ACS PRF | ACS
All e-Annual Reports
42161-GB5
Nanoparticle Platforms for Controlled Adsorption and Behavior in Protein Monolayer Electrochemistry
Our original proposal focused on utilizing nanoparticle films to create more effective protein monolayer electrochemistry (PME) systems. PME is a strategy for studying fundamental interactions of biomolecules at interfaces and involves the attachment of proteins to synthetic surfaces called self-assembled monolayers (SAMs) in order to study their adsorption and electroactivity.[1-3] Unfortunately, PME has certain limitations including a lack of molecular-level control over interactions at the protein/surface interface.[4] In PME, proteins experience a range of surface environments and display non-ideal electrochemistry, a phenomenon easily observed with cytochrome c (Cc) at carboxylic acid SAMs.[5,6] We proposed a novel, alternative platform for PME using specialized nanoparticles called Monolayer-Protected Clusters (MPCs).[7] MPCs are targeted because their properties can be easily tailored to exhibit specific and diverse molecular-level properties, including hydrophobic, coulombic, and interfacial flexibility properties – important factors for effective protein adsorption.[8-11] Our hypothesis, that MPCs can be engineered, tethered to a surface, and used to promote and control the immobilization and adsorbed behavior of proteins.
In any PME system, including the MPC films investigated here, there are two major areas of interest: the modifying layer on the electrode and the protein/substrate interface. The former contributes to the background charging current, while the latter is a key component of the analytical signal from the protein, the faradaic current.[12] Our previous progress report showed that the background signal is highly dependent on the architecture of the MPC film. Specifically, the use of covalent linkages featuring low levels of polarizable functional groups allow for a MPC film with an inherently lower dielectric constant. Films linked in this manner were better able to discriminate against the background charging current. In the second year of our study,† we focused our attention on the electron transfer signal from adsorbed Cc. A specific goal of the research was to determine if rational design of individual MPCs would translate into greater molecular level control at the protein adsorption interface while still generating voltammetry with acceptable signal-to-noise ratios.
As a first step in exploring this aspect of the MPC platforms, we altered the number of protein binding ligands per MPC in the outermost layer of the film before exposure to Cc. Specifically, the ligand composition of the MPCs in the interfacial layer was systematically manipulated to exhibit a range of carboxylic acid terminated alkanethiols which electrostatically immobilize Cc. The voltammetry of Cc at these various MPC films, along with the calculated surface concentration clearly indicate that the protein coverage, as well as voltammetric resolution, drastically improves with increasing number of acid groups incorporated into the MPCs. While not surprising, this result does suggest an ability to control the Cc voltammetry via simple manipulation of the properties of the nanoparticles prior to their incorporation into a film.
Similar experiments were conducted by altering the chainlength of the non-binding ligands in the periphery of individual MPCs. As the chainlengh of the non-binding ligands is increased, the adsorbed Cc exhibits a greater rate of denaturation over time. This observation is consistent with an increase in interfacial hydrophobicity that subsequently causes the protein to unfold upon adsorption. This second set of experiments again establishes that properties of the interface can be directly influenced by engineering the nanoparticles being assembled into films. The signal-to-noise of the Cc voltammetry as well as the stability of the adsorbed protein layer are both highly dependent on the properties of the interfacial layer – a parameter readily controlled during the MPC design phase.
As the main research project of my group, this work has been both successful and popular with students. With Howard Hughes Medical Institute, Dreyfus Foundation, and U. of Richmond Arts & Sciences summer research fellowships supplementing the PRF funding, six undergraduate researchers have significantly contributed to this project. Each student has performed multiple semesters or, in some cases, multiple summers of largely independent research, a fact that is a testament to the project's draw and successful student participation. This past summer was extremely fruitful in terms of experimental results and students learning how to conduct scientific research. Students gained unprecedented self-confidence and a sense of independence in the laboratory. Significant results have been achieved highlighting several interesting aspects of using nanomaterials in PME strategies and prompting us to recently submit the first of two manuscripts on these systems. I am very proud of our efforts on this project and look forward to continued success over the next year (†we have requested and been granted a one year extension on this grant). The next phase of this research will focus on using MPCs to address the inherent non-ideality normally observed in traditional PME systems where protein is directly adsorbed to a self-assembled monolayer modified electrode.[4-6,11]
