Gold-Nanoparticle-Mediated Depolarization of Membrane Potential Is Dependent on Concentration and Tethering Distance from the Plasma Membrane.

The photoactivation of plasma-membrane-tethered gold nanoparticles (AuNPs) for the photothermally driven depolarization of membrane potential has recently emerged as a new platform for the controlled actuation of electrically active cells. In this report, we characterize the relationship between AuNP concentration and AuNP-membrane separation distance with the efficiency of photoactivated plasma membrane depolarization. We show in differentiated rat pheochromocytoma (PC-12) cells that AuNPs capped with poly(ethylene glycol) (PEG)-cholesterol ligands localize to the plasma membrane and remain resident for up to 1 h. The efficiency of AuNP-mediated depolarization is directly dependent on the concentration of the NPs on the cell surface. We further show that the efficiency of AuNP-mediated photothermal depolarization of membrane potential is directly dependent on the tethering distance between the AuNP and the plasma membrane, which we control by iteratively tuning the length of the PEG linker. Importantly, the AuNP conjugates do not adversely affect cell viability under the photoactivation conditions required for membrane depolarization. Our results demonstrate the fine control that can be elicited over AuNP bioconjugates and establishes principles for the rational design of functional nanomaterials for the control of electrically excitable cells.

Quantum Dot-Peptide-Fullerene Bioconjugates for Visualization of in Vitro and in Vivo Cellular Membrane Potential.

We report the development of a quantum dot (QD)-peptide-fullerene (C60) electron transfer (ET)-based nanobioconjugate for the visualization of membrane potential in living cells. The bioconjugate is composed of (1) a central QD electron donor, (2) a membrane-inserting peptidyl linker, and (3) a C60 electron acceptor. The photoexcited QD donor engages in ET with the C60 acceptor, resulting in quenching of QD photoluminescence (PL) that tracks positively with the number of C60 moieties arrayed around the QD. The nature of the QD-capping ligand also modulates the quenching efficiency; a neutral ligand coating facilitates greater QD quenching than a negatively charged carboxylated ligand. Steady-state photophysical characterization confirms an ET-driven process between the donor-acceptor pair. When introduced to cells, the amphiphilic QD-peptide-C60 bioconjugate labels the plasma membrane by insertion of the peptide-C60 portion into the hydrophobic bilayer, while the hydrophilic QD sits on the exofacial side of the membrane. Depolarization of cellular membrane potential augments the ET process, which is manifested as further quenching of QD PL. We demonstrate in HeLa cells, PC12 cells, and primary cortical neurons significant QD PL quenching (ΔF/F0 of 2-20% depending on the QD-C60 separation distance) in response to membrane depolarization with KCl. Further, we show the ability to use the QD-peptide-C60 probe in combination with conventional voltage-sensitive dyes (VSDs) for simultaneous two-channel imaging of membrane potential. In in vivo imaging of cortical electrical stimulation, the optical response of the optimal QD-peptide-C60 configuration exhibits temporal responsivity to electrical stimulation similar to that of VSDs. Notably, however, the QD-peptide-C60 construct displays 20- to 40-fold greater ΔF/F0 than VSDs. The tractable nature of the QD-peptide-C60 system offers the advantages of ease of assembly, large ΔF/F0, enhanced photostability, and high throughput without the need for complicated organic synthesis or genetic engineering, respectively, that is required of traditional VSDs and fluorescent protein constructs.

Complex Förster energy transfer interactions between semiconductor quantum dots and a redox-active osmium assembly.

The ability of luminescent semiconductor quantum dots (QDs) to engage in diverse energy transfer processes with organic dyes, light-harvesting proteins, metal complexes, and redox-active labels continues to stimulate interest in developing them for biosensing and light-harvesting applications. Within biosensing configurations, changes in the rate of energy transfer between the QD and the proximal donor, or acceptor, based upon some external (biological) event form the principle basis for signal transduction. However, designing QD sensors to function optimally is predicated on a full understanding of all relevant energy transfer mechanisms. In this report, we examine energy transfer between a range of CdSe-ZnS core-shell QDs and a redox-active osmium(II) polypyridyl complex. To facilitate this, the Os complex was synthesized as a reactive isothiocyanate and used to label a hexahistidine-terminated peptide. The Os-labeled peptide was ratiometrically self-assembled to the QDs via metal affinity coordination, bringing the Os complex into close proximity of the nanocrystal surface. QDs displaying different emission maxima were assembled with increasing ratios of Os-peptide complex and subjected to detailed steady-state, ultrafast transient absorption, and luminescence lifetime decay analyses. Although the possibility exists for charge transfer quenching interactions, we find that the QD donors engage in relatively efficient Förster resonance energy transfer with the Os complex acceptor despite relatively low overall spectral overlap. These results are in contrast to other similar QD donor-redox-active acceptor systems with similar separation distances, but displaying far higher spectral overlap, where charge transfer processes were reported to be the dominant QD quenching mechanism.