Colloidal quantum dots (QDs) possess numerous physical and optical properties that are ideal for biosensing and multiplexing applications. Fluorescence resonance energy transfer (FRET) is a well-established technique for detecting molecular-scale interactions due to proximity-driven changes in fluorescence. We have shown that QDs are excellent energy donors where dye labeled proteins serve as acceptors, and developed a number of prototype nanosensors for small molecules in solution. Among the more promising potential uses of QD-based FRET nanosensors is the ability to "multiplex" signal channels for parallel detection. Because of their very broad absorption and narrow symmetric emission spectra, QDs are ideal fluorophores for multiplexing applications. In this paper, we describe the benefits of QDs in FRET-based assays (as donors and acceptors) and the potential for signal multiplexing in nanoscale biosensors.
We have previously assembled QD-based fluorescence resonance energy transfer (FRET) sensors specific for the sugar nutrient maltose and the explosive TNT. These sensors utilize several inherent benefits of QDs as FRET donors. In this report, we show that QD-FRET based sensors can also function in the monitoring of proteolytic enzyme activity. We utilize a QD with multiple dye-labeled proteins attached to the surface as a substrate for a prototypical protease. We then demonstrate how this strategy can be extended to detect
protease activity by utilizing a dye-labeled peptide attached to the QD as a proteolytic substrate. Self-assembly of the peptide-dye on the QD brings the dye in close proximity to the QD and result in efficient FRET. Addition of a proteolytic enzyme that specifically recognizes and cleaves the peptide alters the FRET signature of the sensor in a concentration-dependent manner. Both qualitative and quantitative data can be derived from these sensors. The potential benefits of this type of QD sensing strategy are discussed.
Quantum dots (QDs) are a versatile synthetic photoluminescent nanomaterial whose chemical and photo-physical properties suggest that they may be superior to conventional organic fluorophores for a variety of biosensing applications. We have previously investigated QD-fluorescence resonance energy transfer (FRET) interactions by using the E. coli bacterial periplasmic binding protein - maltose binding protein (MBP) which was site-specifically dye-labeled and self assembled onto the QD surface and allowed us to monitor FRET between the QD donor and the acceptor dye. FRET efficiency increased as a function of the number of dye-acceptor moieties arrayed around the QD donor. We used this system to further demonstrate a prototype FRET based biosensor that functioned in the chemical/nutrient sensing of maltose. There are a number of potential benefits to using this type of QD-FRET based biosensing strategy. The protein attached to the QDs surface functions as a biosensing and biorecognition element in this configuration while the QD acts as both nanoscaffold and FRET energy donor. In this report, we show that the sensor design can be extended to target a completely unrelated analyte, namely the explosive TNT. The sensor consists of anti-TNT antibody fragments self-assembled onto the QD surface with a dye-labeled analog of TNT (TNB coupled to AlexaFluor 555 dye) prebound in the fragment binding site. The close proximity of dye to QD establishes a baseline level of FRET and addition of TNT displaces the TNB-dye analog, recovering QD photoluminescence in a concentration dependent manner. Potential benefits of this QD sensing strategy are discussed.
Steady state and time-resolved fluorescence measurements were used to investigate the ability of luminescent quantum dots (QDs) to function as efficient energy acceptors in fluorescence resonance energy
transfer (FRET) binding assays with organic dye donors. Fluorescent dyes, AlexaFluor 488 or Cy3, were used with various QD acceptors in QD-dye-labeled-protein conjugates. Data derived from both sets of
experiments showed no apparent FRET from dye to QD. The collected data were discussed within the framework of a competition between a fast radiative decay rate of the donor excitation and a slower FRET
decay rate. This is due to the long exciton lifetime of the acceptor compared to that of the dye, combined with substantial QD direct excitation.
Colloidal semiconductor quantum dots (QDs) have narrow photoemission bandwidths and broad absorption spectra that are ideal for multiplexing applications. In contrast to organic dyes, which require a complex arrangement of excitation sources and filters to generate multiple signals, many populations of QDs can be simultaneously excited with a single excitation source. In a mixed sample, the narrow and symmetric emission profile of QDs allows simple deconvolution of the composite signal to generate individual QD photoluminescence (PL) contributions. We have shown that CdSe-ZnS core-shell QDs function as efficient energy donors in fluorescence resonance energy transfer (FRET) systems. In this study, we tested several QD-protein bioconjugates, each having a unique PL spectrum (or "color") functioning as independent signal channels, to assess the feasibility of a QD FRET-based multiplexing system. Several populations of QDs were self-assembled with labeled and unlabeled proteins, mixed in solution and excited at single wavelength. The resulting spectra were deconvoluted using the known QD emission profiles to reveal individual contributions of each QD population. QDs coated with dye-labeled protein acceptors showed distinct FRET-induced PL quenching due to the presence of proximal dye acceptors. Steady-state fluorescence results were verified by time-resolved spectroscopic data from the mixed samples where a reduced QD lifetime indicated the presence of proximal dye quencher on one or more QD populations. We will discuss how these findings are used to develop QD-based FRET multiplexed biosensors using a similar strategy where each QD population has surface-bound proteins that are sensitive to a unique molecular target.
We demonstrate the use of a photochromic dye to achieve fluorescence resonance energy transfer (FRET) modulation between a QD donor and the dye acceptor brought in close proximity in a selfassembled QD-protein-dye conjugate. The E. coli maltose binding protein (MBP) appended on its C-terminal with an oligohistidine attachment domain, immobilized onto CdSe-ZnS core-shell QDs was labeled with a sulfo-N-hydroxysuccinimide activated photochromic BIPS molecule (1',3-dihydro-1'-(2-carboxyethyl)-3,3-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2'-(2H)-indoline]). Two different dye-to-MBP-protein ratios of 1:1 and 5:1 were used. The ability of MBP-BIPS to modulate QD photoluminescence was tested by switching BIPS from the colorless spiropyran (SP) to the colored merocyanine (MC) using irradiation with white light (>500 nm) or with UV light (~365 nm), respectively. QDs surrounded by ~20 MBP-BIPS with a dye to protein ratio of 1 showed ~25% loss in their photoemission with consecutive repeated switches, while QDs surrounded by ~20 MBP-BIPS with BIPS to MBP ratio of 5 produced a substantially more pronounced rate of FRET where the QD emission was quenched by ~60%. This result suggests the possibility of using QD-protein conjugates to assemble reversible FRET nanoassemblies where the QD emission can be controlled by changing the properties of the acceptors dyes bound to the protein.