Functionalizing Nanoparticles with Biological Molecules: Developing Chemistries that Facilitate Nanotechnology

Functionalizing Nanoparticles with Biological Molecules: Developing Chemistries that Facilitate Nanotechnolog

Optimizing Protein Coordination to Quantum Dots with Designer Peptidyl Linkers

Semiconductor quantum dots (QDs) demonstrate select optical properties that make them of particular use in biological imaging and biosensing. Controlled attachment of biomolecules such as proteins to the QD surface is thus critically necessary for development of these functional nanobiomaterials. QD surface coatings such as poly­(ethylene glycol) impart colloidal stability to the QDs, making them usable in physiological environments, but can impede attachment of proteins due to steric interactions. While this problem is being partially addressed through the development of more compact QD ligands, here we present an alternative and complementary approach to this issue by engineering rigid peptidyl linkers that can be appended onto almost all expressed proteins. The linkers are specifically designed to extend a terminal polyhistidine sequence out from the globular protein structure and penetrate the QD ligand coating to enhance binding by metal-affinity driven coordination. α-Helical linkers of two lengths terminating in either a single or triple hexahistidine motif were fused onto a single-domain antibody; these were then self-assembled onto QDs to create a model immunosensor system targeted against the biothreat agent ricin. We utilized this system to systematically evaluate the peptidyl linker design in functional assays using QDs stabilized with four different types of coating ligands including poly­(ethylene glycol). We show that increased linker length, but surprisingly not added histidines, can improve protein to QD attachment and sensor performance despite the surface ligand size with both custom and commercial QD preparations. Implications for these findings on the development of QD-based biosensors are discussed

A New Family of Pyridine-Appended Multidentate Polymers As Hydrophilic Surface Ligands for Preparing Stable Biocompatible Quantum Dots

The growing utility of semiconductor quantum dots (QDs) in biochemical and cellular research necessitates, in turn, continuous development of surface functionalizing ligands to optimize their performance for ever more challenging and diverse biological applications. Here, we describe a new class of multifunctional polymeric ligands as a stable, compact and high affinity alternative to multidentate thiolated molecules. The polymeric ligands are designed with a poly­(acrylic acid) backbone where pyridines are used as anchoring groups that are not sensitive to degradation by air and light, along with short poly­(ethylene glycol) (PEG) pendant groups which are coincorporated for aqueous solubility, biocompatibility and colloidal stability. The percentages of each of the latter functional groups are controlled during initial synthesis along with incorporation of carboxyl groups which serve as chemical handles for subsequent covalent modification of the QD surface. A detailed physicochemical characterization indicates that the multiple pyridine groups are efficiently bound on the QD surface since they provide for relatively small overall hydrodynamic sizes along with good colloidal stability and strong fluorescence over a wide pH range, under high salt concentration and in extremely dilute conditions at room temperature under room light over extended timeframes. Covalent conjugation of dyes and metal-affinity coordination with functional enzymes to the QD surfaces were also demonstrated. Biocompatibility and long-term stability of the pyridine polymer coated QDs were then confirmed in a battery of relevant assays including cellular delivery by both microinjection and peptide facilitated uptake along with intracellular single QD tracking studies and cytotoxicity testing. Cumulatively, these results suggest this QD functionalization strategy is a viable alternative that provides some desirable properties of both compact, discrete ligands and large amphiphilic polymers