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1 Supplementary information doi: /nchem.334 Sensing of Proteins in Human Serum using Nanoparticle-Green Fluorescence Protein Conjugates MRINMOY DE, 1 SUBINOY RANA, 1 HANDAN AKPINAR, 1 OSCAR R. MIRANDA, 1 ROCHELLE R. ARVIZO, 1 UWE H. F. BUNZ 2 AND VINCENT M. ROTELLO* 1 1 Department of Chemistry, University of Massachusetts, 710 North Pleasant Street, Amherst, MA 01003, USA 2 School of Chemistry and Biochemistry, Georgia Institute of Technology, 770 State Street, Atlanta, Georgia 30332, USA * rotello@chem.umass.edu nature chemistry 1

2 Synthesis of ligands and nanoparticles. Scheme S1. Synthesis of ligands. General procedure: Compound 2 bearing ammonium end groups were synthesized by the reaction of 1,1,1-triphenyl-14,17,20,23-tetraoxa-2- thiapentacosan-25-yl methanesulphonate (1) with corresponding substituted N,N-dimethylamines with stirring for 48 hours at ~35 o C. The trityl protected thiol ligand (2) was dissolved in dry dichloromethane (Methylene Chloride, DCM) and an excess of trifluoroacetic acid (TFA, ~20 equivalents) was added. The color of the solution was turned into yellow immediately. Subsequently, triisopropylsilane (TIPS, ~1.2 equivalents) was added to the reaction mixture. The reaction mixture was stirred for ~5 h under Ar-atmosphere at room temperature. The solvent, most of the TFA and TIPS were distilled off under reduced pressure. The pale yellow residue was further dried in high vacuum. The product (L) formation was quantitative and their structure was confirmed by NMR and mass spectroscopy. The yields were >95%. Compound L1: 1 H NMR (400MHz, CDCl 3, TMS): 3.95 (br, 2H, -CH 2 O-), (m, 14H, - CH 2 O- + -OCH 2 -(CH 2 N)-), 3.49 (t, 2H, -CH 2 N-), 3.25 (s, 9H, -N(CH 3 ) 3 -), 2.90 (s, 3H, -CH 3 SO - 3-), 2.52 (q, 2H, -CH 2 S-), (m, 4H, (SCH 2 )CH 2 + -CH 2 (CH 2 O)-), (m, 15H, -SH + - CH 2 -). 13 C NMR (400 MHz, CDCl 3 ) (ppm): 71.58, 70.56, 70.49, 70.34, 70.24, 70.15, 70.04, 65.13, 54.72, 39.25, 31.07, 29.76, 29.65, 29.62, 29.57, 29.53, 29.51, 29.27, 28.56, ESI-MS (m/z): calculated for C 22 H 48 NO 4 S + [M + ], ; found nature chemistry 2

3 Compound L2: 1 H NMR (400MHz, CDCl 3, TMS): 3.95 (br, 2H, -CH 2 O-), (m, 14H, - CH 2 O- + -OCH 2 -(CH 2 N)-), 3.46 (t, 2H, -CH 2 N-), (m, 2H,-NCH 2 -), 3.19 (s, 6H, -(CH 3 ) 2 N-), 2.87 (s, 3H, -CH 3 SO - 3-), 2.52 (q, 2H, -CH 2 S-), (m, 6H, -(NCH 2 )CH 2 -) + (SCH 2 )CH CH 2 (CH 2 O)-), (m, 21H, -SH + -(NCH 2 CH 2 -)CH 2 -) + -CH 2 -), 0.89 (t, 3H, - CH 3 -). 13 C NMR (400 MHz, CDCl 3 ) (ppm): 71.59, 70.54, 70.51, 70.44, 70.33, 70.15, 70.00, 66.42, 64.82, 63.50, 51.90, 34.07, 31.18, 29.59, 29.54, 29.52, 29.49, 29.09, 28.39, 26.07, 25.84, 24.68, 22.71, 22.37, ESI-MS (m/z): calculated for C 27 H 58 NO 4 S + [M + ], ; found Compound L3: 1 H NMR (400MHz, CDCl 3, TMS): 4.78 (br, 1H, -CHOH(CH 2 OH)-), 4.59 (br, 1H, - CH 2 OH-), (m, 1H, -CHOH(CH 2 OH)-), 4.43 (d and br, 2H,-NCH 2 -), 3.95 (d and br, 2H, - CH 2 O-), (d and br, 2H, -CH 2 -OH), (m, 14H, -CH 2 O- + -OCH 2 -(CH 2 N)-), 3.48 (t, 2H, -CH 2 N-), 3.34 (s, 6H, -(CH 3 ) 2 N-), 2.99 (s, 3H, -CH 3 SO - 3-), 2.52 (q, 2H, -CH 2 S-), (m, 4H, + (SCH 2 )CH 2 + -CH 2 (CH 2 O)-), (m, 15H, -SH + -CH 2 -). 13 C NMR (400 MHz, CDCl 3 ) (ppm): 71.65, 70.45, 70.37, 70.19, 69.97, 68.07, 66.79, 66.47, 64.89, 64.81, 64.42, 63.83, 60.42, 53.59, 53.15, 45.41, 42.76, 39.51, 34.14, 29.55, 29.30, 29.16, 29.02, 28.74, 28.59, 28.46, 26.08, 24.74, ESI-MS (m/z): calculated for C 24 H 58 NO 4 S + [M + ], ; found Compound L4: 1 H NMR (400MHz, CDCl 3, TMS): 3.96 (br, 2H, -CH 2 O-), (m, 1H, H Cyclo ), (m, 14H, -CH 2 O- + -OCH 2 -(CH 2 N)-), 3.46 (t, 2H, -CH 2 N-), 3.12 (s, 6H, -(CH 3 ) 2 N-), 2.89 (s, 3H, -CH 3 SO - 3-), 2.52 (q, 2H, -CH 2 S-), 2.28 (d, 2H, H Cyclo ), 2.01 (d, 2H, H Cyclo ), (m, 4H, - (SCH 2 )CH 2 + -CH 2 (CH 2 O)-), 1.47 (q, 2H, H Cyclo ), 1.33 (t, 3 J = 8.0 Hz, 1H, -SH), (m, 14H, - CH2-), 1.16 (q, 2H, H Cyclo ) 1.04 (td, 1H -CHC-), 0.86 (s, 9H, -C(CH 3 ) 3 -). 13 C NMR (400 MHz, CDCl 3 ) (ppm): 74.52, 71.71, 70.58, 70.54, 70.38, 70.31, 70.09, 64.90, 62.26, 49.05, 46.79, 39.61, 34.16, 32.30, 29.60, 29.34, 29.19, 28.65, 28.49, 27.49, 26.46, 26.24, 26.16, 25.73, 25.64, ESI-MS (m/z): calculated for C 31 H 64 NO 4 S + [M + ], ; found Compound L5: 1 H NMR (400MHz, CDCl 3, TMS): 7.82 (d, 2H, H Ar ), (m, 3H, H Ar ), 4.24 (br, 2H, -CH 2 O-), 3.78 (s, 6H, -(CH 3 ) 2 N-), (m, 14H, -CH 2 O- + -OCH 2 -(CH 2 N)-), (m, 2H, -CH 2 N-), 2.87 (s, 3H, -CH 3 SO - 3-), 2.52 (q, 2H, -CH 2 S-), (m, 4H, -(SCH 2 )CH CH 2 (CH 2 O)-), (m, 15H, -SH + -CH 2 -). 13 C NMR (400 MHz, CDCl 3 ) (ppm): , , , , , 71.71, 70.60, 70.50, 70.40, 70.19, 70.03, 69.90, 65.32, 56.47, 39.61, 34.13, 29.64, 29.60, 29.52, 29.17, 29.04, 28.69, 28.47, 26.07, ESI-MS (m/z): calculated for C 27 H 50 NO 4 S + [M + ], ; found nature chemistry 3

4 Figure S MHz 1 H NMR spectrum of compound L1 in CDCl 3 (D, 99.8%). nature chemistry 4

5 Figure S MHz 13 C NMR spectrum of compound L1 in CDCl 3 (D, 99.8%). nature chemistry 5

6 Relative Intensity m/z Figure S3. ESI-MS spectrum of compound L2. nature chemistry 6

7 Figure S MHz 1 H NMR spectrum of compound L2 in CDCl 3 (D, 99.8%). nature chemistry 7

8 Figure S MHz 13 C NMR spectrum of compound L2 in CDCl 3 (D, 99.8%). nature chemistry 8

9 Relative Intensity m/z Figure S6. ESI-MS spectrum of compound L2. nature chemistry 9

10 Figure S MHz 1 H NMR spectrum of compound L3 in CDCl 3 (D, 99.8%). nature chemistry 10

11 Figure S MHz 13 C NMR spectrum of compound L3 in CDCl 3 (D, 99.8%). nature chemistry 11

12 Relative Intensity m/z Figure S9. ESI-MS spectrum of compound L3. nature chemistry 12

13 Figure S MHz 1 H NMR spectrum of compound L4 in CDCl 3 (D, 99.8%). nature chemistry 13

14 Figure S MHz 13 C NMR spectrum of compound L4 in CDCl 3 (D, 99.8%). nature chemistry 14

15 Relative Intensity m/z Figure S12. ESI-MS spectrum of compound L4. nature chemistry 15

16 Figure S MHz 1 H NMR spectrum of compound L5 in CDCl 3 (D, 99.8%). nature chemistry 16

17 Figure S MHz 13 C NMR spectrum of compound L5 in CDCl 3 (D, 99.8%). nature chemistry 17

18 Relative Intensity m/z Figure S15. ESI-MS spectrum of compound L5. nature chemistry 18

19 Scheme S2. Fabrication of cationic gold nanoparticles. General procedure: 1-Pentanethiol coated gold nanoparticles (d = ~2 nm) were prepared according to the previously reported protocol. 1 Place-exchange reaction 2 of compound Ls (s = 1, 2, 3, 4, 5) dissolved in DCM with pentanethiol-coated gold nanoparticles (d~2 nm) was carried out for 3 days at room temperature and the DCM was then evaporated under reduced pressure. The residue was dissolved in a small amount of distilled water and dialyzed (membrane MWCO = 1,000) to remove excess ligands, acetic acid and other salts present with the nanoparticles. After dialysis, the particles were lyophilized to afford a brownish solid. The nanoparticles are redispersed in ionized water (18 M -cm). 1 H NMR spectra in D 2 O showed substantial broadening of the proton signals and no free ligands were observed. nature chemistry 19

20 Figure S16. Fluorescence titration curves for the complexation of GFP with four different cationic gold nanoparticles (NP1, NP2, NP4, NP5). The changes of fluorescence intensity at 510 nm were measured following the addition of cationic nanoparticles (0-100 nm for 5 mm sodium phosphate buffer (inset) and 0-2 M for serum solution) with an excitation wavelength of 475 nm. Figure S17. Change in fluorescence responses ( I) of GFP (100 nm, 200 L) in absence and presence of various protein solutions (25 nm). The intensities were recorded at 510 nm with an excitation wavelength of 475 nm. Each fluorescence response value is an average of six parallel measurements. nature chemistry 20

21 Table S1. Binding constants (K S ), Gibbs free energy changes (- G) and binding stoichiometries (n) between GFP and various cationic nanoparticles (NP1-NP5) as determined from fluorescence titration. Nanoparticle K S / 10 9 M -1 G / kj mol -1 n NP NP NP NP NP Table S2. Training matrix of fluorescence response patterns of NP-GFP sensor array (NP1-NP5) against five serum proteins (25 nm) in 5 mm sodium phosphate buffer ph 7.4. Protein NP1 NP2 NP3 NP4 NP5 HSA HSA HSA HSA HSA HSA IgG IgG IgG IgG IgG IgG Fibrinogen Fibrinogen Fibrinogen Fibrinogen Fibrinogen Fibrinogen Antitrypsin Antitrypsin Antitrypsin Antitrypsin Antitrypsin Antitrypsin Transferrin Transferrin Transferrin Transferrin Transferrin Transferrin nature chemistry 21

22 Table S3. LDA classification accuracy of protein analytes by using the individual GFP-nanoparticle complexes as sensor in 5 mm sodium phosphate buffer, ph 7.4. The values are considered from the Jacknifed classification matrix based on LDA analysis of the 6 replicated data listed in Table S1. The maximum classification accuracy was obtained 100% using all five GFP-nanoparticle combinations. Proteins NP1 NP2 NP3 NP4 NP5 All HSA IgG Fibrinogen Antitrypsin Transferrin Total Table S4. Training matrix of fluorescence response patterns of NP-GFP sensor array (NP1-NP5) against five serum proteins (500 nm) in human serum. Protein NP1 NP2 NP3 NP4 NP5 HSA HSA HSA HSA HSA HSA IgG IgG IgG IgG IgG IgG Fibrinogen Fibrinogen Fibrinogen Fibrinogen Fibrinogen Fibrinogen Antitrypsin Antitrypsin Antitrypsin Antitrypsin Antitrypsin Antitrypsin Transferrin Transferrin Transferrin Transferrin Transferrin Transferrin nature chemistry 22

23 Table S5. LDA classification accuracy of protein analytes by using the individual GFP-nanoparticle complexes as sensor in human serum. The values are considered from the Jacknifed classification matrix based on LDA analysis of the 6 replicated data listed in Table S3. The maximum classification accuracy was obtained 97% using all five GFP-nanoparticle combinations. Proteins NP1 NP2 NP3 NP4 NP5 All HSA IgG Fibrinogen Antitrypsin Transferrin Total Table S6. Detection and identification of unknown serum proteins in 5 mm sodium phosphate buffer, ph 7.4 using LDA. # Fluorescence response pattern Identification Verification NP1 NP2 NP3 NP4 NP Antitrypsin -Antitrypsin Fibrinogen Fibrinogen HSA HSA IgG IgG Transferrin Transferrin Antitrypsin -Antitrypsin Fibrinogen Fibrinogen HSA HSA IgG IgG Transferrin Transferrin Antitrypsin -Antitrypsin Fibrinogen Fibrinogen HSA HSA IgG IgG Transferrin Transferrin Antitrypsin -Antitrypsin Fibrinogen Fibrinogen HSA HSA Transferrin IgG Transferrin Transferrin Antitrypsin -Antitrypsin Fibrinogen Fibrinogen HSA HSA IgG IgG Transferrin Transferrin Transferrin Transferrin Antitrypsin -Antitrypsin Fibrinogen Fibrinogen HSA HSA IgG IgG nature chemistry 23

24 Table S7. Detection and identification of unknown serum proteins in serum using LDA. # Fluorescence response pattern Identification Verification NP1 NP2 NP3 NP4 NP Transferrin Transferrin Antitrypsin -Antitrypsin Antitrypsin -Antitrypsin Fibrinogen Fibrinogen IgG -Antitrypsin HSA HSA HSA HSA IgG IgG IgG IgG Fibrinogen Fibrinogen Transferrin Transferrin HSA HSA HSA HSA Antitrypsin -Antitrypsin IgG IgG Transferrin Transferrin Transferrin Transferrin IgG IgG Antitrypsin HSA HSA HSA Fibrinogen Fibrinogen Fibrinogen Fibrinogen Transferrin Transferrin Antitrypsin -Antitrypsin Fibrinogen Fibrinogen Transferrin Transferrin IgG IgG HSA HSA Antitrypsin -Antitrypsin Fibrinogen Fibrinogen nature chemistry 24

25 Table S8. Training matrix of the fluorescence responses of NP-GFP sensor array (NP1-NP5) against two serum proteins at different concentrations in undiluted serum. Proteins NP1 NP2 NP3 NP4 NP5 HSA 500 nm HSA 500 nm HSA 500 nm HSA 500 nm HSA 500 nm HSA 500 nm HSA 1 M HSA 1 M HSA 1 M HSA 1 M HSA 1 M HSA 1 M HSA 2 µm HSA 2 µm HSA 2 µm HSA 2 µm HSA 2 µm HSA 2 µm IgG 500 nm IgG 500 nm IgG 500 nm IgG 500 nm IgG 500 nm IgG 500 nm IgG 1 M IgG 1 M IgG 1 M IgG 1 M IgG 1 M IgG 1 M IgG 2 µm IgG 2 µm IgG 2 µm IgG 2 µm IgG 2 µm IgG 2 µm nature chemistry 25

26 Table S9. Training matrix of the fluorescence responses of NP-GFP sensor array (NP1-NP5) against mixtures of serum proteins (HSA & IgG 250 nm each; HSA & IgG 500 nm each) and individual proteins (HSA and IgG 500 nm each) in undiluted serum. Protein(s) NP1 NP2 NP3 NP4 NP5 HSA0.5 M HSA0.5 M HSA0.5 M HSA0.5 M HSA0.5 M HSA0.5 M IgG0.5 M IgG0.5 M IgG0.5 M IgG0.5 M IgG0.5 M IgG0.5 M HSA0.5-IgG0.5 M HSA0.5-IgG0.5 M HSA0.5-IgG0.5 M HSA0.5-IgG0.5 M HSA0.5-IgG0.5 M HSA0.5-IgG0.5 M HSA0.25-IgG0.25 M HSA0.25-IgG0.25 M HSA0.25-IgG0.25 M HSA0.25-IgG0.25 M HSA0.25-IgG0.25 M HSA0.25-IgG0.25 M (1) Brust, M., Walker, M., Bethell, D., Schiffrin, D.J. & Whyman, R. Synthesis of Thiol-Derivatized Gold Nanoparticles in a 2-Phase Liquid-Liquid System. J. Chem. Soc., Chem. Commun., (1994). (2) Hostetler, M.J., Templeton, A.C. & Murray, R.W. Dynamics of place-exchange reactions on monolayer-protected gold cluster molecules. Langmuir 15, (1999). nature chemistry 26