Ziegler, Jörg. Towards single cell proteomics. 2013, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_10317
In biochemical sensors binding events are detected, when target molecules diffuse close enough to interact with specific recognition elements. To develop a fast and sensitive immunosensor, we benefit from short diffusion times and capillarity in microchannels. We fabricated an on-chip immunochemical surface assay which is performed within a microfluidic system. Using such a chip, the concentration of CRP in human blood serum was determined within eleven minutes. We were able to detect less than 1 ng/mL of CRP using only 1 microlitre of sample. To further reduce the sample volume towards single cells, we first structured surfaces with nanometer-sized patterns to separate, handle and culture individual cells. Pillar arrays with a height of 1 micrometer, aspect ratios of 1:5 and a top diameter of 120 nm were fabricated in silicon and were used as a master to produce a PDMS intermediate on which PLLA replicas were casted. At an inter-pillar distance of 200 nm, we could show how individual cells grow along the lines of cones replicated in PLLA. To handle the liquid content of individual cells and to detect single molecules within such heterogeneous analytes, we developed a method to prepare total content sample for electron microscopy. The method combines microfluidic-based in-line negative staining for TEM as well as desalting for mass measurements by STEM. The main advantages are the lossless sample preparation by liquid contact writing of micro-patterns on EM grids and excellent staining at physiological pH. To detect low molecular weight single molecules label-free with a very high specificity, we propose to immobilize arrays of single DARPins on a very flat surface and to discriminate their bound and unbound state by height measurements using AFM. Arrays of immobilization islands for single DARPins were fabricated by EUV-IL, EBL and a newly developed direct immobilization method. By EUV-IL and a glancing angle metal deposition step 20 nm sized gold immobilization islands could be fabricated. By EBL and thermal annealing, arrays of 5 nm sized gold islands have been achieved. These islands are in the size range of single DARPin molecules. To functionalize only the gold islands with DARPins, the surrounding silicon dioxide surface has to be protected against non-specific DARPin adsorption. However PEG molecules for efficient passivation are often to long and the small immobilization islands might be buried by PEG. Therefore we used a photoresist mask and a chemical linker to directly immobilize single DARPins onto silicon dioxide. On the same chip, the pattern size of the mask was varied and besides several mm sized lines full of DARPins, arrays of single immobilized DARPins could be produced. On such arrays, single binding events between DARPins and their corresponding target proteins were detected and bound and unbound DARPins could be discriminated. The developed methodologies and the engineered surfaces are promising tools for the analysis towards single cell proteomics and their further development might result in valuable methods for systems biology.
Keywords: microfluidics, pillar arrays, nano-dot array, single cell, cell growth, AFM, TEM, STEM, XIL, EBL, GLAD, thermal annealing, immunoassay, DARPin.
|Committee Members:||Plückthun, Andreas|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Structural Biology (Engel)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||145 S.|
|Last Modified:||30 Jun 2016 10:52|
|Deposited On:||06 Mar 2013 15:11|
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