SUPPLEMENTARY INFORMATION DOI: 10.1038/NMAT2811 NATURE MATERIALS | www.nature.com/naturematerials 1 Quantum Dot Dopamine Bioconjugates Function as Redox Coupled Assemblies for in Vitro and Intracellular pH Sensing Igor L. Medintz, Michael H. Stewart, Scott A. Trammell, Kimihiro Susumu, James B. Delehanty, Bing C. Mei, Joseph S. Melinger, Juan B. Blanco-Canosa, Philip E. Dawson and Hedi Mattoussi Synthesis of dopamine isothiocyantate (4-(2′-isothiocyanatoethyl)-1,2-benzenediol). Methyl alcohol (GFS Chemicals), carbon disulfide (Acros), 3-hydroxytyramine hydrochloride (dopamine-salt, Aldrich), triethylamine (Aldrich), tetrahydrofuran (Acros), and 30% hydrogen peroxide (Fisher) were purchased from commercial sources and used as received. 4-(2′-isothiocyanatoethyl)-1,2-benzenediol was synthesized according to a published procedure with slight modification [1]. Triethylamine (0.60 mL, 4.3 mmol) was added to 3-hydroxytyramine hydrochloride (0.750 g, 3.96 mmol) partially dissolved in tetrahydrofuran (10 mL) with stirring. Methyl alcohol was slowly added to dissolve the 3-hydroxytyramine hydrochloride, forming a clear solution. Carbon disulfide (1.21 mL, 20.1 mmol) was added to the mixture, which was stirred for 30 minutes at 28 °C under a nitrogen atmosphere. The dark reaction mixture was cooled to 0 °C and 30% hydrogen peroxide (1.26 mL, 12.3 mmol) was added dropwise by syringe. The solution was immediately acidified with concentrated hydrochloric acid and then concentrated in vacuo. The resulting mixture was filtered and rinsed through with DI water. The filtrate was extracted with ethyl acetate (3 × 50 mL) and the combined organic layers were dried over Na 2 SO 4 , filtered, and the solvent was removed affording the crude product as an oil. A 1 H NMR spectrum of the product matched the spectrum reported in the literature [1]. Cyclic voltammetry. Glassy carbon working electrodes were purchased from Bioanalytical Systems, Inc., BAS, (West Lafayette, IN). The carbon electrodes were polished to a mirror finish as recommended by the manufacture using 0.5 micron Al 2 O 3 .
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SUPPLEMENTARY INFORMATION · components (% of total amplitude) using FluoFit (Picoquant, Berlin Germany). Cell growth and microinjection. COS-1 cell lines (ATCC, Manassas, VA) were
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Laviron’s 9-member square scheme describing a 2-electron, 2-proton redox couple and as applied to a generalized catechol.
Generalized scheme. Reduction proceeds from left to right and protonation from top to bottom. M is the fully deprotonated oxidized from and V is the fully protonated reduced form. Adapted from ref [9].
Scheme as applied to the catechol portion of dopamine [9].
Calculation of the driving force of electron transfer as a function of pH between the QD and quinone.
Using equations 1-3[10], we estimated the one-electron reduction potentials for the redox couples Q/Q.- and Q.-/QH2 from the equilibrium constant of semiquinone formation, Kf, in conjunction with the two-electron formal reduction potentials measured from our cyclic voltammetry data as a function of pH:
Emi(Q/Q.-) = Emi(Q/QH2) + RT/2FlnK’fi Supp Eq 1
Emi(Q.-/QH2) = Emi(Q/QH2) - RT/2FlnK’fi Supp Eq 2 Kf = K’fi[Ks/(Ks +(H+)][(Ka1Ka2+ Ka1 (H+) + (H+)2)/ Ka1Ka2] Supp Eq 3 where R, T, and F represent the gas constant, the absolute temperature, and the Faraday constant, respectively. Emi = formal reduction potential at pH = i.; Kf = the equilibrium constant of semiquinone formation K’fi = the equilibrium constant of semiquinone formation at pH = i; Ka1 = 1st acid dissociation constant; Ka2 = 2nd acid dissociation constant; Ks = acid dissociation constant for the semiquinone. The values used are pKa1 = 9.6; pKa2 = 12.5; pKs = 5, pKf = -0.92 (estimated from quinone data) [10,11]. To estimate the drive force ∆Go for the electron transfer reactions we used the Weller equation[12].
∆Go = e(E(D)-E(A) – U* - e2/4πεoεa Supp Eq 4
∆Go is the standard Gibbs energy change, E(D) and E(A) are the one electrode reduction potentials of the donor, D, and acceptor, A, and e2/4πεoεa is the energy gained by bring the two radical ions to the encounter distance a in a solvent of dielectric constant ε (in our estimate this energy term is small and was dropped from the calculation). U* is the singlet-singlet excitation energy of the chromophore.
The results of the calculations demonstrating that the electron transfer we describe is indeed quite favorable are summarized in the following 2 plots.
Supporting References [1] Tajima, H. and Li, G. (1997) Synthesis of hydroxyalkyl isothiocyanates. Synlett, 7, 773-774. [2] Clapp et al., (2007) Two-photon excitation of quantum dot-based fluorescence resonance energy transfer and its applications. Advanced Materials 19, 1921-1926. [3] Medintz et al., (2007) A reagentless biosensing assembly based on quantum dot donor Förster resonance energy transfer Advanced Materials 17, 2450-2455. [4] Delehanty et al., (2006) Self-assembled quantum dot-peptide bioconjugates for selective intracellular delivery. Bioconjugate Chemistry 17, 920-927. [5] Medintz et al.., (2008) Intracellular delivery of quantum dot-protein cargos mediated by cell penetrating peptides. Bioconjugate Chemistry, 19, 1785–1795. [6] Minaschek, et al., (1989) Quantitation of the volume of liquid injected into cells by means of pressure. Experimental Cell Research 183, 434-442. [7] BCECF, MP 01150, Invitrogen Corporation, Revised 24 April 2006, (www.Invitrogen.com). [8] Wang, et al., (2002). Study on fluorescence property of dopamine and determination of dopamine by fluorimetry. Talanta 57, 899-907. [9] Laviron, E. (1984). Electrochemical reactions with protonations at equilibrium. 10. The kinetics of the parabenzoquinone hydroquinone couple on a platimum-electrode. Journal of Electroanalytical Chemistry 164, 213-227. [10] Wardman, P. (1989) Reduction potentials of one-electron couples involving free-radicals in aqueous solution. J. Phys. Chem. Ref. Data 18, 1637-1755. [11] Deakin, M. R.; Wightman, R. M. (1986) The kinetics of some substituted catechol/ortho-quinone couples at a carbon paste electrode. J. Electroanal. Chem. 206,167-177. [12] Grellman.K.H; Watkins, A. R.; Weller, A. (1972) Electron-transfer mechanism of fluorescence quenching in polar solvents. 2. Tetracyanoethylene and tatrcyanobenzene as quenchers. J. Phys. Chem. 76, 3132.
Supporting Table 1. pH comparison of lifetime data for 550 nm QD vs. ratio of assembled dopamine labeled peptide. Average lifetimea τAv in nanoseconds (Normalized as a %)
a Amplitude weighted as described in Supporting Information.
Supporting Table 2. Comparison of lifetime data for 550 nm QDs self-assembled with 60 equivalents dopamine-peptide and exposed to different pH buffers vs. a Cy5 free dye internal control. Average lifetimea τAv in nanoseconds (%)
Comparison of normalized quenchingefficiency of increasing ratios ofdopamine-labeled peptide assembled to550 nm QDs at different pHs (refer tomanuscript Figure 2)
350000 M 5e-7 M (2) 1e-6 M (4)1e-5 M (40) 1e-4 M (400)5e-4 M (2,000)0.001 M (4,000)0.01 M (40,000)
pH 8
Wavelength (nm)480 500 520 540 560 580 600 620
PL (A
U)
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350000 M 5e-7 M (2) 1e-6 M (4)1e-5 M (40) 1e-4 M (400)5e-4 M (2,000)0.001 M (4,000)0.01 M (40,000)
pH 7
Wavelength (nm)480 500 520 540 560 580 600 620
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U)
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350000 M 5e-7 M (2) 1e-6 M (4)1e-5 M (40) 1e-4 M (400)5e-4 M (2,000)0.001 M (4,000)0.01 M (40,000)
pH 6
Wavelength (nm)480 500 520 540 560 580 600 620
PL (A
U)
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350000 M 5e-7 M (2) 1e-6 M (4)1e-5 M (40) 1e-4 M (400)5e-4 M (2,000)0.001 M (4,000)0.01 M (40,000)
pH 5
Wavelength (nm)480 500 520 540 560 580 600 620
PL (A
U)
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400000 M 5e-7 M (2) 1e-6 M (4)1e-5 M (40) 1e-4 M (400)5e-4 M (2,000)0.001 M (4,000)0.01 M (40,000)
pH 4
Wavelength (nm)480 500 520 540 560 580 600 620
PL (A
U)
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400000 M 5e-7 M (2) 1e-6 M (4)1e-5 M (40) 1e-4 M (400)5e-4 M (2,000)0.001 M (4,000)0.01 M (40,000)
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Quenching efficiency of increasing concentrations of dopamine mixed with 550 nm QDs in different pH buffers. The numbers in parenthesis indicated the fold excess of free dopamine over QD.
Excited state lifetimes of 550 nm QDs at pH 4.8 and pH 6.5 assembled with the indicated increasing ratio of dopamine-labeled peptides (refer to manuscript Figure 3)
3.0580 nm QD with ~60 dopamine peptide/QDLinear fit580 nm QD with no peptide Linear fit
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(A) pH sensing with 580 nm QDsassembled with dopamine peptide ascompared to QDs alone (B), both in thepresence of a Cy5 dye internal standard.(C) Ratios of Cy5/QD PL for bothconfigurations.
(D) pH sensing with 550 nm QDsassembled with dopamine peptide in thepresence of a Cy5 dye internal standard.(E) Ratio of Cy5 PL /QD PL plottedoverlapping that of dopamine peptide 1/Efto highlight the corresponding linearity.For E, a reciprocal plot would be similar toManuscript Figure 4B
(A) Micrographs from COS-1 cells microinjected with 550 nm QD dopamine-conjugates in PBS at pH 6.5and exposed to PBS pH 11.5 buffer supplemented with or without Nystatin over time. (B) Normalized andaveraged PL intensities from (A) vs. time.
BCECF loaded COS-1 cells and confirmation of Nystatin-induced alkalosis. Representative cellularmicrographs of COS-1 cells loaded with BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein,acetoxymethyl ester) using the manufacturers recommended protocol (Invitrogen). The buffer was switchedto PBS pH 10 supplemented with 200 µg/mL Nystatin and fluorescence monitored over time. As intracellularpH increases the dye becomes more fluorescent as expected. After 30 min in the absence of Nystatin at pH10, the cells showed fluorescence similar to the 0 min point (data not shown).
COS-1 cells microinjected with 550 nm PEG methoxy QD with ~60-dopamine peptide/QDmonitored over time with continuous maximal UV illumination (20 msec shutter exposure).
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0 sec + 10 sec + 20 sec + 40 sec + 60 sec
COS-1 cells loaded with BCECF and monitored over time with continuous UV illumination(20 msec shutter exposure, excitation intensity attenuated with a neutral density filter).
(A) Micrographs from COS-1 cells microinjected with 550 nm QD dopamine-conjugates prequenched inPBS at pH 10.1 and exposed to PBS pH 5.4 buffer supplemented with Nystatin over time. (B) Close-up ofthe cells boxed in (A) at 0, 5 and 30 min after Nystatin-buffer addition. (C) Normalized and averaged PLintensities from (A) vs. time.