In situ investigations of biological molecules using vibrational sum-frequency-generation spectroscopy

Abstract

The molecular-level understanding of biological molecules on solid surfaces is critical in areas including medicine, biologically-based industry, and the development of biotechnologies. In order to gain further knowledge of the orientation and organization of biological molecules adsorbed on surfaces, we used the label-free, interface-specific technique of sum-frequency generation (SFG) spectroscopy. This technique has the distinct advantage of being able to be operated in situ as well as ex situ, allowing for direct comparison of changes in biological molecules between these two states. Films of surface-bound single-stranded DNA (ssDNA) on gold were chosen as model biological systems due to their numerous applications in genetic profiling, nano-assembly, and bio-computing, as well as their relative simplicity as biomolecular layers. Sensitivity and proof-of-principle tests on simple surface-bound, short alkane chains demonstrated the ability of SFG spectroscopy to detect molecular concentrations low enough to be useful in the investigation of biological molecules and to accurately detect the interactions of water with model hydrophobic and hydrophilic self-assembled monolayers. Investigations of multilayers of thymine, adenine, and cytosine nucleobases alone revealed a high degree of order in the thymine layers, with the signals from the methyl group unique to this base clearly visible. Films of both thiolated and non-thiolated surface-bound DNA in air showed little and moderate orientation, respectively, with the methylene stretches of the sugar-phosphate backbone dominating the spectra. Comparison of the changes in signal intensity among thymine, adenine, and cytosine ssDNA films in air and in H2O revealed differences in their solubility, which agreed with current ex situ knowledge of the manner in which these differing DNA types adsorb on gold surfaces. These experiments also revealed the appearance of nucleobase-specific spectra upon exposure to water, which was tied to the higher mobility of the sugar-phosphate backbone under these conditions. Investigations of hybridized ssDNA films in air using SFG spectroscopy indicated that the hybridization process in surface-bound DNA molecules does not necessarily correspond with an increase in molecular order, as is known to happen with DNA molecules in solution, and furthermore that even gentle processing of such hybridized samples for ex situ analysis can significantly disrupt the hybrid structure. These results were confirmed using near-edge absorbtion fine structure spectroscopy. Finally, the results obtained from the model DNA films were applied to a more complex biomolecule, fibronectin, on gold surfaces. Experiments showed that SFG spectroscopy could detect a fibronectin film even under a layer of fixed cells. Further tests on living cells over alkanethiol self-assembled monolayers confirmed this observation. These results give new information on the orientation and organization of DNA films on solid surfaces both in and ex situ, and show how this knowledge can be applied to more complex biological systems. Furthermore, this work contributes to a knowledge base for the application of SFG spectroscopy to future questions in which the label-free, in situ knowledge of surface-bound biological molecules is of critical importance.