multifunctional silicon 3D viaduct microelectrodes for continuous-flow cell manipulation by railing and cell transfection under a modulated activation. The microelectrodes serve as tracks along which cells can rail under dielectrophoretic force and hydrodynamic drag. A modulated activation was introduced to achieve railing efficiency up to 80%. Furthermore, cell electroporation is realized through sinewave bursts applied with varying amplitude, duration and interval. Successful transfection is demonstrated by delivering 3 kDa dextran molecules into HEK293 cells with > 60% efficiency. The multifunctional 3D viaduct electrodes show great potential for continuous-flow rapid cell railing and sorting and efficient transfection.

We report on electrokinetic manipulation of submicron particles in a device with microfabricated Si electrodes featuring segmented sidewalls acting as tracks guiding particles under hydrodynamic drag. Electrohydrodynamic drag, both AC electroosmosis in low-conductive medium and electrothermal flow in high-conductive medium, gives rise to fluid rolls that deliver particles to tracks where they are held under the influence of dielectrophoretic forces. Particles are being railed along tracks under pressure-driven flow to a downstream junction where tracks act as bridges, keeping particles on course within the main channel causing their post-junction enrichment. This outcome, however, is strongly coupled to electrode sidewall contours as demonstrated here. We also report on particle oscillations under strong AC electroosmosis. Specifically, particles in deionized water can be seen bouncing off tracks or circulating with convective rolls in and out of space beneath tracks at an oscillation frequency and amplitude depending on the activation frequency. These results collectively draw attention to the functional use of electrode sidewall contours for continuous-flow manipulation of particles, which are rarely explored in microfluidics and yet could lead to more effective designs for particle separation and enrichment. © 2021, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

A compact microcalorimeter (μC) integrated on a fully suspended membrane has been demonstrated for the rapid characterization of the liquid thermal characteristics. The assay do not need thermostat and the active volume is less than 280 pL. The testing with propan-2-ol (IPA) shows the feasibility of the assay. © 2020 CBMS-0001