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. Typical organic fluorescent dyes have narrow excitation spectra and broad emission spectra that exhibit red tailing.

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 biological detection and DNA analysis schemes by functionalizing the surface of ZnSe-based quantum dots. ZnSe emits in the blue part of the visible spectrum and QDots of this material exhibit size-tunable emission in the UV, violet and blue parts of the spectrum (at wavelengths between 360 and 460nm).

ZnSe quantum dots were synthesized by using two techniques:

(1) Injection of precursors in a hot coordinating solvent [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.

(2) Synthesis using a microemulsion as template [7-9]. The synthesis was carried out by dissolving zinc acetate in the aqueous dispersed phase of a microemulsion consisting of water as the dispersed phase and p-xylene as the continuous phase with a tri-block copolymer being the surfactant. Hydorgen selenide gas was bubbled through the microemulsion and converted the zinc acetate to ZnSe. By adding acetate salts of Mn and Cu to the aqueous phase we synthesized (Zn,Mn)Se and (Zn,Cu)Se quantum dots that emit in the orange and green parts of the spectrum, respectively.

The formation of water-dispersible fluorescent quantum dots was accomplished by using surface ligand exchange reactions with mercapto-carboxylic acids. 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-based quantum dots with oligonucleotides was found to increase their fluorescence emission intensity up to fivefold. 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. 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. In another series of experiments DNA hybridization was studied using quantum dots functionalized with oligonucleotides and complementary free oligonucleotides in solution. An increase in the emission intensity of the quantum dots was again observed upon hybridization. A series of experiments has also been performed using free oligonucleotides that are not exactly matched to the ones atatched to the quantum dots. A dependence of the fluorescence intensity on the length and location of the hybridized part was observed. Ongoing experiments in our laboratory are focusing on the development of multiplexed assays for the simultaneous detection of multiple DNA probes functionalized with quantum dots of different sizes and composition that emit at different wavelengths. Applications of these phenomena in the development of DNA detection strategies will be discussed.

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

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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.

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9. G.N. Karanikolos, et al., Nanotechnology, 17, 3121-3128 (2006)

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