Recent interest in quantum dots (QDs) stems from the plethora of potential applications that arises from their tunable absorption and emission profiles, high absorption cross sections, resistance to photobleaching, functionalizable surfaces, and physical robustness. The emergent use of QDs in biological imaging exploits these and other intrinsic properties. For example, quantum confined Stark effect (QCSE), which describes changes in the photoluminescence (PL) of QDs driven by the application of an electric field, provides an inherent means of detecting changes in electric fields by monitoring QD emission and thus points to a ready mean of imaging membrane potential (and action potentials) in electrically active cells. Here we examine the changing PL of various QDs subjected to electric fields comparable to those found across a cellular membrane. By pairing static and timeresolved PL measurements, we attempt to understand the mechanism driving electric-field-induced PL quenching and ultimately conclude that ionization plays a substantial role in initiating PL changes in systems where QCSE has traditionally been credited. Expanding on these findings, we explore the rapidity of response of the QD PL to applied electric fields and demonstrate changes amply able to capture the millisecond timescale of cellular action potentials.
Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.
Nanosensors employing quantum dots (QDs) with appended biofunctional moieties offer tremendous promise for disease surveillance/diagnostics and chemical/biological threat activity. Their small size permits cell penetration and their inherent photochemical properties are well-suited for rapid, optical measurement. The effectiveness of enzymes immobilized on QDs, however, are not completely understood, hindering development of chemical/biological sensors and remediation materials. Here, we analyze enzyme effectiveness for the neutralization of a simulant nerve agent when attached to two distinctly-sized QDs. Two sizes of QDs, 525 or 625 nm, were appended with DHLA ligands to improve aqueous stability and prevent aggregation. Various molar ratios of de novo phosphotriesterase trimer (PTE<sub>3</sub>) were rapidly self-assembled via spontaneous metal coordination of the PTE oligohistidine tag onto the Zn<sup>2+</sup>-rich QD surface. PTE catalyzes the detoxification of organophosphate pesticides (e.g, paraoxon, an analog of sarin) to p-nitrophenol whose absorbance can be measured at 405 nm. The optimal ratio of PTE<sub>3</sub> to 525 nm and 625 nm QD’s was determined to be 12 and 24, respectively. The enhanced enzyme performance in both cases is most likely due to increased enzyme-substrate interactions from improvements in enzyme orientation, enzyme density, and substrate diffusion on or near the QD. Development of these nansosensors as optical-based biosensors (e.g., within compact microfluidic devices) may greatly improve the sensitivity of conventional biological/chemical detection schemes.
Single particle tracking has provided a wealth of information about biophysical processes such as motor protein transport and diffusion in cell membranes. However, motion out of the plane of the microscope or blinking of the fluorescent probe used as a label generally limits observation times to several seconds. Here, we overcome these limitations by using novel non-blinking quantum dots as probes and employing a custom 3D tracking microscope to actively follow motion in three dimensions (3D) in live cells. Signal-to-noise is improved in the cellular milieu through the use of pulsed excitation and time-gated detection.
For nanomaterials to realize their full potential in disease diagnosis and drug delivery applications, one must be able to
exert fine control over their cellular delivery, localization and long-term fate in biological systems. Our laboratory has
been active in developing methodologies for the controlled and site-specific delivery of a range of nanomaterials (e.g.,
quantum dots, colloidal gold, nematic liquid crystals) for cellular labeling, imaging and sensing. This talk will highlight
several examples from these efforts and will demonstrate the use of peptide- and protein-mediated facilitated delivery of
nanomaterials to discrete cellular locations including the endocytic pathway, the plasma membrane and the cellular
cytosol. The implications of the ability to exert fine control over nanomaterial constructs in biological settings will be
discussed with a particular focus on their use in nanoparticle-based theranostics.
There is considerable research in the area of manipulating light below the diffraction limit, with potential applications ranging from information processing to light-harvesting. In such work, a common problem is a lack of efficiency associated with non-radiative losses, e.g., ohmic loss in plasmonic structures. From this point of view, one attractive method for sub-wavelength light manipulation is to use Förster resonance energy transfer (FRET) between chromophores. Although most current work does not show high efficiency, biology suggests that this approach could achieve very high efficiency. In order to achieve this goal, the geometry and spacing of the chromophores must be optimized. For this, DNA provides an easy means for the self-assembly of these complex structures. With well established ligation chemistries, it is possible to create facile hierarchical assemblies of quantum dots (QDs) and organic dyes using DNA as the platform. These nanostructures range from simple linear wires to complex 3-dimensional structures all of which can be self-assembled around a central QD. The efficiency of the system can then be tuned by changing the spacing between chromophores, changing the DNA geometry such that the donor to acceptor ratio changes, or changing the number of DNA structures that are self-assembled around the central QD. By exploring these variables we have developed a flexible optical system for which the efficiency can be both controlled and optimized.
CdSe/ZnS semiconductor quantum dots (QDs) are ideal materials for biological sensing and cellular imaging applications due to their superior photophysical properties in comparison to fluorescent proteins or dyes and their ease of conjugation to biological materials. We have previously developed a number of <i>in vitro</i> FRET based biosensors in the laboratory for detection of proteases and biological and chemical agents. We would like to expand these biosensing capabilities into cellular systems, requiring development of QD cellular delivery techniques. Peptide mediated cellular delivery of QDs is ideal as peptides are small, easily conjugated to QDs, easily manipulated and synthesized, and can be designed with “handles” for further chemical conjugation with other cargo. Here we discuss four cell delivery peptides that facilitate QD uptake in live cells. Understanding these peptides will help us design better nanoparticle cellular delivery systems and advance our capabilities for <i>in vivo</i> biosensing.
Biocompatible nanoparticles have recently attracted significant attention due to increasing interest in their use for
biological sensing, cellular labeling and in vivo imaging. Semiconductor quantum dots (QDs) with good colloidal
stability as well as small hydrodynamic sizes are particularly useful within these applications. We have developed a
series of dihydrolipoic acid (DHLA) based surface ligands to enhance the colloidal stability and biocompatibility of
water soluble QDs. Modification of DHLA with poly(ethylene glycol) derivatives provided the QDs with extended
colloidal stability over a wide pH range and under high salt concentrations, which contrasts with the limited colloidal
stability provided by DHLA alone. Functionalization of the PEG termini enabled one to have easy access to the QD
surface and construct a variety of stable QD-biomolecules conjugates. A series of DHLA-based compact ligands with
zwitterionic character has also been explored to develop compact sized QDs without sacrificing the colloidal stability.
Despite their smaller sizes than the PEG analogs, the QDs coated with the zwitterionic ligands still have excellent
colloidal stability and minimize nonspecific interactions in biological environments. Recent studies of thiol-based
multidentate ligands and ligand exchange methods further improved the colloidal stability and fluorescence quantum
Currently there is considerable interest in using bioconjugated nanoparticles for <i>in vivo</i> imaging, biosensing and
theranostics. Luminescent CdSe/ZnS core shell semiconductor quantum dots (QDs) have unique optical properties
and bioconjugation capabilities that make them ideal prototypes for these purposes. We have previously described
the metal-affinity association between the imidazole groups of terminal hexahistidine residues of peptides and
proteins and the ZnS shell of quantum dots as a useful bioconjugation technique. We have also demonstrated that
QDs labeled with an oligohistidine-tagged cell penetrating peptide (CPP) derived from the HIV TAT-protein could
undergo specific endocytosis-mediated cellular uptake in both HEK293T/17 and COS-1 cells. However, the QDs
were predominantly sequestered in the endosomes. This remains a significant hindrance to future potential cellular
imaging applications which require the QDs to access other subcellular organelles. Here we describe the testing of
several cytosolic QD delivery modalities including microinjection, the commercial cytosolic delivery agent PULSin,
and the cytosolic delivery peptide Palm-1. Palm-1, a palmitylated peptide that is capable of both cellular uptake and
rapid endosomal escape in multiple cell lines without concomitant toxicity, is shown to be the superior method for
cytosolic delivery of QDs. Potential intracellular applications for this peptide are discussed.
We demonstrate Förster resonance energy transfer (FRET) through DNA photonic wires self-assembled around a
central CdSe/ZnS semiconductor quantum dot (QD). The central QD acts as a nanoscaffold and FRET donor to a
series of acceptor dyes along a DNA strand. By utilizing a DNA intercalating dye, altering the location of the dyes
and using a series of increasingly red-shifted dyes along the DNA, we are able to track the efficiency of energy
transfer through the DNA photonic structure via steady-state spectroscopy. Data suggests that limiting factors for
efficient energy transfer are the sub-obtimal photophysical properties of acceptor dyes, including low quantum yields.
These issues may be addressed with improved configurations of QDs, DNA and dyes. The development of biophotonic wire assemblies utilizing the superior photophysical properties of QDs will have widespread application