Toggling Between Blue and Red Emitting Fluorescent Silver Nanoclusters

Transcription

1 Supporting Information Toggling Between Blue and Red Emitting Fluorescent Silver Nanoclusters Uttam Anand, Subhadip Ghosh and Saptarshi Mukherjee * Department of Chemistry, Indian Institute of Science Education and Research Bhopal ITI Campus (Gas Rahat) Building, Govindpura, Bhopal , Madhya Pradesh, India saptarshi@iiserb.ac.in; Fax: ; Tel.: Authors share equal contribution

2 Experimental Details: (a) Instrumentation Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry studies were carried out using ABI Sciex 5800 MALDI-TOF/TOF and sinapinic acid as the matrix. Transmission Electron Microscope (TEM) images were obtained from JEOL 2100F operating at 200 kv. Carbon coated copper grid was used as support material for sample imaging. Absorption data was obtained using Perkin-Elmer Lambda 25 spectrophotometer by scanning in the range from 200 nm to 800 nm. Steady-state fluorescence measurements were recorded on a Horiba Jobin Yvon Fluorolog The fluorescence spectra were measured with a 2 mm path length quartz cuvette. Both the emission and the excitation slits were kept at 5 nm. For fluorescence lifetime measurements, the Ag 9 :HSA were excited at 375 nm and Ag 14 :HSA at 470 nm using N-375L and N-470L picosecond diodes (IBH-NanoLED), respectively. The emission was collected at magic angle polarization using a Hamamatsu MCP Photomultiplier (Model R-3809U-50). The timecorrelated single photon counting (TCSPC) set up consist of an Ortec 9327 pico-timing amplifier. The data was collected with a PCI-6602 interface card as a multi-channel analyzer. Full Width at Half Maximum (FWHM) for the 375 nm and 470 nm excitation was about 145 ps and 155 ps, respectively. (b) Materials: Human serum albumin (HSA) was purchased from Sigma Aldrich (Germany). Silver nitrate (AgNO 3 ), sodium borohydride (NaBH 4 ) and sodium hydroxide (NaOH) were purchased from Rankem (India) and 30% Hydrogen peroxide (H 2 O 2 ) was purchased from

3 MERCK (India). All the chemicals were of analytical grade and were used without further purification. Mili-Q water was used throughout the experiments. (c) Synthesis of Ag:HSA NCs: (i) Synthesis of blue emitting Ag 9 :HSA: Synthesis of Ag 9 :HSA nanoclusters was carried out under optimized conditions by adding 5ml aqueous solution of AgNO 3 (10mM) to HSA solution (0.75 mm) under vigorous stirring. After 2 minutes, 0.3 ml of NaOH (1000 mm) was added to the reaction mixture in order to maintain the reaction ph~11 thereby activating the reducing ability of HSA (rendered by the 18 tyrosine residues in HSA). The mixture was incubated at 37 C for 10 hours under constant vigorous stirring. The Ag 9 :HSA formation was indicated by the change in the colour of the solution from colourless to yellow (stable for months) when viewed under ordinary visible light. (ii) Synthesis of red emitting Ag 14 :HSA: In a typical synthesis, Ag 14 :HSA nanoclusters were prepared via conventional borohydride reduction procedure by adding 5ml aqueous solution of AgNO 3 (10mM) to HSA solution (0.75 mm) under vigorous stirring at room temperature. After 2 minutes, 0.3 ml of NaOH (1000 mm) was introduced to the reaction mixture followed by addition of NaBH 4 (10mM) drop-wise until the colour of the solution turns from colourless to reddish brown indicating nanocluster formation. The reaction takes about 2 minutes for completion which is much faster compared to Ag 9 :HSA nanoclusters preparation. (iii) Inter-conversion of Ag:HSA NCs: The Ag 9 :HSA when treated with 10 µl (per ml of Ag 9 :HSA), 150 mm NaBH 4 drop-wise turns red having spectral properties very similar to Ag 14 :HSA. The Ag 14 :HSA when treated with 15 µl (per ml of Ag 14 :HSA) 30% H 2 O 2 yields Ag 9 :HSA will almost 100% recovery.

4 Figure S1: Normalized absorption profiles of HSA (black) and different synthesized Ag:HSA nanoclusters as marked in the figure. The absorption profiles for HSA alone differ markedly when the Ag:HSA NCs are formed.

5 Figure S2: Fluorescence anisotropy decays measured to calculate the rotational anisotropy time (τ rot ). (a) represents the anisotropy decay for the Ag 9 :HSA and (b) represents the anisotropy decay for the Ag 14 :HSA. The decay of τ rot for Ag 14 :HSA remains virtually constant throughout the lifetime of the fluorophore and hence accounts for the exceptionally larger value of residual anisotropy (r ). This further exemplifies the fact that the size of Ag 14 :HSA NCs are larger than the Ag 9 :HSA NCs.

6 Figure S3: Transmission Electron Microscope images for (a) Ag 9 :HSA exhibiting a wider distribution of the NCs (b) a zoomed in version of Ag 9 :HSA and (c) a zoomed in version of Ag 14 :HSA NCs.

7 Figure S4: Fluorescence emission spectra of the Ag:HSA nanoclusters at two different temperatures, 25 C and 37 C. The lower fluorescence intensity at 25 C (especially for the Ag 9 :HSA) signifies a lower quantum yield at that temperature compared to the physiological temperature (37 C).

8 Figure S5: Fluorescence emission spectra of Ag 9 :HSA (black) and Ag 9 :HSA when converted from Ag 14 :HSA (blue) when excited at 380 nm. The same samples were also excited at 480 nm and they do not exhibit any fluorescence; thereby proving that the Ag 9 :HSA species are almost exclusively formed having emission maximum at 460 nm. The red spectra of Ag 9 :HSA (excited at 480 nm) was multiplied by 4 so as to make it visible and differentiate it from the cyan spectra (converted Ag 9 :HSA).

9 Figure S6: Fluorescence emission spectra of Ag 14 :HSA (black, excited at 380 nm and red, excited at 480 nm). For both the excitation wavelengths, there is only one peak centred at 620 nm proving the exclusive existence of Ag 14 :HSA. Fluorescence spectra of Ag 14 :HSA when converted from Ag 9 :HSA (blue, excited at 480 nm and cyan, excited at 380 nm). The cyan spectra shows an emission peak of much lower intensity at around 460 nm showing the presence of a small fraction of Ag 9 :HSA besides the major contribution from the red emitting Ag:HSA nanoclusters.

10 Figure S7: Normalized fluorescence excitation spectra of the various Ag:HSA nanoclusters. The resemblance of the excitation spectra signifies that even after conversion from Ag 9 :HSA to Ag 14 :HSA and vice-versa, there are no major structural alterations encountered.

11 Figure S8: Fluorescence lifetime decays for the converted Ag 9 :HSA and Ag 14 :HSA. The scattered red and blue points represent the actual decay profile while the solid green lines represent a tri-exponential fit to that decay. The IRF is the Instrument Response Function, i.e. the contribution from the laser diodes which was deconvulated after fitting. Since the IRFs from both the laser diode sources (N-375L and N-470L) were almost similar, the IRF for N-375L laser diode has been shown here. The inset represents the Y-axis in log-scale to depict the differences at longer time.

12 Figure S9: Fluorescence anisotropy decays measured to calculate the rotational anisotropy time (τ rot ). (a) represents the anisotropy decay for the blue emitting Ag:NC converted from Ag 14 :HSA and (b) represents the anisotropy decay for the red emitting Ag:NC converted from Ag 9 :HSA. The decay of τ rot for the red emitting Ag:NC converted from Ag 9 :HSA remains virtually constant throughout the lifetime of the fluorophore and hence accounts for the exceptionally larger value of residual anisotropy (r ).

13 Figure S10: Two-photon excitation and emission spectra for (a) Ag 9 :HSA excited at 760 nm and (b) Ag 14 :HSA excited at 960 nm. The intense fluorescence for both the Ag 9 :HSA and Ag 14 :HSA are obtained by near-ir excitation which is extremely useful for delicate biological samples owing to the low energy of excitation.

14 Figure S11: Figure represents the importance of the synthesized Ag:HSA nanoclusters as photo-luminescent markers. The glass slide has IISERB written and viewed (a) under ordinary white light (b) under UV light when Ag 9 :HSA was used as the marker and (c) under UV light when Ag 14 :HSA was used as the marker.