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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]

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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.

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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.

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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

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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.

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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.

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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).

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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).

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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.

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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.

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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