University of Twente Student Theses


Parallel sampling from individual cells on a microchip : towards a parallel single cell analysis platform

Brink, E.T.G. van den (2011) Parallel sampling from individual cells on a microchip : towards a parallel single cell analysis platform.

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Abstract:Cell populations are heterogeneous: processes are not synchronized in a cell population and individual cells are at different stages of the cell cycle, for instance. Consequently, conventional analysis methods provide averaged information about the cell population as an ensemble and this does not give useful information about the state of individual cells. A single cell analysis approach looks more attractive in that respect; however, the analysis of a single cell in a population appears to be a biased approach as one cannot extrapolate this information to the state of the population. Therefore, a more relevant approach consists of analyzing cells of a population in an individual manner, so as to collect information not only at the single cell level but also at the population level. This approach reveals the population heterogeneity, which is thought to be indicative of disease development. In this work, a microfluidic platform is described for this purpose. This microchip is intended as a first prototype to enable proof-of-principle experiments towards actual parallel single cell analysis. The whole analysis process consists of four steps. First, individual living cells are trapped individually in a controlled and reproducible way. Second, the plasma membrane is permeabilized, either transiently or irreversibly. Third, the cell content of individual cells is extracted in a controlled way and fourth, the analysis is performed on the extracted biomolecules. A PDMS microsystem is developed to perform the first three steps of the analysis protocol. The microsystem contains an array of 16 trapping structures for the immobilization of 16 individual cells in parallel by accurate application of a negative pressure across these structures. A single cell trapping efficiency of > 90% is demonstrated with the aforementioned protocol and optimal dimensions of the trapping structures. Trapping is fast, controllable, reproducible, efficient and scalable. Cells are permeabilized through their exposure to a plug of chemicals, such as digitonin (3.5 min incubation) for reversible permeabilization or lithium dodecylsulphate (LiDS) for irreversible lysis (10-20 s exposure). Cell permeabilization is monitored via the release of calcein out of the cells and the entry of PI, two membrane-impermeable dyes. Interestingly, the way the cell is trapped has a high impact on this permeabilization step, while the cell trapping mode cannot be controlled. Alternatively, cell lysis is demonstrated using an electric field; there, the cell trapping mode has no detectable influence on the permeabilization process. Finally, an electroosmotic flow (EOF) is established in the individual side channels located behind the trapped cells for extraction of the cell content in there. This last step is illustrated with the controlled extraction of calcein out of the cells, for both reversible and irreversible chemical permeabilization.
Item Type:Essay (Master)
Faculty:EEMCS: Electrical Engineering, Mathematics and Computer Science
Subject:53 electrotechnology
Programme:Electrical Engineering MSc (60353)
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