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IMPORTANT: PDMS based miniaturized micro-cell counters using electrical detection technology

Development in the area of Bio-Microelectromechanical (Bio-MEMS) research aims at performing multiple biotechnological steps on chip. One of the important applications of this research is single cell analysis requiring fast and high throughput cell counting and size sorting stations. Both electrical and optical detection methods have been used in such applications. With optical methods, particles are excited at specific wavelengths and fluorescence intensities are monitored at specific emission wavelengths. Electrical methods are based on the Coulter principle, which records impedance of current by the passage of a particle through a narrow pore. The advantage of a fluorescence-based assay is the ability of integrating known fluorescent markers specific to the cells being studied. The coulter principle on the other hand is considered to be the most reliable method of estimating cell volume. When required, both methods can be effectively combined.
In this project we will use PDMS based soft lithography technology to fabricate a fluidic channel. We shall optimize the channel dimension using the flow pattern and electrical current impedance of micron sized beads. We plan to test either hydrodynamic flow focussing or physical focussing using small features in the same flow channel based on the particle size. The detection module will be based on small signal manipulation using lockin technique. Metal electrode (aluminium or gold) pads will be used as sensing electrodes to detect conductivity changes. Once the flow and the detection techniques are optimized using micron size beads, we shall test biologically relevant cell types. We shall use CHO (Chinese hamster ovary) cells (15 microns in size) and Drosophila imaginal disc cells, which are approximately 3 to 5 microns in size. CHO cells will be used as a testing bed for the technique, since they are larger are readily detectable under low magnification microscopes. Imaginal discs are epithelial sac like structures that grow within the fruit fly (Drosophila melanogaster) larva and give rise to the fly’s adult structures, such as the wings, legs, etc. Imaginal disc growth is the subject of study of many labs, the world over. They form an ideal system to study tissue growth in vivo. To aid these studies, it will become extremely useful to develop reliable methods of estimating cell size and number during larval growth stages. One of the disadvantages of the system is the small size of each tissue. However, in this laboratory methods have been developed that allow the preparation of cell suspensions of single imaginal-discs. It is expected that these cell suspensions can be run through a micro-counter for analysis. The aim is to develop micro-counters that can estimate cell number per given volume, and the distribution of cell sizes in each preparation.