Modified silicon-based surfaces provide good home to proteins

Nov 13 2006

Silicon, whether in its elemental form as silicon wafers or in its oxidized form, known as silicon dioxide or silica, is proving to be a valuable material for biomedical nanotechnology, in large part because of the biocompatibility and chemical inertness of these substances.

Now, two teams of investigators have shown that at the nanoscale, these two forms of silicon interact with proteins in ways that have practical applications in cancer screening and drug synthesis, among others.

Mauro Ferrari, Ph.D., of the University of Texas Health Sciences Center at Houston, and Lance Liotta, Ph.D., of George Mason University, led a collaborative effort aimed at using modified nanoengineered silicon as a substrate for constructing protein microarrays. While this current work aimed to evaluate how best to design the silicon surface for optimal array performance, the investigators are now testing silicon-substrate protein microarrays as tools for diagnosing cancer and predicting therapeutic outcome. The results of the design studies appear in the journal Biomaterials.

The majority of protein microarrays, including those that have shown some potential at diagnosing cancer, use nitrocellulose as the substrate to which array developers attach a variety of protein capture agents, such as antibodies and aptamers. When a mixture of proteins obtained from tumor biopsies is applied to one of these arrays, certain proteins stick to the capture agents and the analyst will visualize this binding using fluorescence techniques. Nitrocellulose, however, fluoresces to a small degree, and this background fluorescence limits the inherent sensitivity of such assays – a weak fluorescent signal from a capture protein may be indistinguishable from the background fluorescence emitted by the nitrocellulose.

To eliminate this background fluorescence, the investigators turned to nanoengineered silicon as the substrate for attaching protein capture agents. Silicon has no fluorescence, and experiments by this team showed clearly that there was no background signal observed even when a silicon surface was irradiated and imaged for five minutes. In contrast, nitrocellulose produced a noticeable background signal under the same conditions.

While native silicon will bind proteins to some extent, the researchers found that they needed to nanoengineer the surface of the silicon substrate in order to mimic the excellent protein-binding characteristics of nitrocellulose. The investigators used a technique known as reactive ion etching (RIE), which uses positive ions in a plasma to bombard a silicon surface over a range of times, followed by one of five chemical treatments to create a well-defined surface roughened by evenly distributed nanoscale pits. After comprehensive comparison of surface characteristics and protein-binding properties, the investigators found several combinations of RIE conditions and chemical surface treatment that would produce silicon surfaces with protein-binding properties similar to, and in a few cases superior to, those of nitrocellulose.

The goal of Eric Ackerman, Ph.D., and his colleagues at the Pacific Northwest Laboratory, was to not only create a substrate that would bind proteins, but one that would create a local environment that would allow them to retain their biological activity. To create such a material, Ackerman and his co-workers started with nanoporous silica, and their efforts led to the development of a material that actually increased the activity of the immobilized enzymes.

In the vast majority of cases, surface-immobilized enzymes have less activity than the free enzyme. Immobilized enzymes have potential use in pharmaceutical synthesis and as biosensors for disease-related proteins and biowarfare agents. This work appears in the journal Nanotechnology.

The researchers created their nanoporous silica supports through a combination of established procedures and new chemical techniques that they developed to add either a positive or negative charge to the pores. They then incubated the resulting nanoscale silica structures with a solution of the enzyme that they wanted to immobilize. The investigators measured the activity of the immobilized enzymes using standard assay technologies.

The results of the enzyme activity assays showed clearly that the local environment within the nanoscale pores had a beneficial effect on all three of the enzymes that the investigators studied. The researchers observed enhanced activity only when they immobilized enzymes within silica that had nanoscale pores that had received chemical treatment. Untreated nanoporous silica and chemically treated standard silica did not produce this enhancement. The researchers offered several potential explanations, but conceded that the mechanisms involved remain a mystery and need further study.

The work with nanoporous silica is detailed in a paper titled, “Characterization of functionalized nanoporous supports for protein confinement.” This paper was published online in advance of print publication. An abstract is available at the journal’s website. View abstract.

The work on modified silicon surfaces, which was funded by the National Cancer Institute, is detailed in a paper titled, “Physicochemically modified silicon as a substrate for protein microarrays.” This paper was published online in advance of print publication. An abstract is available through PubMed. View abstract.