Article

Cell-based biosensors based on light-addressable potentiometric sensors for single cell monitoring.

Biosensor National Special Laboratory, Key Laboratory of Biomedical Engineering of the Ministry of Education, Department of Biomedical Engineering, PO Box 1590, Zhejiang University, Hangzhou 310027, China.
Biosensors & Bioelectronics (Impact Factor: 6.45). 04/2005; 20(9):1757-63. DOI: 10.1016/j.bios.2004.06.037
Source: PubMed

ABSTRACT Cell-based biosensors incorporate cells as sensing elements that convert changes in immediate environment to signals for processing. This paper reports an investigation on light-addressable potentiometric sensor (LAPS) to be used as a possible cell-base biosensor that will enable us to monitor extracellular action potential of single living cell under stimulant. In order to modify chip surface and immobilize cells, we coat a layer of poly-L-ornithine and laminin on surface of LAPS chip on which rat cortical cells are grown well. When 10 microg/ml acetylcholine solution is administrated, the light pointer is focused on a single neuronal cell and the extracellular action potential of the targeted cell is recorded with cell-based biosensor based on LAPS. The results demonstrate that this kind of biosensor has potential to monitor electrophysiology of living cell non-invasive for a long term, and to evaluate drugs primarily.

1 Bookmark
 · 
193 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: New methods for functional assays of chemical receptors are highly essential for the research of chemical signal transduction mechanisms and for the development of chemical biosensors. This study described a novel bioengineered cell-based biosensor for label-free functional assays of chemical receptors by localized extracellular acidification measurement with a light-addressable potentiometric sensor (LAPS). A human taste receptor, hT2R4, and an olfactory receptor of Caenorhabditis elegans (C. elegans), ODR-10, were selected as models of chemical receptors, which were expressed on the plasma membrane of human embryonic kidney (HEK)-293 cells. The specific ligand binding function of expressed chemical receptors was monitored by localized extracellular acidification measurement using LAPS chip with a movable focused laser illuminating on the desired single cell. The function of expressed olfactory receptors was further validated using MDL12330A, which can specifically inhibit the activity of adenylyl cyclase. The obtained results indicate that both of chemical receptors were successfully expressed in HEK-293 cells and can be functionally assayed by this bioengineered cell-based biosensor that shows dose-dependent responses to the target ligands of chemical receptors. This bioengineered cell-based biosensor exhibits the sensitivity of 1.0mV/s for hT2R4 assays, and 9.8mV/s for ODR-10 assays. The negative control cells without any chemical receptor expression show no response to all the chemical stimuli tested. All the results demonstrate this bioengineered cell-based biosensor can be used to detect the interactions between chemical receptors and their ligands. This provides a valuable and promising approach for label-free functional assays of chemical receptors as well as for the research of other GPCRs.
    Biosensors & Bioelectronics 11/2013; 54C:623-627. · 6.45 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Lab on a chip (LOC) systems provide interesting and low-cost solutions for key studies and applications in the biomedical field. Along with microfluidics, these microdevices make single-cell manipulation possible with high spatial and temporal resolution. In this work we have designed, fabricated and characterized a versatile and inexpensive microfluidic platform for on-chip selective single-cell trapping and treatment using laminar co-flow. The combination of co-existing laminar flow manipulation and hydrodynamic single-cell trapping for selective treatment offers a cost-effective solution for studying the effect of novel drugs on single-cells. The operation of the whole system is experimentally simple, highly adaptable and requires no specific equipment. As a proof of concept, a cytotoxicity study of ethanol in isolated hepatocytes is presented. The developed microfluidic platform controlled by means of co-flow is an attractive and multipurpose solution for the study of new substances of high interest in cell biology research. In addition, this platform will pave the way for the study of cell behavior under dynamic and controllable fluidic conditions providing information at the individual cell level. Thus, this analysis device could also hold a great potential to easily use the trapped cells as sensing elements expanding its functionalities as a cell-based biosensor with single-cell resolution.
    Biosensors & Bioelectronics 05/2014; 61C:298-305. · 6.45 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: For monitoring of human cellular response to repetitive bacterial stimulations (e.g., Pseudomonas aeruginosa in a lysate form), we devised a chemiluminescent immuno-analytical system for toll-like receptor 1 (TLR1) as marker present on cell surfaces (e.g., A549). Upon stimulation, TLR1 recognizes pathogen-associated molecular patterns of the infectious agent and are then up-regulated via activation of the nuclear factor-κB (NF-κB) pathway. In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder. The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum. The major factors affecting activation were the stimulation dose of the bacterial lysate, stimulation timing during starvation, and up- and down-regulation time intervals. Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution. This immuno-analysis for TLRs could be unique to acquire accumulated response of the human cells to repeated stimulations and, therefore, can eventually apply to persistency testing of the cellular regulation in screening of anti-inflammatory substances.
    Scientific Reports 08/2014; 4(6011). · 5.08 Impact Factor