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Inkjet-Printing Patterned Chip on Sticky Superhydrophobic Surface for High-Efficiency single-cell array Trapping and Real-Time Observation of cellular Apoptosis

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ACS APPLIED MATERIALS & INTERFACES 2018年第37期10卷 31054-31060页

作者： Sun, Yingnan Song, Wenhua Sun, Xiaohan Zhang, Shusheng Linyi Univ Shandong Prov Key Lab Detect Technol Tumor Makers Coll Chem & Chem Engn Linyi 276005 Shandong Peoples R China

single-cell assays have broad applications in cellular studies, tissue engineering, fundamental studies of cell cell interactions, and understanding of cell-to-cell variations. Most existing methods for micron-sized cell patterning are still based on lithography-based micro fabrication process. Thus, exploiting new mask-free strategies while maintaining high-precision single-cell patterning is still a great challenge. Here, we presented a facile, low-cost, and mask-free approach for constructing high-resolution patterning on sticky superhydrophobic (SH) substrates based on inkjet printing with ordinary precision. In this work, the SH surface with both high contact angle and relatively high contact angle hysteresis can not only obtain high-resolution spots but also avoid droplets bouncing behavior. We improved the feature size of printed protein spots as small as 4 mu m, which is much smaller than protein spots used for single-cell trapping. Moreover, with the assistance of a narrow microchannel, the inkjet-printing patterned chip with fibronectin ink allows for fast and high-efficiency trapping of multiple single-cell arrays. Using this method, single-cell occupancy could reach approximately 81% within 30 min on subcellular-sized patterning chip, and there was no significant effect on cell viability. As a proof of concept, this chip has been applied to study the real-time apoptosis of single cells and demonstrated the potential in cells' heterogeneity analysis.

关键词： microdroplet array superhydrophobic surface inkjet printing single-cell array

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single cell array Enclosed with a Photodegradable Hydrogel in Microwells for Image-Based cell Classification and Selective Photorelease of cells

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ACS APPLIED BIO MATERIALS 2020年第9期3卷 5887-5895页

作者： Yamaguchi, Satoshi Takagi, Risa Hosogane, Tsuyoshi Ohashi, Yuki Sakai, Yoko Sakakihara, Shouichi Iino, Ryota Tabata, Kazuhito, V Noji, Hiroyuki Okamoto, Akimitsu Univ Tokyo Dept Chem & Biotechnol Grad Sch Engn Tokyo 1138656 Japan Univ Tokyo Res Ctr Adv Sci & Technol Tokyo 1538904 Japan Osaka Univ Inst Sci & Ind Res Ibaraki Osaka 5670047 Japan Inst Mol Sci Okazaki Aichi 4448787 Japan Univ Tokyo Grad Sch Engn Dept Appl Chem Tokyo 1138656 Japan Japan Scieucc & Technol Agcy PRESTO Kawaguchi Saitama 3510198 Japan

single cell arrays provide an accurate classification of analyte cells through an image-based analysis of cellular phenotypes. Light-guided cell retrieval from a single cell array is a promising approach for the rapid and simple sorting of difficult to distinguish cells. In this study, we developed a single cell array enclosed with a photodegradable hydrogel in microwells to enable both comprehensive image-based single cell analysis and light-guided cell retrieval. In this system, individual cells became trapped in the microwells together with the photodegradable hydrogel at a high cell density on a chip regardless of cell type, adhesiveness, and motility. Fluorescence-stained model cells and vaccinated dendritic cells were identified by microscopic imaging and then selectively released through the light-induced degradation of the cell-embedding hydrogels. The target cells were selectively retrieved with a purity of >95% from the cell mixture through rapid photorelease, and the retrieved cells were confirmed to grow normally. Our results provide proof-of-principle that the photoresponsive microwell array serves as a versatile tool for image-based cell sorting in cellular researches and the manufacturing processes of high-performance cells.

关键词： single-cell array photodegradable lzydrogel microwell image-based cell sorting dendritic cell

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Fabrication of polyHEMA grids by micromolding in capillaries for cell patterning and single-cell arrays

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JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS 2015年第7期103卷 1375-1380页

作者： Ye, Fang Ma, Binghe Gao, Jie Xie, Li Wei, Chen Jiang, Jin Northwestern Polytech Univ Minist Educ Key Lab Micro Nano Syst Aerosp Xian 710072 Peoples R China Northwestern Polytech Univ Sch Mech Engn Xian 710072 Peoples R China Northwestern Polytech Univ Sch Life Sci Inst Special Environm Biophys Key Lab Space Biosci & Biotechnol Xian 710072 Peoples R China

Control of cell adhesion and growth by microfabrication technology and surface chemistry is important in an increasing number of applications in biotechnology and medicine. In this study, we developed a method to fabricate (2hydroxyethyl methacrylate) (polyHEMA) grids on glass by micromolding in capillaries (MIMIC). As a non-fouling biomaterial, polyHEMA was used to inhibit the nonspecific bonding of cells, whereas the glass surface provided a cell adhesive background. The polyHEMA chemical barrier was directly obtained using MIMIC without surface modification, and the microchannel networks used for capillarity were easily achieved by reversibly bonding the polydimethylsiloxane (PDMS) mold and the glass. After fabrication of the polyHEMA micropattern, individual cytophilic microwells surrounded by cytophobic sidewalls were presented on the glass surface. The polyHEMA micropattern proved effective in controlling the shape and spreading of cells, and square-shaped mouse osteoblast MC3T3-E1 cells were obtained in microwell arrays after incubation for 3 days. Moreover, the widths of the microwells in this micropattern were optimized for use as single-cell arrays. The proposed method could be a convenient tool in the field of drug screening, stem cell research, and tissue engineering. (C) 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 13751380, 2015.

关键词： MIMIC polyHEMA chemical micropattern cell patterning single-cell array

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On-chip technology for single-cell arraying, electrorotation-based analysis and selective release

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ELECTROPHORESIS 2019年第14期40卷 1830-1838页

作者： Keim, Kevin Rashed, Mohamed Z. Kilchenmann, Samuel C. Delattre, Aurelien Goncalves, Antonio F. Ery, Paul Guiducci, Carlotta Ecole Polytech Fed Lausanne Lab Life Sci Elect Stn 17STI CLSEBM2-107 CH-1015 Lausanne Switzerland

This paper reports a method for label-free single-cell biophysical analysis of multiple cells trapped in suspension by electrokinetic forces. Tri-dimensional pillar electrodes arranged along the width of a microfluidic chamber define actuators for single cell trapping and selective release by electrokinetic force. Moreover, a rotation can be induced on the cell in combination with a negative DEP force to retain the cell against the flow. The measurement of the rotation speed of the cell as a function of the electric field frequency define an electrorotation spectrum that allows to study the dielectric properties of the cell. The system presented here shows for the first time the simultaneous electrorotation analysis of multiple single cells in separate micro cages that can be selectively addressed to trap and/or release the cells. Chips with 39 micro-actuators of different interelectrode distance were fabricated to study cells with different sizes. The extracted dielectric properties of Henrietta Lacks, human embryonic kidney 293, and human immortalized T lymphocytes cells were found in agreements with previous findings. Moreover, the membrane capacitance of M17 neuroblastoma cells was investigated and found to fall in in the range of 7.49 +/- 0.39 mF/m(2).

关键词： Dielectrophoretic trapping Electrorotation single-cell analysis single-cell array single-cell release

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Microfluidic single-cell Trapping and Cultivation for the Analysis of Host-viral Interactions

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BIOTECHNOLOGY AND BIOPROCESS ENGINEERING 2021年第2期26卷 179-187页

作者： Ganguly, Reya Lee, Byungjin Kang, Solib Kim, Yong Sic Jeong, Seong-Geun Kim, Jae Seong Park, So Young Yohei, Yamauchi Lee, Chang-Soo Chungnam Natl Univ Dept Chem Engn & Appl Chem Daejeon 34134 South Korea Univ Bristol Sch Cellular & Mol Med Bristol Avon England

The isolation of single cells and their further cultivation in confined chambers are essential to the collection of statistically reliable temporal information in cell-based biological experiments. In this work, we present a hydrodynamic single-cell trapping and culturing platform that facilitates biological analysis and experimentation of virus infection into host cells. To find the optimum design of the cell trap at the microscale, we evaluated hook traps with different widths and trap intervals to obtain a high trapping efficiency of a single cell. The proposed design leverages the stochastic position of the cells as they flow into the structured microfluidic channels, where hundreds of single cells are then arrayed in nanoliter chambers for simultaneous cell-specific data collection. Optimum design is used to devise and implement a hydrodynamic cell-trapping mechanism that is minimally detrimental to the cell viability and retains a high trapping efficiency (90%), with the capability of reaching high fill factors (90%) in short loading times (10 min) in a 450-trap device. Finally, we perform an analysis of host-viral interactions under the treatment of a drug concentration gradient as a proof of concept.

关键词： single-cell analysis single-cell array hydrodynamic trapping linear drug gradient host-viral interaction

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A time-coded multi-concentration microfluidic chemical waveform generator for high-throughput probing suspension single-cell signaling

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Chinese Chemical Letters 2022年第6期33卷 3091-3096页

作者： Yiran Guo Peng Chen Zhaolong Gao Yiwei Li Shunji Li Xiaojun Feng Bi-Feng Liu The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory Systems Biology ThemeDepartment of Biomedical EngineeringCollege of Life Science and TechnologyHuazhong University of Science and TechnologyWuhan 430074China The Key Laboratory of Ministry of Education for Image Processing and Intelligent Control School of Artificial Intelligence and AutomationHuazhong University of Science and TechnologyWuhan 430074China

The cellular response to the complex extracellular microenvironment is highly dynamic in time and type of extracellular *** reconstructing this process and analyzing the changes in receptor conformation on the cell membrane surface and intracellular or intercellular signaling has been a major challenge in analytical chemistry and biophysical *** this paper,a time-coded multiconcentration microfluidic chemical waveform generator was developed for the dynamic signaling probing with single-cell array of high temporal resolution,high throughput,and multi-concentration combination *** on innovative microchannel structure,sophisticated external control methods and multiplexing technology,the system not only allowed for temporally sequential permutations of the four concentrations of stimuli(time code),but also generated pulsed and continuous waveforms at different frequencies in a highly controllable ***,the single-cell trap array was set up to efficiently capture cells in suspension,dramatically increasing throughput and reducing experiment preparation *** maximum frequency of the platform was 1 Hz,and one cell could be stimulated at multiple *** show the ability of the system to investigate rapid biochemical events in high throughput,pulse stimulation and continuous stimulation of different frequencies and different time codes,combined with four concentrations of histamine(HA),were generated for probing G protein-coupled receptor(GPCR)signaling in He La ***,statistical analysis was performed for the mean peak height and mean peak area of the cellular *** believe that the time-coded multi-concentration microfluidic chemical waveform generator will provide a novel strategy for analytical chemistry,biophysics,cell signaling,and individualized medicine applications.

关键词： Microfluidics Dynamic stimulation Time code High-throughput single-cell array

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A New cell Separation Method Based on Antibody-Immobilized Nanoneedle arrays for the Detection of Intracellular Markers

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NANO LETTERS 2017年第11期17卷 7117-7124页

作者： Kawamura, Ryuzo Miyazaki, Minami Shimizu, Keita Matsumoto, Yuta Silberberg, Yaron R. Sathuluri, Ramachandra Rao Iijima, Masumi Ruroda, Shun'ichi Iwata, Futoshi Kobayashi, Takeshi Nakamura, Chikashi Natl Inst Adv Ind Sci & Technol Biomed Res Inst Cent 51-1-1 Higashi Tsukuba Ibaraki 3058565 Japan Tokyo Univ Agr & Technol Dept Biotechnol & Life Sci 2-24-26 Naka Cho Koganei Tokyo 1848588 Japan Osaka Univ Inst Sci & Ind Res ISIR Sanken Dept Biomol Sci & React 8-1 Mihogaoka Ibaraki Osaka 5670047 Japan Shizuoka Univ Dept Mech Engn 3-5-1 Johoku Hamamatsu Shizuoka 4328561 Japan AIST Res Ctr Ubiquitous MEMS & Micro Engn 1-2-1 Namiki Tsukuba Ibaraki 3058564 Japan Saitama Univ Dept Chem Sakura Ku 255 Shimo Okubo Saitama 3388570 Japan

Focusing on intracellular targets, we propose a new cell separation technique based on a nano needle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nano needles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nano needles in the NNA. cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.

关键词： cell separation intracellular marker nanoneedle array single-cell array iPS cells high-throughput

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Evaluating single-cell DNA Damage Induced by Enhanced Radiation on a Gold Nanofilm Patch

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ACS APPLIED MATERIALS & INTERFACES 2017年第42期9卷 36525-36532页

作者： Qiao, Yong Zhou, Yuanshuai Xiao, Tongqian Zhang, Zhiwei Ma, Liyuan Su, Ming Suo, Guangli Chinese Acad Sci CAS Key Lab Nanobio Interface Suzhou Inst Nanotech & Nanobion Changzhou 215123 Jiangsu Peoples R China Northeastern Univ Dept Chem Engn Boston MA 02115 USA Wenzhou Med Univ Wenzhou Inst Biomat & Engn Wenzhou 325001 Zhejiang Peoples R China Univ Chinese Acad Sci Beijing 100049 Peoples R China

Although radiotherapy is a general oncology treatment and is often synergistically applied with surgery and chemotherapy, it can cause side effects during and after treatment. Gold nanoparticles were studied as a potential material to enhance radiation to induce damage in cancer cells. However, few studies have been conducted to examine the effects of gold nanofilm on cell impairment under X-ray treatment. This paper describes.a microfabrication-based single-cell array platform to evaluate DNA damage induced by enhanced X-ray radiation on gold nanofilm patches (GNFPs). Cancer cells were patterned on GNFPs of different diameters and thicknesses, where each cell was attached on one GNFP. The end-point DNA damage induced by X-ray was examined in situ at the single-cell level using a halo assay. The preliminary data demonstrated that the enhancement of DNA damage was significantly related to the area and thickness of the GNFP. This platform may be hopefully used to establish the mathematical relationships among DNA damage, X-ray dosage, and thickness and area of the GNFP, and further contribute to radiation dosage screening for personalized radiotherapy.

关键词： radiation therapy gold nanofilm patch single-cell array halo assay photoelectrons

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Micropatterning of single cell arrays using the PEG-Silane and Biotin-(Strept)Avidin System with photolithography and chemical vapor deposition

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SENSORS AND ACTUATORS B-CHEMICAL 2013年 188卷 340-346页

作者： Ren, Dahai Xia, Yiqiu Wang, Jun You, Zheng Tsinghua Univ Dept Precis Instrument Key Lab Precis Measurement Technol & Instruments Beijing 100084 Peoples R China

Bio-MEMS technique of organizing cells in single cell arrays makes it easier to observe cells' individual characteristics and behaviors, which is of benefit for basic cell research and high throughout drug screening. We utilized photolithography and chemical vapor deposition (CVD) to pattern hydrophobic hexamethyldisilazane (HMDS) islands and hydrophilic polyethylene glycol (PEG)-Silane regions on 25 mm 25 mm glass slides. Compared with methods presently used, the photoresist wells can be more stable, and clear HMDS island arrays can be formed. We adopted a better and more powerful medium, the Biotin-(Strept)Avidin System to fix cells on the specified regions of the substrate. This is more efficient than the former medium, antibodies and antigens. Moreover, using a biotinylating cell surface, we produced more biotins on the surface of the cells and made it easier to capture cells and avoid washing away fixed cells. By changing the concentration of cell suspension for seeding, we found a suitable concentration (5 x 10(6) cells/ml), at which the cell occupation was greater than 90%. By comparing various diameters of streptavidin islands, optimal diameters (14-20 mu m) were found to capture single human promyelocytic leukemia cells (HL-60). With all optimal parameters, single cell arrays were formed. The ratio of islands capturing only one cell was approximately 77%, which is better than similar approaches. (C) 2013 Elsevier B.V. All rights reserved.

关键词： Bio-MEMS single-cell array Photolithography Micropatterning Drug screening

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Receding meniscus induced docking of yeast cells inside microfluidic channels at single-cell level 10th

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World Congress on Medical Physics and Biomedical Engineering

作者： Park, Min Cheol Hur, Jae Young Kwon, Keon Woo Park, Jee Won Park, Sang-Hyun Suh, Kahp Y. Seoul Natl Univ Sch Mech & Aerosp Engn Seoul South Korea Seoul Natl Univ Sch Biol Sci & Res Cent Funct Cellul Seoul South Korea

ISBN: (纸本)9783540368397

We present a simple receding meniscus induced method to capture non-adherent yeast cells onto microwells inside microfluidic channels. Microwells were fabricated by capillary molding onto glass substrate using a UV curable polyurethane acrylate (PUA) solution, leading to well-defined, robust microstructures. A cell suspension of the budding yeast, Saccharomyces cerevisiae, was introduced into microfluidic channels by capillary filling and a receding meniscus was subsequently generated by evaporation. As the meniscus receded, one to five yeast cells were spontaneously captured onto microwells by lateral capillary force created at the thin region of the meniscus. Using this cell-based platform, we observed the response of yeast cells upon stimulation by a mating pheromone (alpha-factor) by monitoring the expression of green fluorescent protein (GFP) with time. It was observed that alpha-factor triggered the expression of GFP at 60 min after stimulation and the fluorescence intensity was sustained for additional 60 min without changes.

关键词： receding meniscus capillary molding lateral capillary force single-cell array green fluorescent protein

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