Showing papers by "Michael H. Stewart published in 2011"

Monitoring botulinum neurotoxin a activity with peptide-functionalized quantum dot resonance energy transfer sensors

Abstract: Botulinum neurotoxins (BoNTs) are extremely potent bacterial toxins that contaminate food supplies along with having a high potential for exploitation as bioterrorism agents. There is a continuing need to rapidly and sensitively detect exposure to these toxins and to verify their active state, as the latter directly affects diagnosis and helps provide effective treatments. We investigate the use of semiconductor quantum dot (QD)-peptide Forster resonance energy transfer (FRET) assemblies to monitor the activity of the BoNT serotype A light chain protease (LcA). A modular LcA peptide substrate was designed and optimized to contain a central LcA recognition/cleavage region, a unique residue to allow labeling with a Cy3 acceptor dye, an extended linker-spacer sequence, and a terminal oligohistidine that allows for final ratiometric peptide-QD-self-assembly. A number of different QD materials displaying charged or PEGylated surface-coatings were evaluated for their ability to self-assemble dye-labeled LcA peptide substrates by monitoring FRET interactions. Proteolytic assays were performed utilizing either a direct peptide-on-QD format or alternatively an indirect pre-exposure of peptide to LcA prior to QD assembly. Variable activities were obtained depending on QD materials and formats used with the most sensitive pre-exposure assay result demonstrating a 350 pM LcA limit of detection. Modeling the various QD-peptide sensor constructs provided insight into how the resulting assembly architecture influenced LcA recognition interactions and subsequent activity. These results also highlight the unique roles that both peptide design and QD features, especially surface-capping agents, contribute to overall sensor activity.

Terbium to quantum dot FRET bioconjugates for clinical diagnostics: influence of human plasma on optical and assembly properties.

Abstract: Forster resonance energy transfer (FRET) from luminescent terbium complexes (LTC) as donors to semiconductor quantum dots (QDs) as acceptors allows extraordinary large FRET efficiencies due to the long Forster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma.

Quantum dots as a FRET donor and nanoscaffold for multivalent DNA photonic wires

Abstract: We demonstrate Forster 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 in nanotechnology.

Quantum dots used as single-cell alkalinity sensors

Abstract: Luminescent semiconductor quantum dots (QDs) have become popular for biological imaging, labeling, and sensing due to their bright photoluminescence (PL).1 These QDs are characterized by an excited state—reached after absorbing energy from a source at a given wavelength—and a ground state, achieved when the energy absorbed in the form of a photoluminescent emission is released. Such PL emissions can be tuned according to the size of QDs by what is known as quantum confinement effects. Luminescent QDs show higher performance than other light-emitting sensors: they are excited with high efficiency over a broad range of wavelengths and produce narrow PL emission profiles on returning to their ground state. Thus, when QDs are mixed with materials, such as biological cells, they can be selectively excited by choosing a wavelength that other materials do not absorb. In addition, QDs are more resistant to photobleaching—they are not destroyed by exposure to light—than organic dyes traditionally used in biological analysis. Consequently, they remain emissive longer under continuous excitation. In applications where QDs are used as sensors, a change in QD PL signals the presence of target species or analytes. Currently, detection of analytes in biological systems using QDs is commonly performed using Förster resonance energy transfer (FRET).2 This technique measures the degree of energy transfer from QDs to proximal organic dyes—known as acceptor dyes due to their ability to absorb energy—which changes according to the analyte. Here, we report the creation of biosensors that exploit QD optical properties, which are inherently sensitive to charge-transfer (CT) processes.3 Until now, CT-based sensing with QDs has received far less attention than FRET-based methods because it is more complex. CT processes involving oxidation and reduction (redox) are ubiquitous in biological systems. An important class of molecules that undergo redox processes in such systems are catechols. The catecholamine neurotransmitter dopamine is the Figure 1. Photoluminescence (PL) mechanism of quantum dot (QD)dopamine conjugates in acidic and basic conditions. hv: Photon. His6: Hexahistidine. e , h: Charge carriers. ET: Electron transfer. CB: Conduction band. VB: Valence band.