Biosynthesis of luminescent quantum dotsin an earthworm
S. R. Stu¨rzenbaum
1†
, M. Ho¨ckner
1†
, A. Panneerselvam
2
, J. Levitt
2
, J-S. Bouillard
2
, S. Taniguchi
2
,L-A. Dailey
4
, R. Ahmad Khanbeigi
4
, E. V. Rosca
4
, M. Thanou
4
, K. Suhling
2
, A. V. Zayats
2
and M. Green
2,3,5
*
The synthesis of designer solid-state materials by living organ-isms is an emerging field in bio-nanotechnology. Key examplesinclude the use of engineered viruses as templates for cobaltoxide (Co
3
O
4
) particles
, superparamagnetic cobalt–platinumalloy nanowires
and gold–cobalt oxide nanowires
for photo-voltaic and battery-related applications. Here, we show thatthe earthworm’s metal detoxification pathway can be exploitedto produce luminescent, water-soluble semiconductorcadmiumtelluride (CdTe) quantum dots that emit in the green region of the visible spectrum when excited in the ultraviolet region.Standard wild-type
Lumbricus rubellus
earthworms wereexposed to soil spiked with CdCl
2
and Na
2
TeO
3
salts for 11days. Luminescent quantum dots were isolated from chlorago-genous tissues surrounding the gut of the worm, and were suc-cessfully used in live-cell imaging. The addition of polyethyleneglycol on the surface of the quantum dots allowed for non-targeted, fluid-phase uptake by macrophage cells.
In recent years, semiconductor quantum dots have been usedin various medical and biological imaging applications becausetheir optical properties are considered to be superior to those of organic dyes. However, to make them hydrophilic and compatiblewith biological systems, their surfaces need to be derivatized. Thisprocess is challenging and often reduces the optical quality of thematerials. It is therefore desirable to develop alternative methodsof preparing biologically compatible luminescent nanomaterials.The biosynthesis of semiconductor nanomaterials was firstreported in unicellular yeast, which was shown to be capable of pro-ducing cadmium sulphide (CdS) crystallites in response to challengeby a cadmium salt
. Bacteria or fungi have also been used for thebiosynthesis of CdS, but few investigations have focused on lumines-cent properties
. Cadmium telluride (CdTe)
, lead sulphide(PbS)
and cadmium selenide (CdSe)
have been synthesizedusing bacteria, yeast or fungi, and their reported emission profilesresemble, to some degree, those of their chemically synthesizedcounterparts. It is also known that inorganic granules of HgSe (tie-mannite) form in various animals as a detoxification mechanism
.However, the biosynthesis of luminescent quantum dots has notbeen achieved in higher organisms (metazoans).The earthworm
L. rubellus
can accumulate cadmium to levelsin excess of one-thousandth of its total dry bodyweight
. This isachieved using an isolation strategy that relies on metallothio-neins—a family of cysteine-rich proteins—to transport heavy metals to the chloragogenous tissues (cells that resemble the liverin vertebrates) where toxins are neutralized (Fig. 1a–c)
.Although the cadmium detoxification system in earthworms iswell established, little is known regarding the uptake, trafficking and storage dynamics of tellurium. Previous reports have shownthat,
in vitro
, tellurium interacts strongly with mammalian metallo-thioneins
and that tellurium also binds efficiently with seleno-proteins through metal–cysteine coordination
. Equivalentbenchtop chemistry is used in the synthesis of high-quality CdTequantum dots, where thiol-based capping agents react withcadmium precursors before nanoparticle synthesis and are foundto be an essential surface species in the resulting luminescentmaterials
. We suggest that once transported to the chloragogenoustissue, the tellurium precursor reacts in a similar manner to theselenium analogue, as recently described
. We propose that thetellurite was reduced by glutathione reductase, reduced nicotina-mide adenine dinucleotide phosphate (NADPH) and glutathione(GSH) via the GS–Te–SG complex, to H
2
Te, a common Te precur-sor, and thus able to react with the available Cd
2
þ
:4GSH
+
2H
+
+
TeO
32
−
(
GS
)
2
–Te
+
GSSG
+
3H
2
O
(
GS
)
2
–Te
+
NADPH
+
H
+
−−−−−
glutathionereductase
GSH
+
GSTeH
+
NADP
+
GSH
+
GSTeH
GSSG
+
H
2
TeH
2
Te
+
CdCl
2
CdTe
+
2HClEarthworms exposed for 11 days to standard soil spiked with CdCl
2
and Na
2
TeO
3
were shown to transport the precursors, via metallatedmetallothionein complexes, to the chloragogenous tissue located atthe coelomic surfaces of the worm gut, where they reacted to formquantum dots before isolation. The particles demonstrated anabsorption band-edge of
450 nm with a slight excitonic featureat
360 nm, consistent with CdTe quantum dots (Fig. 1d). Theobserved green emission from the quantum dots (Fig. 1d, bottominset) was determined to be near the band-edge, with a Stokesshifted emission maxima at
520 nm and a second weaker com-ponent at
460 nm from residual autofluorescence (Fig. 1d). Theemission quantum yield was calculated to be 8.3% (againstRhodamine 6G). Following storage for several days under ambientconditions, the main quantum dot emission was found to fade visibly, although autofluorescence remained detectable by spectro-scopy (Fig. 1d, top inset).
1
Analytical and Environmental Sciences Division, King’s College London, 3rd Floor, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK,
2
Department of Physics, King’s College London, Strand, London WC2R 2LS, UK,
3
Department of Imaging Chemistry and Biology, Divisions of ImagingScience and Biomedical Engineering, King’s College London, 4th floor, Lambert Wing, St Thomas Hospital, London SE1 7EH, UK,
4
Institute of PharmaceuticalScience, King’s College London, 5th Floor, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK,
5
Department of Chemistry, King’s CollegeLondon, Hodgkin Building, Guy’s campus, London SE1 1UL, UK;
†
These authors contributed equally to this work.
*
e-mail:mark.a.green@kcl.ac.uk
LETTERS
PUBLISHED ONLINE: 23 DECEMBER 2012 |DOI: 10.1038/NNANO.2012.232
NATURE NANOTECHNOLOGY
| VOL 8 | JANUARY 2013 |www.nature.com/naturenanotechnology
57
Although autofluorescence varies between earthworm species
,the autofluorescence in coelomocytes and chloragogen cells in
L. rubellus
has been shown to be minimal
. The emission may be due to an unknown material, such as a cadmium complex formed in the blood
, or a metal–metallothionein complex
.Significant to note is the fact that a negative control with wormsexposed for 11 days to standard soil spiked with only CdCl
2
didnot yield fluorescent nanoparticles, although minor autofluores-cence was again observed. Emission lifetime measurements exhib-ited two decay components, with a long-lived component(4.54 ns) typical of CdTe quantum dots and a shorter-livedspecies (0.07 ns), possibly of organic origin and attributed to thediminutive levels of cellular autofluorescence (Fig. 1e) or excitationlaser scattering.Dark-field microscopy of the evaporated solution isolated fromthe chloragogen cells identified nanoparticulate materials (Fig. 2a)with an elemental composition consistent with CdTe (Fig. 2b) asdetermined by energy-dispersive X-ray spectroscopy. Iron was alsodetected (blood), as well as chlorine (cadmium precursor) andsulphur (attributed to a thiol-containing cellular species coordi-nated to the particle surface). The presence of the bright band-edge green emission correlates with the thiol capping of CdTequantum dots
. We propose the hypothesis that simple sulphur-containing amino acids, peptides or proteins mayact asthe passivat-ing agent, yielding water-soluble particles while contributing to theemitting state, although the actual capping agent has yet to be ident-ified. The authors of the seminal paper that described CdS particlesprepared from yeast
suggested glutamyl peptides were used tosequester the cadmium salts, which ultimately passivated the par-ticle surface through cysteinyl thiolates.High-resolution electron microscopy analyses (Fig. 2c,d)confirmed the presence of crystalline nanoparticles with anaverage diameter of 2.33
+
0.59 nm (100 particles measured) andclear lattice fringes, while selective area electron diffraction(SAED) demonstrated diffuse rings consistent with CdTe nano-particles (Fig. 2e).Several imaging applications were used to explore the utility of the biosynthesized quantum dots. Initial attempts to label J774.1macrophage-like cells with the isolated quantum dots were unsuc-cessful (Supplementary Fig. S1a); however, the addition of dithio-lated polyethylene glycol to a sample of freshly isolated CdTeparticles resulted in increased particle stability in the cell culturemedium (Supplementary Fig. S2) and allowed for the non-targeted,fluid-phase uptake of particles into live cells, to be visualized overthe course of 20 min (Fig. 3a).Further experiments showed that the quantum dots did notnecessarily require the addition of a polyethylene glycol ligand forsuccessful labelling. The imaging of an ovarian cancer cell line(IGROV-1) was achieved without the addition of further ligands,using the particle’s native passivation obtained during synthesis,which was seemingly sufficient to allow uptake into the cellularcytoplasm (Fig. 3b). Further images obtained at a
z
stack level,which corresponded to the middle of the cell, confirmed uniformstaining within the entire cellular cytoplasm (Supplementary
0.00.51.01.52.02.53.03.5200300400500600700800
300400500600700800
Wavelength (nm)
Wavelength (nm)
I n t e n s i t y ( a . u . )
I n t e n s i t y ( a . u . )
0110,0001,00010010
C o u n t s
10203040Time (ns)
P A C
abcde
Figure 1 |
Schematics of the earthworm used and optical characterization of the quantum dots.
a
, The earthworm
Lumbricus rubellus.
The gut sectionfollowing the thickened glandular clitellum is termed the posterior alimentary canal (PAC).
b
, Cross-section of the posterior section of the earthworm. Thechloragogenous tissue surrounds the PAC (indicated by arrows).
c
, The chloragogenous tissue harvested for analyses is also the predominant location ofmetallothionein accumulation, as determined by immunoperoxidase histochemistry performed with an earthworm-specific metallothionein antibody
(notethat the metallothionein-positive choragogen cells are stained red, as indicated by arrows). These figures highlight the anatomy of the worm and the regionswhere the metallothionein is generated and where the quantum dots are synthesized.
d
, Absorption (dotted line) and emission (solid line) spectra of CdTequantum dots isolated from choragogen cells. Inset: (top) emission spectra for original (blue) and aged (7 days, black) CdTe particles and (bottom)photograph of original CdTe after harvesting, under 365 nm excitation.
e
, Emission lifetime measurements of emission from freshly isolated CdTe particles,showing two emissive species: bi-exponential fit corresponds to
t
1
¼
0.07
+
0.004 ns and
t
2
¼
4.54
+
0.03 ns. These data confirm the optical propertieswere consistent with nanocrystalline CdTe.
LETTERS
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58
Fig. S3). The cells showed negligible autofluorescence, so thefluorescence inFig. 3b can be convincingly attributed to the CdTequantum dots. This suggests that CdTe nanoparticles isolateddirectly from the chloragogen tissues have associated biologicalligands that provide the required colloidal stability in this cell line.Furthermore, these ligands can easily be exchanged for dithiol–PEG to enhance the stability of the nanoparticle.The sensitivity of the photophysical properties of simple thiol-capped quantum dots to temperature, pH, presence and concen-tration of salts in biological systems is well described
and theuse of polyethylene glycol as a non-targeted surface stabilizer isroutine. Thus, the performance of the CdTe particles isolatedfrom chloragogen cells in cell-imaging studies is comparable tomany commercially available systems. This demonstrates that theparticles are suitable imaging agents and can undergo surfaceexchange reactions for specific imaging applications.We suggest that the chloragogen cells are ideally positionedto allow the precursors to form luminescent CdTe quantum dots
in vivo
, a process that is by no means trivial. The analogousreaction
ex vivo
requires an additional reducing agent in the pres-ence of a thiol-capping agent to form luminescent nanoparticulatematerials
, whereas this entirely biosynthetic strategy results inthe Te(
IV
) species being reduced to Te(
II
)
in vivo
using a previously highlighted mechanism
. Notably, the worms remained aliveduring the 11-day quantum dot production period, but were sub-sequently killed for quantum dot harvesting.In conclusion, we were able to generate luminescent, water-soluble CdTe quantum dots in a living animal by highjacking itsintrinsic heavy-metal detoxification strategy. The resultant particleswere easy to harvest, demonstrated optical characteristics typical of quantum-confined semiconductor materials and were utilized forrelevant cell imaging applications. These particles are comparableto thiol-capped CdTe prepared by typical benchtop methods and,if extended to other technically important semiconducting materials, this method promises to be a convenient source of biologically compatible luminescent probes.
100
µ
m14
µ
m
ab
Figure 3 |
Cell imaging using the CdTe quantum dots isolated from thechloragogenous tissues.
a
, Confocal laser scanning microscopic images oflive J774.1 murine macrophage-like cells after 20 min exposure to CdTequantum dots (green) modified with dithiolated polyethylene glycol.
b
, Confocal microscope image of IGROV-1 cells incubated with untreatedCdTe particles for 3 h at 37
8
C: green emission from quantum dots isolatedin the cellular cytoplasm, blue emission from the nuclear stain dye4
′
,6-diamidino-2-phenylindole in the cell nucleus. The data confirm theusefulness of the biosynthesized particles in imaging applications.
50
abc
4030FeFeOCuCuCICdCdTeTeTeFeCuSiS201002.04.06.08.010.0Energy (keV)
C o u n t s
EDX HAADF detector point 1200 nm20 nm
de
5 nm220111311
Figure 2 |
Microscopy analysis of the CdTe particles isolated from the chloragogenous cells.
a
, Dark-field microscopy of CdTe particles isolated fromchloragogen cells.
b
, Energy-dispersive X-ray spectroscopy analysis of CdTe quantum dots.
c
,
d
, High-resolution electron microscopy of CdTe quantum dots.
e
, SAED pattern for CdTe and assigned Miller indices. These data confirm the nanocrystalline identity and structure of the particles as CdTe quantum dots.
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