A one-step molded microfluidic chip featuring a two-layer silver-PDMS microelectrode for dielectrophoretic cell separation.

Dielectrophoresis (DEP) is a powerful technique for label-free cell separation in microfluidics. Easily-fabricated DEP separators with low cost and short turnaround time are in extremely high demand in practical applications, especially clinical usage where disposable devices are needed. DEP separators exploiting microelectrodes made of conducting polydimethylsiloxane (PDMS) composites enable the construction of advantageous 3D volumetric electrodes with a simple soft-lithography process. Yet, existing devices incorporating microelectrodes in conducting PDMS generally have their fluidic sidewalls constructed using a different material, and consequently require extra lithography of a sacrificial layer on the semi-finished master for molding the electrode and fluidic sidewalls in separate steps. Here we demonstrate a novel microfluidic DEP separator with a 3D electrode and fluidic structure entirely integrated within silver-PDMS composites. We develop a further simplified one-step molding process with lower cost using a readily-available and reusable SU8 master, eliminating the need for the additional lithography step in existing techniques. The uniquely designed two-layer electrode exhibits a spatially non-uniform electric field that enables cell migration in the vertical direction. The electrode upper layer then offers a harbor-like region for the trapping of the target cells that have drifted upwards, which shelters them from being dragged away by the main flow streams in the lower layer, and thus allows higher operation flow rate. We also optimize the upper layer thickness as a critical dimension for protecting the trapped cells from high drag and show easy widening of our device by elongation of the digits. We demonstrate that the elongated digits involving more parallel flow paths maintain a high capture efficiency of 95.4% for live cells with 85.6% purity in the separation of live/dead HeLa cells. We also investigate the device feasibility in a viability assay for cells post anti-cancer drug treatment.

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 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.

Fast Single-Cell Patterning for Study of Drug-Induced Phenotypic Alterations of HeLa Cells Using Time-of-Flight Secondary Ion Mass Spectrometry.

A facile single-cell patterning (ScP) method was developed and integrated with time-of-flight secondary ion mass spectrometry (TOF-SIMS) for the study of drug-induced cellular phenotypic alterations. Micropatterned poly(dimethylsiloxane) (PDMS) stencil film and centrifugation-assisted cell trapping were combined for the preparation of on-surface single-cell microarrays, which exhibited both high site occupancy (>90%) and single-cell resolution (>97%). TOF-SIMS is a surface-sensitive mass spectrometry and is increasingly utilized in biological studies. Here we demonstrated, for the first time, its successful application in high-throughput single-cell analysis. Drug-induced phenotypic alterations of HeLa cells in the early stage of apoptosis were investigated using TOF-SIMS. The major molecular sources of variations were analyzed by principle component analysis (PCA).

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.

Machine learning classification of cellular states based on the impedance features derived from microfluidic single-cell impedance flow cytometry.

Mitosis is a crucial biological process where a parental cell undergoes precisely controlled functional phases and divides into two daughter cells. Some drugs can inhibit cell mitosis, for instance, the anti-cancer drugs interacting with the tumor cell proliferation and leading to mitosis arrest at a specific phase or cell death eventually. Combining machine learning with microfluidic impedance flow cytometry (IFC) offers a concise way for label-free and high-throughput classification of drug-treated cells at single-cell level. IFC-based single-cell analysis generates a large amount of data related to the cell electrophysiology parameters, and machine learning helps establish correlations between these data and specific cell states. This work demonstrates the application of machine learning for cell state classification, including the binary differentiations between the G1/S and apoptosis states and between the G2/M and apoptosis states, as well as the classification of three subpopulations comprising a subgroup insensitive to the drug beyond the two drug-induced states of G2/M arrest and apoptosis. The impedance amplitudes and phases used as input features for the model training were extracted from the IFC-measured datasets for the drug-treated tumor cells. The deep neural network (DNN) model was exploited here with the structure (e.g., hidden layer number and neuron number in each layer) optimized for each given cell type and drug. For the H1650 cells, we obtained an accuracy of 78.51% for classification between the G1/S and apoptosis states and 82.55% for the G2/M and apoptosis states. For HeLa cells, we achieved a high accuracy of 96.94% for classification between the G2/M and apoptosis states, both of which were induced by taxol treatment. Even higher accuracy approaching 100% was achieved for the vinblastine-treated HeLa cells for the differentiation between the viable and non-viable states, and between the G2/M and apoptosis states. We also demonstrate the capability of the DNN model for high-accuracy classification of the three subpopulations in a complete cell sample treated by taxol or vinblastine.

High-quality genome assembly and annotation of Clonostachys chloroleuca strain Cc878 using Oxford Nanopore long-read sequencing.

As a noteworthy biocontrol fungus, Clonostachys chloroleuca currently lacks a high-quality reference genome. Here, we present the first high-quality genome assembly of C. chloroleuca strain Cc878 achieved through Oxford Nanopore Long-Read sequencing. The nuclear genome of Cc878 was assembled into four contigs, totaling 59.38 Mb.

Hyphal editing of the conserved premature stop codon in CHE1 is stimulated by oxidative stress in Fusarium graminearum.

Although genome-wide A-to-I editing mediated by adenosine-deaminase-acting-on-tRNA (ADAT) occurs during sexual reproduction in the presence of stage-specific cofactors, RNA editing is not known to occur during vegetative growth in filamentous fungi. Here we identified 33 A-to-I RNA editing events in vegetative hyphae of Fusarium graminearum and functionally characterized one conserved hyphal-editing site. Similar to ADAT-mediated editing during sexual reproduction, majority of hyphal-editing sites are in coding sequences and nonsynonymous, and have strong preference for U at -1 position and hairpin loops. Editing at TA437G, one of the hyphal-specific editing sites, is a premature stop codon correction (PSC) event that enables CHE1 gene to encode a full-length zinc fingertranscription factor. Manual annotations showed that this PSC site is conserved in CHE1 orthologs from closely-related Fusarium species. Whereas the che1 deletion and CHE1TAA (G438 to A) mutants had no detectable phenotype, the CHE1TGG (A437 to G) mutant was defective in hyphal growth, conidiation, sexual reproduction, and plant infection. However, the CHE1TGG mutant was increased in tolerance against oxidative stress and editing of TA437G in CHE1 was stimulated by H2O2 treatment in F. graminearum. These results indicate that fixation of the premature stop codon in CHE1 has a fitness cost on normal hyphal growth and reproduction but provides a benefit to tolerance against oxidative stress. Taken together, A-to-I editing events, although rare (not genome-wide), occur during vegetative growth and editing in CHE1 plays a role in response to oxidative stress in F. graminearum and likely in other fungal pathogens.

Study of cell and drug interactions based on dual-mode detection using SPR and fluorescence imaging.

The investigation of the interactions between cells and drugs forms a crucial aspect of biological and clinical medical studies. Generally, single-cell or local-cellular studies require a microscopic imaging system with high magnifications, which suffers from low detection throughputs and poor time responses. The study presented in this paper combined SPR and fluorescence to achieve cell localization, real-time monitoring of cell images and quantitative analysis of drugs. In order to obtain more comprehensive, accurate and real-time data, a dual-mode system based on surface plasmon resonance (SPR) and fluorescence was constructed based on a 4× magnification lens. This enables simultaneous studies of an entire cell and a specific region of the cell membrane. An adaptive adjustment algorithm was established for distorted SPR images, achieving temporal and spatial matching of the dual-mode detection. The combination of SPR and fluorescence not only achieved micro-detection but also complemented the qualitative or quantitative limitations of SPR or fluorescence method alone. In system characterization, the response signal of SPR was noticed to increase with the increasing concentration of EGF in stimulated cells. It indicated that this platform could be employed for quantitative detection of the cell membrane region. Upon addition of EGF, a peak in the SPR curve was observed, and the cells in the corresponding SPR image turned whiter. This indicated that the platform can simultaneously monitor the SPR response signal and image changes. The response time of fluorescence in EGF testing was several seconds earlier than SPR, revealing that signal transduction first occurred in the whole cell and then propagated to the cell membrane region. The inhibitory ability of Gefitinib on cells was verified in a fast and real-time manner within 20 min. The results indicated that the detection limit of this method was 20 IU/mL for EGF and 10 µg/mL for Gefitinib. In conclusion, this study demonstrates the advantages of SPR and fluorescence dual-mode techniques in the analysis of cell-drug interactions, as well as their strong potential in drug screening.

Unveiling the A-to-I mRNA editing machinery and its regulation and evolution in fungi.

A-to-I mRNA editing in animals is mediated by ADARs, but the mechanism underlying sexual stage-specific A-to-I mRNA editing in fungi remains unknown. Here, we show that the eukaryotic tRNA-specific heterodimeric deaminase FgTad2-FgTad3 is responsible for A-to-I mRNA editing in Fusarium graminearum. This editing capacity relies on the interaction between FgTad3 and a sexual stage-specific protein called Ame1. Although Ame1 orthologs are widely distributed in fungi, the interaction originates in Sordariomycetes. We have identified key residues responsible for the FgTad3-Ame1 interaction. The expression and activity of FgTad2-FgTad3 are regulated through alternative promoters, alternative translation initiation, and post-translational modifications. Our study demonstrates that the FgTad2-FgTad3-Ame1 complex can efficiently edit mRNA in yeasts, bacteria, and human cells, with important implications for the development of base editors in therapy and agriculture. Overall, this study uncovers mechanisms, regulation, and evolution of RNA editing in fungi, highlighting the role of protein-protein interactions in modulating deaminase function.

Single-cell impedance cytometry of anticancer drug-treated tumor cells exhibiting mitotic arrest state to apoptosis using low-cost silver-PDMS microelectrodes.

Chemotherapeutic drugs such as paclitaxel and vinblastine interact with microtubules and thus induce complex cell states of mitosis arrest at the G2/M phase followed by apoptosis dependent on drug exposure time and concentration. Microfluidic impedance cytometry (MIC), as a label-free and high-throughput technology for single-cell analysis, has been applied for viability assay of cancer cells post drug exposure at fixed time and dosage, yet verification of this technique for varied tumor cell states after anticancer drug treatment remains a challenge. Here we present a novel MIC device and for the first time perform impedance cytometry on carcinoma cells exhibiting progressive states of G2/M arrest followed by apoptosis related to drug concentration and exposure time, after treatments with paclitaxel and vinblastine, respectively. Our results from impedance cytometry reveal increased amplitude and negative phase shift at low frequency as well as higher opacity for HeLa cells under G2/M mitotic arrest compared to untreated cells. The cells under apoptosis, on the other hand, exhibit opposite changes in these electrical parameters. Therefore, the impedance features differentiate the HeLa cells under progressive states post anticancer drug treatment. We also demonstrate that vinblastine poses a more potent drug effect than paclitaxel especially at low concentrations. Our device is fabricated using a unique sacrificial layer-free soft lithography process as compared to the existing MIC device, which gives rise to readily aligned parallel microelectrodes made of silver-PDMS embedded in PDMS channel sidewalls with one molding step. Our results uncover the potential of the MIC device, with a fairly simple and low-cost fabrication process, for cellular state screening in anticancer drug therapy.

A Light-Driven Integrated Bio-Capacitor with Single Nano-Channel Modulation.

Bioelectronics, an emerging discipline formed by the biology and electronic information disciplines, has maintained a state of rapid development since its birth. Amongst the various functional bioelectronics materials, bacteriorhodopsin (bR), with its directional proton pump function and favorable structural stability properties, has drawn wide attention. The main contents of the paper are as follows: Inspired by the capacitive properties of natural protoplast cell membranes, a new bio-capacitor based on bR and artificial nanochannels was constructed. As a point of innovation, microfluidic chips were integrated into our device as an ion transport channel, which made the bio-capacitor more stable. Meanwhile, a single nanopore structure was integrated to improve the accuracy of the device structure. Experiments observed that the size of the nanopore affected the ion transmission rate. Consequently, by making the single nanopore's size change, the photocurrent duration time (PDT) of bR was effectively regulated. By using this specific phenomenon, the original transient photocurrent was successfully transformed into a square-like wave.

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 enumeration of colorectal cancer cells from lymphocytes performed at a high cell-loading density by using interdigitated ring-array microelectrodes.

We report the label-free enumeration of human colorectal-carcinoma cells from blood lymphocytes by using interdigitated ring-array microelectrodes; this enumeration was based on the dielectrophoretic selection of cells. Because of the novel design of the device, a continuous flow of cells is uniformly distributed into parallel streams through 300 rings (~40 µm in diameter each) that are integrated into the electrode digits. Using this array, 82% of cancer cells were recovered and 99% of blood lymphocytes were removed. Most of the cancer cells recovered were viable (94%) and could be cultivated for >8 days, during which period they retained their normal cell morphology and proliferation rates. The recovery rate correlated closely with cancer-cell loadings in spiked samples and this relationship was linear over a range of at least 2 orders of magnitude. Importantly, because of the 3D structure of the rings, these results were obtained at a high cell-loading concentration (10(7)cells/mL). The rings could be further optimized for use in accurate label-free identification and measurement of circulating tumor cells in cancer research and disease management.