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.

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.

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.

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.

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.

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.

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.

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.

Pressure-Driven Chromatographic Separation Modes in Self-Enclosed Integrated Nanocapillaries.

This study presents pressure-driven chromatographic separation characteristics of integrated nanofluidic channels (nanocapillaries) featuring distinct cross-sectional geometries: cylindrical, triangular, and rectangular profiles. Cylindrical and triangular nanocapillaries are self-enclosed robust conduits realized through standard semiconductor processing techniques using low-resolution photolithography. Specifically, capillaries in nominal radius 300 and 500 nm have been investigated for chromatographic separation in comparison to 750 nm deep nanoslits as well as triangular capillaries featuring an inscribed circle about 500 nm in radius. Chromatograms have been obtained from 10 mm long nanocapillaries under various modes: normal- and reversed-phase, ion-valence, and hydrodynamic chromatography. The van Deemter plots based on the linear mobile phase velocity for 300 nm radius capillaries and 750 nm deep slits show excellent agreement with the plate heights theoretically predicted. The minimum plate heights achieved are typically below 2 µm and the theoretical plate numbers are in the order of 105 plates/m for the most chromatography modes investigated in the pressure range up to 100 psi. A comparatively high resolving power is achieved with cylindrical nanocapillaries especially those 300 nm in radius. Self-enclosed robust nanocapillaries demonstrated here could facilitate a pressure-driven chromatographic analysis of extremely low-volume samples (e.g., single cell).

Continuous-Flow Electrokinetic-Assisted Plasmapheresis by Using Three-Dimensional Microelectrodes Featuring Sidewall Undercuts.

We present a novel plasmapheresis device designed for a fully integrated point-of-care blood analysis microsystem. In the device, fluidic microchannels exhibit a characteristic cross-sectional profile arising from distinct three-dimensional (3D) microelectrodes featuring sidewall undercuts readily integrated through a single-mask process. The structure leverages mainly electrothermal convective rolls that efficiently manifest themselves in physiological fluids and yet have received inadequate attention for the application of plasmapheresis due to concerns over Joule heating. Using this device, we show that such convective rolls not only lead to plasma extraction at a high yield and purity but also deliver plasma at an acceptable quality with no evidence of hemolytic stress or protein denaturation. Specifically, plasma from 1.5 µL of whole blood diluted to 4% hematocrit in a high-conductivity buffer (1.5 S/m) is extracted in a continuous flow at a fraction of 70% by using a peak voltage of ±10 Vp applied at 650 kHz; the extracted plasma is nearly 99% pure, as shown by a rigorous assessment using flow cytometry. The plasmas obtained using this device and using conventional centrifugation and sedimentation are of comparable quality as revealed by absorbance and circular dichroism spectra despite thermal gradients; however, these gradients effectively drive electrothermal bulk flows, as assessed using the microparticle image velocity technique. The device achieves high target molecule recovery efficiency, delivering about 97% of the proteins detected in the plasma obtained using sedimentation. The utility of the extracted plasma is further validated based on the detection of prostate-specific antigen at clinically relevant levels.

Dielectrophoretic isolation of cells using 3D microelectrodes featuring castellated blocks.

We present 3D microelectrodes featuring castellated blocks for dielectrophoretically isolating cells. These electrodes provide a more effective dielectrophoretic force field than thin-film surface electrodes and yet immobilize cells near stagnation points across a parabolic flow profile for enhanced cell viability and separation efficiency. Unlike known volumetric electrodes with linear profiles, the electrodes with structural variations introduced along their depth scale are versatile for constructing monolithic structures with readily integrated fluidic paths. This is exemplified here in the design of an interdigitated comb array wherein electrodes with castellated surfaces serve as building blocks and form digits with an array of fluidic pores. Activation of the design with low-voltage oscillations (±5 Vp, 400 kHz) is found adequate for retaining most viable cells (90.2% ± 3.5%) while removing nonviable cells (88.5% ± 5%) at an increased throughput (5 × 10(5) cells h(-1)). The electrodes, despite their intricate profile, are structured into single-crystal silicon through a self-aligned etching process without a precision layer-by-layer assembly.

Label-free specific detection of femtomolar cardiac troponin using an integrated nanoslit array fluidic diode.

We demonstrate here for the first time the utility of an integrated nanofluidic diode for detecting and quantifying physiologically relevant macromolecules. Troponin T, a key human cardiac protein biomarker, was selectively and rapidly detected free of labels for concentrations down to 10 fg/mL (∼ 0.3 fM) in buffer as well as 10 pg/mL (∼ 300 fM) in untreated human serum. This ultrasensitive detection arises from monolithic integration of a unique nanofluidic diode structure that is highly robust and amenable to site-specific surface modification. The structure features a planar nanoslit array where each nanoslit is defined at a nominal width of 70 nm over a micrometer-scale silicon trench without the use of high-resolution patterning techniques. Through vapor deposition, a glass layer is placed at a nonuniform thickness, tapering the trench profile upward and contributing to the triangular nanoslit structure. This asymmetric profile is essential for ionic current rectification noted here at various pH values, ionic strengths, and captured target species, which modulate the surface-charge density within the sensitive region of the nanoslit. The nanoslit, unlike nanopores, offers only 1D confinement, which appears to be adequate for reasonable rectification. The measurements are found in quantitative agreement with the diode simulations for the first time based on a pH- and salt-dependent surface-charge model.

Fast DNA sieving through submicrometer cylindrical glass capillary matrix.

Here, we report on DNA electrophoresis through a novel artificial sieving matrix based on the highly regular submicrometer cylindrical glass capillary segments alternatingly arranged with wells. Such round capillaries pose a higher-order confinement resulting in a lower partition coefficient and greater entropic energy barrier while limiting the driving field strength to a small fraction of the applied electric field. In return, the separation can be performed at high average field strengths (up to 1.6 kV/cm) without encountering the field-dependent loss of resolving power. This leads to fast DNA sieving as demonstrated here on the capillaries of 750 nm in diameter. The 600 bp to 21 kbp long chains are shown to resolve within 4 min after having undergone a fairly limited number of entropic barriers (128 in total). The capillary matrix also exhibits a critical field threshold below which DNA bands fail to launch, and this occurs at a considerably greater magnitude than in other matrixes. The submicrometer capillaries are batch-fabricated on silicon through a fabrication process that does not require high-resolution advanced lithography or well-controlled wafer bonding techniques to define their critical dimension.

Induced hydraulic pumping via integrated submicrometer cylindrical glass capillaries.

Here, we report on a micropump that generates hydraulic pressure owing to a mismatch in EOF rates of microchannels and submicrometer cylindrical glass capillaries integrated on silicon. The electrical conductance of such capillaries in the dilute limit departs from bulk linear behavior as well as from the surface-charge-governed saturation in nanoslits that is well described by the assumption of a constant surface charge density. The capillaries show rather a gradual decrease in conduction at low salt concentrations, which can be explained more aptly by a variable surface charge density that accounts for chemical equilibrium of the surface. The micropump uses a traditional cross-junction structure with ten identical capillaries integrated in parallel on a side arm and each with a 750 nm diameter and 3 mm length. For an applied voltage of 700 V, a hydraulic pressure up to 5 kPa is generated with a corresponding flow velocity nearly 3 mm/s in a straight field-free branch 20 µm wide, 10 µm deep, and 10 mm long. The micropump utility has been demonstrated in an open tubular LC of three fluorescently labeled amino acids in just less than 20 s with minimal plate height values between 3 and 7 µm. The submicrometer capillaries are self-enclosed and produced through a unique process that does not require high-resolution advanced lithography or wafer-bonding techniques to define their highly controlled precise structures.

Microfluidic-based metal enhanced fluorescence for capillary electrophoresis by Ag nanorod arrays.

As metal nanorods show much higher metal enhanced fluorescence (MEF) than metal nanospheres, microfluidic-based MEF is first explored with Ag nanorod (ND) arrays made by oblique angle deposition. By measuring the fluorescein isothiocyanate (FITC) solution sandwiched between the Ag NDs and a piece of cover slip, the enhancement factors (EFs) are found as 3.7 ± 0.64 and 6.74 ± 2.04, for a solution thickness at 20.8 µm and 10 µm, respectively. Because of the strong plasmonic coupling between the adjacent Ag NDs, only the emission of the fluorophores present in the three-dimensional NDs array gets enhanced. Thus, the corresponding effective enhancement factors (EEFs) are revealed to be relatively close, 259 ± 92 and 340 ± 102, respectively. To demonstrate the application of MEF in microfluidic systems, a multilayer of SiO2 NDs/Ag NDs is integrated with a capillary electrophoresis device. At a microchannel depth of 10 µm, an enhancement of 6.5 fold is obtained for amino acids separation detection. These results are very encouraging and open the possibility of MEF applications for the Ag ND arrays decorated microchannels. With the miniaturization of microfluidic devices, microfluidic-based MEF by Ag ND arrays will likely find more applications with further enhancement.

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.

Microchannel plate as a novel bipolar electrode for high-performance enrichment of anions.

Microchannel plate (MCP), a high-porosity glass membrane used as an electron multiplier in analytical/scientific instruments for the detection of energetic photons and charged particles is demonstrated here as a highly effective bipolar electrode (BPE) for electrokinetic focusing of anions. Assembled between a pair of microfluidic channels filled with an electrolyte buffer and subjected to a sufficient bias potential, MCP supports faradaic reactions, owing to its semiconducting characteristics. Thousands of microcapillary tubes fused together define MCP and act in unison such that each microcapillary serves as a tiny BPE surrounding an infinitesimal element of bulk electrolyte with a large surface-area-to-volume ratio and hence performs highly effective as compared to a planar electrode inlaid into a microchannel. This performance has been validated here where concentration enrichment of a fluorescent tracer has been demonstrated at a remarkable rate of up to 175-fold/s exceeding those reported for planar BPEs. We attribute such high performance to the rapid onset of ion-depletion zone and subsequent steep field gradient, signifying the high-porosity structure of MCP as an effective BPE.

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.

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.