If you thought microarrays were tiny, brace yourself for the next small thing: nanoarrays. In last week’s Sciencexpress, Chad Mirkin and colleagues from Northwestern University and the University of Chicago describe their protein arrays with feature sizes of 100 to 350 nanometers, producing an array about 200,000 times more dense than the yeast proteome array most recently reported in the literature. Besides higher density and novel detection methods, these chips introduce “a fundamentally new way of studying biorecognition,” said Mirkin, director of Northwestern’s Institute for Nanotechnology.
The researchers used an atomic force microscope “like a quill pen” to deposit organic molecules with affinity for proteins in dots or grids on a gold layer. After passivating the remaining surface with another organic solution that repels proteins, they immersed the chip in a protein solution, so proteins stuck only to the features. The process goes by the name of dip-pen nanolithography, a type of soft lithography, and can also be used to array oligonucleotides or other types of biomolecules, Mirkin said.
With feature sizes in the range of the wavelength of light, optical detection methods can no longer be used, he said. “The good news here is that by going small, you can start using scanning probe methods, and screening the arrays based upon height and stickiness and shape changes that accompany a biomolecular binding event on one of the active features of the array,” said Mirkin. In the paper, he and his colleagues determined the binding of an antibody in a mixture of four different proteins to another antibody immobilized on the chip surface by measuring the increase in height using atomic force microscopy.
As an example of how the arrays could be used to study molecular recognition, the scientists immobilized a cell adhesion protein, added live cells and assayed for cellular binding, “proving that small features can support cell adhesion,” said Mirkin. In the future he wants to use the nanochips to find “anatural receptors” for biomolecules such as proteins, by screening them against arrays of simple organic molecules that together form a unique pattern. “If you wanted to develop new receptors for cells, you can make patterns using the dip-pen nanolithography process, and you can discover in combinatorial format what the best receptor is for a given biostructure of interest,” he said. These synthetic receptors may then be used “on any sort of signal transduction device,” he added.
Though the current study was just a proof of concept, “dip-pen nanolithography is now quickly becoming a routine type of technique, a technique that can be adopted by anybody,” Mirkin said. All a researcher needs is a conventional atomic force microscope with closed-loop scanning and a single commercially available type of tip. “It becomes easier and faster if you have access to software that we have,” he added. A Chicago-based company called NanoInk, of which Mirkin is a co-founder, is currently commercializing the dip-pen process, he said, “and they will be selling and distributing all of the writers and software within a three to six months time frame.” Also, the company wants to scale up the process from one pen to up to a thousand.
Mirkin sees the main proteomics applications of the technology in “very high density arrays and new screening procedures that get the information out quicker.” But even his nanoarrays are facing the same problems that other protein chips do: “There is always the issue of how the proteins react once they are deposited onto surfaces,” said Mirkin.