Femtosecond (fs) laser pulsed excitation of plasmonic nanoparticle (NP)−biomolecule conjugates is a promising method to locally heat biological materials. Studies have demonstrated that fs pulses of light can modulate the activity of DNA or proteins when attached to plasmonic NPs; however, the precision over subsequent biological function remains largely undetermined. Specifically, the temperature the localized biomolecules "experience" remains unknown. We used 55 nm gold nanoparticles (AuNPs) displaying double-stranded (ds) DNA to examine how, for dsDNA with different melting temperatures, the laser pulse energy fluence and bulk solution temperature affect the rate of local DNA denaturation. A universal "template" single-stranded DNA was attached to the AuNP surface, and three dye-labeled probe strands, distinct in length and melting temperature, were hybridized to it creating three individual dsDNA-AuNP bioconjugates. The dye-labeled probe strands were used to quantify the rate and amount of DNA release after a given number of light pulses, which was then correlated to the dsDNA denaturation temperature, resulting in a quantitative nanothermometer. The localized DNA denaturation rate could be modulated by more than threefold over the biologically relevant range of 8−53 °C by varying pulse energy fluence, DNA melting temperature, and surrounding bath temperature. With a modified dissociation equation tailored for this system, a "sensed" temperature parameter was extracted and compared to simulated AuNP temperature profiles. Determining actual biological responses in such systems can allow researchers to design precision nanoscale photothermal heating sources.

Schematic of a tetrameric β-galactosidase enzyme attached to and displaying 625 nm emitting QDs coated with a CL4 ligand via each of the 4 pendent His6 tags.

AuNCs engage in energy transfer by a non-Förster process although many of the same photophysical requirements are needed.

DNA nanotechnology has proven to be a powerful strategy for the bottom-up preparation of colloidal nanoparticle (NP) superstructures, enabling the coordination of multiple NPs with orientation and separation approaching nanometer precision. To do this, NPs are often conjugated with chemically modified, single-stranded (ss) DNA that can recognize complementary ssDNA on the DNA nanostructure. The limitation is that many NPs cannot be easily conjugated with ssDNA, and other conjugation strategies are expensive, inefficient, or reduce the specificity and/or precision with which NPs can be placed. As an alternative, the conjugation of nanoparticle-binding peptides and peptide nucleic acids (PNA) can produce peptide-PNA with distinct NP-binding and DNA-binding domains. Here, we demonstrate a simple application of this method to conjugate semiconductor quantum dots (QDs) directly to DNA nanostructures by means of a peptide-PNA with a six-histidine peptide motif that binds to the QD surface. With this method, we achieved greater than 90% capture efficiency for multiple QDs on a single DNA nanostructure while preserving both site specificity and precise spatial control of QD placement. Additionally, we investigated the effects of peptide-PNA charge on the efficacy of QD immobilization in suboptimal conditions. The results validate peptide-PNA as a viable alternative to ssDNA conjugation of NPs and warrant studies of other NP-binding peptides for peptide-PNA conjugation.

The interfacing of nanoparticle (NP) materials with cells, tissues, and organisms for a range of applications including imaging, sensing, and drug delivery continues at a rampant pace. An emerging theme in this area is the use of NPs and nanostructured surfaces for the imaging and/or control of cellular membrane potential (MP). Given the important role that MP plays in cellular biology, both in normal physiology and in disease, new materials and methods are continually being developed to probe the activity of electrically excitable cells such as neurons and muscle cells. In this Review, we highlight the current state of the art for both the visualization and control of MP using traditional materials and techniques, discuss the advantageous features of NPs for performing these functions, and present recent examples from the literature of how NP materials have been implemented for the visualization and control of the activity of electrically excitable cells. We conclude with a forward-looking perspective of how we expect to see this field progress in the near term and further into the future.

Supporting MethodsTransmission Electron Microscopy. TEM was carried out using a JEOL 2200-FX analytical high-resolution transmission electron microscope at 200 kV accelerating voltage. Samples were prepared by spreading a drop (5~10 µl) of the filtered QDs (0.25 µm Millipore syringe filters) onto ultrathin carbon/holey support film on a 300 mesh Au grid (Ted Pella, Inc.). The concentration was typically ~ 1 µM. Individual particle sizes were measured using a Gatan Digital Micrograph (Pleasanton, CA) and size estimates were made from the analysis of at least ≥100 nanoparticles.Agarose Gels. Electrophoretic mobility shift assays (EMSAs) were performed on DHLA-QDs assembled with increasing ratios of each Pro n peptide. PEGylated QDs do not migrate on gels.Agarose gels were run at a 1 µM QD concentration with varying peptide to QD ratios in 1% agarose (low EEO) gels (w/v) in 1× TBE (Tris/borate/EDTA pH 8.5) buffer at 150-200 mV.Detection was based on QD fluorescence with UV excitation and images were typically collected after 5, 10, 15, and 20 min.

Dynamic rearrangement of DNA nanostructures provides a straightforward yet powerful mechanism for sequence-specific sensing and potential signaling of such interactions.

Progress has been made using B-form DNA duplex strands to template chromophores in ordered molecular aggregates known as J-aggregates. These aggregates can exhibit strong electronic coupling, extended coherent lifetimes, and long-range exciton delocalization under appropriate conditions. Certain cyanine dyes such as pseudoisocyanine (PIC) dye have shown a proclivity to form aggregates in specific DNA sequences. In particular, DX-tiles containing nonalternating poly­(dA)–poly­(dT) dinucleotide tracks (AT-tracks), which template noncovalent PIC dye aggregates, have been demonstrated to exhibit interesting emergent photonic properties. These DNA-based aggregates are referred to as J-bits for their similarity to J-aggregates. Here, we assemble multifluorophore DX-tile scaffolds which template J-bits into both contiguous and noncontiguous linear arrays. Our goal is to understand the relay capability of noncontiguous J-bit arrays and probe the effects that orientation and position have on the energy transfer between them. We find that linearly contiguous J-bits can relay excitons from an initial AlexaFluor 405 donor to a terminal AlexaFluor 647 acceptor across a distance of up to 16.3 nm. We observed a maximum increase in energy transfer of 41% in the shortest scaffold and an 11% increase in energy transfer across the maximum distance. However, in nonlinear arrays, exciton transfer is not detectable, even when off-axis J-bit-to-J-bit transfer distances were <2 nm. These results, in conjunction with the previous work on PIC–DNA systems, suggest that PIC–DNA-based systems may currently be limited to simple 1-D designs, which prevent isolating J-bits for enhanced energy-transfer characteristics until further understanding and improvements to the system can be made.