Supporting information for the paper:
Understanding the Evolution of Luminescent Gold Quantum Clusters in Protein Templates Kamalesh Chaudhari, †,‡ Paulrajpillai Lourdu Xavier, ‡ Thalappil Pradeep*,‡ †Department of Biotechnology and ‡DST Unit of Nanoscience (DST UNS), Department of Chemistry, Indian Institute of Technology Madras, Chennai 600 036, India *Address correspondence to
[email protected] 1
Table of contents Sl. No.
Title
Page No.
Time dependent MALDI MS of NLf in the presence and absence of 1
4 NaOH.
2
Experimental details to check the reversibility of NLf mass spectrum.
5
Change in percentage contribution of Au25@NLf, Au13@NLf and NLf 3
6 with time.
4
Photograph of AuQC@NLf in visible and UV light at different times.
7
Concentration dependent time evolution of the excitation spectra of 8
5 AuQC@NLf. Concentration dependent time evolution of the emission spectra of 6
9 AuQC@NLf.
7
Comparison between MALDI MS of AuQC@NLf and AuQC@(NLf)2.
10
8
Time evolution of the MALDI MS of AuQC@NLf after 3 days.
11
9
Saturation effect at 0 h for varying molar ratios of Au3+:NLf.
12
Luminescence spectrum of Au3+ and NLf mixture in the absence of 10
13 NaOH. Time evolution of the MALDI MS of AuQC@NLf in the absence of
11
14 NaOH.
12
XPS in the S 2p region.
15
Change in the emission peak position with time for different molar 13
15 ratios of Au3+:NLf.
2
Sl. No.
Title
Page No.
Change in the emission peak position with time when pH of the 14
16 sample was maintained constant. MALDI MS of AuQC@NLf with time for different molar
15
17 combinations of Au3+:NLf at constant pH.
16
MALDI MS of the clusters synthesized by one step approach.
18
17
Photograph of the clusters synthesized by one step approach.
19
Time dependent UV-Vis spectra for different molar combinations of 20
18 Au3+:NLf (250-350 nm). Time dependent UV-Vis spectra for different molar combinations of 19
21 3+
Au :NLf (450-800 nm). Schematic for the synthesis of AuQC@NLf by three different 20
22 approaches. MALDI MS and PL data of AuQC@NLf synthesized by two step
21
23 approach. UV-Vis spectra of AuQC@NLf synthesized by three different
22
24 approaches.
23
MALDI MS data of the clusters synthesized with multistep approach.
25
24
UV-Vis spectra of AuQC@BSA at various stages of evolution.
26
25
PL spectra of AuQC@BSA at various stages of evolution.
27
3
Supporting information 1. Time dependent MALDI MS of NLf in the presence and absence of NaOH.
NLf in DI water
A
NLf at pH ~12.4
B
24 h
12 h
8h
4h
12 h
Normalised intensity
Normalised intensity
24 h
8h
4h
0h
0h
75k
80k
85k
90k
95k
100k
75k
80k
85k
m/z
90k
95k
100k
m/z
Figure S1. Time dependent MALDI MS data of (A) NLf in DI water (pH ~ 7) (average peak position ~ 83,338 Da) and (B) NLf at pH ~12.4 (average peak position ~ 82,842 Da). Peak difference is m/z 496, which proves that there is downshift in mass of NLf by ~500 at alkaline pH.
4
Experimental details to check reversibility of m/z value of NLf at basic pH To check the reversibility of the downshift in mass caused by alkaline pH, we have performed an experiment in which MALDI MS of NLf samples at neutral pH and basic pH (5% NaOH) was monitored after 12 h. Then pH of NLf (in 5% NaOH) was reverted to neutral by adding HCl and MALDI MS analysis was done after 12 h. Averaging of 5 MS of all three samples have shown that downshift in mass of NLf is not reversible. Values obtained are as below, NLf in DI water – m/z 83805 NLf at basic pH - m/z 83199 NLf at basic pH after reverting the pH – m/z 83112
5
Table 1. Change in percentage contribution of Au25@NLf, Au13@NLf and NLf with time. This relative quantification is obtained by peak fitting of the MALDI MS data. % of NLf and AuQC@NLf after 12 h of reaction Peak
Cluster
FWHM
Percentage contribution, %
1
Au25@NLf
5670.775
88.82
2
Au13@NLf
1773.100
4.04
3
NLf
1624.000
7.14
% of NLf and AuQC@NLf after 72 h of reaction Peak
Cluster
FWHM
Percentage contribution, %
1
Au25@NLf
5130.995
45.26
2
Au13@NLf
3508.015
7.12
3
NLf
2903.470
47.61
% of NLf and AuQC@NLf (after 48 h) when extra Au3+ was added to the parent reaction after 24 h Peak
Cluster
FWHM
Percentage contribution, %
1
Au25@NLf
6643.915
79.80
2
Au13@NLf
3517.271
---
3
NLf
3506.934
20.19
6
Supporting information 2. Photograph of AuQC@NLf in visible and UV
48 h
24 h
12 h
8h
4h
0h
NLf+Au
NaOH added →
UV
Visible
NLf
light at different times.
Figure S2. Photograph taken in visible and UV light to show the enhancement in luminescence intensity as a function of time in AuQC@NLf system indicating the evolution of clusters.
7
Supporting information 3. Concentration dependent time evolution of the excitation spectra of AuQC@NLf. 50M 0h 4h 8h 12 h 24 h 48 h
Intensity (a.u.)
40M 30M 20M
B
0h 4h 8h 12 h 24 h 48 h
12M
Intensity (a.u.)
A
9M
6M
3M
10M 0
0
280
320
360
400
280
Wavelength (nm)
360
400
440
480
Wavelength (nm)
6.0M 0h 4h 8h 12 h 24 h 48 h
4.0M
2.0M
5M
D
0h 4h 8h 12 h 24 h 48 h
4M
Intensity (a.u.)
Intensity (a.u.)
C
320
3M 2M 1M
0.0
0 300
400
500
600
300
Wavelength (nm)
Intensity (a.u.)
E
400
500
600
Wavelength (nm)
1M
0h 4h 8h 12 h 24 h 48 h
900k
600k
300k
0 300
400
500
600
Wavelength (nm)
Figure S3. Incubation time dependent excitation spectra collected for the emission wavelength of 670 nm, indicating the evolution of cluster upon the interaction of Au3+ with NLf. NLf concentration was kept constant 150 µM while Au3+ concentration was varied as follows: (A) 0.5 mM, (B) 1.5 mM., (C) 2.5 mM, (D) 3.5 mM, and (E) 4.5 mM. Secondary peak at 335 nm is removed in these spectra. 8
Supporting information 4. Concentration dependent time evolution of the emission spectra of AuQC@NLf. 300k 250k 200k
Intensity (a.u.)
B
0h 4h 8h 12 h 24 h 48 h
2.0M 0h 4h 8h 12 h 24 h 48 h
1.5M
Intensity (a.u.)
A
150k 100k
1.0M
500.0k
50k 0
0.0
400
500
600
700
400
Wavelength (nm)
C
0
2M
1M
0 400
500
600
700
Wavelength (nm)
400
500
600
700
Wavelength (nm)
750k 0h 4h 8h 12 h 24 h 48 h
600k
Intensity (a.u.)
0h 4h 8h 12 h 24 h 48 h
3M
Intensity (a.u.)
Intensity (a.u.)
0h 4h 8h 12 h 24 h 48 h
1M
E
700
4M
D
2M
600
Wavelength (nm)
4M
3M
500
450k 300k 150k 0 400
500
600
700
Wavelength (nm)
Figure S4. Time dependent emission spectra collected indicating evolution of the cluster as a function of incubation time of Au3+ with NLf in alkaline pH. NLf concentration was kept constant (150 µM) and Au3+ concentration was varied as (A) 0.5 mM, (B) 1.5 mM., (C) 2.5 mM, (D) 3.5 mM, and (E) 4.5 mM. Samples were excited at 370 nm. 9
Supporting information 5. Comparison between MALDI MS of AuQC@NLf and AuQC@(NLf)2. B
A
#(Au = 37
#(Au) = 25
12 h 12 h
#(Au) = 11
Normalized intensity
Normalized intensity
monomer
#(Au) = 20
dimer 0h
0h
60k
65k
70k
75k
80k
85k
m/z
90k
95k 100k
160k
170k
180k
190k
m/z
Figure S5. MALDI MS of (A) AuQC@NLf and (B) AuQC@(NLf)2 at 0 and 12 h. The number of Au atoms added in the monomer and the dimer are shown with vertical lines. Note that in both the cases, the same mass window is shown.
10
Supporting information 6. Time evolution of the MALDI MS of AuQC@NLf after 3 days. ~month 3
Normalized intensity
day 7
day 6
day 5
day 4
day 3
75k
80k
85k
90k
m/z
95k
100k
Figure S6. No considerable change was observed after 2 days when mass spectra were monitored for AuQC@NLf (17:1 molar ratio of Au3+:NLf). Reaction was carried out at alkaline pH (5% NaOH).
11
Supporting information 7. Saturation effect at 0 h for varying molar ratios of Au3+:NLf.
4.5 mM
Intensity normalized
3.5 mM
2.5 mM
1.5 mM
0.5 mM 75k
80k
85k
90k
95k
100k
m/z Figure S7. MALDI MS data showing saturation effect at 0 h when varying concentrations of Au3+ were added to the NLf solution (150 µM).
12
Supporting information 8. Luminescence spectrum of Au3+ and NLf mixture in the absence of NaOH.
Normalized intensity
3+ Au + NLf (2.5 mM) (150 µM)
400
450
500
550
600
650
Wavelength (nm) Figure S8. Luminescence spectrum of the reaction product when cluster synthesis was carried out in the absence of NaOH (pH ~ 7). Au3+:NLf molar ratio was 17:1. In this case, only emission from the protein was seen.
13
Supporting information 9. Time evolution of the MALDI MS of AuQC@NLf in the absence of NaOH.
A
B
8h
4h
72 h
Intensity normalized
Intensity normalized
12 h
60 h
48 h ~ 86.4 kDa
0h
75k
80k
85k
90k
m/z
95k
24 h
100k
75k
80k
85k
90k
95k
100k
m/z
Figure S9. Mass spectra slight shift in mass of protein indicating the binding of Au3+ ions as a function of incubation time of Au3+ with NLf in the absence of NaOH. In the absence of alkaline pH, protein was observed to bind to 13- 14 Au ions and form Au+ - protein complex (see XPS data in Figure 5).
14
Supporting information 10. XPS of the S 2p region. B
Intensity (a.u.)
Intensity (a.u.)
A
160
165
170
164
Binding energy (eV)
168 172 Binding Energy (eV)
Figure S10. XPS of S 2p (A) before addition of NaOH and (B) 24 h after addition of NaOH. The region shows higher binding energy peaks due to sulfate, sulfite and sulfonate species as a result of X-ray beam induced damage.
Supporting information 11. Change in the emission peak position with time for different molar ratios of Au3+:NLf. 2.5 mM
Peak position (nm)
680
1.5 mM 670
0.5 mM
660
3.5 mM
650
4.5 mM
640
10
20
30 Time (h)
40
50
Figure S11. Emission peak positions for different Au3+ concentrations by keeping NLf concentration constant (150 µM) plotted against time, peak position shows blue shift with increasing concentration of Au3+ against NLf 15
Supporting information 12. Change in the emission peak position with time when pH of the sample was maintained constant. 661 nm
3.5M 0.5 mM 1.5 mM 2.5 mM 3.5 mM 4.5 mM
Intensity (a.u.)
3.0M 2.5M 2.0M
670 nm
1.5M
675 nm
1.0M
650 nm
500.0k
673 nm
0.0 400
500
600
700
Wavelength (nm) Figure S12. PL spectra of AuQC@NLf with different Au3+:NLf ratios. NLf concentration was kept constant (150 µM) and Au3+ concentration was varied from 0.5 mM-4.5 mM. The pH of the solution was kept constant at 12.4±0.03.
16
Supporting information 13. MALDI MS of AuQC@NLf with time for different molar combinations of Au3+:NLf at constant pH.
82k
84k
86k
88k
90k
1h 24 h 48 h
80k
82k
m/z 3.5 mM
Intensity normalized
86k
88k
90k
92k
m/z
1h 24 h 48 h
80k 82k 84k 86k 88k 90k 92k 94k 96k
2.5 mM
1h 24 h 48 h
80k
82k
84k
86k
88k
90k
92k
94k
m/z
E
4.5 mM
Intensity normalized
D
84k
C
Intensity normalized
Intensity normalized
1h 24 h 48 h
80k
1.5 mM
B
Intensity normalized
0.5 mM
A
1h 24 h 48 h
80k
84k
m/z
88k
92k
96k
100k
m/z
Figure S13. MALDI MS data of AuQC@NLf as time progresses for varying Au3+:NLf ratios. NLf concentration was kept constant (150 µM) and Au3+ concentration was varied as (A) 0.5 mM, (B) 1.5 mM, (C) 2.5 mM, (D) 3.5 mM, and (E) 4.5 mM. The pH of the solution was kept constant at 12.4±0.03. No noticeable change was observed in these mass spectra as compared to pH maintained samples. The occurrence of free protein is seen only at 48 h.
17
Supporting information 14. MALDI MS of the clusters synthesized by one step approach.
Au40 5 mM
Normalized intensity
Au39 Au34 Au30
Au26 Au25
75k
80k
85k
90k
m/z
4.5 mM
4 mM
3.5 mM
3 mM 2.5 mM
95k
100k
Figure S14. MALDI MS data of the clusters synthesized by one step approach. Spectra were collected after 48 h of reaction. Bigger sized clusters were seen upon addition of Au3+ above a concentration of 2.5 mM in the beginning of the reaction.
18
Supporting information 15. Photograph of the clusters synthesized by one
UV
Visible
step approach.
2.5
3
3.5 4 4.5 Au3+ (mM)
5
Figure S15. Photograph of the clusters synthesized by one step approach taken in the visible and UV light to show variations in the luminescence intensity of AuQC@NLf by one step approach.
19
Supporting information 16. Time dependent UV-Vis spectra for different molar combinations of Au3+:NLf. 1.0
B
3+
Au 0.5 mM 0h 4h 8h 12 h 24 h 48 h
0.8 0.6
Absorbance (a.u.)
Absorbance (a.u.)
A
0.4 0.2 0.0
1.5 3+
Au 1.5 mM 0h 4h 8h 12 h 24 h 48 h
1.0
0.5
0.0 250
275
300
325
350
260
Wavelength (nm)
280
D
3+
Absorbance (a.u.)
Absorbance (a.u.)
Au 2.5 mM 0h 4h 8h 12 h 24 h 48 h
Au 3.5 mM 0h 4h 8h 12 h 24 h 48 h
2
1
0
0 260
280
300
320
340
250
Wavelength (nm)
E
340
3
3+
1
320
Wavelength (nm)
C 2
300
275
300
325
350
Wavelength (nm)
3
Absorbance (a.u.)
3+
Au 4.5 mM 0h 4h 8h 12 h 24 h 48 h
2
1
0 250
275
300
325
350
Wavelength (nm)
Figure S16. Time dependent UV-Vis spectra upon interaction of Au3+ with NLf. NLf concentration was kept constant (150uM) and Au3+ concentration was varied as (A) 0.5 mM, (B) 1.5 mM, (C) 2.5 mM, (D) 3.5 mM, and (E) 4.5 mM.
20
Supporting information 17. Time dependent UV-Vis spectra for different molar combinations of Au3+:NLf. A
0.5 mM
0h 4h 8h 24 h 48 h
622
484
B
1.5 mM
0h 4h 8h 12 h 24 h 48 h
672
Absorbance (a.u.)
Absorbance (a.u.)
772
483 514
771
485 515 504
755
486
584 722
500
600
C
700
800
500
600
700
800
Wavelength (nm)
800
0h 4h 8h 12 h 24 h 48 h
728 762 750
500
600
700
800
Wavelength (nm) 0h 4h 8h 12 h 24 h 48 h
4.5 mM
Absorbance (a.u.)
700
3.5 mM
0h 4h 8h 12 h 24 h 48 h
506
600
Wavelength (nm)
Absorbance (a.u.)
Absorbance (a.u.)
2.5 mM
E
500
D
Wavelength (nm)
712 508
489
500
600
700
800
Wavelength (nm)
Figure S17. Time dependent UV-Vis spectra upon interaction of Au3+ with NLf; showing multiple features which are changing with time. NLf concentration was kept constant (150 µM) and Au3+ concentration was varied as (A) 0.5 mM, (B) 1.5 mM, (C) 2.5 mM, (D) 3.5 mM, and (E) 4.5 mM. 21
Scheme 1. Schematic for the synthesis of AuQC@NLf by three different approaches. One step synthesis Reaction was began with different concentrations of Au3+ HAuCl4 (1 mL, 1 mM)
Two step synthesis Final Au3+ concentration was adjusted to,
HAuCl4 (1 mL, 3 mM) NLf (1 mL, 25 mg/mL)
+
HAuCl4 (1 mL, 5 mM) HAuCl4 (1 mL, 7 mM)
+
NaOH (100 µL, 1M)
24 h
24 h
3 mM 3.5 mM
3 mM HAuCl4 (1 mL, 9 mM)
24 h 3.5 mM
4M
24 h
4 mM 4.5 mM 5 mM
4 mM
I670 nm
3M
24 h 4.5 mM
2M
24 h 1M 0
One step Two step Multistep
2.5 3.0 3.5 4.0 4.5 5.0 3+ Au (mM)
5 mM Multi step synthesis Final Au3+ concentration was adjusted after each 24 h
Scheme 1. Schematic showing different approaches used for attaining monodispersed clusters with enhanced luminescence.
22
Supporting information 18. MALDI MS and PL data of AuQC@NLf synthesized by two step approach. 4M
C
2.5 mM
3M 2 mM
2M
1M
0
0.0
B
0.5
1.0
1.5
2.0
2.5
3+
Visible
Intensity normalised
Intensity at 670 nm
A
1.5 mM
1 mM
0.5 mM
Conc. of Au added to AuQC@NLf (mM) 0 mM
UV
Concentration of gold added to AuQC@NLf (mM)
75k
80k
85k
90k
m/z
95k
100k
Figure S18. AuQC@NLf were synthesized with the 17:1 molar ratio of Au3+:NLf and after 24 h of reaction final concentration of Au3+ was adjusted from 3-5 mM to interact with the emerged free proteins (lost gold ions and became free during the reaction). (A) Bar diagram shows that 1 mM Au3+ is enough to consume the free protein and form additional clusters to enhance luminescence. (B) Photograph taken in visible and UV light to show enhanced luminescence. (C) MALDI MS shows relative reduction in the free NLf peak after addition of Au3+ and cluster growth.
23
Supporting information 19. UV-Vis spectra of AuQC@NLf synthesized by three different approaches. A Absorbance (a.u.)
Absorbance (a.u.)
3+
Au added to NLf 2.5 mM 3 mM 3.5 mM 4 mM 4.5 mM 5 mM
One step 400
300
450
500
550
600
650
Wavelength (nm)
400
500
600
Wavelength (nm)
B
3+
0 mM 1 mM 2 mM
Absorbance (a.u.)
Absorbance (a.u.)
Au added to AuQC@NLf 0.5 mM 1.5 mM 2.5 mM
Two step 400
300
450
500
550
600
Wavelength (nm)
400
500
600
Wavelength (nm)
C Absorbance (a.u.)
Absorbance (a.u.)
3+
Final concentration of Au 3 mM 3.5 mM 4 mM 4.5 mM 5 mM
Multi step 400
300
450
400
500
550
600
Wavelength (nm)
500
650
600
Wavelength (nm) Figure S19. UV-Vis spectra of AuQC@NLf measured after 48 h for three different approaches, (A) one step, (B) two step and (C) multi step. 24
Supporting information 20. MALDI MS data of the clusters synthesized with multistep approach.
Normalized intensity
4.5 mM
75k
4.0 mM
3.5 mM
3.0 mM
2.5 mM
80k
85k
90k
95k
100k
m/z Figure S20. MALDI MS data of the clusters synthesized with multistep approach.
25
Supporting information 21. UV-Vis spectra of AuQC@BSA at various stages of evolution.
Absorbance (a.u.)
BSA 0 h 3+ BSA + Au 0 h After adding NaOH 0h 4h 8h 12 h 24 h 3+ 24 h after adding extra Au 0.0 mM 0.5 mM 1.0 mM
300
400
500
600
Wavelength (nm) Figure S21. UV-Vis spectra of AuQC@BSA at various stages of evolution.
26
Supporting information 22. PL spectra of AuQC@BSA at various stages of evolution.
Luminescence intensity
2.0M
I677 nm
1.5M 1.0M 500.0k
1.0 mM
0.5 mM
0.0 mM
24 h
12 h
8h
4h
0h
BSA + Au3+
BSA 0 h
0.0
After adding NaOH 24 h after adding extra Au3+
Figure S22. PL spectra of AuQC@BSA at various stages of evolution. Average emission peak position was found to be 677 nm for AuQC@BSA, hence PL intensity was compared at 677 nm. It can be seen that as emergence of parent protein takes place after 8 h, PL intensity decreases. Then after adding extra Au3+ to consume parent protein, increase in the PL intensity was observed.
27