Trapping cells on a stretchable microwell array for single-cell analysis

There is a need for a technology that can be incorporated into routine laboratory procedures to obtain a continuous, quantitative, fluorescence-based measurement of the dynamic behaviors of numerous individual living cells in parallel, while allowing other manipulations, such as staining, rinsing, and even retrieval of targeted cells. Here, we report a simple, low-cost microarray platform that can trap cells for dynamic single-cell analysis of mammalian cells. The elasticity of polydimethylsiloxane (PDMS) was utilized to trap tens of thousands of cells on an array. The PDMS microwell array was stretched by a tube through which cells were loaded on the array. Cells were trapped on the array by removal of the tube and relaxation of the PDMS. Once that was accomplished, the cells remained trapped on the array without continuous application of an external force and permitted subsequent manipulations, such as staining, rinsing, imaging, and even isolation of targeted cells. We demonstrate the utility of this platform by multicolor analysis of trapped cells and monitoring in individual cells real-time calcium flux after exposure to the calcium ionophore ionomycin. Additionally, a proof of concept for target cell isolation was demonstrated by using a microneedle to locally deform the PDMS membrane in order to retrieve a particular cell from the array.     

Prediction analysis and quality assessment of microwell array images

Microwell arrays are widely used for the analysis of fluorescent‐labelled biomaterials. For rapid detection and automated analysis of microwell arrays, the computational image analysis is required. Support Vector Machines (SVM) can be used for this task. Here, we present a SVM‐based approach for the analysis of microwell arrays consisting of three distinct steps: labeling, training for feature selection, and classification into three classes. The three classes are filled, partially filled, and unfilled microwells. Next, the partially filled wells are analyzed by SVM and their tendency towards filled or unfilled tested through applying a Gaussian filter. Through this, all microwells can be categorized as either filled or unfilled by our algorithm. Therefore, this SVM‐based computational image analysis allows for an accurate and simple classification of microwell arrays.     

Ultra-thin temperature controllable microwell array chip for continuous real-time high-resolution imaging of living single cells

Single-cell imaging, a powerful analytical method to study single-cell behavior, such as gene expression and protein profiling, provides an essential basis for modern medical diagnosis. The coding and localization function of microfluidic chips has been developed and applied in living single-cell imaging in recent years. Simultaneously, chip-based living single-cell imaging is also limited by complicated trapping steps, low cell utilization, and difficult high-resolution imaging. To solve these problems, an ultra-thin temperature-controllable microwell array chip (UTCMA chip) was designed to develop a living single-cell workstation in this study for continuous on-chip culture and real-time high-resolution imaging of living single cells. The chip-based on ultra-thin ITO glass is highly matched with an inverted microscope (or confocal microscope) with a high magnification objective (100 × oil lens), and the temperature of the chip can be controlled by combining it with a home-made temperature control device. High-throughput single-cell patterning is realized in one step when the microwell array on the chip uses hydrophilic glass as the substrate and hydrophobic SU-8 photoresist as the wall. The cell utilization rate, single-cell capture rate, and microwell occupancy rate are all close to 100% in the microwell array. This method will be useful in rare single-cell research, extending its application in the biological and medical-related fields, such as early diagnosis of disease, personalized therapy, and research-based on single-cell analysis.     

Rapid assessment of drug response in cancer cells using microwell array and molecular imaging

Selection of personalized chemotherapy regimen for individual patients has significant potential to improve chemotherapy efficacy and to reduce the deleterious effects of ineffective chemotherapy drugs. In this study, a rapid and high-throughput in vitro drug response assay was developed using a combination of microwell array and molecular imaging. The microwell array provided high-throughput analysis of drug response, which was quantified based on the reduction in intracellular uptake (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose) (2-NBDG). Using this synergistic approach, the drug response measurement was completed within 4 h, and only a couple thousand cells were needed for quantification. The broader application of this microwell molecular imaging approach was demonstrated by evaluating the drug response of two cancer cell lines, cervical (HeLa) and bladder (5637) cancer cells, to two distinct classes of chemotherapy drugs (cisplatin and paclitaxel). This approach did not require an extended cell culturing period, and the quantification of cellular drug response was 4–16 times faster compared with other cell-microarray drug response studies. Moreover, this molecular imaging approach had comparable sensitivity to traditional cell viability assays, i.e., the MTT assay and propidium iodide labeling of cellular nuclei;and similar throughput results as flow cytometry using only 1,000–2,000 cells. Given the simplicity and robustness of this microwell molecular imaging approach, it is anticipated that the assay can be adapted to quantify drug responses in a wide range of cancer cells and drugs and translated to clinical settings for a rapid in vitro drug response using clinically isolated samples.      

Nanofluidics for single-cell analysis

Living single-cell analysis is vital for cell biology, disease pathology, drug discovery and medical treatment. It is of great significance to reveal the law of creature and to explore the mechanism of serious disease. The conventional single cell analysis focuses on a large number of cells or cell lysis, in order to obtain the average information about cells. Therefore, it fails to analyze the real-time and continuous data of differences between the individual cells, thus limiting the developme...     

Optical microwell array for large scale studies of single mitochondria metabolic responses

Microsystems based on microwell arrays have been widely used for studies on single living cells. In this work, we focused on the subcellular level in order to monitor biological responses directly on individual organelles. Consequently, we developed microwell arrays for the entrapment and fluorescence microscopy of single isolated organelles, mitochondria herein. Highly dense arrays of 3-μm mean diameter wells were obtained by wet chemical etching of optical fiber bundles. Favorable conditions for the stable entrapment of individual mitochondria within a majority of microwells were found. Owing to NADH auto-fluorescence, the metabolic status of each mitochondrion was analyzed at resting state (Stage 1), then following the addition of a respiratory substrate (Stage 2), ethanol herein, and of a respiratory inhibitor (Stage 3), antimycin A. Mean levels of mitochondrial NADH were increased by 29 % and 35 % under Stages 2 and 3, respectively. We showed that mitochondrial ability to generate higher levels of NADH (i.e., its metabolic performance) is not correlated either to the initial energetic state or to the respective size of each mitochondrion. This study demonstrates that microwell arrays allow metabolic studies on populations of isolated mitochondria with a single organelle resolution.

A silicone-based stretchable micropost array membrane for monitoring live-cell subcellular cytoskeletal response

External forces are increasingly recognized as major regulators of cellular structure and function, yet the underlying mechanism by which cells sense forces and transduce them into intracellular biochemical signals and behavioral responses ('mechanotransduction') is largely undetermined. To aid in the mechanistic study of mechanotransduction, herein we devised a cell stretching device that allowed for quantitative control and real-time measurement of mechanical stimuli and cellular biomechanical responses. Our strategy involved a microfabricated array of silicone elastomeric microposts integrated onto a stretchable elastomeric membrane. Using a computer-controlled vacuum, this micropost array membrane (mPAM) was activated to apply equibiaxial cell stretching forces to adherent cells attached to the microposts. Using the mPAM, we studied the live-cell subcellular dynamic responses of contractile forces in vascular smooth muscle cells (VSMCs) to a sustained static equibiaxial cell stretch. Our data showed that in response to a sustained cell stretch, VSMCs regulated their cytoskeletal (CSK) contractility in a biphasic manner: they first acutely enhanced their contraction to resist rapid cell deformation ('stiffening') before they allowed slow adaptive inelastic CSK reorganization to release their contractility ('softening'). The contractile response across entire single VSMCs was spatially inhomogeneous and force-dependent. Our mPAM device and live-cell subcellular contractile measurements will help elucidate the mechanotransductive system in VSMCs and thus contribute to our understanding of pressure-induced vascular disease processes.     

Mesh-integrated microdroplet array for simultaneous merging and storage of single-cell droplets

We constructed a mesh-grid integrated microwell array which enables easy trapping and consistent addition of droplets. The grid acts as a microchannel structure to guide droplets into the microwells underneath, and also provides open access for additional manipulation in a high-throughput manner. Each droplet in the array forms a stable environment of pico-litre volume to implement a single-cell-based assay.     

Microfluidic techniques for dynamic single-cell analysis

Dynamic single-cell analysis is a very important and frontier research field of single-cell analysis. Microfluidic techniques have become new and effective tools for precise, high-throughput, automatic analysis of single-cell dynamic process. This review aims to give an overview of dynamic single-cell analysis methods based on microfluidic platforms, with emphasis on the recent developments of microfluidic devices and its application to real-time dynamic monitoring of the signal molecules release from single living cell with temporal and spatial resolution, dynamic gene expression in single cells, the cell death dynamic events at the level of a single cell, and direct cell—cell communication between individual cell pairs.     

Polysulfone/pyrene membranes: a new microwell assay platform for bioapplications

The use of PSU‐Py prepared by click chemistry as a platform in membrane‐bottom microwell plates for oxidase and hydrolase/oxidase‐based enzyme assays is studied. For the GOx assay, the postulated fluorescence mechanism is based on the consumption of glucose by dissolved oxygen and GOx in the microwell plates covered with the PSU‐Py membrane. For the AG‐GOx assay, maltose is used as AG substrate and hydrolyzed to glucose which is then oxidized by the GOx activity. It is shown that the PSU‐Py membrane acts as a fluorescence indicator of the enzymatic reactions, and both GOx and AG/GOx enzyme assays are successfully applied for glucose, maltose and acorbose analysis in the range 0.125–2.0 × 10^?3 M glucose, 0.05–0.5 × 10^?3 M maltose, and 0.0125–0.1 mg · mL^?1 acorbose, respectively.

     

Flexible and stretchable electrodes for next generation polymer electronics: a review

Transparent conductive electrodes play a significant role in the fabrication and development of optoelectronic devices. As next generation optoelectronic devices tend towards mobile and wearable devices, the added attribute of flexibility or stretchability for these electrodes becomes increasingly important. However, mechanical requirements aside, transparent conductive electrodes must still retain high transparency and conductivity, with the metrics for these parameters being compared to the standard, indium tin oxide. In the search to replace indium tin oxide, two materials that have risen to the forefront are carbon nanotubes and silver nanowires due to their high transparency, conductivity, mechanical compliance, and ease of fabrication. This review highlights recent innovations made by our group in electrodes utilizing carbon nanotubes and silver nanowires, in addition to the use of these electrodes in discrete devices and integrated systems.     

A single-cell drug efflux assay in bacteria by using a directly accessible femtoliter droplet array

Active efflux of drugs, such as antibiotics, from a cell is one of the major mechanisms that cause multi-drug resistance in bacteria. Here we report a method to assess drug efflux activity in individual Escherichia coli cells enclosed and isolated in a directly accessible femtoliter droplet array with a fluorogenic compound. The inhibitory effect of a chemical compound on an exogenously expressed efflux pump system from pathogenic bacteria was easily detected at the single-cell level. We also present a proof-of-principle experiment to screen for the gene encoding a drug efflux pump by collecting individual droplets containing single cells in which the drug efflux activity was restored after introduction of the exogenous gene from pathogenic bacteria. Our approach will be a useful tool to screen novel pump inhibitors and efflux pump genes, and to overcome infectious diseases caused by multi-drug-resistant bacteria.     

Development of a microfluidic platform for single-cell secretion analysis using a direct photoactive cell-attaching method

A precise understanding of individual cellular processes is essential to meet the expectations of most advanced cell biology. Therefore single-cell analysis is considered to be one of possible approach to overcome any misleading of cell characteristics by averaging large groups of cells in bulk conditions. In the present work, we modified a newly designed microchip for single-cell analysis and regulated the cell-adhesive area inside a cell-chamber of the microfluidic system. By using surface-modification techniques involving a silanization compound, a photo-labile linker and the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer were covalently bonded on the surface of a microchannel. The MPC polymer was utilized as a non-biofouling compound for inhibiting non-specific binding of the biological samples inside the microchannel, and was selectively removed by a photochemical reaction that controlled the cell attachment. To achieve the desired single-macrophage patterning and culture in the cell-chamber of the microchannel, the cell density and flow rate of the culture medium were optimized. We found that a cell density of 2.0 × 10(6) cells/ml was the appropriate condition to introduce a single cell in each cell chamber. Furthermore, the macrophage was cultured in a small size of the cell chamber in a safe way for 5 h at a flow rate of 0.2 μl/min under the medium condition. This strategy can be a powerful tool for broadening new possibilities in studies of individual cellular processes in a dynamic microfluidic device.     

Plasticizer and catalyst co-functionalized PEDOT:PSS enables stretchable electrochemical sensing of living cells

Recently, stretchable electrochemical sensors have stood out as a powerful tool for the detection of soft cells and tissues, since they could perfectly comply with the deformation of living organisms and synchronously monitor mechanically evoked biomolecule release. However, existing strategies for the fabrication of stretchable electrochemical sensors still face with huge challenges due to scarce electrode materials, demanding processing techniques and great complexity in further functionalization. Herein, we report a novel and facile strategy for one-step preparation of stretchable electrochemical biosensors by doping ionic liquid and catalyst into a conductive polymer (poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate), PEDOT:PSS). Bis(trifluoromethane) sulfonimide lithium salt as a small-molecule plasticizer can significantly improve the stretchability and conductivity of the PEDOT:PSS film, and cobalt phthalocyanine as an electrocatalyst endows the film with excellent electrochemical sensing performance. Moreover, the functionalized PEDOT:PSS retained good cell biocompatibility with two extra dopants. These satisfactory properties allowed the real-time monitoring of stretch-induced transient hydrogen peroxide release from cells. This work presents a versatile strategy to fabricate conductive polymer-based stretchable electrodes with easy processing and excellent performance, which benefits the in-depth exploration of sophisticated life activities by electrochemical sensing.

A facile strategy for constructing stretchable sensors with excellent mechanical, electrochemical and biocompatible performance is developed, and in situ inducing and monitoring of stretch-evoked H₂O₂ release from cells has been successfully achieved.     

MEMS microwell and microcolumn arrays: novel methods for high-throughput cell-based assays

Although the cell-based assay is becoming more popular for high throughput drug screening and the functional characterization of disease-associated genes, most researchers in these areas do not use it because it is a complex and expensive process. We wanted to create a simple method of performing an on-chip cell-based assay. To do this, we used micro-electro-mechanical systems (MEMS) to fabricate a microwell array chip comprised of a glass substrate covered with a photoresist film patterned to form multiple microwells and tested it in two reverse transfection experiments, an exogenous gene expression study and an endogenous gene knockdown study. It was used effectively in both. Then, using the same MEMS technology, we fabricated a complementary microcolumn array to be used as a drug carrier device to topically apply drugs to cells cultured in the microwell array. We tested the effectiveness of microwell-microcolumn on-chip cell-based assay by using it in experiments to identify epidermal growth factor receptor (EGFR) activity inhibitors, for which it was found to provide effective high throughput and high content functional screening. In conclusion, this new method of cell-based screening proved to be a simple and efficient method of characterizing gene function and discovering drug leads.     

A modular single-cell pipette microfluidic chip coupling to ETAAS and ICP-MS for single cell analysis

Accurate single-cell capture is a crucial step for single cell biological and chemical analysis. Conventional single-cell capturing often confront operational complexity, limited efficiency, cell damage, large scale but low accuracy, incompetence in the acquirement of nano-upgraded single-cell liquid. Flow cytometry has been widely used in large-scale single-cell detection, while precise single-cell isolation relies on both a precision operating platform and a microscope, which is not only extre...     

QSNPlite, a software system for quantitative analysis of SNPs based on capillary array SSCP analysis

We present a newly developed software called "QSNPlite" that comprehensively interprets the data of SSCP and sequencing analyses obtained from capillary array electrophoresis systems used in the quantitative characterization of SNPs. QSNPlite assists in the genotyping of individuals with SNPs and in estimating the allele frequencies of SNPs using pooled DNA. We show that this estimation is accurate (mean absolute error, 1.4%) by comparing the results of the pooled analysis using QSNPlite with the true frequencies based on the allele counting after performing individual genotypings. The QSNPlite program runs on Windows XP and can be used to determine the allele frequencies of SNPs among a large number of individuals, such as in association studies of disease-responsible genes using the candidate gene approach.