An alternative molecular recognition approach was developed for sensing small molecule analytes using the differential binding of an allosteric transcription factor (TF, specifically TetR) to its cognate DNA as the molecular recognition element coupled with fluorescent resonance energy transfer (FRET) to yield an internally calibrated optical signal transduction mechanism. Sensors were evaluated comprising Cy5-modified DNA (FRET acceptor) with either a tdTomato-TetR fusion protein (FP-TF) or quantum dot-TetR conjugate (QD-TF) as the FRET donor by measuring the ratio of acceptor and donor fluorescence intensities (FA/FD) with titrations of a derivative of the antibiotic tetracycline, anhydrous tetracycline (aTc). A proof-of-concept FRET-based biosensor was successfully demonstrated through the modulation of FA/FD signal intensities based on varying analyte concentrations. Sensor design parameters affecting overall signal-to-noise ratio and sensitivity of the sensors are also identified.
Cadmium-free near infrared (NIR) emitting quantum dots (QDs) have significant potential for multiplexed tissue-depth imaging applications in the first optical tissue window (i.e., 650 – 900 nm). Indium phosphide (InP) chemistry provides one of the more promising cadmium-free options for biomedical imaging, but the full tunability of this material has not yet been achieved. Specifically, InP QD emission has been tuned from 480 – 730 nm in previous literature reports, but examples of samples emitting from 730 nm to the InP bulk bandgap limit of 925 nm are lacking. We hypothesize that by generating inverted structures comprising ZnSe/InP/ZnS in a core/shell/shell heterostructure, optical emission from the InP shell can be tuned by changing the InP shell thickness, including pushing deeper into the NIR than current InP QDs. Colloidal synthesis methods including hot injection precipitation of the ZnSe core and a modified successive ion layer adsorption and reaction (SILAR) method for stepwise shell deposition were used to promote growth of core/shell/shell materials with varying thicknesses of the InP shell. By controlling the number of injections of indium and phosphorous precursor material, the emission peak was tuned from 515 nm to 845 nm (2.41 – 1.47 eV) with consistent full width half maximum (FWHM) values of the emission peak ~0.32 eV. To confer water solubility, the nanoparticles were encapsulated in PEGylated phospholipid micelles, and multiplexing of NIR-emitting InP QDs was demonstrated using an IVIS imaging system. These materials show potential for multiplexed imaging of targeted QD contrast agents in the first optical tissue window.
High-quality core/shell CdSe/xCdS quantum dots (QDs) ranging from 3 to 20 nm in diameter were synthesized for use as Förster Resonance Energy Transfer (FRET) donors. gNQDs are carefully characterized for size, emission, absorption, QY, and brightness in both organic and aqueous solution. FRET has been verified in optimally designed systems that use short capping ligands and donor-acceptor pairs that have well-matched emission and absorption spectra. The interplay between shell thickness, donor-acceptor distance, and particle brightness is systematically analyzed to optimize our biosensor design.
Quantum dots (QDs) are semiconductor nanocrystals with extensive imaging and diagnostic capabilities, including the
potential for single molecule tracking. Commercially available QDs offer distinct advantages over organic fluorophores,
such as increased photostability and tunable emission spectra, but their cadmium selenide (CdSe) core raises toxicity
concerns. For this reason, replacements for CdSe-based QDs have been sought that can offer equivalent optical
properties. The spectral range, brightness and stability of InP QDs may comprise such a solution. To this end,
LANL/CINT personnel fabricated moderately thick-shell novel InP QDs that retain brightness and emission over time in
an aqueous environment. We are interested in evaluating how the composition and surface properties of these novel QDs
affect their entry and sequestration within the cell. Here we use epifluorescence and transmission electron microscopy
(TEM) to evaluate the structural properties of cultured Xenopus kidney cells (A6; ATCC) that were exposed either to
commercially available CdSe QDs (Qtracker® 565, Invitrogen) or to heterostructured InP QDs (LANL). Epifluorescence
imaging permitted assessment of the general morphology of cells labeled with fluorescent molecular probes (Alexa
Fluor® ® phalloidin; Hoechst 33342), and the prevalence of QD association with cells. In contrast, TEM offered unique
advantages for viewing electron dense QDs at higher resolution with regard to subcellular sequestration and
compartmentalization. Preliminary results show that in the absence of targeting moieties, InP QDs (200 nM) can
passively enter cells and sequester nonspecifically in cytosolic regions whereas commercially available targeted QDs
principally associate with membranous structures within the cell. Supported by: NIH 5R01GM084702.
Fluorescence resonance energy transfer (FRET) between a quantum dot (QD) and the pH-sensitive fluorescent protein
mOrange has been used to develop a fluorescent pH-indicator that is bright and photostable enough for applications in
fluorescence imaging, including the tracking of molecules through endocytic pathways. As the molar extinction
coefficient of mOrange increases with pH, the ratio of the mOrange emission to the QD emission (F<sub>A</sub>/F<sub>D</sub>) increases
sharply, producing greater than 10-fold increases in the F<sub>A</sub>/F<sub>D</sub> ratio between pH 4.5 and 7.5. This probe has been
thoroughly characterized and it intracellular imaging potential explored.
Efficient Fluorescence (or Förster) Resonance Energy Transfer (FRET) pairs between fluorescent proteins and quantum
dots (QDs) have a significant potential for ultrasensitive biochemical assays in disease detection and diagnosis. We have
developed such FRET pairs using commercially available QDs as donors and fluorescent protein as acceptor, with
polyhistidine-chelation as the means of bioconjugation. In this study we compared two brands of QDs with different
surface coatings and found that the FRET pair containing EviTags from Evident Technology produced a higher FRET
efficiency due to the shorter donor-acceptor distance. The polyhistidine binds directly to the ZnS capping layer of the
EviTags, whereas the carboxyl QDots from Invitrogen, although having a higher quantum yield, require the addition of
Ni<sup>2+</sup> to the solution in order to facilitate chelation-mediated binding to outer surface of the polymer coating. These
findings have significant implications to QD-based FRET assay design.