Jay Nadeau1, Jeremiah Kloefper1, Michael Wong2, Galen Stucky2, Kenneth Nealson1 1 Jet Propulsion Lab

1
Jay Nadeau1, Jeremiah Kloefper1, Michael Wong2,
Galen Stucky2, Kenneth Nealson11 Jet Propulsion
Laboratory, California Institute of Technology2
Department of Chemistry, University of
California, Santa Barbara
Quantum dots for off-on biological labeling
Center for Life Detection
Quenching adenine, guanine, and
tryptophan broaden absorption and quench
luminescence
Quenching reversal seen with 3-8 hr of
incubation with live microorganisms
Goal to create in situ labels for bacteria and
cells that indicate metabolic and redox processes
Quantum dots uncapped CdSe nanocrystallites
synthesized by simple process1, water
solubilized as in previous studies2
B
A, Fully quenched red QD-adenine conjugates show
a strong signal after incubation with S. aureus
for 3 hours (transferrin serves as a positive
control). The tryptophan signal is blue-shifted
and broader. B, The tryptophan signal depends on
the chirality of the molecule and whether the
bacteria are live or heat-killed (dead)
(metabolic inhibition with EDTA is similar to
heat-killing). C,Subtracting bacterial
autofluorescence from green QD-tryptophan labeled
samples shows a blue-shifted signal for
L-tryptophan (live L-tryp-auto), and a double
Gaussian peak for D-tryptophan (live
D-tryp-auto). Dead bacteria show no difference
between L- and D-tryptophan (live L-tryp-auto, D
overlaps) and no signal in the wavelengths of the
QDs.
Fluorescence emission and absorbance spectra for
adenine- conjugated QDs. gQD, rQD green and red
QDs gAd, rAd adenine conjugates Ad adenine
alone
Quenching color dependence location of QD band
gap determines persistence of quenching
Epifluorescence images
B
B
A, emission spectra of 4 independent preparations
of QDs (excitation 400 nm) B, absorbance spectra
of green- and red-emitting QDs (emission spectra
overlayed as dashed lines for comparison)
a,Yellow-fluorescent quantum dots (QDs),
solubilized, in phosphate-buffered saline
(PBS). b,Conjugated to tryptophan. There is some
slight yellow color left, but it's weak and
blue-shifted. c,Conjugated to adenine. The blue
that you see is nonspecific crystal fluorescence.
All yellow has vanished. d, Adenine-QDs Staph.
aureus, 3 hours incubation. e, Tryptophan-QDs
Staph. aureus, 3 hours incubation.
f,Tryptophan-QDs Staph. aureus, same image as
e after 1-2 min of photobleach. The QD signal
remains. g, Transferrin-QDs Staph. aureus, 3
hours incubation (positive control). Note that
there are some QDs that aren't associated with
organisms, particularly the area on the upper
left corner. This is not the case for adenine or
tryptophan. All images same gain/exposure time
dots photographed at 100X magnification, bacteria
at 1000X 100 watt Hg lamp illumination with DAPI
filter (Ex. 330-380 nm, 435 LP out).
Early results Specific binding to
microorganisms can be achieved by conjugating to
biomolecules
A, Energy levels of our QD samples relative to
redox potentials of some commonly used dyes and
molecules. Red lines reduction potentials of
naphthoquinone (NQ), benzoquinone (bq), and
anthraquinone (AQ). In a previous study, the
first two compounds were able to quench QD
fluorescence, while the last was not3. Blue
lines oxidation potentials of molecules used in
this study. GUA, guanine (pH 12) AD, adenine
(pH 7) trp, tryptophan (pH 6). Black, band edge
positions for the QD samples used in this study,
calculated from absorbance spectra. The valence
band edge of the red QDs is very close to the
redox potential of adenine. B,Quenching of green
QDs remains after 1 wk of storage in the dark
(green dark) slight loss of quenching is seen
under ambient lighting (green light). Red QDs
show strong fluorescence signals under both
conditions, but greater in QDs exposed to light
(all samples normalized to optical density before
conjugation).
B
A, emission spectra of green QDs conjugated to
human holo- and apo-ransferrin, and of red QDs
conjugated to wheat germ agglutinin (WGA).
Increased photoluminescence due to surface
passivation is the only effect seen. B, WGA is
specific for Gram positive bacteria, binding to
Staphylococcus aureus but not to E. coli. (Left)
spectrum at 400 nm excitation (right),
epifluorescence image of stained S. aureus. C,
transferrin is taken up only by organisms
possessing the receptor, such as human pathogens
and this eukaryotic yeast. Scale bars 10 mm.
References
1. Wong, M.S. and Stucky, G.D. The facile
synthesis of nanocrystalline semiconductor
quantum dots. (Full ref?) 2. Chan, W. C., and
Nie, S. 1998. Quantum dot bioconjugates for
ultrasensitive nonisotopic detection. Science
2812016-8. 3. Burda, C., Green, T. C., Link, S.,
and El-Sayed, M. A. 1999. Electron shuttling
across the interface of CdSe nanoparticles
monitored by femtosecond laser spectroscopy. J
Phys Chem B 1031783-1788.