WO2010028217A1 - Poly(ethylene glycol) methyl ether carbodiimide coupling reagents for the biological and chemical functionalization of water soluble nanoparticles - Google Patents

Abstract

Reagents and methods for synthesizing water-soluble nanoparticles have been developed. In an aspect, poly(ethylene glycol) methyl ether carbodiimide is used as a coupling reagent for the biological and chemical functionalization of water-soluble nanoparticles.

Description

POLY(ETHYLENE GLYCOL) METHYL ETHER CARBODIIMIDE COUPLING REAGENTS FOR THE BIOLOGICAL AND CHEMICAL FUNC TION ALIZ ATION OF

WATER SOLUBLE NANOPARTICLES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 61/094,477, filed September 5, 2008, the entire disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to reagents and methods for the biological and chemical functionalization of water soluble nanoparticles.

BACKGROUND OF THE INVENTION

[0003] Many types of metal and semiconductor nanoparticles (NPs) are created via colloidal synthetic methods which render the materials hydrophobic. Such NPs are dispersed in water through surface organic cap exchange or by amphiphilic polymer encapsulation; often, water solubility is achieved via the presence of carboxylic acid functionalities on the solubilizing agents. While this renders the material water soluble, subsequent functionalization of the systems can be very difficult. A method to derivatize carboxylic acid coated NPs is to conjugate chemical and biological moieties containing amine functionality to the NP surface using the water soluble activator l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). However, the excess use of this reagent appears to cause complete and permanent precipitation of the NPs.

[0004] Metallic and semiconductor nanoparticles (NPs, or quantum dots) have attracted enormous attention over the past several decades due to their unique size dependent physical and optical properties. Semiconductor NPs may be luminescent and display narrow and size tunable emission spectra, high quantum yields, and exceptional photostability. Water solubilized NPs can be functionalized to produce especially useful systems that have found applications in imaging, tracking, sensing, and labeling in biology. See Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C; Mikulec, F. V.; Bawendi, M. G. Self- assembly of CdSe-ZnS Quantum Dot Bioconjugates Using an Engineered Recombinant Protein. J. Am. Chem. Soc. 2000, 122, 12142-12150; Willard, D. M.; Carillo, L. L.; Jung, J.; Van Orden, A. CdSe-ZnS Quantum Dots as Resonance Energy Transfer Donors in a Model Protein-protein Binding Assay. Nano Lett. 2001, 1, 469-474; Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. In vivo Imaging of Quantum Dots Encapsulated in Phospholipid Micelles. Science 2002, 298, 1759-1762; West, J. L.; Halas, N. J. Engineered Nanomaterials for Biophotonics Applications: Improving Sensing, Imaging, and Therapeutics. Ann. Rev. Biomed. Eng. 2003, 5, 285-292; Cang, H.; Xu, C. S.; Montiel, D.; Yang, H. Guiding a Confocal Microscope by Single Fluorescent Nanoparticles. Opt. Lett. 2007, 32, 2729-2731; Cang, H.; Wong, C. M.; Xu, C. S.; Rizvi, A. H.; Yang, H. Confocal Three Dimensional Tracking of a Single Nanoparticle with Concurrent Spectroscopic Readouts. App. Phys. Lett. 2006, 88, 223901; Howarth, M.; Takao, K.; Hayashi, Y.; Ting, A. Y. Targeting Quantum Dots to Surface Proteins in Living Cells with Biotin Ligase. P. N. A. S. 2005, 102, 7583-7588; Snee, P. T.; Somers, R. C; Nair, G.; Zimmer, J. P.; Bawendi, M. G.; Nocera, D. G. A Ratiometric CdSe/ZnS Nanocrystal pH Sensor. J. Am. Chem. Soc. 2006, 128, 13320-13321; Yezhelyev, M. V.; Qi, L.; O'Regan, R. M.; Me, S.; Gao, X. Proton-Sponge Coated Quantum Dots for siRNA Delivery and Intracellular Imaging. J. Am. Chem. Soc. 2008, 130, 9006-9012; and Rajan, S. S.; Liu, H. Y.; Vu, T. Q. Ligand-bound Quantum Dot Probes for Studying the Molecular Scale Dynamics of Receptor Endocytic Trafficking in Live Cells. ACS Nano 2008, 2, 1153-1166.

[0005] However, these applications would not exist were it not for the development of water solubilization and chemical and biological conjugation methods for the generally hydrophobic materials that are created using colloidal synthetic procedures. Since the problem with the conversion of the hydrophobic semiconductor NPs into water soluble systems was first addressed around 1998 (see e.g., Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Semiconductor Nanocrystals as Fluorescent Biological Labels. Science 1998, 281, 2013- 2016; and Chan, W. C. W.; Me, S. M. Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection. Science 1998, 281, 2016-2018), several methods have been developed to water solubilize NPs. See e.g., Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Quantum Dots for Live Cells, In vivo Imaging, and Diagnostics. Science 2005, 307, 538-544. Generally, these methods involve cap exchange or encapsulation. Cap exchange involves stripping the native trioctylphosphine/triocylphosphine oxide (TOP/TOPO) surface ligands and replacing them with bifunctional surfactants such as mercapto-acids. It has been found that encapsulation of NPs in amphiphilic polymers is highly robust; the use of a 40% octylamine modified poly(acrylic acid) polymer is fairly ubiquitous in this regard. See Pellegrino, T.; Manna, L.; Kudera, S.; Liedl, T.; Koktysh, D.; Rogach, A. L.; Keller, S.; Radler, J.; Natile, G.; Parak, W. J. Hydrophobic Nanocrystals Coated with an Amphiphilic Polymer Shell: A General Route to Water Soluble Nanocrystals. Nano Lett. 2004, 4, 703-707; and Wu, X. Y.; Liu, H. J.; Liu, J. Q.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N. F.; Peale, F.; Bruchez, M. P. Immunofluorescent Labeling of Cancer Marker Her2 and Other Cellular Targets with Semiconductor Quantum Dots. Nat. Biotechnol 2003, 21, 41-46.

[0006] The solubilization of NPs in water is unfortunately only half the problem when synthesizing functional materials - the nanoparticles must further be derivatized with chemical or biological vectors before they serve a useful purpose. See e.g., Chen, Y.; Thakar, R.; Snee, P. T. Imparting Nanoparticle Function with Size-Controlled Amphiphilic Polymers. J. Am. Chem. Soc. 2008, 130, 3744-3745; Medintz, I. L.; Berti, L.; Pons, T.; Grimes, A. F.; English, D. S.; Alessandrini, A.; Facci, P.; Mattoussi, H. A Reactive Peptidic Linker for Self-assembling Hybrid Quantum Dot-DNA Bioconjugates. Nano Lett. 2007, 7, 1741-1748; Fernandez- Arguelles, M. T.; Yakovlev, A.; Sperling, R. A.; Luccardini, C; Gaillard, S.; Medel, A. S.; Mallet, J. M.; Brochon, J. C; Feltz, A.; Oheim, M.; Parak, W. J. Synthesis and Characterization of Polymer-coated Quantum Dots with Integrated Acceptor Dyes as FRET -based Nanoprobes. Nano Lett. 2007, 7, 2613-2617; Guo, W. Z.; Li, J. J.; Wang, Y. A.; Peng, X. G. Conjugation Chemistry and Bioapplications of Semiconductor Box Nanocrystals Prepared via Dendrimer Bridging. Chem. Mater. 2003, 15, 3125-3133.

[0007] Water soluble NPs are often synthesized using systems with carboxylic acid terminated functionality. One approach is to cross link the organic shell with a primary amine containing chemical or biological vector using a carbodiimide activator such as l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC). However, quenching caused by the precipitation of carboxylic acid coated NPs by the use of excess EDC has been observed. See e.g., Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C; Mikulec, F. V.; Bawendi, M. G. Self-assembly of CdSe-ZnS Quantum Dot Bioconjugates Using an Engineered Recombinant Protein. J. Am. Chem. Soc. 2000, 122, 12142-12150; Chen, Y.; Thakar, R.; Snee, P. T. Imparting Nanoparticle Function with Size-Controlled Amphiphilic Polymers. J. Am. Chem. Soc. 2008, 130, 3744-3745; Clapp, A. R.; Goldman, E. R.; Mattoussi, H. Capping of CdSe-ZnS Quantum Dots with DHLA and Subsequent Conjugation with Proteins. Nat. Protoc. 2006, 1, 1258-1266; and Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Quantum Dot Bioconjugates for Imaging, Labelling and Sensing. Nat. Mater. 2005, 4, 435-446.

[0008] The NP aggregation can cause the loss of an entire sample if the material is exposed to excess EDC (on the order of 10⁴ per NP by mole), and there are indications that this precipitation is permanent.

BRIEF SUMMARY OF THE INVENTION

[0009] An embodiment of the invention is the compound poly(ethylene glycol) methyl ether carbodiimide of various masses determined by the length of the poly(ethylene glycol) polymer.

[00010] An embodiment of the invention is a composition comprising a nanoparticle and poly(ethylene glycol) methyl ether carbodiimide which functionalizes the nanoparticle.

[00011] An embodiment of the invention is a method for making the compound poly(ethylene glycol) methyl ether carbodiimide comprising exposing amine functional poly(ethylene glycol) methyl ether to ethyl isothiocyanate, followed by exposing to HgO.

[00012] An embodiment of the invention is a method for making the compound poly(ethylene glycol) methyl ether carbodiimide comprising exposing amine functional poly(ethylene glycol) methyl ether to ethyl isocyanate, followed by exposure to p- toluenesulfonyl chloride in the presence of triethylamine.

[00013] An embodiment of the invention is a method for making a functional water-soluble nanoparticle comprising exposing amine functional poly(ethylene glycol) methyl ether to ethyl isothiocyanate, followed by exposing to HgO to form a poly(ethylene glycol) methyl ether carbodiimide, and combining the poly(ethylene glycol) methyl ether carbodiimide with a nanoparticle.

BRIEF DESCRIPTION OF THE DRAWINGS

[00014] FIG. 1 is a graph of absorption versus wavelength, and depicts dynamic light scattering characterization of the size of water soluble CdSe/ZnS NPs.

[00015] FIG. 2 is a graph of absorption versus wavelength, and depicts absorption spectra of water soluble CdSe/ZnS NPs before and after exposure to EDC. [00016] FIG. 3 is a graph of % initial emission versus time, and depicts the fluorescence intensity of CdSe/ZnS NPs over time after exposure to the carbodiimide reagents disclosed herein compared to the initial emission.

[00017] FIG. 4 is graph of absorption versus wavelength, and depicts the absorption spectra of CdSe/ZnS - TAMRA cadaverine dye before and after dialysis using IOOK MW cutoff filters.

[00018] FIG. 5 is a graph of absorption versus wavelength, and depicts the absorption spectra of CdSe/ZnS and TAMRA cadaverine dye before and after dialysis using IOOK MW cutoff filters.

[00019] FIG. 6 is a graph of absorption versus wavelength, and depicts the normalized absorption spectra of TAMRA cadaverine dyes bound to CdSe/ZnS NPs after subtraction of the CdSe/ZnS component.

[00020] FIG. 7 is a picture of bottles depicting A) visible light image of CdSe/ZnS NP samples after coupling to amine functional TAMRA dye as a function of the MPEG 350 CD to dye ratio, and B) the emission under UV light excitation of the same samples.

[00021] FIG. 8 is a graph of normalized emission versus wavelength, and depicts the emission spectra of a CdS/ZnS - fluorescein cadaverine dye conjugate as a function of pH.

[00022] FIG. 9 is a graph of absorption versus diameter, and depicts the gel permeation chromatography trace of aqueous CdSe/ZnS NPs and amine functional MPEG 750 / NP conjugate.

[00023] FIG. 10 is a graph of absorption versus diameter, and depicts the gel permeation chromatography trace of aqueous CdSe/ZnS NPs and streptavidin conjugate as a function of the streptavidin to NP ratio used during the synthesis.

[00024] FIG. 11 is a graph of emission versus wavelength, and depicts the emission of CdSe/ZnS - streptavidin conjugates, wherein the sample was prepared using a 100^χ excess of protein to NP.

[00025] FIG. 12 is a picture of CdSe/ZnS NPs precipitated by EDC.

[00026] FIG. 13 is a graph of absorbance versus wavelength, and depicts absorption spectra of blank and tetramethylrhodamine-5-carboxamide cadaverine functionalized Fe₂O₃ nanoparticles. [00027] FIG. 14 is a graph of absorbance versus wavelength, and depicts absorption spectra of blank and streptavidin functionalized CdSe/ZnS nanoparticles, showing residual absorption at 280 nm that is likely due to streptavidin.

[00028] FIG. 15 is a 13C NMR spectrum of carbodiimide functional poly(ethylene glycol) methyl ether 350, 0→80 ppm.

[00029] FIG. 16 is a 13C NMR spectrum of carbodiimide functional poly(ethylene glycol) methyl ether 350, 140→150 ppm.

[00030] FIG. 17 is mass spectrum of amine functional poly(ethylene glycol) methyl ether 750.

[00031] FIG. 18 is mass spectrum of amine functional poly(ethylene glycol) methyl ether 350.

[00032] FIG. 19 is the proton NMR spectra of cellulose and biotinylated cellulose. DETAILED DESCRIPTION OF THE INVENTION

[00033] Embodiments of the invention relate to reagents and methods to eliminate precipitation of nanoparticles (NPs). An embodiment of the invention includes at least one carbodiimide compound that does not cause precipitation of nanoparticles, and which can be used to functionalize an amphiphilic polymer coated NP with a high yield.

[00034] An embodiment of the invention includes a method of functionalizing a nanoparticle system, including but not limited to a semiconductor nanoparticle system, using a carbodiimide reagent. The carbodiimide reagent does not cause precipitation even at high loading levels and can be used to efficiently functionalize carboxylic acid coated NPs.

[00035] EDC Induced NP Precipitation

[00036] It has been observed that the coupling reagent l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (known as EDC) often results in precipitation of water solubilized NPs. To demonstrate this effect, the size distribution of a sample of water soluble CdSe/ZnS NPs using Dynamic Light Scattering (DLS) before and after exposure to excess EDC (~3^χ10⁵ per NP by mole) was examined. As shown in Figure 1 , the NPs initially have a radius of about 10 nm (a secondary peak appears at about 45 nm which, which is believed to be attributed to dust particles as confirmed with gel permeation chromatography). After exposure to EDC and incubation overnight, the NPs were clearly precipitated due to their "snowflake" appearance and loss of emission intensity which was verified in the DLS data shown in the inset of Figure 1. In other words, after exposure to excess EDC (~3^χ 10⁵ per NP by mole), the precipitation of the NPs is evident as the average size of the particulates increases to -1550 nm. There is no peak at 10 nm while the small peak near -100 nm is attributed to small dust particulates. The absorption and emission of the samples before and after EDC precipitation is shown in Fig. 2.

[00037] The average size increased -150 fold due to the induced precipitation of the NPs which can be seen in the absorption spectrum of the sample which is dominated by scattering as shown in Figure 2.

[00038] As a result of the chemical exposure, the emission intensity of the NPs is quenched by 65% relative to the untreated sample. A sample of CdSe/ZnS NPs using EDC was precipitated and stored for seventeen months, and none of the material appeared to have resolubilized, although it was difficult to imagine that the EDC had not hydro lyzed over the 17 months. Thus, the precipitation appears permanent, which would likely be the case if the amphiphilic NP polymer coating was lost.

[00039] Figure 2 depicts absorption spectra of water soluble CdSe/ZnS NPs before and after exposure to EDC. The agglomerization characterized from DLS measurements are reflected by the scattering absorption features in the sample after exposure to EDC. See Inset a of Figure 2. The fluorescence emission spectra of CdSe/ZnS NPs before and after exposure to EDC reveal that the emission intensity of the NPs is reduced by 65% due to the EDC induced precipitation. These samples were also characterized by DLS (Figure 1).

[00040] MPEG CD Synthesis

[00041] Conventional current methods to water solubilize hydrophobic colloidally synthesized NPs involve the utilization of systems with carboxylic acid moieties. As such, the derivatization of water soluble NPs through highly stable amide bonds should be simple and efficient through the use of amine functional vectors with a carbodiimide activating agent. While this strategy has been employed, a survey of the literature indicates that EDC is often used sparingly, and many researchers have to re-filter their samples after exposure of aqueous NPs to this reagent. As noted and demonstrated herein, exposure to excess EDC causes complete precipitation of the sample, and there are indications that this precipitation is permanent. When the precipitation of NPs with EDC was examined, it was initially hypothesized that residual amine surfactants, generally used in the synthesis of NPs such as CdSe/ZnS, allowed the NPs to crosslink, which was the source of the observed precipitation. CdSe/ZnS was synthesized without the use of amines, at any step, according to some original procedures (see Dabbousi, B. O.; RodriguezViejo, J.; Mikulec, F. V.; Heine, J. R.; Mattoussi, H.; Ober, R.; Jensen, K. F.; Bawendi, M. G. (CdSe)ZnS Core-shell Quantum Dots: Synthesis and Characterization of a Size Series of Highly Luminescent Nanocrystallites. J. Phys. Chem. B 1997, 101, 9463-9475; and Murray, C. B.; Norris, D. J.; Bawendi, M. G. Synthesis and Characterization of Nearly Monodisperse CdE (E = S, Se, Te) Semiconductor Nanocrystallites. J. Am. Chem. Soc. 1993, 115, 8706-8715). After water solubilizing the NPs using octylamine modified PAA amphiphilic polymers and purification, severe precipitation of the materials was observed after exposure to excess EDC.

[00042] At this point, the chemical reactivity of the carbodiimide seemed less responsible for the observed precipitation as there is no reaction that the EDC could cause that would allow NPs to precipitate in water. A focus was directed on the water solubilizing moiety of EDC: the terminal cationic N,N-dimethylamine functionality. A question could be raised whether the negative charge of the NPs and the positively charged coupling agent wasn't in fact responsible for the observations; as such, an approach to synthesize non-polar carbodiimide reagents was undertaken. To this end, water soluble carbodiimides were synthesized where the water solubilizing moiety was neither ionic nor reactive with the carbodiimide functionality. In this regard, poly(ethylene glycol) methyl ether (MPEG) was found to satisfy these requirements. Two MPEG carbodiimide derivatives of various molecular weights (i.e., 350 grams/mole and 750 grams/mole) were synthesized and a bifunctional PEG carbodiimide derivative was synthesized as discussed in the examples below, and an investigation made whether these reagents would still precipitate aqueous NP suspensions of various compositions.

[00043] Time resolved precipitation studies of NPs exposed to carbodiimide reagents

[00044] To study the dynamics of exposure of an aqueous suspension of CdSe/ZnS NPs to the carbodiimides EDC and MPEG 350 CD, it is noted that it is demonstrated herein that precipitation of fluorescent materials reduces the emission intensity. Solutions of the carbodiimide reagents EDC and MPEG 350 CD were injected into aqueous CdSe/ZnS NP suspensions while monitoring the fluorescent emission intensity over time.

[00045] Shown in Figure 3 are the normalized emission intensities after correction for the dilution that occurs after reagent injection. More specifically, Figure 3 shows the fluorescence intensity of CdSe/ZnS NPs over time after exposure to the carbodiimide reagents discussed herein compared to the initial emission. Carbodiimide functional poly(ethylene glycol) methyl ether 350 (MPEG 350 CD) was used in this study. The amounts of EDC added to the samples were 10% (B), 33% (C) and 50% (D) by mole relative to the amount of MPEG 350 CD (A, 1 xlO⁵ per NP).

[00046] The PEG 350 CD sample (Ix IO⁵ molar excess / NP) shows some CdSe/ZnS brightening; however, this effect is slight and is very likely due to an increase in signal simply from fluorescence waveguiding to the detector via dilution. The sample emission intensity remains relatively constant, which indicates that the nanoparticles are stable in solution. In a separate experiment, injection of a 10% molar equivalent of EDC to MPEG 350 CD into a fresh 3 mL CdSe/ZnS sample of the same concentration causes a similar, slight increase in the CdSe/ZnS signal; however, over time, the emission intensity decreases. When exposed to 33% and 50% molar equivalents, there is a clear and immediate loss of emission intensity which could be associated with the precipitation of the NPs. After monitoring the emission, the presence of EDC-induced precipitated NPs was confirmed visually as the sample had a "snowdome" like appearance under ambient lighting (see Figure 12). Consequently, exposure to high concentrations of neutral MPEG 350 CD does not cause precipitation of the NPs under these conditions, which was confirmed with DLS measurements. Precipitation of NPs was never visually observed when exposing NPs to MPEG 750 CD as well. The efficacy of using the carbodiimide reagent as an activator for forming amide bonds with NPs and other materials is discussed in the following sections.

[00047] It is interesting to note that the EDC precipitation data reveal two correlated, yet distinctly separated, dynamical processes occurring after exposure to the reagent. After addition of EDC, a second order kinetic drop in NP emission is observed, which is followed by a second quenching event after some critical time. While it is difficult to know what can cause this type of dynamic, a hypothesis could be made that the initial event is due to multiple EDC molecules associating with the NP amphiphilic polymer coating or perhaps even directly with the trioctylphosphine oxide coated ZnS surface. This also accounts for the clear second order nature of the dynamic as seen in Figure 3. This interaction may result in a loss of polymers coating such that the local (surface NP associated) concentration of polymers falls below the critical micelle concentration (CMC). A drop below the CMC would explain the sudden initiation of the second event, i.e., the mass agglomeration and quenching of CdSe/ZnS NPs as they have lost too many surfactant polymers to remain stable in an aqueous solution. This would also explain the apparent permanent precipitation of NPs caused by EDC as noted previously.

[00048] Amide bond formation with MPEG CD

[00049] The carbodiimide functionalization of amphiphilic carboxylic acid polymer coated NPs with primary amine functional chemical and biological vectors are a desirable method. The amide bond is much more stable within biological systems than esters; further, most proteins have primary amine functionality on their surface. As discussed above, there are serious issues with the use of excess commercially available EDC reagent that limits the ability to create highly multifunctional NP systems. Having established that neutral carbodiimide reagents of the present invention do not cause NP precipitation, it was examined whether the carbodiimide functionality was effective for activating carboxylic acid groups for amide bond formation.

[00050] First, the coupling of 40% octylamine modified poly(acrylic acid) CdSe/ZnS NPs with an amine functional dye represents a model system as the reaction efficiency is easy to calculate using simple UV7VIS spectroscopy; further, the demonstration of energy transfer between properly matched NP fluorescent donor to dye acceptor is a second observable way to attest to the conjugation efficiency. To accomplish this, 540 nm emitting CdSe/ZnS NPs were synthesized, which is an ideal donor for 545 nm absorbing TAMRA cadaverine dye. Generally, 1 mL of a stock NP solution was consistently exposed to a -140 fold molar excess of dye with respect to the nanoparticle; optimization of the method was accomplished by varying the amount of MPEG CD reagent used relative to the dye and varying the amount of time that the coupling was allowed to occur. The absorption spectra before and after removing the unreacted dye were used to calculate reaction yields as discussed in the experimental section. The data for two reagents, MPEG 750 and 350 CD, are shown in Figures 4 and 5.

[00051] Figure 4 depicts the absorption spectra of CdSe/ZnS - TAMRA cadaverine dye before (A) and after (B) dialysis using IOOK MW cutoff filters. Compared to the bare NPs (C), there is clearly retention of dye which must be bound to the NP via carbodiimide coupling. This example represents the highest yield that was achieved with the MPEG 750 CD reagent (23%).

[00052] Figure 5 depicts the absorption spectra of CdSe/ZnS and TAMRA dye before (A) and after (B) dialysis using IOOK MW cutoff filters. This example represents the highest yield that was achieved with the MPEG 350 CD reagent (93%), which was a significant improvement compared to the best results using MPEG 750 CD (Figure 4).

[00053] The first reagent studied was the larger MPEG 750 CD. Initially, it was found that a significant excess of this carbodiimide to dye was necessary to observe an appreciable yield. Generally, using a 100 fold excess and stirring overnight gave -7% yield; the use of more reagent up to 300^χ did not seem to affect the results. Similar efficiency was observed when coupling TAMRA cadaverine to Fe₂O₃ NPs as shown in Figure 13. Next, the efficiency of coupling the dye to CdSe/ZnS with a 100 fold excess of MPEG 750 CD as a function of time was studied, and it was found that stirring for three days gave the highest observed yield (23%). The data from this sample is shown in Figure 4. It was a surprise to see that the reagent was active for so long after exposure to water, which led to a belief that the length of the MPEG moiety was "protecting" the carbodiimide function from water and perhaps from reacting with the NP polymer surface. This led to the conclusion that the polymer functionality of MPEG 750 CD is too long and suppresses amide bond formation. As such, a much smaller reagent with a shorter PEG functionality, MPEG 350 CD, was synthesized and studied.

[00054] Initial results with MPEG 350 CD clearly demonstrated that this was a highly effective reagent for coupling amine functional dyes to amphiphilic polymer coated NPs. First, it was found that the optimum MPEG 350 CD to dye ratio was 230^χ, over twice that observed with the larger MPEG carbodiimide. The efficiencies were substantially improved as well with a 93% coupling yield using a 23Ox excess reagent as evident from the data in Figure 5; in fact, time dependent experiments were not performed as they did not seem necessary. This led to the conclusion that steric hindrances lowered the reaction yields observed with the MPEG 750 CD reagent. Another interesting observation was made at this point - i.e., the dye spectra appeared to red shift with increasing reaction yield. The spectra of the dye in two constructs, with dye to NP ratios of 33:1 (B) and another with 130:1 (A), reveal that the absorption of the dye has been noticeably altered in the shape and absorption maximum as shown in Figure 6.

[00055] Figure 6 depicts the normalized absorption spectra of TAMRA dyes bound to CdSe/ZnS NPs after subtraction of the CdSe/ZnS component. Data are taken from a sample with -33 dyes / NP (B) and -130 dyes / NP (A). Clearly, the higher loading levels have affected the dye's photophysical properties. The 33:1 conjugate was prepared with MPEG 750 CD and the 130:1 loaded sample was prepared with MPEG 350 CD. It can be postulated that perhaps the dye is being encapsulated within the interior of the polymer in the high loading conditions, which can change the absorptive properties of the dye. See Fernandez- Arguelles, M. T.; Yakovlev, A.; Sperling, R. A.; Luccardini, C; Gaillard, S.; Medel, A. S.; Mallet, J. M.; Brochon, J. C; Feltz, A.; Oheim, M.; Parak, W. J. Synthesis and Characterization of Polymer- coated Quantum Dots with Integrated Acceptor Dyes as FRET-based Nanoprobes. Nano Lett. 2007, 7, 2613-2617. It can also be postulated that the dyes begin to associate with one another at very high loading concentrations, which would likely red shift their absorptions.

[00056] The particular CdSe/ZnS NP and dye were chosen such that they may undergo Fluorescent Resonant Energy Transfer (FRET) {see Fόrster, T. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Physik 1948, 437, 55-75) from the NP donor to the TAMRA dye acceptor. While the emission spectra show suppressed NP emission with concomitant enhanced dye emission, it was discovered that energy transfer is clearly visible to the eye as shown in Figure 7. Not only can the presence of the dye be observed from the increase of the pink color as more MPEG 350 CD was used in the synthesis of the coupled construct, the emission from the TAMRA dye becomes dominant as the loading levels of dye were increased as confirmed with fluorometry.

[00057] Figure 7 depicts A) visible light images of CdSe/ZnS NP samples after coupling to amine functional TAMRA dye as a function of the MPEG 350 CD to dye ratio (the color becomes a darker shade of pink in the samples from left to right). B) depicts the emission under UV light excitation of the same samples (the control is green, 1 :1 is light green, 10:1 is yellow, and 50:1 is orange). A significant amount of dye is coupled using -50:1 ratio of carbodiimide reagent to dye as evident from energy transfer from the NP to the emissive dye.

[00058] Next, the synthesis of chemically sensitive functional NPs was investigated by the coupling of a CdS/ZnS NP donor to a pH sensitive amine functional fluorescein dye acceptor. As shown in Figure 8, the system displays a ratiometric response to pH most likely due to the variation of FRET efficiencies due to the change in the local chemical environment, as has been reported earlier. See Snee, P. T.; Somers, R. C; Nair, G.; Zimmer, J. P.; Bawendi, M. G.; Nocera, D. G. A Ratiometric CdSe/ZnS Nanocrystal pH Sensor. J. Am. Chem. Soc. 2006, 128, 13320-13321; and Chen, Y.; Thakar, R.; Snee, P. T. Imparting Nanoparticle Function with Size-Controlled Amphiphilic Polymers. J. Am. Chem. Soc. 2008, 130, 3744-3745. It was found that MPEG CD is a versatile reagent and can be used to derivatize several NP systems to create a variety of functional materials. It was found that using a bi-functional carbodiimide PEG reagent caused NP precipitation, however this was likely due to the activation of carboxylic acid functionalities on separate NPs with the same reagent. Addition of excess base resolubilized the NPs.

[00059] Figure 8 depicts the emission spectra of a CdS/ZnS - fluorescein cadaverine dye conjugate as a function of pH. The inset shows a scheme of the coupled construct. As the pH is increased from 6.0 to 7.0 and then 8.0, the emission peak located at about 490 nm decreased, while the emission peak located at about 535 nm increased.

[00060] While the conjugation of NPs and dyes was used to optimize the procedure for MPEG CD coupling conditions, most research involving NPs revolves around biological imaging. To this end, the synthesis of biologically compatible materials is important and as such two relevant systems were examined. First, poly(ethylene glycol) coating of NPs will decrease non-specific adsorption of proteins in biological systems and may prevent precipitation due to high salt concentrations or changes in pH. See Liu, W.; Howarth, M.; Greytak, A. B.; Zheng, Y.; Nocera, D. G.; Ting, A. Y.; Bawendi, M. G. Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging. J. Am. Chem. Soc. 2008, 130, 1274-1284; Uyeda, H. T.; Medintz, I. L.; Jaiswal, J. K.; Simon, S. M.; Mattoussi, H. Synthesis of Compact Multidentate Ligands to Prepare Stable Hydrophilic Quantum Dot Fluorophores. J. Am. Chem. Soc. 2005, 127, 3870-3878; and Zimmer, J. P.; Kim, S. W.; Ohnishi, S.; Tanaka, E.; Frangioni, J. V.; Bawendi, M. G. Size Series of Small Indium Arsenide -Zinc Selenide Core-shell Nanocrystals and Their Application to In vivo Imaging. J. Am. Chem. Soc. 2006, 128, 2526-2527.

[00061] As a quantity of the amine derivative of poly (ethylene glycol) methyl ether (MW 750) had been synthesized for this study, it was examined whether the carbodiimide functional MPEG 350 CD was effective for PEGylation of amphiphilic PAA polymer encapsulated CdSe/ZnS NPs. Following the optimized procedures for the use of MPEG 350 CD, an attempt was made conjugate as many as -1000 MPEGs per NP. Unfortunately, it is difficult to estimate the reaction yield in this system; however, the presence of MPEG 750 on the surface of the NPs via analysis with GPC was verified as shown in Figure 9.

[00062] Figure 9 depicts gel permeation chromatography trace of aqueous CdSe/ZnS NPs and amine functional MPEG 750 / NP conjugate. The increase in size is indicative of the coating of the NPs with the poly(ethylene glycol) methyl ether. [00063] The PEGylated NPs were found to have an increase in diameter from 13.7 nm for the unfunctionalized blank NPs to 15.2 nm for the PEGylated NPs. Further, the PEGylated NPs were found to be more resistant to precipitation by salts such as calcium chloride. Consequently, the MPEG 350 CD reagent is effective for the coating of NPs with more than just amine functional dyes.

[00064] The synthesis of NP / protein conjugates is important for using nanoparticles to image biological systems. As such, the conjugation of CdSe/ZnS NPs with excess streptavidin under various protein to NP ratios was investigated. During the initial phase of this study, the coupling of aqueous solutions of NPs with low streptavidin to NP ratios (l→5^χ) using MPEG 350 CD was examined. After stirring overnight and subsequent dialysis, it was found that a large amount of material was precipitating. As avoiding NP losses was the purpose of this study and since it has been demonstrated that MPEG 350 CD disclosed herein is safe to use in this regard, it was examined whether the tetrameric nature of streptavidin was at fault - essentially, whether the protein was cross linking NPs together. Analysis with gel permeation chromatography (GPC) confirmed that the majority of materials eluted within the dead volume (>90 nm diameter), which suggested that the NPs were highly cross linked. In the next round of optimization, the streptavidin to NP ratio was increased from 10:1, to 50:1 and 100:1 and repeated the coupling using excess PEG 350 CD. No precipitation observed and analysis with GPC under these conditions revealed that the formation of large aggregates was suppressed at high (100:1) streptavidin to NP loading ratios. As shown in Figure 10, the highest protein loading (B) has the smallest peak in the dead volume, which increased as the loading ratio became smaller. After dialysis and measuring the UV7VIS spectra of the streptavidin and blank NPs, it was determined that ~2 proteins were conjugated to every NP (see Figure 14). While this may seem to reveal a low efficiency, it is unlikely that 100 proteins could be coupled to a single NP; as such, the excess protein simply blocks the formation of (-NP-streptavidin-)_x aggregates.

[00065] Figure 10 depicts gel permeation chromatography traces of aqueous CdSe/ZnS NPs and streptavidin conjugate as a function of the streptavidin to NP ratio used during the synthesis. The data are normalized to the unaggregated peak near 20 nm diameter. Clearly, a large excess of protein is needed to prevent agglomeration as evident by the increase in the dead volume peak as the protein to NP ratio is lowered. [00066] After a method of preventing the precipitation of the NPs during the coupling of the protein was developed, it was examined how to verify the activity of streptavidin on the surface of the nanoparticles via their interaction with biotin. An inexpensive assay was developed by measuring the emission of a solution of functionalized NPs that was passed though cellulose and biotinylated cellulose columns. A control sample was made by diluting the original solution into the same volume as the effluent from the columns and comparing the fluorescence under identical conditions. As shown in Figure 11, there is some loss of emission intensity when streptavidin coupled CdSe/ZnS NPs are run through the cellulose control column, which is likely due to non-specific adsorption to the cellulose. However, when run through an identical column containing biotinylated cellulose, there is no emission from the effluent and a bright orange (CdSe/ZnS) band was observed at the very top of the column. Consequently, the MPEG 350 CD coupling of the protein to the NP appears to not have adversely affected the binding of the protein towards biotin.

[00067] Figure 11 depicts emission of CdSe/ZnS - streptavidin conjugates: this sample was prepared using a 100^χ excess of protein to NP. The control sample was made by diluting the NP - protein constructs to the same volume as the effluent collected. Less emission is observed from NP - protein conjugates after running through a cellulose column, most likely due to nonspecific adsorption; however, no emission is observed if the column contains biotin.

[00068] The derivatization of nanoscopic material systems is an active area of research. Some of the synthetic methods developed to create chemical and biological functional NP systems include pre-reaction of the NP water solubilizing polymers (see Fernandez- Arguelles, M. T.; Yakovlev, A.; Sperling, R. A.; Luccardini, C; Gaillard, S.; Medel, A. S.; Mallet, J. M.; Brochon, J. C; Feltz, A.; Oheim, M.; Parak, W. J. Synthesis and Characterization of Polymer- coated Quantum Dots with Integrated Acceptor Dyes as FRET-based Nanoprobes. Nano Lett. 2007, 7, 2613-2617), water solubilizing NPs using amphiphilic polymer backbones with built in chemical handles {see Chen, Y.; Thakar, R.; Snee, P. T. Imparting Nanoparticle Function with Size-Controlled Amphiphilic Polymers. J. Am. Chem. Soc. 2008, 130, 3744-3745), formation of disulfide bonds (see Medintz, I. L.; Berti, L.; Pons, T.; Grimes, A. F.; English, D. S.; Alessandrini, A.; Facci, P.; Mattoussi, H. A Reactive Peptidic Linker for Self-assembling Hybrid Quantum Dot-DNA Bioconjugates. Nano Lett. 2007, 7, 1741-1748), or by electrostatically conjugating systems through salt bridges (see Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C; Mikulec, F. V.; Bawendi, M. G. Self- assembly of CdSe-ZnS Quantum Dot Bioconjugates Using an Engineered Recombinant Protein. J. Am. Chem. Soc. 2000, 122, 12142-12150). While these methods have proved effective, they generally require a level of synthetic expertise which may be difficult to reproduce for researchers relying on commercially available water soluble NPs. Fixing the problems with precipitation using commercially available carbodiimide coupling reagents is thus a significant step in this regard, especially as carbodiimide coupling of carboxylic acid and primary amine groups to form stable amide bonds is a well established protocol.

[00069] As disclosed herein, it has been shown that NP precipitation and quenching problems associated with the use of the ubiquitous coupling reagent EDC is not due to the carbodiimide functionality; rather, it is due to the net positive charge of the chemical. There are some interesting dynamics observed in the precipitation of NPs caused by EDC, which suggests that the NPs are stripped of their polymer coating causing them to permanently precipitate from solution. Replacement of the N,N-dimethylamine functionality of EDC with poly(ethylene glycol) methyl ether creates a carbodiimide coupling reagent which is highly effective and does not cause NP aqueous suspensions to precipitate. This reagent can create a variety of interesting functional systems, including magnetic and emissive NPs, fluorescent chemical sensors, and biologically tailored CdSe/ZnS nanoparticles, in high yield. Further, the reagent can be synthesized in two steps using commercially available amine functional poly(ethylene glycol) methyl ether.

[00070] Examples [00071] Materials

[00072] Dyes such as fluorescein-5-carboxamide cadaverine and tetramethylrhodamine-5- carboxamide cadaverine were purchased from Anaspec (San Jose, CA); streptavidin was purchased from the same source. Poly-acrylic acid (1800 MW), octylamine, biotin, cellulose, thionyl chloride, sodium azide, triphenylphosphine and general solvents were purchased from Sigma- Aldrich (St. Louis, MO). The water soluble carbodiimide l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Advanced Chemtech (Louisville, Kentucky). Concentrated hydrochloric acid and sodium hydroxide was purchased from Fisher Scientific (Pittsburgh, PA). The majority of chemicals were used as received. [00073] As nanoparticle synthesis has become a general practice among many groups, it is noted that the methods reported in the following can serve as guides for researchers that are not familiar with these procedures: Dabbousi, B. O.; RodriguezViejo, J.; Mikulec, F. V.; Heine, J. R.; Mattoussi, H.; Ober, R.; Jensen, K. F.; Bawendi, M. G. (CdSe)ZnS Core-shell Quantum Dots: Synthesis and Characterization of a Size Series of Highly Luminescent Nanocrystallites. J. Phys. Chem. B 1997, 101, 9463-9475; Fisher, B. R.; Eisler, H. J.; Stott, N. E.; Bawendi, M. G. Emission Intensity Dependence and Single-exponential Behavior in Single Colloidal Quantum Dot Fluorescence Lifetimes. J. Phys. Chem. B 2004, 108, 143-148; Hines, M. A.; Guyot-Sionnest, P. Synthesis and characterization of strongly luminescing ZnS-Capped CdSe nanocrystals. J. Phys. Chem. 1996, 100, 468-471; Peng, Z. A.; Peng, X. G. Formation of High- quality CdTe, CdSe, and CdS Nanocrystals Using CdO as Precursor. J. Am. Chem. Soc. 2001, 123, 183-184; and Jana, N. R.; Chen, Y. F.; Peng, X. G. Size- and Shape-controlled Magnetic (Cr, Mn, Fe, Co, Ni) Oxide Nanocrystals via a Simple and General Approach. Chem. Mater. 2004, 76, 3931-3935.

[00074] Octylamine Modified Polyacrylic Acid

[00075] Use of amphiphilic polymer encapsulation as a method to impart water solubility to hydrophobic NPs has employed a system composed of 40% octylamine modified poly(acrylic acid) (PAA). First, 5.0 grams (70 mmol) commercially available 1800 Molecular Weight dry PAA powder was diluted into -70 mL dry N,N-dimethylformamide (DMF). Next, 5.325 g (28 mmol) EDC was dissolved into the same solution, stirring for 0.5 hours followed by the addition of 4.6 mL (28 mmol) of octylamine. The solution was stirred at room temperature overnight before reduction of the solvent under vacuum. Next, distilled water was added to precipitate the polymer which was subsequently isolated by centrifugation; the excess water was discarded. Next, an aqueous sodium hydroxide solution of ~1.7 g (42.5 mmol) NaOH in 40 mL H₂O was added and the solution was shaken overnight to resolubilize the polymer in water. The crude modified solution was washed by ethyl acetate (3 x 2OmL) which can improve the clarity of the aqueous polymer suspension substantially, especially when using a RAFT synthesized PAA backbone. See Chen, Y.; Thakar, R.; Snee, P. T. Imparting Nanoparticle Function with Size-Controlled Amphiphilic Polymers. J. Am. Chem. Soc. 2008, 130, 3744- 3745; and Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H. Living Free-radical Polymerization by Reversible Addition-fragmentation Chain Transfer: The RAFT Process. Macromol. 1998, 31, 5559-5562. A dilute solution of HCl was added to the aqueous layer until the pH was below 5 to precipitate the polymer again. The solid product was collected by centrifugation and dried under vacuum. The product was slowly and carefully neutralized using a 0.1 M NaOH solution and lyophilized for storage and subsequent use.

[00076] Nanoparticle Water Solubilization

[00077] The nanoparticles used herein were all initially hydrophobic; as such, an amphiphilic polymer coating was used to render them water soluble. First, the NPs in growth solution were precipitated with the addition of a few drops of isopropyl alcohol and excess methanol inside of a pre-weighed glass vial. Materials synthesized in 1-octadecene required more isopropyl alcohol. The flocculate was centrifuged and the supernatant was removed. The precipitation was performed again for some samples by redispersing the NPs in hexanes followed by isopropyl and methyl alcohol addition followed by centrifugation and vacuum drying. Some NPs become soluble in alcohol solution after one precipitation; these samples were dried after the first isolation step. Generally, ~10 mg bare NPs were collected to which 50 mg amphiphilic PAA polymers were added with 5 mL chloroform and two to three drops of methanol. The mixture was sonicated until the polymer solubilized. After being dried under vacuum, the sample was dissolved in basic water (pH 8-10) to form an aqueous solution of NPs, which were often clear. However, some samples of CdSe/ZnS and all iron oxide NPs require additional cleaning through 0.1 μm filters. Dialysis was then performed using IOOK molecular weight cutoff filters from Millipore (Amicon Ultra 15, cat. UFC910024). It is found that using smaller molecular weight cutoff filters results in some uncoupled polymer remaining in solution. The solutions were dialyzed with deionized water (DI) which tends to be somewhat acidic (pH ~6). Another method to water solubilize NPs is to expose the precipitated NCs to excess, amphiphilic caps such as but not limited to mercaptoacetic acid, 11- mercaptoundecanoic acid, di-hydrolipoic acid or cystine. Subsequent de-protonation renders the cap exchanged NCs hydrophilic. The invention described herein will also functionalize such NPs which are also known to precipitate in the presence of EDC.

[00078] Synthesis of Polyethylene glycol) methyl ether amine (MPEG 350 amine)

[00079] The synthesis of MPEG 350 amine is the first step in the synthesis of the carbodiimide functional material. Unfortunately, the purchase of amine functional poly(ethylene glycol) methyl ether is highly cost prohibitive; as such, a reported procedure to synthesize the material was used as disclosed in Liu, W.; Howarth, M.; Greytak, A. B.; Zheng, Y.; Nocera, D. G.; Ting, A. Y.; Bawendi, M. G. Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging. J. Am. Chem. Soc. 2008, 130, 1274-1284, although they are repeated herein for completeness. Neat poly(ethylene glycol) methyl ether, average MW 350 g/mol, (10 g, 28.6 mmol) was degassed and stirred at 80 ⁰C for 1 hour to remove traces of water. The reaction solution was purged with N₂ and cooled in an ice bath before 3.0 ml (41 mmol) thionyl chloride was added drop wise. The solution was warmed to room temperature and stirred for 2 hours to convert the hydroxide group to chloride. Thionyl chloride was removed under vacuum; the pressure of the vacuum system was monitored to assure removal of the excess reagent. The product was then diluted with 10.0 mL DMF and the solvent was removed again under reduced pressure at room temperature to aid in the removal of residual trace amounts of thionyl chloride. Addition and removal of DMF was repeated three times total which took ~4 days. Next, 75 mL DMF together with 2.83 g (43.5 mmol) sodium azide was added and stirred overnight at 85 ⁰C with aluminum foil coating the flask to protect the material from light. (Warning: sodium azide is a highly toxic contact explosive). The DMF was reduced under vacuum before 60 mL dichloromethane (DCM) was added. A solid precipitate was removed with a glass fritted filter or by centrifugation. The DCM was then removed with vacuum to yield the intermediate azide. The sample was dissolved in 90 mL tetrahydrofuran (THF), and 8.26 g (31.5 mmol) triphenylphosphine was added and stirred at room temperature for 4 hours before 1.2 mL water was added and continued stirring for another day. After THF was removed, 200 mL water was added. The resulting solution was washed with excess toluene twice and was subsequently dried and removed under vacuum to yield a yellow oil. Samples were analyzed using NMR and mass spectrometry, as shown in Figures 15 and 16 (NMR) and Figures 17 and 18 (mass spectrometry). Two other amine functional reagents were synthesized, including a methyl ether MPEG with a molecular weight of 750 and a bifunctional amine starting from poly(ethylene glycol) with a molecular weight of 400. These materials are stored at 4 ⁰C in an airtight container. Despite these precautions, hydrolysis of the product is observed after several months of storage which is not unexpected given the reactivity of the carbodiimide functionality.

[00080] Carbodiimide functional polyethylene glycol) methyl ether (MPEG 350 CD)

[00081] Two forms of neutral carbodiimide reagents starting with amine functional poly(ethylene glycol) methyl ether with molecular weights of 350 and 750 were synthesized as outlined in Scheme 1. Another bifunctional carbodiimide was created starting with bis-amine poly(ethylene glycol). The methods reported herein are based in part according to the synthesis of EDC first reported in Sheehan, J. C; Cruickshank, P. A.; Boshart, G. A Convenient Synthesis of Water - Soluble Carbodiimides. J. Org. Chem. 1961, 26, 2525-2528. Approximately 3 g (8.6 mmol) of amine functionalized MPEG 350 was dried under vacuum in a septum sealed round bottom flask. Next, the sample was dissolved into 5 mL anhydrous DMF after which an excess of ethyl isothiocyanate (~1.1 mL, 12.6 mmol) was dripped into the MPEG amine solution slowly with a syringe while stirring. The reaction was allowed to continue overnight. The next day the DMF was removed under vacuum after which a suspension of 4.65 g (21.5 mmol) of HgO (yellow) in 10 mL dichloromethane was added. After stirring again overnight, an additional 1.0 g of HgO was added and the solution was again allowed to stir overnight. The yellow mercuric oxide turns green due to HgS formation after which the majority of the byproduct was removed with centrifugation. Some samples appeared to react slowly with the HgO; in these cases, the samples were filtered and recharged with an additional 1.0 g of HgO in DCM and stirred overnight. This process was repeated until no more dark green byproducts were observed. To purify the materials, the MPEG-CD can be dissolved into cold ether which causes the HgS to precipitate. In a few cases, a residual dark green color was observed which was removed by dissolving the product in water and removing a green insoluble material with centrifugation and filtration. The excess water was then removed immediately under vacuum at room temperature to prevent the hydrolysis of the carbodiimide functionality. The samples may also be purified by flash chromatography over alumina using methanol as the mobile phase. Reaction yields are typically on the order of 90%. The known method of dehydration of a urea intermediate (formed from the reaction of poly(ethylene glycol) methyl ether amine with ethyl isocyanate, for example) with an excess of p-toluenesulfonic acid to form the carbodiimide also reported by Sheehan et al. is effective in this regard. [00082]

A ethyl

*~ isothiocyanate

S m MH- C-NH^/X

HgO ,,-._s , _{s r} JM^ h 3 V. xro- m

B s Ns V' i NKTC m

[00083] Scheme 1. A: Synthetic method employed to synthesize carbodiimide functional poly(ethylene glycol) methyl ether (MPEG CD). Inset B: the structure of the bi- functional PEG carbodiimide.

[00084] Biotinylated Cellulose

[00085] To confirm the activity of the protein in streptavidin coated NPs (see below), these systems were examined by chromatography through a biotinylated cellulose column. The synthesis of biotinylated cellulose was performed according to McCormick, D. B. Specific Purification of Avidin by Column Chromatography on Biotin-Cellulose. Anal. Biochem. 1965, 13, 194-198. This material was analyzed using NMR spectroscopy, see Figure 19.

[00086] Nanoparticle Functionalization

[00087] Several functional materials were synthesized, and pertinent examples presented herein. Most of the research on PEG CD was conducted to optimize the reaction yield between amphiphilic polymer coated NPs in water and amine functional materials. For convenience, CdSe/ZnS - tetramethylrhodamine-5-carboxamide (TAMRA) cadaverine conjugates were synthesized, as the optical characterization of NP - dye coupling is highly robust. The coupling of other dyes, amine functional MPEG and streptavidin, was also investigated.

[00088] During the course of work on CdSe/ZnS NP - TAMRA dye coupling, an approach was developed that has an approximate 93% reaction yield, which is reported here. A 1 mL quantity of aqueous solution of 1.38xlO^~6 M 540 nm emitting CdSe/ZnS suspensions in slightly acidic (pH ~6) DI water was transferred to a clean glass vial. Next, 11.4 mg of MPEG 350 CD was added to the sample which was stirred for 0.5 hours. Next, 20 μL of a 1 mL DMF solution containing 5 mg of tetramethylrhodamine-5-carboxamide cadaverine (194 nmoles, approximately 140^χ excess relative to the NPs) was added and the solution was basified by adding 3 rnL of a pH 8 NaOH solution- buffer was not used as the purpose of this study was to examine coupling reactions under conditions with few electrolytes. The solutions were then stirred overnight and were subsequently purified using dialysis centrifuge filters. The amount of MPEG 350 CD reagent was approximately 230^χ excess relative to the dye. The amount and identity of PEG CD reagents to dye ratio was varied and optimized over the course of this work, with this method resulting in the highest yields.

[00089] Once the procedure for conjugating amine functional dyes to NPs was optimized, the coupling to proteins was examined. To 0.768 mL of a 6.3 x 10^"7 M solution of 580 nm emitting CdSe/ZnS NPs was added 0.1891 g of a 0.048 M solution of MPEG 350 CD in DI water. After stirring 0.5 hours, 5 mg of streptavidin was diluted into 0.8 mL of a pH 8 NaOH solution from which 0.4 mL was added and stirred overnight- excess material was removed with a IOOK MW centrifuge filter the next day. Other loading ratios of streptavidin to NPs were investigated as discussed below. The targeted ratios in this experiment were 100^χ excess of streptavidin proteins per NP, with a 200^χ excess of MPEG 350 CD to the moles of streptavidin.

[00090] Optical Characterization: Reaction Yields

[00091] Absorption spectroscopy was used to determine the reaction yields for creating CdSe/ZnS NP / dye conjugates. After stirring a solution of NPs, PEG-CD reagents, and TAMRA-cadaverine dye overnight, the absorption spectrum of a known portion of the solution was measured using a Cary 400 UV7VIS. Next, excess dye was removed using IOOK MW cutoff centrifuge dialysis filters from Millipore until no dye was observed from the liquid effusate. The UV/VIS absorption spectrum was then measured from the purified sample. As the concentrations of NPs in the first and second measurements were recorded, the absorption spectra from the solution before and after dialysis were normalized by their NP concentrations and plotted together. The NP blank spectrum (also normalized by concentration) is then directly subtracted from each spectrum leaving dye only absorption features. The yield of reaction is then calculated as the integrated dye absorption after dialysis divided by the integrated absorption before dialysis. [00092] Optical Characterization: EDC Precipitation

[00093] To demonstrate the effect of excess EDC on NPs, a blank 6.IxIO^"8 M sample (3 mL, 0.18 nmol) of water soluble CdSe/ZnS NPs were characterized with Dynamic Light Scattering (DLS) and Gel Permeation Chromatography (GPC). Next, the sample was incubated with 10 mg (52 μmol) of EDC overnight; the next day the sample sizes were re-characterized with DLS as the aggregates that formed overnight were too large to be characterized with GPC. DLS measurements were also made on MPEG CD 350 exposed CdSe/ZnS samples which revealed no agglomerization of the NPs. The relative emission intensities were also quantified. The absorption and emission of these samples were also characterized. This work demonstrated that EDC causes precipitation that can be characterized via fluorescence quenching.

[00094] First, 3 mL of a 1.5x 10^"7 M solution (0.45 nmol) of CdSe/ZnS NPs was placed in a vial inside the optical cavity of a Fluorolog-3 (Horiba Jobin-Yvon) while continuously stirring. The emission of the NPs was monitored at the fluorescence maximum when a 1 mL solution of 0.048 M (48 μmol) MPEG 350 CD in de-ionized water was swiftly injected. This procedure was repeated with fresh NP solutions being exposed to 1 mL of 0.0047 M, 0.014 M, and 0.024 M (4.7, 14, and 24 μmol) EDC solutions in de-ionized water. The emission of the NPs was monitored in kinetic mode for ~90 minutes with a 1 second integration time. The fluorescence data was corrected for the dilution of the NPs after the injection by dividing the signal by the dilution factor. Accurate dilution factors were calculated from the mass of the sample before and after the injection of the carbodiimide solutions.

[00095] Biological Conjugate Characterization

[00096] To verify the activity of the streptavidin conjugates, the retention of these materials on a biotinylated cellulose column was examined. Approximately 2 grams of cellulose and biotinylated cellulose was mixed with pH 8 phosphate buffered water and loaded into a small pipette plugged with glass wool. Phosphate buffer was passed through the column until the packing material settled. Next, blank and streptavidin coupled CdSe/ZnS NPs of known emission intensity were injected into the column from which several milliliters of effusate were collected. Next, the emission of the effluent was measured and normalized for the dilution that occurs as a result of running through the column. All streptavidin coupled samples were run through the biotinylated column; the 100:1 streptavidin to NP sample was also analyzed through a pure cellulose column to measure non-specific adsorption losses. The NP -protein conjugates were also studied using gel permeation chromatography in a GE Healthcare Atka Prime FPLC using pH 8 phosphate buffered water through a Superose 6 10/300 GL column.

[00097] FIG. 12 is a picture of CdSe/ZnS NPs precipitated by EDC. The precipitation is shown by the holiday "snowdome"- like appearance.

[00098] FIG. 13 is a graph of absorbance versus wavelength, and depicts absorption spectra of blank and tetramethylrhodamine-5-carboxamide cadaverine functionalized Fe₂O₃ nanoparticles. The inset shows the emission of the sample under irradiation with a UV light source; the sample does not appear very bright under these conditions as the dye has very little absorption at UV wavelengths.

[00099] FIG. 14 is a graph of absorbance versus wavelength, and depicts absorption spectra of blank and streptavidin functionalized CdSe/ZnS nanoparticles, showing residual absorption at 280 nm that is likely due to streptavidin, the strength of which suggests that there are about 2 proteins per NP.

[000100] FIG. 15 is a ¹³C NMR spectrum of carbodiimide functional poly(ethylene glycol) methyl ether 350, 0→80 ppm. Four distinct species are observed, which corresponds to the unique carbons at both ends of the functional polymer.

[000101] FIG. 16 is a ¹³C NMR spectrum of carbodiimide functional poly(ethylene glycol) methyl ether 350, 140→150 ppm. Based on data for other carbodiimide reagents, the peak a 141.0 ppm is the central carbon in the carbodiimide functionality.

[000102] FIG. 17 is mass spectrum of amine functional poly(ethylene glycol) methyl ether 750. Masses are for the positive (polymer + H⁺) ion fragments. The difference in the peaks is 44.0 amu, corresponding to OCH₂CH₂ polymer subunits. The variety of peaks is likely the result of the polydispersity in the original sample.

[000103] FIG. 18 is a mass spectrum of carbodiimide functionalized poly(ethylene glycol) methyl ether 350. Masses are for the positive (polymer + Na⁺) ion fragments. One series of the smaller fragments results from the hydro lyzed product (such as mass peak 609.3 amu and ± amu fragments) may be due to the starting material.

[000104] FIG. 19 is the proton NMR spectra of cellulose and biotinylated cellulose. The resonances near 3.3 ppm can be associated with biotin, however, other biotin around 2.0 ppm to 1.0 ppm resonances are missing. This may be due to line broadening caused by the fact that these biotin protons are near to the ester that conjugates the biotin to the cellulose which does not dissolve well in neat DMSO.

[000105] Although systems and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable systems and methods are described above. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed above are illustrative only and not intended to be limiting.

[000106] Any improvement may be made in part or all of the method steps and systems components. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.

Claims

What is claimed is:

1. The compound poly(ethylene glycol) methyl ether carbodiimide.

2. The compound of claim 1, wherein the poly(ethylene glycol) methyl ether carbodiimide comprises a poly portion having a molecular weight of about 350 to about 750.

3. The compound of claim 1, wherein the poly(ethylene glycol) methyl ether carbodiimide comprises a poly portion having a molecular weight of about 350.

4. A composition comprising a nanoparticle and poly(ethylene glycol) methyl ether carbodiimide.

5. The composition of claim 4, wherein the poly(ethylene glycol) methyl ether carbodiimide comprises a poly portion having a molecular weight of about 350 to about 750.

6. The composition of claim 4, wherein the poly(ethylene glycol) methyl ether carbodiimide comprises a poly portion having a molecular weight of about 350.

7. The composition of claim 4, wherein the nanoparticle is coated with an amphiphilic polymer or cap exchanged with an amphiphilic molecule.

8. The composition of claim 7, wherein the amphiphlic polymer or cap comprises carboxylic acid.

9. A method for making the compound poly(ethylene glycol) methyl ether carbodiimide comprising: exposing amine functional poly(ethylene glycol) methyl ether to ethyl isothiocyanate, followed by exposing to HgO.

10. The method of claim 9, wherein the amine functional poly(ethylene glycol) methyl ether comprises a poly portion having a molecular weight of about 350 to about 750.

11. The method of claim 9, wherein the amine functional poly(ethylene glycol) methyl ether comprises a poly portion having a molecular weight of about 350.

12. A method for making a water-soluble nanoparticle comprising: exposing amine functional poly(ethylene glycol) methyl ether to ethyl isothiocyanate, followed by exposing to HgO to form a poly(ethylene glycol) methyl ether carbodiimide, and combining the poly(ethylene glycol) methyl ether carbodiimide with a nanoparticle.

13. The method of claim 12, wherein the poly(ethylene glycol) methyl ether carbodiimide comprises a poly portion having a molecular weight of about 350 to about 750.

14. The method of claim 12, wherein the poly(ethylene glycol) methyl ether carbodiimide comprises a poly portion having a molecular weight of about 350.

15. The method of claim 12, wherein the nanoparticle comprises an amphiphilic polymer coating prior to combining the poly(ethylene glycol) methyl ether carbodiimide with the nanoparticle.

16. The method of claim 15, wherein the amphiphlic polymer or cap comprises carboxylic acid.

17. A method for making the compound poly(ethylene glycol) methyl ether carbodiimide comprising: exposing amine functional poly(ethylene glycol) methyl ether to ethyl isothiocyanate, followed by exposing to p-toluenesulfonyl chloride in the presence of triethylamine.

18. A method for making a water-soluble nanoparticle comprising: exposing amine functional poly(ethylene glycol) methyl ether to ethyl isothiocyanate, followed by exposing to p-toluenesulfonyl chloride in the presence of triethylamine to form a poly(ethylene glycol) methyl ether carbodiimide, and combining the poly(ethylene glycol) methyl ether carbodiimide with a nanoparticle.

19. A semiconductor nanoparticle system comprising a semiconductor nanoparticle and poly(ethylene glycol) methyl ether carbodiimide.

20. The semiconductor nanoparticle system of claim 19, wherein the poly(ethylene glycol) methyl ether carbodiimide comprises a poly portion having a molecular weight of about 350 to about 750.

21. The semiconductor nanoparticle system of claim 19, wherein the poly(ethylene glycol) methyl ether carbodiimide comprises a poly portion having a molecular weight of about 350.

22. The semiconductor nanoparticle system of claim 19, wherein the nanoparticle is coated with an amphiphilic polymer or cap.

23. The semiconductor nanoparticle system of claim 22, wherein the amphiphlic polymer or cap comprises carboxylic acid.