Development of an Electrochemical Cellular Chip for Real-time Monitoring of Single-cell Secretion

Autor: Ching-Yu Chang, 張景裕
Rok vydání: 2009
Druh dokumentu: 學位論文 ; thesis
Popis: 97
In this study, we intended to develop a chip which can be used for single-cell manipulation and analysis. A microwell electrode (MWE) which was constructed by sequential microfabrication processes was proposed to meet these requirements. With the cooperation of an Indium Tin Oxide (ITO) cover on the top of the chip, a vertical electrophoretic force was employed to trap/repel the living cells into/out the MWE with a DC voltage biased between MWE and ITO cover. Moreover, a flow channel was defined by a double-side adhesive tape, and thus a hydraulic force was used to remove the additional trapped cells around the MWE. By a cooperation of the electrophoretic and hydraulic forces, an individual cell can be accommodated inside the addressed MWE. According to the simulation results, the depth of the MWE (30 �慆 diameter) was optimized to be 25 �慆, and then the trapped cell can be stabilized in a sufficient low flow velocity area (LFVA) in a continuous flow. A technique for single-cell manipulation was successfully developed. The electrochemical characteristics of the MWE were investigated with potassium ferricyanide (K3Fe(CN)6), and then the steady-state current was measured by calculating the difference between baseline and diffusion-limit current in a sigmoid voltammogram. The effective electrode radius was estimated based on the theorem of ultramicroelectrode (UME) with a given constants, known K3Fe(CN)6 concentration, and the measured steady current. The calculated radius was found to be in a good agreement with the design value. A positive feedback phenomenon was observed when the ITO cover was attached onto the MWE, and thus an enhanced current was measured. However, the enhanced factor was not consistent with a theoretical value; the difference was supposed to the uncertainty of the tape thickness. ALP-beads (10 �慆 diameter), with alkaline phosphatase (ALP) coating, was employed to validate the sensitivity of the MWE for trace chemical detection. The amperograms (+0.3 V vs. Ag/AgCl) of a MWE with blank and ALP-bead were compared in p-aminophenylphosphate (PAPP) solution. A larger current response was found in the amperogram of ALP-bead, and the elevated current suggested an evidence of trace catalytic reaction by the ALP-bead. The sensitivity of the MWE to a trace signal was confirmed to be applicable for the detection of single-cell secretion. The MWE was employed for the living cell detection, and three kinds of cells including pSEAP-HeLa, KU-812, and neutrophil were tested. The related findings and results were illustrated as the followings: (1) The recombinant pSEAP-HeLa cell which can continuously secret the secreted alkaline phosphatase (SEAP) was measured in PAPP solution. The secretion activity was real-time monitored by an amperogram (+0.3 V vs. Ag/AgCl), and a wave-like current was found in the detected amperogram. The secreted SEAP was supposed to catalyze the PAPP substrate to produce the wave-like current, and provided an evidence of cell secretion. (2) KU-812, a human basophilic cell line, was employed for the exocytosis detection. The exocytosis of histamine vesicles was induced with high K+ solution, and then a spiked amperogram (+0.3 V vs. Ag/AgCl) was detected. (3) Primary neutrophils which were isolated from peripheral blood of volunteers were measured with the MWE to detect the reactive oxygen species (ROS) and myeloperoxidase (MPO) after phorbol 12-myristate 13-acetate (PMA) stimulation. Amperogram of the activated neutrophil was monitored in phosphate buffer saline (PBS) solution, and the ROS release was confirmed by the spiked current. Voltammograms and amperograms of the activated neutrophil in 3, 3’, 5, 5’-tetramethylbenzidine (TMB) solution verified the MPO release. A significant redox current was observed in the voltammogram of activated cells, and several reductive spiked current appeared in the amperogram when the MWE was biased at -0.25 V. The MWE device presents some feasible applications for the single-cell manipulation and analysis. The manipulating technique can be employed to address the functionalized beads which were coated with enzyme, antibody, or DNA on a specific position, and then the problem of addressable modification in conventional method can be solved. Moreover, the MWE provides an effective method for the preparation of cell array. This device also demonstrates possibilities to integrate the chip with a confocal microscopy, and the combined technology might provide a simultaneous detection of intra- and extracellular species.
Databáze: Networked Digital Library of Theses & Dissertations
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