(213f) Development of Novel Clinical Diagnostic Tools Using Zinc Selenide Quantum Dots as Fluorescent Labels

Fluorescent labeling of biological molecules is a technique used widely for analytical purposes in biotechnology and bioengineering. It typically involves the use of an organic dye linked to a moiety that selectively bonds a particular biological molecule, allowing the detection of the latter by the fluorescence of the dye.

Semiconductor nanocrystals or quantum dots have emerged as a new class of fluorescent markers with distinct advantages over the traditional organic dyes [1-5]. Their attractive properties include a narrow, symmetric, and strong emission that is size-tunable, continuous excitation by any wavelength smaller than the emission wavelength, resistance to photobleaching, as well as excellent optical and chemical stability that allows their use in lengthy experiments, both in vitro and in vivo. The ability to synthesize different populations of quantum dots with narrow emission spectra permits multiplexing, a property that is very important for simultaneous detection of several analytes, that would be very tedious and expensive if done sequentially.

The focus of this work is the development of new strategies for functionalizing the surface of II-VI nanocrystals and their use in biological sensing and DNA analysis. Highly luminescent ZnSe quantum dots have been synthesized by modifying a reported liquid-phase technique that utilizes a hot coordinating solvent in which the nanocrystals are grown by injection of suitable precursors [6]. The synthesis of ZnSe quantum dots is carried out in a stirred batch reactor containing liquid hexadecylamine at 260 degrees C. The precursors are diethylzinc diluted in heptane and selenium powder dispersed in trioctylphosphine. The mixture of reactants is injected into the batch reactor and the time of reaction is used to control the size and emission wavelength of the quantum dots. Capping of the ZnSe quantum dots with a ZnS layer to obtain a core-shell structure was found to increase their quantum yield and, as a result, their luminescence intensity, without significantly affecting their emission wavelength.

The formation of water-dispersible fluorescent quantum dots was accomplished by using surface ligand exchange reactions with mercapto-carboxylic acid. The resulting ZnSe quantum dots are stable in aqueous solutions and luminescent over a period of several days, thus suitable for biological sensing applications.

Conjugation of water-dispersible ZnSe and (ZnSe)ZnS core-shell quantum dots with oligonucleotides was found to increase their fluorescence emission intensity about 5 times. Quantum dots conjugated with longer DNA strands exhibited stronger fluorescence emission intensity than the ones conjugated with shorter strands, up to a limit of about 50 bases. At the same time, the stability of quantum dots in water was improved. Hybridization kinetics of water-dispersible quantum dots functionalized with complementary short oligonucleotides were also investigated. Ina nother series of experiments DNA hybridization was studied using quantum dots functionalized with oligonucleotides and free oligonucleotides in solution. Upon hybridization, a significant increase (up to 3 times) in the fluorescence intensity of the quantum dots was detected accompanied by a measurable red shift (up to 6nm) in the wavelenght of the fluorescence peak. Applications of these phenomena in the development of DNA detection strategies will be discussed.

Conjugation of water-dispersible ZnSe and (ZnSe)ZnS core-shell quantum dots with bovine serum albumin (BSA) was found to increase their fluorescence emission intensity and to dramatically imrpove their fluorescence lifetime in an aqueous environment (up to several months).

Ongoing experiments in our laboratory aim to develop multiplexed assays for the simultaneous detection of multiple DNA probes functionalized with quantum dots of different sizes and emitting at different wavelengths. Substitution of fluorescent proteins by quantum dot labels in ELISA-type immunoassays is also under investigation aiming to improve both the sensitivity and range of detection of such assays. Successful attainment of these goals will have important implications for the use of quantum dots in high-throughput clinical diagnostic applications, such as real time PCR, DNA microarrays, and immunodiagnostics. We will discuss advances in these efforts using fluorescent ZnSe quantum dots.

References

1. M. Bruchez, M. Moronne, P. Gin, S. Weiss and A.P. Alivisatos, Science 281, 2013 (1998).

2. W.C.W. Chan and S. Nie, Science 281, 2016 (1998).

3. M.-Y. Han, X. Gao, J. Z. Su, and S. Nie, Nature Biotechnology 19, 631 (2001).

4. W. C. W. Chan, D. J. Maxwell, X. Gao, R. E. Bailey, M. Han and S. Nie, Current Opinion in Biotechnology, 13, 40 (2002).

5. X. Gao, L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, L. Chung, and S. Nie, Current Opinion in Biotechnology, 16, 63, 2004.

6. M. Hines and P. Guyot-Sionnest, J. Phys. Chem. B, 102(19), 3655 (1998).