The interaction of biomolecules with gold nanoparticles: from amine-driven binding to covalent capture

The aim of this PhD thesis was to study possible ways of interaction between gold nanoparticles (AuNPs) and biological molecules. I have approached this problem in two ways: a) by addressing the interaction of non-functionalized nanoparticles with a molecule through the Au-N “bond” formation; b) by functionalizing AuNPs with proper ligands able to “covalently capture” a target protein. In spite of the fact that most protein interact with “naked” AuNPs through the amino groups present on their surface, very little is known about the nature of this interaction. The Au-N interaction is much weaker than the most popular Au-S one. Because of this, I have set up experiments aimed at addressing i) how the Au-N interaction correlates with the properties of an amino group (its pKa, level of substitution); ii) the possible mode of bindings of amino groups to the nanoparticles and its dependence on the AuNPs size; iii) the kinetics of these processes. I have used ethanol as the solvent throughout most of my experiments because, being a less competitive solvent than water, it allowed me to more precisely tune the strength of the Au-N interaction. Of course, this choice will require, at a later stage, to check the transferability of the information acquired to the natural biological solvent. The results showed that there is a very good correlation between amine pKa and its affinity to the AuNPs surface and that there are at least three different modes of binding of the amines. They are characterized by quite different kinetics and percentages of amines bound to the gold nanoparticle surface. The Brönsted plots of the logarithm of the apparent affinity constants of the amines for the AuNPs surface vs their pKa were linear with slopes in the -0.4 - -0.6 interval indicating that the same properties that control the interaction of an amine with a proton control also that with the surface Au atoms. The ability of amines to interact with the AuNPs surface has prompted me to study the interaction of peptides presenting free amines at their termini. Thus, I have discovered that peptide sequences functionalized with primary amines at the N- and C-terminus are able to induce the aggregation of AuNPs in ethanol following their folding into a helical conformation. Random coil peptides are unable to induce such an aggregation process. I have observed that the aggregation can be monitored spectrophotometrically by following the shift of the surface plasmon resonance (SPR) band of the nanoparticles and confirmed denaturation of the peptides results in diminished crosslinking ability. I then examined how the helicity parameter 222/208 correlates with the shift of the SPR band to longer wavelength and I found a reasonably good linear correlation. All the date I have obtained support the existence of a relationship between the amount of helical content of a peptide sequence and its ability to induce aggregation of the AuNPs. I also have tried to find mild passivating agents, soluble in an aqueous solution, that could be easily replaced by more stable ones in a controlled way. The most obvious choice was to rely on the Au-N bond in view of the expertise I had acquired on the matter. By using glucosamine phosphate (GAP) as a natural and inexpensive passivating agent, quite serendipitously, I discovered that this compound was leading to the formation of nanowires. Indeed, in an aqueous solution devoid of any surfactant, I was able to obtain, under aerobic conditions and substoichiometric nanoparticle passivation (i. e. the concentration of passivating agent is lower than the concentration of surface Au atoms), Au-nanowires of controlled length and reasonably narrow dimensional distribution starting from AuNPs. Since the challenge of obtaining plasmonic nanosystems absorbing light in the near infrared is always open because of the interest that such systems pose in applications such as nanotherapy or nanodiagnostics, I explored more in detail the initial results I had obtained. I discovered that oxygen was required to induce the process and that glucosamine phosphate was oxidized to glucosaminic acid phosphate and H2O2 was also produced. I could establish that the process leading to the nanosystems comprises nanoparticles growth, their aggregation into necklace-like aggregates, and the final fusion into nanowires. Control experiments in an anaerobic environment confirmed that the fusion requires the consumption of H2O2. I could passivate the nanowires with an organic thiol, lyophilize and resuspend them in water without losing their dimensional and optical properties. By adjusting the length of the nanowires, I could also tune the position of their broad surface plasmon band in the range 630 to >1350 nm. Finally, I have started investigating the way to functionalize AuNPs with a suitable targeting agent to obtain the “covalent capture” of an enzyme. The strategy requires the preparation of a thiolated molecule featuring an irreversible inhibitor of the target protein. I have selected as my target Urokinase, also called urinary plasminogen activator (uPA or u-PA), a serine protease. Elevated expression levels of urokinase have been found to be correlated with tumor malignancy. This makes urokinase an attractive drug target, and, so, inhibitors have been sought to be used as anticancer agents. Based on published results, concerning the structure of the irreversible inhibitor, The chemical structure of Ligand 2 can be divided into four parts: 1) the hydrophilic oligo(ethylene glycol) moiety (blue) bearing thiol, as a water soluble linker to be anchored on the nanoparticles surface; 2) general linker (orange), which acts as a building block for coupling the capture unit; 3) inhibitor unit, which ensures high selectively for the target protein; 4) capture unit that leads to phosphorylation of serine. I have also used Ligand 1 assuming that it should be able, when used to co-passivate the AuNPs to tune their solubility in water and prevent, in a biological fluid, unspecific interactions with plasma proteins.