Cell chip array for microfluidic proteomics enabling rapid in situ assessment of intracellular protein phosphorylation.
Ontology highlight
ABSTRACT:
SUBMITTER: Faley SL
PROVIDER: S-EPMC3112183 | biostudies-other | 2011 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:There is growing recognition that lipids play key roles in ion channel physiology, both through the dynamic formation and dissolution of lipid ion channels and by indirect regulation of protein ion channels. Because existing technologies cannot rapidly modulate the local (bio)chemical conditions at artificial bilayer lipid membranes used in ion channel studies, the ability to elucidate the dynamics of these lipid-lipid and lipid-protein interactions has been limited. Here we demonstrate a microfluidic system supporting exceptionally rapid perfusion of reagents to an on-chip bilayer lipid membrane, enabling the responses of lipid ion channels to dynamic changes in membrane boundary conditions to be probed. The thermoplastic microfluidic system allows initial perfusion of reagents to the membrane in less than 1 s, and enables kinetic behaviors with time constants below 10 s to be directly measured. Application of the platform is demonstrated toward kinetic studies of ceramide, a biologically important lipid known to self-assemble into transmembrane ion channels, in response to dynamic treatments of small ions (La(3+)) and proteins (Bcl-x(L) mutant). The results reveal the broader potential of the technology for studies of membrane biophysics, including lipid ion channel dynamics, lipid-protein interactions, and the regulation of protein ion channels by lipid micro domains.
| S-EPMC3343723 | biostudies-literature
Project description:The polymerase chain reaction (PCR) is a robust technique used to make multiple copies of a segment of DNA. However, the available PCR platforms require elaborate and time-consuming operations or costly instruments, hindering their application. Herein, we introduce a sandwiched glass-polydimethylsiloxane (PDMS)-glass microchip containing an array of reactors for the real-time PCR-based detection of multiple waterborne bacteria. The PCR solution was loaded into the array of reactors in a single step utilising capillary filling, eliminating the need for pumps, valves, and liquid handling instruments. Issues of generating and trapping bubbles during the loading chip step were addressed by creating smooth internal reactor surfaces. Triton X-100 was used to enhance PCR compatibility in the chip by minimising the nonspecific adsorption of enzymes. A custom-made real-time PCR instrument was also fabricated to provide thermal cycling to the array chip. The microfluidic device was successfully demonstrated for microbial faecal source tracking (MST) in water.
| S-EPMC6952809 | biostudies-literature
Project description:Portable, low-cost, and quantitative nucleic acid detection is desirable for point-of-care diagnostics; however, current polymerase chain reaction testing often requires time-consuming multiple steps and costly equipment. We report an integrated microfluidic diagnostic device capable of on-site quantitative nucleic acid detection directly from the blood without separate sample preparation steps. First, we prepatterned the amplification initiator [magnesium acetate (MgOAc)] on the chip to enable digital nucleic acid amplification. Second, a simplified sample preparation step is demonstrated, where the plasma is separated autonomously into 224 microwells (100 nl per well) without any hemolysis. Furthermore, self-powered microfluidic pumping without any external pumps, controllers, or power sources is accomplished by an integrated vacuum battery on the chip. This simple chip allows rapid quantitative digital nucleic acid detection directly from human blood samples (10 to 105 copies of methicillin-resistant Staphylococcus aureus DNA per microliter, ~30 min, via isothermal recombinase polymerase amplification). These autonomous, portable, lab-on-chip technologies provide promising foundations for future low-cost molecular diagnostic assays.
| S-EPMC5362183 | biostudies-literature
Project description:Several sophisticated microfluidic devices have recently been proposed for femtosecond laser axotomy in the nematode C. elegans for immobilization of the animals for surgery to overcome time-consuming and labor-intensive manual processes. However, nerve regeneration studies require long-term recovery of the animals and multiple imaging sessions to observe the regeneration capabilities of their axons post-injury. Here we present a simple, multi-trap device, consisting of a single PDMS (polydimethylsiloxane) layer, which can immobilize up to 20 animals at the favorable orientation for optical access needed for precise laser surgery and high-resolution imaging. The new device, named "worm hospital" allows us to perform the entire nerve regeneration studies, including on-chip axotomy, post-surgery housing for recovery, and post-recovery imaging all on one microfluidic chip. Utilizing the worm hospital and analysis of mutants, we observed that most but not all neurodevelopmental genes in the Wnt/Frizzled pathway are important for regeneration of the two touch receptor neurons ALM and PLM. Using our new chip, we observed that the cwn-2 and cfz-2 mutations significantly reduced the reconnection possibilities of both neurons without any significant reduction in the regrowth lengths of the severed axons. We observed a similar regeneration phenotype with cwn-1 mutation in ALM neurons only.
| S-EPMC5575036 | biostudies-literature
Project description:Cancer is a dynamic disease involving constant changes. With these changes, cancer cells become heterogeneous, resulting in varying sensitivity to chemotherapy. The heterogeneity of cancer cells plays a key role in chemotherapy resistance and cancer recurrence. Therefore, for effective treatment, cancer cells need to be analyzed at the single-cell level by monitoring various proteins and investigating their heterogeneity. We propose a microfluidic chip for a single-cell proteomics assay that is capable of analyzing complex cellular signaling systems to reveal the heterogeneity of cancer cells. The single-cell assay chip comprises (i) microchambers (n = 1376) for manipulating single cancer cells, (ii) micropumps for rapid single-cell lysis, and (iii) barcode immunosensors for detecting nine different secretory and intracellular proteins to reveal the correlation among cancer-related proteins. Using this chip, the single-cell proteomics of a lung cancer cell line, which may be easily masked in bulk analysis, were evaluated. By comparing changes in the level of protein secretion and heterogeneity in response to combinations of four anti-cancer drugs, this study suggests a new method for selecting the best combination of anti-cancer drugs. Subsequent preclinical and clinical trials should enable this platform to become applicable for patient-customized therapies.
| S-EPMC8541572 | biostudies-literature
Project description:A hypoxic (low oxygen level) microenvironment and nitric oxide paracrine signaling play important roles in the control of both biological and pathological cell responses. In this study, we present a microfluidic chip architecture for nitric oxide delivery under a hypoxic microenvironment in human embryonic kidney cells (HEK-293). The chip utilizes two separate, but interdigitated microfluidic channels. The hypoxic microenvironment was created by sodium sulfite as the oxygen scavenger in one of the channels. The nitric oxide microenvironment was created by sodium nitroprusside as the light-activated nitric oxide donor in the other channel. The solutions are separated from the cell culture by a 30 µm thick gas-permeable, but liquid-impermeable polydimethylsiloxane membrane. We show that the architecture is preliminarily feasible to define the gaseous microenvironment of a cell culture in the 100 µm and 1 mm length scales.
| S-EPMC7692389 | biostudies-literature
Project description:Coral reefs, and the unique ecosystems they support, are facing severe threats by human activities and climate change. Our understanding of these threats is hampered by the lack of robust approaches for studying the micro-scale interactions between corals and their environment. Here we present an experimental platform, coral-on-a-chip, combining micropropagation and microfluidics to allow direct microscopic study of live coral polyps. The small and transparent coral micropropagates are ideally suited for live-imaging microscopy, while the microfluidic platform facilitates long-term visualization under controlled environmental conditions. We demonstrate the usefulness of this approach by imaging coral micropropagates at previously unattainable spatio-temporal resolutions, providing new insights into several micro-scale processes including coral calcification, coral-pathogen interaction and the loss of algal symbionts (coral bleaching). Coral-on-a-chip thus provides a powerful method for studying coral physiology in vivo at the micro-scale, opening new vistas in coral biology.
| S-EPMC4785229 | biostudies-literature
Project description:This paper presents a microfluidic instrument capable of quantifying single-cell specific intracellular proteins, which are composed of three functioning modules and two software platforms. Under the control of a LabVIEW platform, a pressure module flushed cells stained with fluorescent antibodies through a microfluidic module with fluorescent intensities quantified by a fluorescent module and translated into the numbers of specific intracellular proteins at the single-cell level using a MATLAB platform. Detection ranges and resolutions of the analyzer were characterized as 896.78⁻6.78 × 10⁵ and 334.60 nM for Alexa 488, 314.60⁻2.11 × 10⁵ and 153.98 nM for FITC, and 77.03⁻5.24 × 10⁴ and 37.17 nM for FITC-labelled anti-beta-actin antibodies. As a demonstration, the numbers of single-cell beta-actins of two paired oral tumor cell types and two oral patient samples were quantified as: 1.12 ± 0.77 × 10⁶/cell (salivary adenoid cystic carcinoma parental cell line (SACC-83), ncell = 13,689) vs. 0.90 ± 0.58 × 10⁵/cell (salivary adenoid cystic carcinoma lung metastasis cell line (SACC-LM), ncell = 15,341); 0.89 ± 0.69 × 10⁶/cell (oral carcinoma cell line (CAL 27), ncell = 7357) vs. 0.93 ± 0.69 × 10⁶/cell (oral carcinoma lymphatic metastasis cell line (CAL 27-LN2), ncell = 6276); and 0.86 ± 0.52 × 10⁶/cell (patient I) vs. 0.85 ± 0.58 × 10⁶/cell (patient II). These results (1) validated the developed analyzer with a throughput of 10 cells/s and a processing capability of ~10,000 cells for each cell type, and (2) revealed that as an internal control in cell analysis, the expressions of beta-actins remained stable in oral tumors with different malignant levels.
| S-EPMC6265747 | biostudies-literature
Project description:Diatom frustules are a type of natural biomaterials that feature regular shape and intricate hierarchical micro/nano structures. They have shown excellent performance in biosensing, yet few studies have been performed on flow-through detection. In this study, diatom frustules were patterned into step-through holes and bonded with silicon substrate to form an open-ended filtration array. Then they were fixed into a microfluidic chip with a smartphone-based POCT. Human IgG and FITC-labeled goat-anti-human IgG were adopted to investigate the adsorption enhancement when analyte flowed through diatom frustules. The results indicated up to 16-fold enhancement of fluorescent signal sensitivity for the flow-through mode compared with flow-over mode, at a low concentration of 10.0 μg/mL. Moreover, the maximum flow rate reached 2.0 μL/s, which resulted in a significant decrease in the testing time in POCT. The adsorption simulation results of diatom array embedded in the microchannel shows good agreement with experimental results, which further proves the filtration enrichment effect of the diatom array. The methods put forward in this study may open a new window for the application of diatom frustules in biosensing platforms.
| S-EPMC8469004 | biostudies-literature
Project description:Immunoassay is one of the important applications of microfluidic chips and many methodologies were reported for decreasing sample∕reagent volume, shortening assay time, and so on. Micro-enzyme-linked immunosorbent assay (micro-ELISA) is our method that utilizes packed microbeads in the microfluidic channel and the immunoreactions are induced on the beads surface. Due to the large surface-to-volume ratio and small analytical volume, excellent performances have been verified in assay time and sample∕reagent volume. In order to realize the micro-ELISA, one of the important processes is the immobilization of antibody on the beads surface. Previously, the immobilization process was performed in a macroscale tube by physisorption of antibody, and long time (2 h) and large amount of antibody (or high concentration) were required for the immobilization. In addition, the processes including the reaction and washing were laborious, and changing the analyte was not easy. In this research, we integrated the immobilization process into a microfluidic chip by applying the avidin-biotin surface chemistry. The integration enabled very fast (1 min) immobilization with very small amount of precious antibody consumption (100 ng) for one assay. Because the laborious immobilization process can be automatically performed on the microfluidic chip, ELISA method became very easy. On-demand immunoassay was also possible just by changing the antibodies without using large amount of precious antibodies. Finally, the analytical performance was investigated by measuring C-reactive protein and good performance (limit of detection <20 ng∕ml) was verified.
| S-EPMC2967239 | biostudies-other