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MICROFLUIDIC SYSTEM FOR HIGH-THROUGHPUT PROTEOMICS

Séverine Le Gac,1 Christian Rolando,1 Stephen Arscott,1,2 Christian Druon,2 Pierre Tabourier2
USTL, FR

Keywords: microfluidics, microfabrication, integrated proteomics

Abstract:
The comparative study of a cell type whole proteome in normal, pathological conditions or under the administration of drugs, which is called differential proteomics, appears as a keytool to understand diseases. To reach this goal, news analytical tools, which are faster, more sensitive and compatible with high-throughput analysis are required. Recently, microsystems have emerged to answer these demands and have been therefore highly developed.
 
This work deals with the development of a microfluidic device for the preparation of protein samples prior to their analysis by electrospray mass spectrometry. Using microsystems in this context favours fast and high-throughput analysis for their identification and comparison of different cell states. Actually, this device is a new tool dedicated to integrated proteomics. Our device includes three main modules, (i) an internal micropump, (ii) an elution column, (iii) an integrated nanoESI nozzle, whose realization reflects our technological choices. The overall device is realized in silicon for its many advantages compared to glass particularly in terms of etching characteristics. Liquids flow through 10µm channels etched in silicon; these channels are closed on their top by a Pyrex® plate bound on the silicon by anodic bonding techniques. First generation microsystems have allowed us to assess basic principles of fabrication such as wet etching and anodic bonding. These microsystems have been connected to a QqTOF mass spectrometer (Q-Star PULSTAR®, Applied Biosystems) through a commercial nanoESI PicoTip® source.
 
To avoid analysis problems induced by salt addition, we have opted for a novel and nonelectrophoretic pump, contrary to most of the microsystems described in the literature. The pumping system here relies on dielectrophoretical principles. The stationary phase chosen for the elution column is a macroporous monolith, which is synthesized in situ. Monoliths present many advantages over other classical techniques like the widely used silica particles : (i) no frits are required, (ii) spatial control is ensured with a photochemical polymerization initiation, (iii) mass transfer resistance is lower and (iv) their morphology, functionality and porosity are easily adjustable. Moreover, these monoliths may also serve as support for enzyme immobilization in a digestion cell. Preliminary studies were performed with in UVtransparent capillaries (i.d. 50-100µm) prepared monoliths, which enables their connection to a LC Packings® nano-LC allowing the retention properties, porosity, mass transfer resistance optimization before their downscaling to microsystems. The nanoESI nozzle is fabricated using microtechnology techniques and mimics classical nano-ESI tips made from pulled glass or fused-silica.
 
The integration of these different modules is under investigation and will be presented.

NSTI Nanotech 2003 Conference Technical Program Abstract

 
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