Harshitha, Chadalavada (2015) Characterization of In-house Developed Electroporator. BTech thesis.
Electroporation is defined as an amplification in the electrical permeability and conductivity of the external cell membrane created by an externally applied electric field. At present substantial quantities of Electroporators are in the business sector, but they prove to be noneconomical and cumbersome to use. In the current project, an in-house slide based, low cost electroporator was designed, fabricated and characterized. Briefly, a circular well of diameter 25 mm was made within a thick glass slide. It serves as a cuvette for the electroporator. The well was connected to electrodes made up of copper for supplying high voltage electrical impulse. The electronics of in-house electroporator comprised of five circuits.Firstly, a 555 Timer circuit to generate low amplitude pulses. Secondly, a 7805 regulator circuit to stabilize the voltage.Thirdly, a 12 Volt relay circuit to switch on/off the pulse generation. Fourth, the Microcontroller, 89C52 which can be programmed to control the pulses.Finally,a Transformer circuit with 1:100 turns ratio inorder to magnify pulse intensity necessary for the poration of the outer cell membrane. Thus, a wide range of variations in pulse amplitude and duration enables the Electroporator to be suitable for different cell types and molecules of different sizes. In the second phase, this Electroporator was characterized for precision in delivery of high voltage pulse for a short duration so that the cells would remain viable. The cells used were NIH3T3 Mouse embryonic fibroblast cells (NCCS,Pune) and the viability was estimated using MTT assay. The cells were first cultured.By keeping the pulse duration constant, the amplitude was varied. Next, keeping the the amplitude constant, the pulse duration was varied. In each case, MTT assay test was carried out for the cells which have experienced the electrical impulse. At each condition, the cells remained significantly viable. The cells were found to be most viable when subjected to 752V pulsating DC voltage with pulse duration 5 µs. The in-house electroporator was found to be effective in delivering the required voltage for required duration. Such a device would reduce the work process in research centers proper characterization for transfection.
|Item Type:||Thesis (BTech)|
|Uncontrolled Keywords:||Electroporation, 555 Timer Circuit, Voltage, Pulse Width, Cell Culture, MTT Assay|
|Subjects:||Engineering and Technology > Biomedical Engineering|
|Divisions:||Engineering and Technology > Department of Biotechnology and Medical Engineering|
|Deposited By:||Mr. Sanat Kumar Behera|
|Deposited On:||25 May 2016 10:16|
|Last Modified:||25 May 2016 10:16|
|Supervisor(s):||Nayak, B P|
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