The Effects of Solvent Purity on Highly Photoluminescent Nanocrystals (CdSe/ZnS Quantum Dots) Used as Biological/Biomedical Imaging Probes Kevin

Song Carmel High School, Carmel, IN 46032 INTRODUCTION Developments in the synthesis, characterization, and surface modification of nanoscale structures have expedited their usage in biological and medical applications.1-4 Semiconductor nanocrystals are widely used as probes for labeling cells and tissues owing to their intense, tunable fluorescence.5-9 Quantum dots (QDs) are semiconductor nanocrystals whose photophysical properties originate from the quantum confinement effect.10,11 QDs have been used for many applications including light emitting diodes (LEDs), photovoltaic cells, fluorescent dyes, antimicrobial agents, and cancer detection.12-16 The quantum confinement effect occurs when the dimensions of a crystal semiconductor become smaller than the distance between a valence band electron and its departed conduction band hole.10 A semiconductor nanocrystal whose radius is smaller than the exciton Bohr radius forms a QD with discrete energy states. Because band gap size is inversely related to nanocrystal size, QDs can be synthesized to emit light at various wavelengths on the visible light spectrum.11 This quality, in addition to their excellent luminescence and photostability, gives QDs a distinct advantage over traditional organic dyes for biological imaging applications.

Figure 1. Band gap size is inversely related to nanocrystal size; QDs can be synthesized to emit at various wavelengths on the visible light spectrum.2

The small size and promising photophysical properties of QDs have made them attractive optical labels in wide field single molecule fluorescence microscopy (SMFM) applications.18 However, due to the inconsistent quality of recently synthesized QD products, QDs that fluoresced dimly and were unsuitable for SMFM were frequently encountered. The quality and consistency of CdSe nanocrystals are normally greatly influenced by the presence of certain impurities.19 The synthesis of semiconductor nanoparticles in reverse micelles was introduced in the 1980s.20-24 During that early synthetic era, the Steigerwald group discovered that poorly crystalline CdSe nanoparticles prepared in reverse micelles could be ripened and recrystallized in n-Bu3P/n-Bu3PO mixtures.25 The phosphine-oxide component was found necessary to the process. Shortly thereafter, Murray, Norris, and Bawendi introduced an organometallic synthesis of CdE nanocrystals (E = S, Se, and Te) using n-octyl3P (TOP)/TOPO solvent mixtures, which constituted a breakthrough in the preparation of semiconductor nanocrystals.26 Subsequently, tri-n-octylphosphine oxide (TOPO) and TOPO mixtures have been employed as solvents in a wide range of nanocrystal syntheses.27-41 More recently, greener syntheses of QDs employing non-coordinating solvents have emerged.42-48 However, TOPO remains an important option for the synthesis of semiconductor nanocrystals.26-41,49-55 The likely participation of adventitious TOPO impurities in nanocrystal growth has been recognized for several years. TOPO is available from research-chemical suppliers in nominal 90%-purity (technical) and 99%-purity grades (hereafter referred to as 90% TOPO and 99% TOPO, respectively). Wang and co-workers identified such impurities in the main QD coordinating solvent, TOPO, by 31P NMR.55 They incorporated these impurities into their thermal syntheses of QDs, quantum wires (QWs), and quantum rods (QRs). These authors also concluded that one of the impurities responsible for CdSe nanocrystal growth is di-n-octylphosphinic acid (DOPA).56 2

Figure 2. CdSe/ZnS QDs fluorescing under a handheld UV lamp. As QD radius increases, so does the wavelength of light emitted by the QD, which results in a redder color of visible light.11

Based on the studies of Wang and co-workers, it is hypothesized that increasing the purity of TOPO and adding DOPA during sonochemical QD synthesis will cause more intense QD fluorescence, more efficient photon emissions, and greater numbers of photons emitted per QD molecule. The goal of this study is to test this hypothesis. QD products were sonochemically synthesized from differing grades of solvent and were characterized by fluorescence spectroscopy, UV-visible spectroscopy, and confocal fluorescence correlation spectroscopy (FCS). This research could provide a good reference for the related synthetic reproducibility of the growth of CdSe QDs. MATERIALS AND METHODS Materials Tri-n-octylphosphine (TOP, 90%, Strem Chemicals), selenium powder (Se, 99.99%, Aldrich), cadmium acetate (Cd(OAc)2, 99.9%, Aldrich), and hexadecylamine (HDA, 90%, Aldrich) were used as received. Technical grade tri-n-octylphosphine oxide (TOPO, 90%) was purchased from Strem Chemicals (batch number B4751105). Purification of TOPO

Distillation and recrystallization were performed to improve the purity of 90% technical grade TOPO. Distillation had already been accomplished according to a literature procedure.57 For recrystallization, 30 g TOPO was transferred into a 500 ml Erlenmeyer flasks. Another flask that contained acetonitrile (Aldrich) was kept warm in a water bath at 7075 C. Acetonitrile (7.5 ml) was added to the TOPO and the flask was gently hand-shaken. The supersaturated solution was left to cool to room temperature. After the TOPO had precipitated, the flasks of solution and acetonitrile were placed in an ice bath. To extract any remaining acetonitrile, the TOPO was vacuum-filtered and stored in an unheated vacuum oven overnight. Two grades of purified TOPO were assayed: (1) once-distilled and twice-recrystallized TOPO; and (2) undistilled, oncerecrystallized TOPO. TOPO straight from the manufacturer was also utilized for comparison. Each grade of TOPO was used to synthesize QDs. Sonochemical QD Core Synthesis All syntheses of QDs were carried out with ultrasonic sonochemical methods as described in literature.18 These methods have many advantages over conventional, thermally-intensive techniques, including (1) lower temperature; (2) shorter time; and (3) greater control over nanocrystal growth. Because sonication duration and power are directly controllable, it is possible to stop the further formation and growth of nanocrystals once they have reached a specific size that corresponds to a specific color of emitted visible light.

I Cd(OAc)2 + Se
)

((CH2)7CH3)3
P
)

II
))

((CH2)7CH3)3
P H3C P N H O P

))

18W, 15min

TOP, TOPO, HDA

H3C H CdSe 2N H3C

N H O P

(CH2)15CH3 ((CH2)7CH3)3

8W, 10min

H2N CdSe

(CH2)15CH3 ((CH2)7CH3)3

ZnC6H10O2S4, TBP ZnS

((CH2)3CH3)3

Figure 3. Schematic of sonochemical CdSe QD core synthesis and ZnS shelling.57

For all QD CdSe core syntheses, 2.5 mL tri-n-octylphosphine (TOP) (Strem Chemicals) was injected into a disposable borosilicate test tube containing 0.30 g selenium powder (Aldrich), which was then sonicated at 10 watts (W) using a rod sonicator (Branson Ultrasonics) until all Se powder was dissolved to form tri-n-octylphosphine selenide (TOPSe). 7.450 g TOPO (Strem Chemicals) was combined with 2.35 g hexadecylamine (HDA) (Aldrich) and 0.240 g cadmium acetate (Cd(OAc)2) (Aldrich) in a sidearm test tube. The mixture was melted using a heat gun on low settings, and kept purged under argon gas while being sonicated at 14 W until 100 C was reached. After the TOPO/HDA/Cd(OAc)2 mixture was cooled to 60 C, the TOPSe was added to the mixture in the sidearm tube and sonicated at 18 W until the CdSe QDs turned dark red in color (after about 15 minutes). Triplicates of 4 ml aliquots of these unshelled QDs were then collected.

Figure 4. Tri-n-octylphosphine oxide (TOPO), the main coordinating solvent used in QD synthesis.

ZnS Shelling of QD Cores To improve their fluorescence intensity, photon emission efficiency (quantum yield) and shelf life (by protecting against photobleaching and oxidation), QD were shelled with ZnS, a semiconductor material with a larger band gap than CdSe.58 Specifically, zinc ethylxanthate (0.1 g ZnC6H10O2S4) synthesized as described previously59, and 2 ml tri-n-butylphosphine (TBP) (Strem Chemicals) were vortexed in a disposable test tube, added to the test tube containing the

QD cores, and sonicated for 10 min at 8 W. The temperature was maintained above 100 C, but was kept lower than the maximum temperature reached (170200 C) during QD core formation. Incorporation of DOPA QD products that incorporated DOPA were synthesized to compare the differences between QDs made with and without DOPA. DOPA had already been synthesized following a method described in the literature.57 To incorporate DOPA into QD synthesis, 0.04 mol% (0.021 g) DOPA was added to the TOPO/HDA/Cd(OAc)2 mixture in the side arm test tube before initial melting and sonication, followed by CdSe core synthesis and ZnS shelling.

Figure 5. Di-n-octylphosphinic acid (DOPA), one of the TOPO impurities believed to improve QD nanocrystal growth.

Hydrophilic Thin-Coating Method Hydrophilic coatings were required for the QDs to be used in aqueous environments. A method which had been previously described was used to functionalize the nonpolar QDs by coating them in 3-mercaptopropionic acid (MPA).62 Specifically, 0.15 g of shelled CdSe/ZnS QDs was put into four disposable test tubes and dispersed in HPLC-grade chloroform (Fisher Scientific). The QDs were then precipitated out of solution by adding a 50/50 acetone/methanol solution, followed by centrifugation, disposal of supernatant, and drying under argon gas. After the QDs were redispersed in chloroform, 0.15 g MPA (Aldrich) and a flake of 46

dimethylaminopyridine (DMAP) (Aldrich) were added to each test tube. The QDs were then vortexed, centrifuged, removed of their supernatant, and dried under argon gas. 100 l of 5 M NaOH (Aldrich) was added to one of the test tubes. The solutions in these test tubes were then combined with each other while vortexing in between transfers. One ml deionized H2O was then added to the QDs, followed by high speed centrifugation at 14,000 rpm for 30 minutes. Biocompatible Lipopolymer Coating Method QDs were functionalized with a lipopolymer coating as described previously.60 The QDs were redispersed in HPLC-grade chloroform, precipitated, and dried in the same way as described in the thin-coating method.61 DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) solution (96.8 L of 25 mg/ml stock, Avanti Polar Lipids) was combined with 600 l DPPE PEG2000 (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]) solution. DSPEPEG2000 Maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[maleimide(polyethylene glycol)-2000]) solution (161 l of 1 mg/ml stock) was also added to the lipid mixture, which was then poured into the test tube containing dry QDs and stirred to redissolve the QDs in solution. The test tube was heated alongside another test tube that contained 1 ml deionized H2O in an 85 C water bath for 15 minutes. The water in the heated test tube was added to the QDs, which were then high-speed centrifuged at 14,000 rpm for 30 minutes.

Figure 6. DSPEPEG2000 Maleimide ((1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000])) allows for QDs to bind onto lipids (picture courtesy of Avanti Polar Lipids).

RESULTS All QDs were synthesized using the same 90% technical grade TOPO (Strem batch number. B4751105). The four compared QD products were as follows: (1) made from distilled, twice-recrystallized TOPO with 0.04 mol% added DOPA (Product A); (2) undistilled, oncerecrystallized TOPO with 0.04 mol% added DOPA (Product B); (3) stock (undistilled, unrecrystallized) TOPO with 0.04 mol% added DOPA (Product C); and (4) stock (undistilled, unrecrystallized) TOPO without DOPA (Product D). Fluorescence Spectra of Synthesized Samples To obtain relative fluorescence intensities, the QD samples were each redispersed in HPLC-grade chloroform at the same concentration and characterized using a Cary Eclipse fluorescence spectrophotometer (Instrument Serial Number el02025045, Scan Software Version: 1.1(132)). The excitation wavelength was set to 400 nm.

Figure 7. Fluorescence spectra of synthesized QD products redispersed in chloroform. The Full Width at Half Maximum (FWHM) of the spectrum for Product B was 33 nm and for Product A was 42 nm.

Absorbance and Emission Spectra of Synthesized Samples Absorbance data were obtained using an Evolution 600 UV-visible spectrophotometer and emission spectra were obtained using the same Cary Eclipse fluorescence spectrophotometer as described previously. To obtain quantum yield values, absorbance data and emission spectra were obtained at three different concentrations (high concentration, medium concentration and low concentration) of each QD Product (A through D) and a Rhodamine 6G standard in ethanol. The absorbance values were taken at the excitation wavelengths for the CdSe/ZnS QDs (400 nm) and Rhodamine (480 nm). The fluorescence spectra for all concentrations were integrated (using Origin software) and plotted against their corresponding products absorbance values (Figure 8 to Figure 12).

Figure 8. The absorbance spectra of Product A at three concentrations

Figure 9. The absorbance spectra of Product B at three concentrations

Figure 10. The absorbance spectra Product C at three concentrations

Figure 11. The absorbance spectra of Product D at three concentrations

Figure 12. The absorbance spectra of Rhodamine at three concentrations

FCS (Fluorescence Correlation Spectroscopy) of Hydrophilically Coated QD Samples Fluorescence correlation spectroscopy was used to determine the hydrodynamic radii of QDs with various hydrophilic surface coatings. Autocorrelation data were acquired using a Zeiss ConfoCor2 fluorescence correlation spectrometer attached to a Zeiss Axiovert 200m Inverted Microscope. The autocorrelation curve generated by the fluorescent nanoparticles diffusing in bulk solution through a confocal excitation volume calculates the time it takes for a molecule to 10

pass through the confocal volume. Through the use of this technique, the particle sizes of four
Count Rate (kHz) hydrophilic nanoparticles were measured. As seen in Figure 13-16, the autocorrelation curves 600

generated by various hydrophilic nanocrystal coatings can be linked to an overall diameter of the 400
300

500

quantum dot. As a comparision, the diffusion of Rhodamine dye was also measured. 200
1 2 3 4 5 Time (seconds) 6 7 8 9 Number 55, Carrier 5, Kinetics 1, REDQDS G (t ) 3.0 2.5 2.0 Count Rate (kHz) 1.5

270 1.0 265

10 100 1000 Time (s) 10000 100000 1e+006

260 Number 55, Carrier 5, Kinetics 1, REDQDS 255

Figure 13. FCS of MPA-coated QDs made from undistilled, recrystallized TOPO and DOPA
1 2 3 4 5 Time (seconds) 6 7 8 9 Number 55, Carrier 5, Kinetics 1, REDQDS G (t ) 1.20 1.15 1.10 1.05
Count Rate (kHz)

1.00
16 14

10

100

1000 Time (s)

10000

100000

1e+006

Number 55, Carrier 5, Kinetics 1, REDQDS

12 10 1 2

Figure 14. FCS of Rhodamine 6G in water

3 4 5 Time (seconds) 6 7 8 9

Number 11, Carrier 1, K inetics 1, RE DQDS G (t ) 1.6 1.5 1.4 1.3

Count Rate (kHz) 1.2

1.1 650 1.0

600
10 100 1000 Time (s) 10000 100000 1e+006

550
Number 11, Carrier 1, K inetics 1, RE DQDS

500 450

Figure 15. FCS of lipopolymer-coated QDs (Product A)

1 2 3 4 5 Time (seconds) 6 7 8 9

Number 22, Carrier 2, Kinetics 1, REDQDS G (t ) 1.4 1.3 1.2 1.1 1.0 10 Number 22, Carrier 2, Kinetics 1, REDQDS 100 1000 Time (s) 10000 100000 1e+006

Figure 16. FCS of MPA-coated QDs made from DOPA and purified TOPO

11

ILLUSTRATIONS

Figure 17. Relative fluorescence quantum yields of synthesized QD products (redispersed in chloroform).

Figure 18. Mean (n = 6 FCS runs of 10 scans per sample) photon counts per molecule for synthesized QD Products A and B (coated in either MPA or lipopolymer) and Rhodamine 6G. The total photon acquisition time was 600 s.

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QD-linked Lipid Probe Tracking

DISTANCE (m)

DISTANCE (m) Figure 19. Snapshots of a fluorescent QD-linked lipid probe were taken by another researcher (snapshot time interval set to 41 msec) and the probes coordinates were plotted. Brownian motion was detected.

DISCUSSION Solvent Impurities Result in More Intense Fluorescence QDs made from undistilled, once-recrystallized TOPO and DOPA (Product B) had the brightest fluorescence (Fig. 7). The second brightest product was synthesized from distilled, twice-recrystallized TOPO and DOPA. The QDs synthesized from commercial TOPO (Strem batch number B4751105) and DOPA had the broadest emission peaks, and even fluoresced from 420 to 550 nm, whereas the other dots did not fluoresce in this range. All four QD products had their emission peaks centered on 600-620 nm. The FWHM of the QDs made from undistilled, once-recrystallized TOPO and DOPA (Product B) was 33 nm. This FWHM was less than the FWHM of 42 nm for the QDs (Product A) made from distilled, twice-recrystallized TOPO and DOPA. This data indicated that solvent impurities resulted in more fluorescent QDs. The fact 13

that more purified (distilled, twice-recrystallized) TOPO and DOPA resulted in less luminescent QDs than less purified (undistilled, once-recrystallized) TOPO and DOPA, suggested that the presence of other unidentified impurities was beneficial. Solvent Impurities Result in Better Quantum Yields Quantum yield is defined as a function of photon emission divided by photon absorbance. After the relative quantum yields of the four QD products were obtained, the efficiencies of the QD products proton emissions were compared. The method used to obtain quantum yields required taking both absorbance and emission spectra for the sample solutions16, but was adapted so that multiple concentrations of each sample (QDs redispersed in chloroform and Rhodamine 6G in ethanol) were analyzed (Figure 8-12). The following formula, adapted from a previously described method, was used:62

In this equation, is the quantum yield, the gradient represents the slope of the least squares regression line of the plot of integrated fluorescence against absorbance, T represents the test sample, S represents the standard sample, and is the refractive index of the solvent. A least squares regression line was calculated (using Microsoft Excel software) for each QD product and Rhodamine. The slopes of these lines, the quantum yield of the Rhodamine standard (0.95), and the refractive indices of chloroform (1.446) and ethanol (1.361) were used to calculate the products relative quantum yields.

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Table 1. Quantum Yield of QD Products (QY of Rhodamine 6G Standard = .95) Sample A B C D Rhodamine Gradient 40905 207380 17590 27938 1000000 Quantum Yield (%) 4.39 22.24 1.89 3.00 95.00

The quantum yield of QDs synthesized from undistilled, once-recrystallized TOPO and DOPA (Product B) was about five times as efficient as that of the QDs made from distilled, twice-recrystallized TOPO and DOPA (Product A) (Fig. 17). The quantum yield of the QDs made from only stock TOPO (no DOPA) was higher than the quantum yield of the QDs made from stock TOPO and DOPA. The results showed that the recrystallization of TOPO and the addition of DOPA made the greatest difference in quantum yield. TOPO Impurities Result in More Photons Emitted per QD The QD samples were characterized by a Zeiss ConfoCor2 microscope using fluorescence correlation spectroscopy (FCS). Since FCS cannot be used to characterize QDs that are redispersed in chloroform, it was necessary to hydrophilically coat them using either one of the MPA surface-exchange or lipopolymer encapsulation methods described previously.14,15 Due to their greater brightness and quantum yield, only Products A (distilled, twice-recrystallized TOPO and DOPA) and B (undistilled, once-recrystallized TOPO and DOPA) were functionalized with MPA and lipopolymer hydrophilic coatings. The functionalized QD products were analyzed by FCS six times (n = 6). A sample containing Rhodamine 6G in water was also

15

characterized in order to compare QD photon counts with that of a conventional fluorescent organic dye. The MPA-coated QDs synthesized from undistilled, once-recrystallized TOPO and DOPA had a photon count per molecule of about 650, which was 13 times higher than that of Rhodamine 6G (Fig. 18). All QD samples, except the lipopolymer-coated ones synthesized from distilled, twice-recrystallized TOPO and DOPA, emitted greater numbers of photons than Rhodamine 6G. The MPA-coated dots were brighter than the lipopolymer-coated QDs. It is intriguing, perhaps, that although Rhodamine 6G has a much higher quantum yield than the QDs, the QDs emit more photons per molecule. This can be explained by the QDs higher extinction coefficient that enables them to recover much faster than Rhodamine 6G does. Biofunctionalized QDs Synthesized from DOPA Track Single Lipid Movement Lipopolymer-coated QDs from product B were used to track lipid movement on a model bilayer with single particle tracking (SMFM) as described previously.60 By incorporating DSPE PEG2000 Maleimide into the lipid-lipopolymer coating mixture, lipopolymer-coated QDs could be linked onto lipids with thiol headgroups (SH-lipids), which were incorporated into a phospholipid lipid bilayer.12

Figure 20. Lipopolymer-coated QD-linked lipid probe.63

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A series of still snapshots of a moving QD-linked lipid were taken by another researcher and the QDs position coordinates were tracked over 41 millisecond intervals when its movement was plotted on a coordinate grid (Fig. 19). QDs synthesized with DOPA and undistilled, once-recrystallized TOPO were more visible than QDs made from unadulterated (undistilled, unrecrystallized) stock TOPO when viewed with SMFM microscopy, in excellent agreement with the spectroscopy results. CONCLUSIONS AND FUTURE WORK In this study, the effects of TOPO purity and DOPA addition on sonochemically synthesized CdSe/ZnS QDs were observed by comprehensive measurements including fluorescence spectroscopy, UV-visible spectroscopy, and confocal fluorescence correlation spectroscopy characterization. Up until now, traditional TOPO (unpurified and with no solvent additives) had been used to produce mediocre products of QDs with their quality varying from solvent batch to solvent batch. The limitations of traditional QDs synthesized from stock TOPO of an average batch were (1) extremely large FWHMs; (2) the inability to fluoresce under a conventional handheld UV lamp; and (3) very low photon counts per molecule. This study has provided strong evidence that impurities such as DOPA resulted in brighter, more efficient QDs. This work has also indicated an ability to produce higher-quality QDs for applications in industry, biological imaging, and medical imaging and diagnosis.12-16 Future Directions The effects of the presence of DOPA on QDs could be expanded in future studies involving different concentrations and degrees of additive purification by distillation or recrystallization. Characterization of TOPO and its impurities could be accurately achieved by utilizing gas chromatography-mass spectrometry (GC-MS) or
31

P NMR.13 Also, atomic force

microscopy18 (AFM) could be used to compare the isotropies of QDs synthesized from stock 17

TOPO and QDs synthesized from TOPO with various incorporated additives. It was reported on September 28, 2010, that scientists at the University of Illinois have developed a nanoneedle that releases quantum dots directly into the nucleus of a living cell when a small electrical charge is applied. Their study generated extremely small-size and high quality QDs that have the potential to be utilized as in vivo optical imaging probes and in a variety of research applications.

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REFERENCES 1. Smith AM, Mohs, Nie S. (2009).Tuning the optical and electronic properties of collodidal nonacrystals by lattice strains. Nat Nanotechnol, 4:56-63. 2. Peng X, Manna L, Yang W, et al. (2000). Shape control of CdSe nanocrystals. Nature, 404:5961. 3. Li JJ, Wang YA, Guo W, et al. (2003). Large-scale synthesis of nearly monodisperse CdSe/CdS core/shell nanocrystals using air-stable reagents via successive ion layer adsorption and reaction. J Am Chem Soc. 125:1256712575. 4. Murray CB, Kagan CR, Bawendi MG. (2000). Synthesis and characterization of monodisperse nanocrystals and close-packed nanocrystal assemblies. Ann Rev Materials Sci. 30:545610. 5. Smith AM, Duan H, Mohs AM, Nie S. (2008), Bioconjugated quantum dots for in vivo molecular and cellular imaging. Adv Drug Deliv Rev. 60:12261240. 6. Nie S, Xing Y, Kim GJ, Simons JW. (2007). Nanotechnology applications in cancer. Annu Rev Biomed Eng. 9:25788. 7. Smith AM, Dave S, Nie S, True L, Gao X. (2006). Multicolor quantum dots for molecular diagnostics of cancer. Expert Rev Mol Diagn. 6:231244. 8. Chan WC, Maxwell DJ, Gao X, Bailey RE, Han M, Ne S. (2002).Luminescent quantum dots for multiplexed biological detection and imaging. Curr Opin Biotechnol. 13:4046. 9. Klostranec JM, Chan WC. (2006).Quantum dots in biological and biomedical research: Recent progress and present challenges. Advanced Materials. 18:19531964. 10. Nandakumar, P., et al. (2000). Quantum size effects on the third order optical nonlinearity of CdS quantum dots in Nafion. Optics Comm. 185: 457-64. 11. Frasco, Manuela F., et al. (2010). Bioconjugated quantum dots as fluorescent probes for bioanalytical applications. Anal. Bioanal. Chem., 396: 229-38. 12. Gao, Xiaohu, et al. (2005). In vivo molecular and cellular imaging with quantum dots. Curr. Op. Biotech. 16: 63-9. 13. Shao, Fangwei, et al. (2008). Monofunctional Carbocyanine Dyes for Bio- and Bioorthogonal Conjugation. Bioconj. Chem., 19: 2487-90. 14. Lu, Zhisong, et al. (2008). Mechanism of Antimicrobial Activity of CdTe Quantum Dots. Langmuir 24: 5445-52.

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15. Lao, U Loi, et al. (2006). Simple Conjugation and Purification of Quantum DotAntibody Complexes Using a Thermally Responsive Elastin-Protein L Scaffold As Immunofluorescent Agents. J. Amer. Chem. Soc.128: 14756-7. 16. Vashist, Sandeep Kumar, et al. (2006). Review of Quantum Dot Technologies for Cancer Detection and Treatment. J. Nanotechnol., Chem. Mater.2: 2-6. 17. Biofunctionalization of Fluorescent Nanoparticles. Biofunctionalization of Nanomaterials Ed. C. Kumar. Vol. 1. Weinheim: Wiley-VCH, 2005. 5-7. 18. Murcia, Michael J., et al. (2006). Facile Sonochemical Synthesis of Highly Luminescent ZnS-Shelled CdSe Quantum Dots. Chemistry of Materials 18: 2219-21. 19. Morris-Cohen, Adam J., et al. (2010). The Effect of a Common Purification Procedure on the Chemical Composition of the Surfaces of CdSe Quantum Dots Synthesized with Trioctylphosphine Oxide. J. Phys. Chem. C, 114: 897-906. 20. Meyer, M.; Wallberg, C.; Kurihara, K.; Fendler, J. H. (1984).Photosensitized charge separation and hydrogen production in reversed micelle entrapped platinized colloidal cadmium sulphide, J. Chem. Soc. Chem. Commun., 90-91. 21. Dannhauser, T.; ONeil, M.; Johansson, K.; Whitten, D.; McLendon, G. (1986), Photophysics of quantized colloidal semiconductors. Dramatic luminescence enhancement by binding of simple amines, J. Phys. Chem., 90: 6074. 22. Petit, C.; Pileni, M. P. (1988), Synthesis of cadmium sulfide in situ in reverse micelles and in hydrocarbon gels, J. Phys. Chem., 92: 2282-2286 23. Steigerwald, M. L.; Alivisatos, A. P.; Gibson, J. M.; Harris, T. D.; Kortan, R.; Muller, A. J.;Thayer, A. M.; Duncan, T. M.; Douglass, D. C.; Brus, L. E. (1988), Surface derivatization and isolation of semiconductor cluster molecules, J. Am. Chem. Soc., 110: 3046-3050. 24. Kortan, A. R.; Hull, R.; Opila, R. L.; Bawendi, M. G.; Steigerwald, M. L.; Carroll, P. J.; Brus, L.E. (1990), Nucleation and growth of cadmium selendie on zinc sulfide quantum crystallite seeds, and vice versa, in inverse micelle media, J. Am. Chem. Soc., 112: 13271332. 25. Bawendi, M. G.; Carroll, P. J.; Wilson, W. L.; Brus, L. E. (1992), Luminescence properties of CdSe quantum crystallites: Resonance between interior and surface localized states, J. Chem. Phys., 96: 946-954. 26. Murray, C. B.; Norris, D. J.; Bawendi, M. G. (1993), Synthesis and characterization of nearly monodisperse CdE (E = sulfur, selenium, tellurium) semiconductor nanocrystallites, J. Am. Chem. Soc., 115: 8706-8715.

20

27. Katari, J .E. B.; Colvin, V. L.; Alivisatos, A. P. X-ray Photoelectron Spectroscopy of CdSe Nanocrystals with Applications to Studies of the Nanocrystal Surface, J. Phys. Chem., 98: 4109-4117. 28. Guzelian, A. A.; Katari, J. E. B.; Kadavanich, A. V.; Banin, U.; Hamad, K.; Juban, E.; Alivisatos, A. P.; Wolters, R. H.; Arnold, C. C.; Heath, J. R.(1996) , Synthesis of SizeSelected, Surface-Passivated InP Nanocrystals, J. Phys. Chem. , 100: 7212-7219. 29. Peng, X.; Wickham, J.; Alivisatos, A. P. (1998), Kinetics of II-VI and III-V Colloidal Semiconductor Nanocrystal Growth: Focusing of Size Distributions, J. Am. Chem. Soc., 120: 5343-5344. 30. Peng, Z. A.; Peng, X.(2001), Formation of High-Quality CdTe, CdSe, and CdS Nanocrystals Using CdO as Precursor, J. Am. Chem. Soc., 123: 183-184. 31. Qu, L.; Peng, X. (2002), Control of Photoluminescence Properties of CdSe Nanocrystals in Growth, J. Am. Chem. Soc., 124: 2049-2055. 32. Mekis, I.; Talapin, D. T.; Kornowski, A.; Haase, M.; Weller, H. (2003) One-Pot Synthesis of Highly Luminescent CdSe/CdS CoreShell Nanocrystals via Organometallic and Greener Chemical Approaches, J. Phys. Chem. B, 107:74547462. 33. Qu, L.; Yu, W. W.; Peng, X., In Situ Observation of the Nucleation and Growth of CdSe Nanocrystals, Nano Lett. 2004, 4: 465-469. 34. Manna, L.; Scher, E. C.; Alivisatos, A. P.(2000), Synthesis of Soluble and Processable Rod-, Arrow-, Teardrop-, and Tetrapod-Shaped CdSe Nanocrystals, J. Am. Chem. Soc., 122: 12700-12706. 35. Li, L.-S; Hu, J.; Yang, W.; Alivisatos, A. P.(2001), Band Gap Variation of Size- and Shape-Controlled Colloidal CdSe Quantum Rods, Nano Lett.,1: 349-351. 36. Hu, J.; Li, L.-S; Yang, W.; Manna, L.; Wang, L.-W; Alivisatos, A. P. (2001), Linearly Polarized Emission from Colloidal Semiconductor Quantum Rods, Science, 292: 20602063. 37. Peng, Z. A.; Peng, X. (2002). Nearly Monodisperse and Shape-Controlled CdSe Nanocrystals via Alternative Routes: Nucleation and Growth, J. Am. Chem. Soc. , 124: 3343-3353. 38. Talapin, D. V.; Nelson, J. H.; Shevchenko, E. V.; Aloni, S.; Sadtler, B.; Alivisatos, A. P. (2007), Seeded Growth of Highly Luminescent CdSe/CdS Nanoheterostructures with Rod and Tetrapod Morphologies, Nano Lett., 7: 2951-2959. 39. Carbone, L. et al. (2007), Synthesis and Micrometer-Scale Assembly of Colloidal CdSe/CdS Nanorods Prepared by a Seeded Growth Approach, Nano Lett., 7: 2942-2950. 21

40. Milliron, D. L.; Hughes, S. M.; Cui, Y.; Manna, L.; Li, J.; Wang L.-W.; Alivisatos, A. P. (2004), Colloidal nanocrystal heterostructures with linear and branched topology ,Nature, 430: 190-195. 41. Carbone, L.; Kudera, S.; Carlino, E.; Parak, W. J.; Giannini, C.; Cingolani, R.; Manna, L. (2006), Multiple Wurtzite Twinning in CdTe Nanocrystals Induced by Methylphosphonic Acid, J. Am.Chem. Soc., 128: 748-755. 42. Puntes, V. F.; Zanchet, D.; Erdonmez, C. K.; Alivisatos, A. P. (2002). Article Synthesis of hcp-Co Nanodisks, J. Am. Chem. Soc., 124:12874-12880. 43. Perera, S. C.; Tsoi, G.; Wenger, L. E.; Brock, S. L. (2003), Synthesis of MnP Nanocrystals by Treatment of Metal Carbonyl Complexes with Phosphines: A New, Versatile Route to Nanoscale Transition Metal Phosphides, J. Am. Chem. Soc., 125: 13960- 13961. 44. Qian, C.; Kim, F.; Ma, L.; Tsui, F.; Yang, P.; Liu, J. (2004), Solution-Phase Synthesis of Single-Crystalline Iron Phosphide Nanorods/Nanowires,,J. Am. Chem. Soc., 126: 11951198. 45. Li, Y.; Malik, M. A.; OBrien, P. (2005),Synthesis of Single-Crystalline CoP Nanowires by a One-Pot MetalOrganic Route, J. Am. Chem. Soc., 127: 16020-16021. 46. Yu, W. W.; Peng, X. (2002), Formation of High-Quality CdS and Other IIVI Semiconductor Nanocrystals in Noncoordinating Solvents: Tunable Reactivity of Monomers, Angew. Chem., Int. Ed. 2002, 41: 2368-2371. 47. L.i, J. J.; Wang, Y. A.; Guo, W.; Keay, J. C.; Mishima, T. D.; Johnson, M. B.; Peng, X. (2003), Large-Scale Synthesis of Nearly Monodisperse CdSe/CdS Core/Shell Nanocrystals Using Air-Stable Reagents via Successive Ion Layer Adsorption and Reaction, J. Am.Chem. Soc., 125: 12567-12575. 48. Xie, R.; Battaglia, D.; Peng, X. (2007), Colloidal InP Nanocrystals as Efficient Emitters Covering Blue to Near-Infrared, J. Am. Chem. Soc., 129: 15432-15433. 49. Yu, H.; Li, J.; Loomis, R. A.; Gibbons, P. C.; Wang, L.-W; Buhro, W. E. (2003), Cadmium Selenide Quantum Wires and the Transition from 3D to 2D Confinement, J. Am. Chem. Soc., 125: 16168-16169. 50. Dong, A.; Yu, H.; Wang, F.; Buhro, W. E. (2007), SolutionLiquidSolid (SLS) Growth of ZnSeZnTe Quantum Wires having Axial Heterojunctions, Nano Lett.,7: 1308-1313. 51. Wang, F.; Buhro, W. E. (2007), Determination of the RodWire Transition Length in Colloidal Indium Phosphide Quantum Rods,J. Am. Chem. Soc., 129: 14381-14387.

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52. Dong, A.; Yu, H.; Wang, F.; Buhro, W. E. (2008), Colloidal GaAs Quantum Wires: SolutionLiquidSolid Synthesis and Quantum-Confinement Studies, J. Am. Chem. Soc., 130: 5954-5961. 53. Sun, J.; Wang, L.-W.; Buhro, W. E. (2008), Synthesis of Cadmium Telluride Quantum Wires and the Similarity of Their Effective Band Gaps to Those of Equidiameter Cadmium Telluride Quantum Dots, J. Am. Chem. Soc., 130: 7997-8005. 54. Liu, H.; Owen, J. S.; Alivisatos, A. P. (2007), Mechanistic Study of Precursor Evolution in Colloidal Group IIVI Semiconductor Nanocrystal Synthesis, J. Am. Chem. Soc, 129: 305-312. 55. Kopping, J. T.; Patten, T. E.,(2008). Identification of Acidic Phosphorus-Containing Ligands Involved in the Surface Chemistry of CdSe Nanoparticles Prepared in Tri-Noctylphosphine Oxide Solvents.J. Am. Chem. Soc. 130: 5689-5698. 56. Wang, Fudong, et al. (2008). The Trouble with TOPO; Identification of Adventitious Impurities Beneficial to the Growth of Cadmium Selenide Quantum Dots, Rods, and Wires. Nano Letters 8: 3521-3. 57. Wang, Fudong, et al. (2009). Spectroscopic Identification of Tri-n-octylphosphine Oxide (TOPO) Impurities and Elucidation of Their Roles in Cadmium Selenide Quantum-Wire Growth. J. Amer. Chem. Soc.131: 4983-94. 58. Reiss, Peter, et al. (2009). Core/Shell Semiconductor Nanocrystals. Small 5: 154-7.
59. Murcia, J, Michael, Sonochemical Synthesis and bioconjugation of highly fluorescent,

water soluble quantum dots for single molecule imaging on phospholipid membranes, PhD. , Dissertation, Purdue university, (2006):38 60. Murcia, Michael J., et al. (2008). Design of Quantum Dot-Conjugated Lipids for LongTerm, High-Speed Tracking Experiments on Cell Surfaces. J. Amer. Chem. Soc.130: 15054-7. 61. Eaton, David F. (1988). Reference Materials for Fluorescence Measurement. Pure & Applied Chemistry 60: 1107-14. 62. Murcia, Michael J., et al. (2008). Fluorescence correlation spectroscopy of CdSe/ZnS quantum dot optical bioimaging probes with ultra-thin biocompatible coatings. Optics Communications 281: 1771-5. 63. Siegel, A.P.*, Murcia, M.J., Ruehe, J., Jordan, R., Naumann, C.A. (2009). Tuneable buckling in polymer-tethered lipid bilayers creates diffusion barriers and a platform for studying hop diffusion. (Poster) Presented at the 237th ACS National Meeting, Salt Lake City, UT.

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