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1 Biosynthesis of Luminescent Quantum Dots in an Earthworm S.R. Stürzenbaum, a# M. Hoeckner, a# A. Panneerselvam, b J. Levitt, b J.-S. Bouillard, b S. Taniguchi, b L.-A. Dailey, d R. Ahmad Khanbeigi, d E. V. Rosca, d M. Thanou, d K. Suhling, b A. V. Zayats, b M. Green, b,c,e * Methods Electron microscopy and energy dispersive X-ray spectroscopy were performed using a Tecnai 20 (acceleration voltage 200 kv) for normal resolution images. Samples were dropped on a copper grid with a carbon amorphous film (Agar Scientific) and dried in ambient conditions. High resolution images and selected area electron diffraction patterns were collected at the University of Leeds EPSRC Nanoscience and Nanotechnology Research Equipment Facility, using a FEI Tecnai F20 200kv FEGTEM fitted with a Gatan Orius SC600 CCD camera and an Oxford Instruments 80mm2 X-Max SDD EDX detector. The absorption spectra were recorded using a Hitachi U-4100 UV-Visible-NIR spectrophotometer in a 1 cm path length quartz cuvette. Emission spectra were obtained using a Perkin Elmer LS 50B spectrometer. For emission lifetime measurements, the sample was placed in a 1 cm path length quartz cuvette (Hellma) and was measured at 23 C using a time-correlated single photon counting system (FluoroCube, Horiba Jobin Yvon). The sample was excited using a nanoled source at 482 nm with a pulse duration of < 200 ps and repetition rate of 1 MHz, and the emission was detected at 495 nm. The fluorescence decay was fitted using a biexponential model in the DAS6 software package (Horiba Jobin Yvon) with one fast lifetime component of 70 ± 10 ps (shorter than the instrumental response of the system) and a longer component with a lifetime of 4.54 ± 0.03 ns. See figure 1e. Live cell imaging J774A.1 cells (derived from BALB/c mice) were chosen as a model macrophage-like cell line due to their durability and high rate of fluid phase uptake. Cells were seeded onto 8- chambered coverglasses at a density of 1x10 6 cells/cm 2 and incubated overnight in cell culture medium (DMEM 4.5 g/l glucose supplemented with 10% (v/v) fetal bovine serum, 1% sodium pyruvate, 1% penicillin/ streptomycin, 1% HEPES buffer and 1% L-glutamine) in a humidified 5% CO 2 : 95% air incubator at 37 C. Prior to imaging experiments, the cell culture medium was removed and replenished with 100 µl fresh cell culture medium. Live cell imaging was performed using a Leica DMIR E2 confocal microscope (Leica Microsystems, Milton Keynes, UK). Quantum dot luminescence was visualised at λ ex = 405 nm; λ em = nm and compared with phase contrast transmission images collected using a separate channel at a magnification of 40x. Images of the untreated cell monolayer was used to choose instrument sensitivity so that cell autofluorescence was at a minimum (gain: 616 V, contrast: -2.8%, laser strength: 55%) and setting were not altered throughout the duration of the experiment. 10 µl of either (A) CdTe particle extract isolated freshly from chloragogen cells of L.rubellus or (B) CdTe particle extract isolated freshly from chloragogen cells of L.rubellus and subsequently mixed with dithiolated PEG (~50 mg/ml 1 NATURE NANOTECHNOLOGY 1

2 final PEG concentration) were carefully added to the cell culture medium and the first image taken (t=0 min), with subsequent images taken at t= 10 and 20 min. IGROV-1 cells were plated in 8 well chamber cover glass slides over night at a density of 30,000 cells/well. The cells were maintained in RPMI-1640 media supplemented with 10% FBS and 1% penicillin/streptomycin at incubated in a humidified environment at 37 C with 5% CO 2. Next day the media was removed and replaced with media containing the dots. The cells were incubated with the dots for three hours at 37 C, after which they were washed three times with PBS. Cells were fixed with 1% paraformaldehyde by incubating 15 minutes at room temperature, washed again 3 times and incubated with PBS containing 50 µg/ml DAPI for nuclear counter staining. Slides were stored in PBS, at 4 C until the next day when they were imaged. Cells were imaged at the Nikon Imaging Centre, core facility at King s College London using a NIKON A1R SI MP confocal microscope. Images were acquired at 40x and 100x objectives using an excitation of 405 nm and emission of 518 nm. Untreated cells were imaged as control at the same imaging setting and showed no or very minimal background fluorescence. Particle size distribution analysis The hydrodynamic size distribution curves of CdTe particles and PEG-modified CdTe particles were measured using photon correlation spectroscopy (Nanosizer, Malvern Instruments, UK). 100 µl of each sample was mixed with 900 µl serum-supplemented cell culture medium and the size distribution curves measured at a scattering angle of 173. The instrument parameters used for measurements were: Estimated refractive index of particles=1.350, refractive index of DMEM /FBS 10% = 1.337, temperature = 37 C, dynamic viscosity of cell culture medium = 0.738x10-3 Pa s. The mixtures were incubated for 30 min at 37 C with particle size measurements taken every 10 minutes. 2 NATURE NANOTECHNOLOGY

3 SUPPLEMENTARY INFORMATION Supplementary Figures Supplementary figure 1 (A) Confocal laser scanning microscopic images of live J774.1 murine macrophage-like cells exposed to CdTe particles isolated directly from chloragogen cells over a range of incubation times (at t= 0, 10 and 20 min). Scale bar is 37.5 µm. Note that no labelling was observed; (B) Microscopic images of live J774.1 murine macrophage-like cells exposed to CdTe particles isolated from chloragogen cells (and subsequently modified with dithiolated polyethylene glycol) over a range of times (at t= 0, 10 and 20 min). Scale bar is 37.5 µm. The images show that the addition of a thiolated polyethylene glycol to the biosynthesised quantum dots results in the successful application in labelling macrophage cells. NATURE NANOTECHNOLOGY 3

4 Supplementary figure 2 The particle size distribution of each sample diluted 1:10 in serum-supplemented cell culture medium was assessed at t= 0, 10 and 20 min to determine whether aggregation of (A) CdTe particles and (B) polyethylene glycol (PEG)-modified CdTe particles occurred upon exposure to cells and could be responsible for luminescence quenching. Although the particle size distribution curves reflect the complexity of the systems, the CdTe particles (A) are marked by a greater degree of change in particle size distribution towards larger particle sizes over time, as compared to the PEG-modified particles (B). 4 NATURE NANOTECHNOLOGY

5 SUPPLEMENTARY INFORMATION Supplementary figure 3 - Compilation of images of CdTe labelled ovarian cancer cell line (IGROV-1) taken at different levels of the z stack. Each step is 0.25 µm. Blue correspond to the cell nucleus stain dye 4',6-diamidino-2-phenylindole, the green correspond to the CdTe quantum dots. NATURE NANOTECHNOLOGY 5