August 8, 2003
Often, biochemists wish they had twenty pairs of hands so they can quickly perform a large number of basic laboratory tests known as assays, on their enzyme sample. These particular tests are quite crucial because they help determine whether their sample still retains its original biological function or has changed into something else.
In an attempt to fully automate this labor-intensive type of testing, scientists and engineers have begun to devise simple-to-use bioanalytical devices called biochips (sometimes described as laboratories on chips), which allows one to carry out hundreds of these assays simultaneously, using very small amounts of material. Our University of Chicago researchers, Milan Mrksich and Stephen Kron, here at the Materials Research Center, and their collaborators, have been hard at work improving the existing biochip fabrication methodology.
Specifically, they have designed a generally applicable, yet highly selective method for patterning a biochip with “active ingredients” (known as ligands). Their central innovation involves the use of the Diels-Alder reaction to immobilize the active species to the self-assembled monolayer (aka SAM-see nugget on dynamic substrates for more info) on the biochip surface (see figure 1). This particular Diels-Alder reaction involves the cyclization of an electrochemically accessible species known as a benzoquinone derivative. This allows one to monitor the extent of incorporation of the active species on the biochip surface. While in previous biochips, one was never sure exactly how much of the active ingredient was accessible for testing, this methodology allows one to precisely determine how much of the active species has been incorporated. And each of the active species are fully accessible for reaction, and exactly one molecule deep.
In addition, Prof. Mrksich and Kron chose a special substrate (the unreactive material making up most of the surface of the biochip where the reaction takes place) for the chip, oligo-ethylene glycol derivatives, which resists non-specific interactions with the chip. A small concentration of these oligomers are derivatized with the benzoquinones which can later undergo the Diels Alder reaction. Because the same inert oligomeric backbone is used for the substrate as well as for the functionalized species, a very regular and uniform spatial arrangement of the active ingredients on the chip can be achieved. Achieving this very high degree of uniformity allowed the researchers to characterize reactions occurring on the biochip surface quantitatively.
In a demonstration of their new fabrication methodology, Prof. Mrksich and Kron deliberately patterned their standard biochip with a number of different ligands, each of which has been shown to react specifically with an enzyme or protein. A number of inert ligands which were not expected to react with nor bind to any of the targeted enzymes or proteins were also included on the chip for comparison (see figure 2).
After dipping the patterned chip in several solutions of different proteins or enzymes, the researchers observed that, in each case, selective binding or selective reaction occurred on the biochip. But only those sites that were specifically designed to react with that particular protein or enzyme reacted. This was evidenced by observing fluorescence (in general, a distinct glow under UV light) from the incorporation of labeled protein or enzyme, exactly where the researchers predicted that it should occur (see figure 3). The cells, which were expected to be inert to the particular protein or enzyme, indeed, did not react, and thus did not fluoresce, confirming the effectiveness of their strategy.
This result represents two important advances in the short history of bio-chips. First, the strategy used to incorporate active groups on the new bio-chips seems to occur very specifically; few side reactions with other chemical groups on the bio-chip are observed. Secondly, while other biochips have experienced problems with “sticky” proteins adsorbing to the materials used to construct the bio-chips, the U of C researchers use of the inert ethylene-glycol oligomers as a substrate minimized these types of side reactions. Thus, the need for complicated blocking procedures (which restrict the chips range of applicability) characteristic of previous biochips are eliminated.
Bio-chips have dramatically increased the range and scope of discovery for biochemists. In fact, some bio-chips have already been utilized in attempts to more readily identify the presence of certain medical conditions which can be detected using biochemical assays. The new bio-chips developed here at University of Chicago are expected provide an even more reliable and cost-effective method for biochemists to perform these and other tests. Their very general method of incorporating active ingredients on the biochip, compatibility with standard commercial equipment, and the absence of side reactions and other processes which can hinder the chip’s range of application, promises rapid development of applications for the U of C peptide chip.
Figure 3 represents a superposition of images of the results (each represented by a different color) of separately dipping identically fabricated biochips in three different solutions. Red: anti-phosphotyrosine antibody bound to phosophotyrosine (position 2). Green: anti-biotin antibody bound to biotin (position 11). Blue: lectin concanavalin A bound to carbohydrates positions 12 and 13).
References:
- "Peptide Chips for the Quantitative Evaluation of Protein Kinase Activity " Benjamin T. Houseman, Joon H. Huh, Stephen J. Kron, Milan Mrksich. Nature Biotechnology. v20, March 2002.
