High-Performance Gel-Free and Label-Free Size Fractionation of Extracellular Vesicles with Two-Dimensional Electrophoresis in a Microfluidic Artificial Sieve.

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.

Correction: Sub-nL thin-film differential scanning calorimetry chip for rapid thermal analysis of liquid samples.

Correction for 'Sub-nL thin-film differential scanning calorimetry chip for rapid thermal analysis of liquid samples' by Sheng Ni et al., Lab Chip, 2023, 23, 1926-1934, https://doi.org/10.1039/D2LC01094A.

Accelerated Electrophoretic Focusing and Purification of DNA Based on Synchronous Coefficient of Drag Alteration.

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.

Nanobead-based single-molecule pulldown for single cells.

Investigation of cell-to-cell variability holds critical physiological and clinical implications. Thus, numerous new techniques have been developed for studying cell-to-cell variability, and these single-cell techniques can also be used to investigate rare cells. Moreover, for studying protein-protein interactions (PPIs) in single cells, several techniques have been developed based on the principle of the single-molecule pulldown (SiMPull) assay. However, the applicability of these single-cell SiMPull (sc-SiMPull) techniques is limited because of their high technical barrier and special requirements for target cells and molecules. Here, we report a highly innovative nanobead-based approach for sc-SiMPull that is based on our recently developed microbead-based, improved version of SiMPull for cell populations. In our sc-SiMPull method, single cells are captured in microwells and lysed in situ, after which commercially available, pre-surface-functionalized magnetic nanobeads are placed in the microwells to specifically capture proteins of interest together with their binding partners from cell extracts; subsequently, the PPIs are examined under a microscope at the single-molecule level. Relative to previously published methods, nanobead-based sc-SiMPull is considerably faster, easier to use, more reproducible, and more versatile for distinct cell types and protein molecules, and yet provides similar sensitivity and signal-to-background ratio. These crucial features should enable universal application of our method to the study of PPIs in single cells.

Continuous-flow label-free size fractionation of extracellular vesicles through electrothermal fluid rolls and dielectrophoresis synergistically integrated in a microfluidic device.

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.

Sub-nL thin-film differential scanning calorimetry chip for rapid thermal analysis of liquid samples.

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 (∼6 µW 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 °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 °C min-1, which is an order of magnitude faster than those of many chip counterparts.

Analyzing protein-protein interactions in rare cells using microbead-based single-molecule pulldown assay.

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.

Ordered surface crack patterns in situ formed under confinement on fluidic microchannel boundaries in polydimethylsiloxane.

We present ordered surface crack patterns discovered in microfluidic channels/chambers in polydimethylsiloxane (PDMS). The cracks are formed in situ under confinement due to compression applied following an oxygen plasma step in a soft lithography process. The crack patterns are noticeable only after fluorescent labeling and vary with fluidic layout as well as material compliance.

Railing Nanoparticles Along Activated Tracks Towards Continuous-Flow Electrokinetic Enrichment from Blood Plasma.

We report on a unique microfluidic device that can enrich nanoparticles in a continuous flow by railing them along activated tracks (electrodes). This was achieved based on dielectrophoretic force and electrohydrodynamic drag (electrothermal rolls and AC electroosmosis) in both low and high conductive media. The results have implication for the isolation of high quality and pure nanoparticles such as exosomes from biofluids for applications in cancer diagnosis and prognosis.

The vision of point-of-care PCR tests for the COVID-19 pandemic and beyond.

Infectious diseases, such as the most recent case of coronavirus disease 2019, have brought the prospect of point-of-care (POC) diagnostic tests into the spotlight. A rapid, accurate, low-cost, and easy-to-use test in the field could stop epidemics before they develop into full-blown pandemics. Unfortunately, despite all the advances, it still does not exist. Here, we critically review the limited number of prototypes demonstrated to date that is based on a polymerase chain reaction (PCR) and has come close to fulfill this vision. We summarize the requirements for the POC-PCR tests and then go on to discuss the PCR product-detection methods, the integration of their functional components, the potential applications, and other practical issues related to the implementation of lab-on-a-chip technologies. We conclude our review with a discussion of the latest findings on nucleic acid-based diagnosis.

Rapid Characterization of Biomolecules' Thermal Stability in a Segmented Flow-Through Optofluidic Microsystem.

Optofluidic devices combining optics and microfluidics have recently attracted attention for biomolecular analysis due to their high detection sensitivity. Here, we show a silicon chip with tubular microchannels buried inside the substrate featuring temperature gradient (∇T) along the microchannel. We set up an optical fluorescence system consisting of a power-modulated laser light source of 470 nm coupled to the microchannel serving as a light guide via optical fiber. Fluorescence was detected on the other side of the microchannel using a photomultiplier tube connected to an optical fiber via a fluorescein isothiocyanate filter. The PMT output was connected to a lock-in amplifier for signal processing. We performed a melting curve analysis of a short dsDNA - SYBR Green I complex with a known melting temperature (TM) in a flow-through configuration without gradient to verify the functionality of the proposed detection system. We then used the segmented flow configuration and measured the fluorescence amplitude of a droplet exposed to ∇T of ≈ 2.31 °C mm-1, determining the heat transfer time as ≈ 554 ms. The proposed platform can be used as a fast and cost-effective system for performing either MCA of dsDNAs or for measuring protein unfolding for drug-screening applications.

Conductance Interplay in Ion Concentration Polarization across 1D Nanochannels: Microchannel Surface Shunt and Nanochannel Conductance.

Ion concentration polarization has received much interest in the past decade for lab-on-a-chip applications, primarily preconcentration of biomolecules and water desalination. Studying the basic phenomenon in microfluidics has also generated new knowledge, which could be pivotal in the design of novel devices. Many studies, however, have focused on designs featuring nanoslits/slots or surface-patterned ion-selective membranes whereas the characteristics of 1D nanochannels are still lacking. Here, we report on ion concentration polarization across highly ordered 1D nanochannels in isolation as well as in array formation. Intriguingly, the nanochannels in isolation exhibit a linear current-voltage characteristic at low salt concentrations despite the confirmed presence of ion-depletion zone, which is associated with the diffusion-limited transport and the consequent nonlinearity in the classical sense. The characteristic in array formation breaks away from the linearity with a peculiar dip in current for a critical salt concentration in the dilute limit. We describe these findings based on the interplay between the nanochannel conductance and the conductance of the neighboring microchannel walls (the so-called surface shunt). Also, the nanochannel transport is identified with the mobility of protons more closely than that of salt cations. We attribute fast transport to phosphorus-doped silicate glass, the nanochannel material known to have very fine pores likely to be populated with protons as a result of moisture and carbon dioxide adsorption from the air. The nanochannels possess a tubular profile 70 nm in nominal diameter and fabricated through thermal reflow of doped glass on silicon without using advanced lithography.

A nanofluidic memristor based on ion concentration polarization.

This paper reports the very first nanofluidic memristor based on the principle of ion concentration polarization (ICP). ICP is induced through highly-ordered cylindrical nanochannels. These so-called nanocapillaries are formed within a glass layer on silicon through a thermal reflow process and low-resolution lithography. The current-voltage plots exhibit characteristic pinched-hysteresis loops and the concurrent tracking of fluorescent charged particles correlates the memristive behaviour to the ICP. The ICP-based nanofluidic memristor could have implications in emerging areas such as integrated fluid-based logic circuits.

Single-Cell Point Constrictions for Reagent-Free High-Throughput Mechanical Lysis and Intact Nuclei Isolation.

Highly localized (point) constrictions featuring a round geometry with ultra-sharp edges in silicon have been demonstrated for the reagent-free continuous-flow rapid mechanical lysis of mammalian cells on a single-cell basis. Silicon point constrictions, robust structures formed by a single-step dry etching process, are arranged in a cascade along microfluidic channels and can effectively rupture cells delivered in a pressure-driven flow. The influence of the constriction size and count on the lysis performance is presented for fibroblasts in reference to total protein, DNA, and intact nuclei levels in the lysates evaluated by biochemical and fluoremetric assays and flow-cytometric analyses. Protein and DNA levels obtained from an eight-constriction treatment match or surpass those from a chemical method. More importantly, many intact nuclei are found in the lysates with a relatively high nuclei-isolation efficiency from a four-constriction treatment. Point constrictions and their role in rapid reagent-free disruption of the plasma membrane could have implications for integrated sample preparation in future lab-on-a-chip systems.

Railing cells along 3D microelectrode tracks for continuous-flow dielectrophoretic sorting.

We demonstrate a unique microfluidic device for continuous-flow cell sorting by railing target cells along physical tracks (electrode sidewalls) based on the combined effect of dielectrophoresis and hydrodynamic drag. The tracks are the raised digits of comb-like structures made of conducting bulk silicon as the electrodes. Unlike other volumetric electrodes, the structures feature a segmented sidewall profile with linear and concave segments forming the tracks and supporting columns, respectively. The interdigitated bulk electrodes lead to a built-in flow chamber in which the digits (tracks) extend downstream at a characteristic angle with respect to the flow, which runs through the passages between the columns. Target cells leaving the passages are levitated and docked against the tracks under positive dielectrophoresis and railed under hydrodynamic drag. Railing efficiency, as high as >95%, is reported against the activation voltage and flow rate for the designs 7°, 16°, and 26° as the track angles. A collection efficiency of about 86% is noted for both target (HCT116) and non-target cells (K562) in the 16° design at a sample flow rate of 8.3 µL min-1 and an activation voltage of 12.5 Vp at 200 kHz. This performance is comparable if not better than those obtained with thin-film surface microelectrodes and yet achieved here at an order of magnitude higher sample flow rate. This enhancement mainly arises from a considerably low drag along the tracks in relation to the chamber top or bottom surface where the thin-film electrodes would be typically placed.

Label-Free Multiplexed Electrical Detection of Cancer Markers on a Microchip Featuring an Integrated Fluidic Diode Nanopore Array.

We introduce an integrated array of glass nanopores on a silicon microchip fabricated in a batch process through low-resolution photolithography and standard semiconductor processing tools. By functionalizing each nanopore against a distinct target, we further demonstrate ultrasensitive, label-free, multiplexed electrical detection of cancer-marker proteins in real time through charge-dependent ionic current rectification. As nanofluidic diode biosensors, the nanopores return rapid results, with a limit of detection reaching concentrations as low as attomolars in assay buffer and femtomolars in undiluted untreated human serum, a rare achievement for this class of nanosensors. Multiplexed detection capability has been demonstrated on proteins carcinoembryonic antigen, α-fetoprotein antigen, and human epidermal growth factor receptor-2, with the assay further scalable to a size that is limited by the readout electronics. The nanopores are also found with a considerably advanced detection limit as well as dynamic range in relation to the nanoslit counterparts, validated by the measurements on cardiac protein troponin T. This highly robust assay platform draws from rich nanopore physics and could provide further enhanced detection through concentration polarization, subsequent target enrichment, and serum desalting, all potentially induced by the nanopores presently redundant in the array. This integration would be crucial for removing major obstacles for the practical use of nanopore-based assays.

A Low-Backpressure Single-Cell Point Constriction for Cytosolic Delivery Based on Rapid Membrane Deformations.

Mechanically deforming biological cells through microfluidic constrictions is a recently introduced technique for the intracellular delivery of macromolecules possibly through transient membrane pores induced in the process. The technique is attractive for research and clinical applications mainly because it is simple, fast, and effective while being free of adverse effects often associated with well-known techniques that rely on field- or vector-based delivery. In this nascent approach, an utmost and crucial role is played by the constriction, often in rectangular profile, and it squeezes cells only in one dimension. The results achieved suggest that the longer the constriction is the higher the delivery performance. Contrary to this view, we demonstrate here a unique constriction profile that is highly localized (point) and yet returns comparably effective delivery. Point constrictions are of a semiround geometry, forcing cells in both dimensions while introducing very little backpressure to the system, which is a silicon-glass platform wherein constrictions are arranged in series along an array of channels. The influence of the constriction size and count as well as treatment pressure on delivery performance is presented on the basis of the flow-cytometric analyses of HCT116 cells treated using dextran as model molecules. Delivery performance is also presented for common mammalian cell lines including NIH 3T3, HEK293, and MDCK. Moreover, the versatility of the platform is demonstrated in gene knockdown experiments using synthetic siRNA as well as on the delivery of proteins. Target proteins in some cells exhibit nondiffusive distribution profile raising the plausibility of mechanisms other than transient membrane pores.

Continuous-Flow Electrophoresis of DNA and Proteins in a Two-Dimensional Capillary-Well Sieve.

Continuous-flow electrophoresis of macromolecules is demonstrated using an integrated capillary-well sieve arranged into a two-dimensional anisotropic array on silicon. The periodic array features thousands of entropic barriers, each resulting from an abrupt interface between a 2 µm deep well (channel) and a 70 nm capillary. These entropic barriers owing to two-dimensional confinement within the capillaries are vastly steep in relation to those arising from slits featuring one-dimensional confinement. Thus, the sieving mechanisms can sustain relatively large electric field strengths over a relatively small array area. The sieve rapidly sorts anionic macromolecules, including DNA chains and proteins in native or denatured states, into distinct trajectories according to size or charge under electric field vectors orthogonally applied. The baseline separation is achieved in less than 1 min within a horizontal migration length of ∼1.5 mm. The capillaries are self-enclosed conduits in cylindrical profile featuring a uniform diameter and realized through an approach that avoids advanced patterning techniques. The approach exploits a thermal reflow of a layer of doped glass for shape transformation into cylindrical capillaries and for controllably shrinking the capillary diameter. Lastly, atomic layer deposition of alumina is introduced for the first time to fine-tune the capillary diameter as well as to neutralize the surface charge, thereby suppressing undesired electroosmotic flows.

On-chip hydrodynamic chromatography of DNA through centimeters-long glass nanocapillaries.

This study demonstrates hydrodynamic chromatography of DNA fragments in a microchip. The microchip contains a highly regular array of nanofluidic channels (nanocapillaries) that are essential for resolving DNA in this chromatography mode. The nanocapillaries are self-enclosed robust structures built inside a doped glass layer on silicon using low-resolution photolithography and standard semiconductor processing techniques. Additionally, the unique nanocapillaries feature a cylindrical inner radius of 600 nm maintained over a length scale of 5 cm. The microchip with bare open nanocapillaries is shown to rapidly separate a digest of lambda DNA in free solution (<5 min under the elution pressure of 60 to 120 psi), relying entirely on pressure-driven flows and, in doing so, avoiding the field-induced DNA aggregations encountered in gel-free electrophoresis. The nanocapillaries, despite their relatively short length, are observed to fractionate DNA fragments reasonably well with a minimum resolvable size difference below 5 kbp. In the chromatograms obtained, the number of theoretical plates exceeds 105 plates per m for 3.5 and 21 kbp long DNA fragments. The relative mobility of fragments in relation to their size is found to be in excellent agreement with the simple quadratic model of hydrodynamic chromatography. The model is shown to estimate greater effective hydrodynamic radii than those of respective fragments being unconfined in bulk solution, implying increased drag forces and reduced diffusion coefficients, which is also a noticeable trend among diffusion coefficient estimates derived from the experimentally obtained plate heights. This robust mass-producible microchip can be further developed into a fully integrated bioanalytic microsystem.

Mechanical Characterization of Microengineered Epithelial Cysts by Using Atomic Force Microscopy.

Most organs contain interconnected tubular tissues that are one-cell-thick, polarized epithelial monolayers enclosing a fluid-filled lumen. Such tissue organization plays crucial roles in developmental and normal physiology, and the proper functioning of these tissues depends on their regulation by complex biochemical perturbations and equally important, but poorly understood, mechanical perturbations. In this study, by combining micropatterning techniques and atomic force microscopy, we developed a simple in vitro experimental platform for characterizing the mechanical properties of the MDCK II cyst, the simplest model of lumen-enclosing epithelial monolayers. By using this platform, we estimated the elasticity of the cyst monolayer and showed that the presence of a luminal space influences cyst mechanics substantially, which could be attributed to polarization and tissue-level coordination. More interestingly, the results from force-relaxation experiments showed that the cysts also displayed tissue-level poroelastic characteristics that differed slightly from those of single cells. Our study provides the first quantitative findings, to our knowledge, on the tissue-level mechanics of well-polarized epithelial cysts and offers new insights into the interplay between cyst mechanics and cyst physiology. Moreover, our simple platform is a potentially useful tool for enhancing the current understanding of cyst mechanics in health and disease.