"The addition of specific biomolecular information to the high-resolution topography recorded by the atomic force microscope is very important to the widespread use of AFM for biological imaging," Jason Hafner told nanotechweb.org. "Currently, conjugation of antibodies or other ligands to the tip through polymer linkers is the limiting step. We wanted to develop a simpler tip conjugation method."
Hafner and colleagues attached crystals of the enzyme lysozyme to AFM probe tips by a process of seeded nucleation. They scraped the tips of the cantilevers across 100 µm crystals of lysozyme. This attached small fragments of the crystals to the cantilevers. Bathing these fragments in a solution of the protein caused them to grow into crystals of the desired size. The researchers kept a careful eye on the whole process using ×5 magnification in an optical microscope with dark field illumination.
After depositing the protein crystals, the researchers used a solution of glutaraldehyde to cross-link the crystals and improve their stability in a range of environments. Prior to cross-linking the crystals dissolved in buffer solution.
When tested on an array of 5 nm diameter colloidal gold particles deposited on mica, the tip provided similar topographic resolution to a standard silicon nitride AFM tip.
"We have shown that cross-linked protein crystals are sufficiently stable to act as a tip material for AFM, and they retain their functional properties," said Hafner. "In addition, one can confirm that there are proteins at the tip by simple visual inspection through a low-power microscope. This method represents a new paradigm in AFM tip functionalization."
To examine the tip's molecular recognition properties, the scientists tested it on a mica surface coated with antilysozyme IgG antibody. They measured force curves at a loading rate of about 5 nN/s, before blocking the antibody sites by adding a free lysozyme solution. The adhesion force was more than 100 pN before blocking of the antibody sites, but dropped to a mean of less than 50 pN after blocking.
Hafner said that they were aiming to create a new tip technology for recognition AFM. "We are continuing along this direction," he said. "The paper demonstrates that cross-linked protein crystals are sufficiently stable for device applications, so we hope it also leads others to consider their use in applications such as cantilever sensing or microfluidic separations."
The team is also investigating the use of protein crystals for fundamental studies of the molecular recognition interaction. "Protein crystals are unique in that they hold the constituent molecules at a well defined orientation, but allow molecular fluctuations necessary for complex interactions," explained Hafner. "By measuring interactions between two crystals, rather than a crystal and an un-oriented protein layer, we may be able to measure recognition interactions as a function of molecular orientation."
The researchers reported their work in Nano Letters.