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

Melting temperatures and recovery of native structure.

<p> <b>The recovery of native structure (rounds 1–3) was calculated from the total change in left and right ellipticity for each cycle of heating and cooling compared to the initial total ellipticity.</b></p

Affinity of sdAbs for <i>Bacillus anthracis</i> strains.

<p>Biotinylated sdAbs were tested for their ability to recognize a panel of <i>B. anthracis</i> strains from a number of geographic locations (acquired as irradiated spore preparation from NMRC).</p

Specificity of sdAbs for whole bacteria.

<p>Bacteria were either grown overnight as live cultures or obtained from either CRP or KPL as killed material. Equivalent optical densities were immobilized to microtiter dishes for analysis of specificity using a direct binding ELISA.</p

Immunoblot analysis of sdAb target protein.

<p><i>B. anthracis</i> Sterne strain cell and spore lysates were separated via SDS-PAGE for immunoblotting using each of the sdAbs.</p

Amino acid alignments of sdAb families.

<p>Families were identified based on amino acid alignments and variability in the complementarity determining regions (CDRs) bracketed in red boxes. Single representatives of each family are shown here. Clones d3x, d9x, G1, H4, and C8x are members of families that are not described in this study.</p

Specificity of sdAbs for <i>Bacillus</i> species spores.

<p>Intact and broken spores were passively immobilized to microtiter plates at equivalent optical densities to assess specificity of sdAbs.</p

Binding kinetics for sdAbs.

a<p>KD values are calculated from kd/ka.</p><p>NA = No binding observed.</p

Maximal current production of fuel cells inoculated with wild-type cells of <i>G. sulfurreducens</i> or strains with the designated genes deleted or the deleted genes complemented via expression of the designated gene on a plasmid.

<p>Values are the average±the standard deviation of triplicates.</p

Current production of <i>Geobacter sulfurreducens</i>.

<p>A. Current production time courses of wild type <i>G. sulfurreducens</i> grown entirely as current harvesting (arrow indicates the switch from original feed of 10 mM acetate to continual feed) and current production time course of fully grown fumarate control biofilms switched to current harvesting of wild type <i>G. sulfurreducens</i>. These data are representative time courses for multiple replicates of each treatment. B–C. Confocal scanning laser microscopy images of fumarate control swapped to current harvesting biofilms of <i>G. sulfurreducens</i> . Metabolically active (green) and inactive (red) cells where differentiated with a LIVE/DEAD kit based on the permeability of the cell membrane. B. 3-D projection, top view; C. slices through biofilm parallel to electrode large panel and perpendicular to electrode top and side panel.</p