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Study of enzyme interactions on microstructured surfaces

Thomas Wilhelm*, Gunther Wittstock

Wilhelm-Ostwald-Institut for Physical and Theoretical Chemistry, University of Leipzig,
Leipzig D-04103 Germany

This is an abstract for a presentation given at the
Eighth Foresight Conference on Molecular Nanotechnology.
There will be a link from here to the full article when it is available on the web.

 

Miniaturisation of analytical systems has been a prevailing trend for the last decades. This trend includes electrochemical sensors and assays which have to meet new requirements set up by high-throughput testing and parallel processing. While the last years have seen considerable progress in DNA chip technology, an equally versatile technology for more delicate enzymes and antibodies is still missing. For the development of this technology, knowledge about enzymatic interactions in microstructured systems is required. Amongst these interactions are enzymatic reaction chains.

Scanning electrochemical microscopy (SECM)[1] offers the possibility to create or modify microstructured enzymatic activity on surfaces and to directly image its functionality using the same instrument. In this work, a reaction chain of the two enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) is examined as a model for multi enzyme microstructures and the effect of structure geometry on the interaction is shown.

GOx oxidises glucose:

glucose + oxygen -> gluconolactone + hydrogen peroxide

HRP reduces hydrogen peroxide:

hydrogen peroxide + ferrocene -> water + ferrocinium

SECM probe reduces ferrocinium:

ferrocinium + electron -> ferrocene

The SECM offers an elegant way to directly image the functionality and local reactivity on surfaces in buffered solutions. For this purpose, the generation-collection mode[2] is well suited: in this mode the probe (collects) oxidises or reduces compounds which are generated by the enzymatic reaction and diffuse from the surface into the solution. Thus, in a reaction chain the activity of HRP, which can be imaged by detection of ferrocinium at the probe, depends on the presence of glucose as a substrate for GOx.

Microstructuring of enzymes can be done with different techniques: soft lithography[3], e.g. microcontact printing, offers an easy approach to parallel large-area modification. The technique can either be used to transfer the enzyme directly onto the surface or indirectly by stamping spacers or inhibitors, followed by modification of the active areas.

Additional tip-induced structuring techniques like local electrochemical desorption[4] in the direct mode of the SECM (in this mode, a potential is applied directly between probe and sample) or the placement of enzyme-modified magnetic microbeads[5] can be used to add further elements under soft conditions which do not harm the existing enzyme activity. The great flexibility of these approaches allows to form a variety of geometries, whose functional characterisation can be performed with another working mode in the same experimental setup.

References:

[1] Bard AJ, Fan F-RF, Mirkin MV (1994) in: Bard AJ (ed) Electroanalytical Chemistry, vol.18, Marcel Dekker, New York Basel Hong Kong, pp. 244-370
[2] Wilhelm T, Wittstock G, Szargan R (1999) Fres J Anal Chem 365: 163-167
[3] Xia YN, Whitesides GM (1998) Angew Chem Int Ed Engl 37: 551-575
[4] Wilhelm T, Wittstock G (2000) Microchim Acta 133: 1-9
[5] Wijayawardhana CA, Wittstock G, Halsall HB, Heinemann WR, Electroanalysis 12: 640-644


*Corresponding Address:
Thomas Wilhelm
Wilhelm-Ostwald-Institut for Physical and Theoretical Chemistry, University of Leipzig,
Linnéstr. 2,
Leipzig D-04103 Germany,
fax: +49-341-9736399,
email: root@secm.de, twilhelm@rz.uni-leipzig.de
web: http://www.secm.de



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