Biomolecular detection has applications in many fields, including environmental monitoring, food quality monitoring, hazardous gas detection, and medical diagnostics. Three volatile analytes are of particular interest; trimethylamine (TMA) is associated with seafood spoilage and Trimethylaminuria [1], acetic acid (AA) is a biomarker for asthma [2], and ammonia (NH₃) is a biomarker for protein degradation [3]. Detection of these molecules requires a highly sensitive and highly specific sensing platform. For the purpose of detecting these molecules, a microcantilever is designed with a nanostructured surface and is functionalized with odorant-binding peptides.

The hammerhead microcantilever shown in Figure 1 was designed and fabricated at the Georgia Institute of Technology. The hammerhead shape provides greater surface area and causes the cantilever to resonant in-plane. Nanostructure is fabricated on the microcantilever surface in order to increase the sensitivity of the device. Silicon dioxide nanostructure is created using glancing angle deposition (GLAD) and increases the overall surface area, allowing more molecules to be captured. Images of the nanostructure taken using a high-resolution scanning electron microscope are shown in Figure 2 and 3.

The odorant-binding peptides are attached to the SiO₂ surface using aminosilane. Validation of the surface chemistry was performed by attaching biotin-streptavadin with a fluorescence tag and viewing under a Fluorescein Isothiocyanate (FITC) light microscope. Further validation is performed by attaching a fluorescence tag to the ammonia-binding peptide and introducing it to a SiO₂ surface. Additionally, Raman spectroscopy was used to show the attachment of the silane. The peptides are modeled using the PEP-FOLD servers [4], and binding interactions are modeled using the AutoDock routines in the PyRx software, Figure 4.

A scanning Kelvin probe (SKP) is used to demonstrate a charge differential between the flat surface and nanostructure-coated surface. In addition, a charge differential is shown between a surface that is functionalized with peptides and one that is not. This charge differential can be used as a numerical indication of the success of the nanostructure and surface chemistry strategies.

The improvement in sensor surface area with added 250 nm nanostructure is estimated to be on the order of a 12-fold increase. BET with Krypton analysis is being used to confirm the amount of nanostructured surface area for both the 250 and 600 nm nanostructure additions.

Peptide-functionalized nanostructured cantilevers are being tested for specific VOC molecular binding characteristics. The relevant parameter is the change (decrease) in resonance frequency with added molecular mass.

Acknowledgements

Research supported by NSF Award #1445488 to KMH. Additional support for RJM provided by the University of Dayton Graduate School.

References

[1] R. A. Chalmers, M. D. Bain, H. Michelakakis, J. Zschocke, R. A. Iles, “Diagnosis and management of trimethylaminuria (FMO3 deficiency) in children,” Journal of Inherited Metabolic Disease, vol. 29, pp. 162-172, 2006.

[2] R. M. Effros, R. Casaburi, J. Su, M. Dunning, J. Torday, J. Biller, R. Shaker, “The effects of volatile salivary acids and bases on exhaled breath condensate pH,” American Journal of Respiratory and Critical Care Medicine, vol. 173, pp. 386-392, 2006.

[3] G. Neri, A. Lacquaniti, G. Rizzo, N. Donato, M. Latino, M. Buemi, “Real-time monitoring of breath ammonia during haemodialysis: use of ion mobility spectrometry (IMS) and cavity ring-down spectroscsopy (CRDS) techniques,” Nephrology Dialysis Transplantation, vol. 27, pp. 2945-2952, 2012.

[4] Shen Y, Maupetit J, Derreumaux P, Tufféry P. “Improved PEP-FOLD approach for peptide and miniprotein structure prediction” J. Chem. Theor. Comput. 2014; 10:4745-4758

Thévenet P, Shen Y, Maupetit J, Guyon F, Derreumaux P, Tufféry P. “PEP-FOLD: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides.” Nucleic Acids Res. 2012. 40, W288-293.