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SUPPLEMENTARY INFORMATIONdoi: 10.1038/nmat2811

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Supporting Information.

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 Na2SO4, filtered, and the solvent was removed affording the crude product as an oil.

A 1H 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 Al2O3.

2 nature materials | www.nature.com/naturematerials

SUPPLEMENTARY INFORMATION doi: 10.1038/nmat2811

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After polishing, the carbon electrodes were rinsed copiously with DI H2O, sonicated for 5

minutes in water and dried under dry N2. 18.2 M de-ionized (DI) H2O was obtained

from a Milli-Q water purification system (Millipore, Billerica, MA).

All electrochemical measurements were performed in a Faraday cage in the 3-

electrode geometry, using a glassy carbon working electrode, a Pt counter electrode, and

a Ag/AgCl, 3 M KCl reference electrode. Measurements were recorded by an

electrochemical workstation Model 750 from CH Instruments (Austin, TX). At each pH,

background voltammograms were recorded before the dopamine or dopamine-labeled

peptide was added to the electrolyte. All voltammograms were collected under ambient

conditions. During each measurement an Ar gas blanket was kept above the solution.

Each voltammogram was initiated at the negative potential limit with a scan rate of 50

mV/s following a 2 sec quiescence period. No signal was obtained in the same potential

range on electrodes not containing peptide. Background subtracted voltammograms were

generated using software provided by CH instruments.

Collection of time-resolved data. Excited-state fluorescence lifetime measurements

were performed using a time-correlated single-photon counting instrumental system

[2,3]. Laser excitation consisted of a synchronously pumped and cavity-dumped dye

laser (305 nm), pumped by the second harmonic of a Nd:YLF laser (527 nm, 100MHz).

The dye laser contained a single plate birefringent filter tuned to produce laser oscillation

at 610 nm and pulse width of ~1 ps FWHM. The dye laser was cavity-dumped at 1 MHz,

and frequency doubled using a potassium dihydrogen phosphate nonlinear crystal.

Sample fluorescence was spectrally filtered with a monochromator (bandpass ~10 nm)

and detected with a cooled microchannel plate PMT (Hamamatsu R2809U-11, Shizuoka

Japan). Temporal response function of the system was measured to a FWHM of ca. 50

ps. Multi-exponential QD PL decay traces (lifetimes) were fitted with an average of 3 - 4

lifetime components as derived from the fractional amplitude of the positive decay

components (% of total amplitude) using FluoFit (Picoquant, Berlin Germany).

Cell growth and microinjection. COS-1 cell lines (ATCC, Manassas, VA) were

cultured in complete growth medium (Dulbecco’s Modified Eagle’s Medium (DMEM)



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supplemented with 4 mM L-glutamine, 1 mM sodium pyruvate, 1% (v/v)

penicillin/streptomycin/actinomycin D-antibiotic/antimycotic, and 10% (v/v) heat-

inactivated fetal bovine serum (FBS). Cells were incubated at 37ºC under 5% CO2

atmosphere and a subculture was performed every 3-4 days [4]. Cells were seeded onto

35 mm coverslip bottom plastic dishes (BD Biosciences, Bedford MA) coated with poly-

D-lysine and 50 µg/mL fibronectin (Sigma-Aldrich, St Louis, MO) in sodium bicarbonate

buffer pH 8.5. Approximately 2 x 104 cells were seeded into the wells and cultured

overnight. Femtoliter aliquots QDs and QD-dopamine conjugates were directly injected

into the adherent COS-1 cells using an InjectMan® NI2 micromanipulator equipped with

a FemtoJet programmable microinjector (Eppendorf, Westbury, NY) [5,6]. During

microinjection, the cells were buffered with Dulbecco’s Modified Eagle Medium

(DMEM) supplemented with 25 mM Hepes pH 7.4 (Invitrogen). Following

microinjection, the cellular media was switched to PBS at the indicated pH ± 200 µg/mL

Nystatin for sensor monitoring. BCECF-acetoxymethyl ester (Invitrogen, Carlsbad, CA)

was loaded into the COS-1 cells following the manufactures recommended protocol [7].

Microscopy. Epifluorescence image collection was carried out using an Olympus IX-71

microscope where samples were excited with a Xe lamp. BCECF emission was collected

with a 500 nm long-pass filter. QD emission was collected with a Qdot 525 cube

(excitation HQ420/20x, dichroic T495, emission ET525/17x, Chroma Technology

Rockingham, VT) and emission from the 680 nm red-fluorescent Fluorophorex 20 nm

nanospheres (FLX, Phosphorex, Inc. Fall River, MA) using a Cy5 cube (excitation

HQ620/60x, dichroic Q660LP, emission HQ700/75m). Differential interference contrast

(DIC) images were collected using a bright light source. Cell images were captured with

a DP71 color digital camera (Olympus, Center Valley, PA) using constant camera

settings over time as indicated. Images were analyzed using DP Manager Software

(Olympus, Center Valley, PA) and Image J (NIH, Bethesda, MD –

http://rsb.info.nih.gov/ij/).

UV-Vis monitored oxidation of dopamine derivative. UV-Vis absorption spectra were

collected on an HP 8453 diode array spectrophotometer using a 1 cm × 1 cm quartz



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cuvette. For this experiment, we synthesized a PEG-dopamine derivative as it removes

both the peptidyl absorption component and prevents cyclization while still allowing us

to monitor changes in bond conjugation.

mPEG750-dopamine structure:

NH

OO

n

O

OH

OH

n ~ 15

A stock solution of mPEG750-dopamine was prepared by dissolving CH3O-PEG750-

dopamine (30 mg, 0.03 mmol) in DI water (1 mL). First, 1 × PBS buffer (1.99 mL) at a

desired pH was added to the cuvette and stock solutions of CH3O-PEG750-dopamine (10

L) were diluted into the cuvette and mixed. Absorption spectra were collected at

various time intervals to monitor the changes in the spectra. This procedure was repeated

using buffers at different pH values. The most apparent changes in the UV-Vis spectra

occurred at more basic pH, and importantly, these changes happened within the first hour

of incubation with nothing significant noted thereafter. This was as expected and has

been previously confirmed experimentally [8]. See Supporting Figure 10 for a

representative set of data.



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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].

OH R

OH

ROH

O

OH R

O-

O- R

O-

O R

O

RO-

O

O R

OH +

OH + R

OH +

ROH

OH +

-H+ +H+

+e-

-e-

+e-

-e-

+e-

-e-

+e-

-e-

+e-

-e-

+e-

-e-

-H+ +H+ -H+ +H+

-H+ +H+ -H+ +H+ -H+ +H+

?

?



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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.



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-1

-0.5

0

0.5

1

1.52 4 6 8 10 12

pH

Pote

ntia

l, V

vs. N

HE CB

Q/Q._

Series5

Q._/QH2

VB

Q/Q.-

Q.-/QH2

Q/QH2

Q/Q.-

Supporting Plot 1. Formal reduction potentials as a function of pH calculated using equations 1-3.

-1

-0.8

-0.6

-0.4

-0.2

0

2 4 6 8 10 12pH

Driv

ing

Forc

e, ∆∆ ∆∆

Go,

eV reduction of Q by the QD

oxidation of QH2 by the QDQH2

Supporting Plot 2. The driving force of electron transfer as a function of pH between the QD and quinone or the QD and catechol.



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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.



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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 %)

Ratio of peptide/QD pH 4.8 pH 6.5 pH 9.3 0 8.92 (100) 10.78 (100) 8.82 (100) 2 8.77 (98.3) 9.97 (92.5) 9.20 (104) 4 8.87 (99.4) 10.62 (98.5) 8.31 (94.2) 8 8.85 (99.2) 10.14 (94.1) 7.69 (87.2)

12 8.56 (96.0) 9.96 (92.4) 7.04 (79.8) 15 8.30 (93.0) 9.59 (89.0) 7.13 (80.8) 20 8.50 (95.3) 9.58 (88.9) 6.49 (73.6) 30 8.28 (92.8) 9.11 (84.5) 5.14 (58.3) 40 8.03 (90.0) 8.55 (79.3) 4.29 (48.6) 50 7.77 (87.1) 8.33 (77.3) 4.12 (46.7) 60 7.26 (81.4) 7.52 (69.8) 3.83 (43.4)

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 (%)

pH QD Cy5 6.5 7.27 (100) 1.09 (100) 7.0 6.70 (92.2) 1.04 (95.4) 7.4 5.90 (81.2) 1.01 (92.7) 8.0 4.84 (66.6) 0.99 (90.8) 8.8 3.47 (47.7) 0.91 (83.5) 9.3 3.04 (41.8) 1.01 (92.7)

10.1 2.70 (37.1) 0.99 (90.8) a Amplitude weighted as described in Supporting Information.



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pH4 6 8 10 12 14

Form

al p

oten

tial (

E f),

V vs

. NHE

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Potential, V vs NHE-0.2 0.0 0.2 0.4 0.6 0.8

Nor

mal

ized

Cur

rent

(uA)

-1.5

-1.0

-0.5

0.0

0.5

1.0

pH 4.3 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 pH 11 pH 12

Potential V vs. NHE0.2 0.3 0.4 0.5 0.6 0.7 0.8

Cur

rent

, µµ µµA

-10

-5

0

5

10

15

pH 4.0pH 6.0pH 6.8pH 7.4pH 8.0pH 9.1

Cyclic voltammograms of dopamine collected at different pH’s

A.

B.

C.Cyclic voltammograms of dopamine-peptide collected at different pH’s

Formal potential of dopamine-peptide at different pH’sD.

Structure of the ligands used to functionalize the QD surfaces and provide aqueous compatibility (Mw~600/750).

SH SH

O

O O OHn(n ~ 12)DHLA–PEG600–OH

SH SH

O

NH

O OCH3n(n ~ 15)DHLA–PEG750–OCH3

Supporting Figure 1.



S-11

pH 5.4

Wavelength (nm)500 520 540 560 580 600 620

PL (A

U)

0

10000

20000

30000

400000 8 12 15 20 30 40 50 75

pH 5.9

Wavelength (nm)500 520 540 560 580 600 620

PL (A

U)

0

5000

10000

15000

20000

25000

30000

350000 8 12 15 20 30 40 50 75

pH 7.0

Wavelength (nm)500 520 540 560 580 600 620

PL (A

U)

0

5000

10000

15000

20000

25000

30000

350000 8 12 15 20 30 40 50 75

pH 8.0

Wavelength (nm)500 520 540 560 580 600 620

PL (A

U)

0

10000

20000

30000

400000 8 12 15 20 30 40 50 75

A.

B.

C.

D.

Comparison of normalized quenchingefficiency of increasing ratios ofdopamine-labeled peptide assembled to550 nm QDs at different pHs (refer tomanuscript Figure 2)

pH 6.5

Wavelength (nm)500 520 540 560 580 600 620

PL (A

U)

0

5000

10000

15000

20000

25000

30000

350000 8 12 15 20 30 40 50 75

E.




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pH 9

Wavelength (nm)480 500 520 540 560 580 600 620

PL (A

U)

0

5000

10000

15000

20000

25000

30000

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)

0

5000

10000

15000

20000

25000

30000

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

PL (A

U)

0

5000

10000

15000

20000

25000

30000

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)

0

5000

10000

15000

20000

25000

30000

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)

0

10000

20000

30000

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)

0

10000

20000

30000

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)

A.

B.

C.

D.

E.

F.

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.




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pH 6.5

Time (ns)0 5 10 15 20 25

Norm

aliz

ed In

tens

ity (A

U)

0.10

1.00024812152030405060

pH 4.8

Time (ns)0 5 10 15 20 25

Norm

aliz

ed In

tens

ity (A

U)

0.10

1.000 24 812152030405060

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)




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mPEG-Dop Spectra 60 min

Wavelength (nm)300 400 500 600

Abso

rban

ce (A

U)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

pH 4 pH 7 pH 9 pH 10 Water (0 min)

mPEG-Dop Spectra 120 min

Wavelength (nm)300 400 500 600

Abso

rban

ce (A

U)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

pH 4 pH 7 pH 9 pH 10 Water (0 min)

A.

B.

C.

Representative changes in mPEG-Dop Abs at various pH’s over time

Time (min)0 20 40 60 80 100 120 140

Diff

. Abs

. at 2

81 n

m

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18pH 4 pH 7 pH 9 pH 10

Changes in mPEG-Dop abs. at 281 nm over time




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Wavelength (nm)500 550 600 650 700

PL (A

U)

0

10000

20000

30000

40000

50000

5.4 5.9 6.5 7.0 7.4 8.0 8.8 9.3 10.1

pH

Cy5

580 nm QD - No peptide

Wavelength (nm)500 550 600 650 700

PL (A

U)

0

10000

20000

30000

40000

5.4 5.9 6.5 7.0 7.4 8.0 8.8 9.3 10.1

pH

580 nm QD w/ ~60 Dopamine peptide

Cy5

pH5 6 7 8 9 10

Ratio

of C

y5/Q

D PL

(664

nm

/578

nm

)

0.5

1.0

1.5

2.0

2.5

3.0580 nm QD with ~60 dopamine peptide/QDLinear fit580 nm QD with no peptide Linear fit

A.

B.

C.

(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.

pH5 6 7 8 9 10

Dop

amin

e-pe

ptid

e 1/

E f

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5R

atio

of C

y5 P

L / Q

D PL

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25Dopamine - peptide 1 / EfQD sensor - ratio Cy5 / QD PL

pH

Wavelength (nm)500 550 600 650 700

PL (A

U)

0

10000

20000

300004.8 5.4 5.9 6.5 7.07.4 8.0 8.8 9.3 10.1

Cy5

640 660 680 700

5000

10000

15000

20000

D.

E.

QD PL

Cy5

(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




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Fluo

resc

ence

0 min + 15 min + 30 min + 60 min + 90 min

2 00 µM

2 00 µM 20 0 µM

20 0 µM 20 0 µM

0 min + 60 min + 90 min + 105 min + 120 minFl

uore

scen

ce- Nystatin

+ Nystatin

Time (min)0 20 40 60 80 100 120

Nor

mal

ized

PL

(%)

0

20

40

60

80

100

120

+ Nystatin- Nystatin

pH 6.5 pH 11.5

2 00 µM 200 µM

B.

(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.




S-17

0 min + 20 min + 30 min

DIC

Fluo

resc

ence

Mer

ged

+ Nystatin- Nystatin

100 µM

100 µM

100 µM

100 µM

100 µM

100 µM

100 µM

100 µM

100 µM

200 µM 100

µM

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).




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0 sec + 30 sec + 60 sec + 90 sec + 120 sec

Fluo

resc

ence

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).

Fluo

resc

ence

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).

100 µM



S-19

DIC

Fluo

resc

ence

Mer

ged

0 min + 5 min + 20 min + 30 minA.

100 µM

Time (min)0 5 10 15 20 25 30

Nor

mal

ized

PL

(%)

80

90

100

110

120

130

B.

C.0 min

+ 5 min

+ 30 min

(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.


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