Supporting InformationInternalization of a Preformed Atomically Precise Silver Cluster in Proteins by Multistep Events and Emergence of Luminescent Counterparts Retaining Bioactivity

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Supporting Information

Internalization of a Preformed Atomically Precise Silver Cluster in Proteins by Multistep Events and Emergence of Luminescent Counterparts Retaining Bioactivity

Debasmita Ghosh,¹ Mohammad Bodiuzzaman,¹ Anirban Som,¹ Sebastian Raja,² Ananya Baksi,¹ Atanu Ghosh,¹ Jyotirmoy Ghosh,¹ Akshayaa Ganesh,² Priyanka Samji,² Sundarasamy Mahalingam,² Devarajan Karunagaran,² and Thalappil Pradeep^1*

1DST Unit of Nanoscience (DST UNS) and Thematic Unit of Excellence, Department of Chemistry, Indian Institute of Technology Madras, Chennai 600036, India

2Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India Table of Contents

Number Description Page

Number SI Protocols for the synthesis of [Ag18HSA] and [Ag13Lf] S2 Figure S1 The EDS spectrum of the precipitate S3

Figure S2 The EDS spectrum of the cluster S4

Figure S3 Total XPS survey spectrum of Ag18@BSA S5 Figure S4 Characterization of the Ag18@HSA cluster S6 Figure S5 MALDI MS of the Ag18@HSA cluster S7 Figure S6 Characterization of Ag13@Lf cluster S8

S2

Table S1 Number of cysteine/methionine, Trp/Tyr/His, and disulfide

bridges present in BSA, HSA, and Lf S9 Figure S7 Time dependent luminescence profile of the Ag18@BSA

cluster S10

Figure S8 Reaction of [Ag18H16(TPP)10]²⁺ with less amount of BSA S11

Figure S9 Reaction of BSA with Ag NP S12

Figure S10 Cell viability with Ag18@BSA S13

Figure S11 Confocal fluorescence imaging of HeLa cells with NCs after 5 h incubation

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Figure S12 Z stacking of all images S15

Figure S13 Phosphatase activity of pure Lf and Ag13@Lf S16

Protocol for the synthesis of [Ag18HSA]:

25 mg HSA was dissolved in 1.6 mL Milli-Q water. Then stirred the solution for 1 min. Under stirring condition, 400 µL of I was added. The addition of I into the HSA solution made the reaction mixture turbid. The turbid solution was centrifuged after 12 h and from the supernatant the brown colored cluster solution was collected. This solution was used for characterization.

Protocol for the synthesis of [Ag13Lf]:

25 mg Lf was dissolved in 1.6 mL Milli-Q water. Then stirred the solution for 1 min. Under stirring condition, 400 µL of I was added. The addition of I into the Lf solution made the reaction mixture turbid. The turbid solution was centrifuged after 12 h and from the supernatant the brown colored cluster solution was collected. This solution was used for characterization.

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Elem Wt % At % --- C K 71.26 79.60 N K 8.68 8.32 O K 12.52 10.50 P K 1.34 0.58 S K 0.77 0.32 AgL 5.44 0.68 Total 100.00 100.00

A)

B)

Figure S1: The EDS spectrum of the precipitate along with the quantification data (A). (B) is the image of the precipitate under visible light (left) and under UV light (right).

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B)

D) E)

C)

Ag L

S K N K

Elem Wt % At % --- C K 70.03 76.35 N K 4.07 3.80 O K 21.57 17.63 NaK 3.62 2.06 S K 0.28 0.11 AgL 0.43 0.05 Total 100.00 100.00

A) B)

D) E)

C)

Ag L

S K N K 50 µm

Ag:S(Wt%) Expected 1.53 Observed 1.53

Figure S2: The EDS spectrum of the Ag₁₈@BSA cluster along with the quantification data (A).

(C–E) are the EDS images of the cluster collected using Ag Lα, S Kα, and N Kα emissions.

Corresponding SEM image of the Ag18@BSA is shown in B. The quantification data suggest that the Ag:S ratio is in good agreement with the theoretical value for Ag₁₈@BSA.

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0 200 400 600 800 1000

0 10k 20k 30k 40k 50k 60k

In te ns ity

Binding Energy (eV)

365 370 375 380

158 160 162 164 166 168

Intensity

Binding Energy (eV) Binding Energy (eV)

3d_5/2

3d_3/2 2p_3/2

S 2p

2p_1/2

Ag 3d

S 2p

Ag 3d

O 1s

C 1s

Figure S3: Total XPS survey spectrum of Ag18@BSA. The insets show the spectra of the cluster in the Ag 3d region and S 2p regions.

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400 500 600 700 800

300.0k 600.0k 900.0k

1.2M Ex 1

Ex 2 Em1

Wavelength (nm)

B)

400 600 800

0.0 0.5 1.0 1.5

400 500 600 700 800

0.009 0.018 0.027 0.036

Wavelength(nm)

Wavelength (nm)

Absorbance

A) visible

UV

Intensity

Figure S4: Characterization of the Ag₁₈@HSA cluster. (A) UV-vis profile of HSA (blue trace) and Ag18@HSA cluster (red trace). (B) Luminescence profile of the Ag18@HSA cluster. Inset shows the color of HSA and the cluster under visible light (top) and under UV light (bottom).

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50k 60k 70k 80k 90k

m/z

HSA

Ag

₁₈

@HSA 18 Ag

Figure S5: MALDI MS of pure HSA (blue trace) collected in linear positive ion mode using sinapic acid as matrix and that of the as-prepared red emitting Ag18@HSA (red trace).

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80000 90000 100000

400 600 800 1000

0.0 0.5 1.0 1.5

400 450 500 550 600

0.00 0.05 0.10

400 450 500 550 600 650 700 200000

400000 600000 800000 1000000 1200000

Wavelength (nm)

Intensity

Wavelength (nm)

Absorbance

m/z 13 Ag

A) B)

C)

Wavelength (nm) 500

450 645

Ag₁₃@Lf Lf

Figure S6: Characterization of Ag13@Lf cluster. (A,B) UV-vis and luminescence spectra (excitation at 365 nm) of Ag₁₃@Lf cluster. C) MALDI MS spectra of the parent protein and the cluster showing the formation of the Ag13@Lf cluster.

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Table S1: Number of cysteine/methionine, Trp/Tyr/His, and disulfide bridges present in BSA, HSA, and Lf.

Cys (C) Met (M) Trp (W) Tyr (Y) His (H) Disulfide

bond

BSA 35 5 2 19 17 17

HSA 35 5 1 16 17 17

Lf 33 3 13 19 10 16

Note: Though the molecular weight of Lf is high than BSA and HSA, the number of His residue present in Lf is less. Thus the ability of Lf to stabilize the small clusters formed at initial stage is expected to be lower than both BSA and HSA. This probably result in the formation of a smaller 13-atom Ag cluster in this case.

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In te ns ity

Wavelength (nm)

0 hour 2 hours 4 hours 12 hours

6 hours 8 hours

400 500 600 700

0 100k 200k 300k

Figure S7: Time dependent luminescence profile of the Ag18@BSA cluster during synthesis.

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400 600 800 1000

0.0 0.5 1.0

A bs or ba nc e

Wavelength (nm)

Figure S8: UV-vis profile of Ag nanoparticle formed in a reaction of [Ag₁₈H₁₆(TPP)₁₀]²⁺ cluster with reduced concentration of protein.

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30k 40k 50k 60k 70k 80k 90k

m/z

Wavelength (nm)

Ag-citrate NP

BSA

After reaction with Ag NP B)

A)

400 600 800 1000

0.0 0.5 1.0 1.5 2.0

Absorbance

[1

⁺

]

[2

⁺

]

[1

⁺

]

[2

⁺

] * *

Figure S9: (A) MALDI MS of BSA before (blue) and after (red) reaction with Ag Nps. The presence of conalbumin in the BSA solution is indicated with an asterisk (*). (B) UV-vis spectrum of Ag Nps.

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100%

80%

60%

40%

20%

0 2 5 8 10

Concentration of Ag

₁₈

@BSA (µM)

C el l v ia bi lit y af te r 2 4 ho ur s

Figure S10: The viability of HeLa cells after treating with different concentrations of Ag18@BSA NCs for 24 hours. The viability was obtained by resazurin assay. The error bar represents standard error of 3 trials.

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5µm

5µm 5µm

5µm

A ) B )

C ) D )

Figure S11: Microscopic images of the HeLa cells after 5 h incubation with NCs. (A) Fluorescence image of NCs in cells (B) Cell nucleus stained with DAPI. (C) Bright-field image of the corresponding fluorescence images. (D) An overlay of these three images.

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Figure S12: Z stacking images of cells shows the internalization of NCs in the HeLa cells.

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0 10 20 30 40 50 60

0.0 0.1 0.2

0.3

_Ag^Lf

13@Lf

A bs or ba nc e at 4 00 n m

Time (min)

Activity Lf 100%

Ag₁₃@Lf 90%

Figure S13: Phosphatase activity of pure Lf and Ag13@Lf clusters as a function of time. Cluster showed 90% catalytic activity.