FLUORESCENCE RESONANCE ENERGY TRANSFER BETWEEN DYES AND WATER-SOLUBLE QUANTUM DOTS WITH AVIDIN AS A BRIDGE

International Journal of Nanoscience Vol. 3, No. 3 (2004) 1 8 c World Scientific Publishing Company FLUORESCENCE RESONANCE ENERGY TRANSFER BETWEEN DYES AND WATER-SOLUBLE QUANTUM DOTS WITH AVIDIN AS A BRIDGE QI-DAN CHEN,, ZHANG-BI LIN,, XING-GUANG SU,, HAO ZHANG, XIAO-HONG HE, BAI YANG and QIN-HAN JIN Institute for Micro Analytical Instrumentation, College of Chemistry Jilin University, Changchun 130023, P. R. China Key Lab for Supramolecular Structure and Materials, College of Chemistry Jilin University, Changchun 130023, P. R. China Department of Molecular Biology, College of Life Science Jilin University, Changchun 130023, P. R. China suxg@mail.jlu.edu.cn Received 16 December 2003 Revised 11 February 2004 3-Mercaptopropyl acid-capped quantum dots (QDs) synthesized in aqueous solution were coupled to avidin-sulforhodamine, also named avidin-texas red (ATR), via electrostatic attraction. An intensity reduction in the fluorescence emission spectrum of QDs and an enhanced fluorescence intensity of the dye were observed on account of fluorescence resonance energy transfer from the QD donors to the dye acceptors. In addition, the fluorescence characteristics of the QD-ATR conjugates were strongly-related to the quantity of ATR, ph value and ionic strength. Keywords: Quantum dots; avidin; organic dyes; fluorescence resonance energy transfer. 1. Introduction Fluorescence resonance energy transfer (FRET) occurs when the electronic excitation energy of a donor chromophore is transferred to an acceptor molecule nearby via a through-space dipole dipole interaction between the donor acceptor pair. 1,2 The FRET process is more efficient when there is an appreciable overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. The strong distance-dependence of the FRET efficiency has been widely-exploited in studying the structure and dynamics of proteins and nucleic acids, in the detection and visualization of intermolecular association, and in the development of intermolecular binding assays. 3 Compared with conventional chemical analysis, FRET-based analytical method has higher sensitivity and more simplicity in These authors contribute equally to this article. Corresponding author. 1

2 Q.-D. Chen et al. detection of ligand-receptor binding by observing merely the enhanced fluorescence of the acceptor. FRET studies involve commonly pairs of organic dye molecules as the donor acceptor complexes. However, in such circumstances, the narrow absorption spectra and the broad emission spectra with long red tails make it difficult to avoid the overlap between the donor and acceptor emission spectra. Some recent reports showed that semiconductor QDs can substitute the dye molecules and have the potential to overcome some of the disadvantages of organic dyes in FRET processes. 3 10 Semiconductor QDs are luminescent inorganic fluorophores currently being widely-used as luminescent biological probes because of their unique size-dependent optical and electronic properties such as having a broad, continuous excitation spectrum, a narrow size-tunable, symmetric emission spectrum, the excellent photochemical stability and the high fluorescence quantum yield. 11 17 The avidin, a glycoprotein found in avian egg white, is a highly-positivelycharged homotetramer (isoelectric point, 10 10.5) with a molecular mass of 67 000 68 000 daltons. 18,19 In this work, an avidin is used as a bridge that binds the QDs and organic dyes sulforhodamine to form donor acceptor complexes. The FRET process between QDs and dye molecules is observed by the changes of their spectra. Furthermore, the influences of the quantity of ATR, ph value and ionic strength on the fluorescence characteristics of the QD-ATR complexes are studied in detail. 2. Experimental 2.1. Apparatus and reagents Fluorescence experiments were performed on a Shimadzu RF-5301 PC spectrofluorophotometer. UV-vis absorption spectra were obtained using a Shimadzu 3100 UV-vis-near-IR recording spectrophotometer. In either experiment a 1 cm quartz cuvette was used to measure the absorption or fluorescence spectrum. All optical measurements were carried out at room temperature under ambient conditions. The disk gel electrophoretic apparatus was provided by Beijing Liuyi Instrument Factory. Analytical reagent grade chemicals and doubly-distilled water (> 18 MΩ cm) were used for preparing all aqueous solutions. 3-Mercaptopropyl acid (MPA) (99+%), tellurium powder ( 200 mesh, 99.8%), CdCl 2 (99+%), NaBH 4 (99%) and coomassie brilliant blue R-250 were purchased from Aldrich Chemical Co. Acrylamide, N,N -methylene-bis-acrylamide, N,N,N N -tetramethyl-ethylenediamine, ammonium persulfate and lyophilized ATR were obtained from Sigma Chemical Co. The ATR powder was dissolved in a 2 mmol/l phosphate buffered saline solution (PBS, ph 7.3) to obtain 10 mg/ml solution and stored at 20 C, dilute only prior to immediate use. Ampholine ph 3 10 was purchased from Pharmacia Fine Chemicals (Sweden).

Fluorescence Resonance Energy Transfer 3 2.2. Preparation of water-soluble CdTe QDs Water-soluble CdTe quantum dots capped with MPA were prepared according to the procedure reported in literature. 20,21 In brief, in the first step, sodium hydrogen telluride (NaHTe) was produced in an aqueous solution by reaction of sodium borohydride (NaBH 4 ) with tellurium powder at a molar ratio of 2:1. In the second step, freshly-prepared oxygen-free NaHTe solution was added to nitrogen-saturated 1.25 10 3 mol/l CdCl 2 aqueous solution at ph 11.4 in the presence of MPA as a stabilizing agent. The molar ratio of Cd 2+ :MPA:HTe was fixed at 1:2.4:0.5. The resulting mixture was then subjected to refluxing to control the size of the CdTe nanocrystals. A luminescence quantum yield of 25% was measured for the CdTe nanoparticles at room temperature by comparing with the fluorescence emission of Rhodamine 6G. 22 QDs with emission maximum at 566 nm (3.2 nm in size) were used throughout the study. 2.3. Preparation of QD-ATR conjugates CdTe-ATR bioconjugates were prepared by mixing the ATR and QDs (3.2 nm, 4.2 10 5 mol/l, referring to Cd 2+ ) in 2 ml of 2 mmol/l PBS buffer solution (ph 7.3) and then incubated for 5 min at room temperature. The resulting solution contained stable QD-ATR conjugates without obvious aggregates was ready for assay. 2.4. Isoelectric focusing of ATR The isoelectric point (pi) of ATR was obtained by polyacrylamide disk gel electrophoresis following the method described in reference, utilizing 7% polyacrylamide containing ampholine (ph 3.5 10). 23 By using the ph gradient curve of blank gel as a reference, the pi of ATR was determined to be 9.5. 3. Results and Discussion 3.1. Spectral analysis Figure 1 shows the absorption and emission spectra obtained from pure CdTe QDs and ATR dispersed in PBS buffer solution, respectively. The right QD size was chosen so as to maximize the spectral overlap of the donor acceptor emission and absorption spectra while still maintaining good spectral resolution of the donor and acceptor emission. The emission maximum of the QDs is at 566 nm, while that of the ATR is at 611 nm. The fluorescence spectra change obviously when QDs conjugate with sulforhodamine, with avidin as the bridge, as shown in Fig. 2. The fluorescence intensity of QDs gradually decreases, meanwhile that of the ATR enhances. This finding indicates that a FRET process occurs due to a specific electrostatic interaction between negatively-charged CdTe QDs and positively-charged ATR.

4 Q.-D. Chen et al. 1.4 1.2 Relative intensity 1.0 0.8 0.6 0.4 0.2 0.0 450 500 550 600 650 700 Wavelength (nm) Fig. 1. The normalized absorption and emission spectra obtained from pure solutions of CdTe QDs ( ) and ATR (- - -). Solutions were prepared in 2 mmol/l PBS buffer (ph 7.32). For more detailed study, the peak ratio of wavelength at 611 nm to 566 nm is plotted as a function of the ATR quantity (Fig. 3). The sharp rise in the line confirms ATR fluorescence enhances with increased ATR quantity. However, when ATR is of sufficient excess (over 18 µg), the line levels off. Although QD-ATR FRET is clearly responsible for the enhanced ATR fluorescence in this study, nevertheless the largest fraction of static quenching not resulting in enhanced ATR fluorescence occurs upon addition of 18 µg ATR. It is apparent that other types of energy transfer mechanisms, in addition to FRET, need to be considered as contributing factors in the quenching of the QD emission. In order to investigate this in detail, a series of solutions with different quantity of unlabeled avidin and the same concentration of QDs as above were analyzed by fluorescence spectroscopy. A phenomenon was observed: with the increase of avidin quantity, the intensity of the QD emission decreased, when the avidin quantity exceeded 18 µg, the intensity of the QD emission did not decrease at all, which indicates that the presence of Texas red is crucial for this type of energy transfer to occur. Previous studies have shown that electron and /or hole acceptor molecules can quench the QD emission in a similar manner. 24,25 Thus, we speculate that nonspecific interactions between the avidin molecules and the QD surfaces, such as QD to avidin electron transfer may be responsible for most of the initial quenching displayed in Fig. 2 and most of the QD surface sites available for nonspecific interaction with avidin are saturated at 18 µg avidin, which would explain why subsequent addition of ATR results mainly in QD-ATR FRET.

Fluorescence Resonance Energy Transfer 5 800 700 A a Fluorescence intensity 600 500 400 300 200 g 100 0 450 500 550 600 650 700 Wavelength (nm) (a) Fluorescence intensity 70 60 50 40 30 20 B f k 10 0 450 500 550 600 650 700 Wavelength (nm) (b) Fig. 2. (A) Fluorescence emission spectra from solutions containing QDs with (a) 0 µg, (b) 3 µg, (c) 6 µg, (d) 9 µg, (e) 12 µg, (f) 15 µg, and (g) 18 µg ATR solutions. To see the change of the spectra clearly, (B) shows the zoomed-in plot. Quantity of ATR: (f) 15 µg, (g) 18 µg, (h) 21 µg, (i) 24 µg, and (j) 27 µg. The lower solid curve (k) is from a free ATR control. All solutions were prepared in 2 mmol/l PBS buffer. An excitation wavelength of 400 nm was used for all samples.

6 Q.-D. Chen et al. 1.8 1.6 611 nm / 566 nm peak rati 1.4 1.2 1.0 0.8 0.6 0.4 0.2-3 0 3 6 9 12 15 18 21 24 27 30 Increased quantity of avidin-texas red (µg) Fig. 3. The peak ratio of wavelength at 611 nm to 566 nm as a function of ATR quantity shows the enhanced fluorescence intensities of the ATR and the decline of that of QDs. 3.2. Effect of the buffer ph When the ph value of the PBS buffer solution was adjusted to a value (such as 11) higher than the isoelectric point of ATR (pi = 9.5), the fluorescence spectrum of the CdTe-ATR solution was shown to be the same as that of free CdTe QDs. The reason for this phenomenon is that the ATR displays negative when ph value of the solution is 11, which inhibits ATR from binding to CdTe QDs via electrostatic attraction. This result is consistent with the previous findings that the interaction between proteins and QDs is charge charge electrostatic attraction. 26 3.3. Effect of the ionic strength It is well-known that the conjugation via electrostatic interaction is not stable enough and can be easily affected by environmental conditions, not only ph value but also ionic strength. In the above-mentioned studies, the analysis was carried out in a low concentration PBS buffer solution (2 mmol/l) with low ionic strength. To examine the influence of higher ionic strength on the interaction between QDs and ATR, the fluorescence spectra of free CdTe and CdTe-ATR solution are recorded as a function of increased NaCl concentration (Fig. 4). It is shown that the peak ratio of wavelength at 611 nm to 566 nm of QD-ATR decreases, whereas that of free QDs shows no change upon salt addition, implying that the ionic strength has a great effect on the binding between QDs and ATR system. This phenomenon is a result of the counter-ion screening effect, which decreases the binding affinity of QDs to ATR. 26

Fluorescence Resonance Energy Transfer 7 1.4 611 nm / 566 nm peak ratio 1.2 1.0 0.8 0.6 0.4 0.2 0.00 0.02 0.04 0.06 0.08 0.10 NaCl concentration (mol / L) Fig. 4. The peak ratio of wavelength at 611 nm to 566 nm shows effect of increased ionic strength on the emission intensity of CdTe ( ) and CdTe-ATR ( ). A series of different volume of 0.5 mol/l NaCl solutions are added to the sample of CdTe and CdTe-ATR conjugates. Each salt concentration is calculated in the resulting solution. 4. Conclusions In summary, a FRET process between water-soluble QDs donor and organic acceptor dyes with avidin as the binding bridge results in a strong enhancement of the dye fluorescence and a decline of the QDs fluorescence. The donor acceptor pair of QDs and dye molecules will find utilities in the design of assays of antibody antigen binding, DNA hybridization, and enzyme substrate interaction, etc. Acknowledgment This work was financially-supported by the National Natural Science Foundation of China (No. 20075009). References 1. B. W. Van Der Meer, G. Coker and S. Y. S. Chen, Resonance Energy Transfer: Theory and Data (VCH, New York, 1994). 2. P. Wu and L. Brand, Anal. Biochem. 218, 1 (1994). 3. D. M. Willard, L. L. Carillo, J. Jung and A. V. Orden, Nano Lett. 1, 469 (2001). 4. C. R. Kagan, C. B. Murray, M. Nirmal and M. G. Bawendi, Phys. Rev. Lett. 76, 1517 (1996). 5. C. R. Kagan, C. B. Murray and M. G. Bawendi, Phys. Rev. B 54, 8633 (1996). 6. C. E. Finlayson, D. S. Ginger and N. C. Greenham, Chem. Phys. Lett. 338, 83 (2001). 7. N. N. Mamedova, N. A. Kotov, A. L. Rogach and J. Studer, Nano Lett. 1, 281 (2001). 8. I. L. Medintz, A. R. Clapp, H. Mattoussi, E. R. Goldman, B. Fisher and J. M. Mauro, Nat. Mater. 2, 630 (2003).

8 Q.-D. Chen et al. 9. D. M. Willard and A. V. Orden, Nat. Mater. 2, 575 (2003). 10. J. K. Jaiswal, H. Mattoussi, J. M. Mauro and S. M. Simon, Nat. Biotech. 21, 47 (2003). 11. M. Bruchez Jr., M. Moronne, P. Gin, S. Weiss and A. P. Alivisatos, Science 281, 2013 (1998). 12. W. C. W. Chan and S. M. Nie, Science 281, 2016 (1998). 13. B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou and A. Libchaber, Science 298, 1759 (2002). 14. M. E. Åkerman, W. C. W. Chan, P. Laakkonen, S. N. Bhatia and E. Ruoslahti, Proc. Natl. Acad. Sci. USA 99, 12617 (2002). 15. X. Y. Wu, H. J. Liu, J. Q. Liu, K. N. Haley, J. A. Treadway, J. P. Larson, N. F. Ge, F. Peale and M. P. Bruchez, Nat. Biotechnol. 21, 41 (2003). 16. J. K. Jaiswal, H. Mattoussi, J. M. Mauro and S. M. Simon, Nat. Biotechnol. 21, 47 (2003). 17. Z. B. Lin, S. X. Cui, H. Zhang, Q. D. Chen, B. Yang, X. G. Su, J. H. Zhang and Q. H. Jin, Anal. Biochem. 319, 239 (2003). 18. N. Green, Biochem. J. 92, 16c (1964). 19. M. Wilchek and E. A. Bayer, Anal. Biochem. 171, 1 (1988). 20. M. Y. Gao, S. Kirstein, H. Möhwald, A. L. Rogach, A. Kornowski, A. Eychmüller and H. Weller, J. Phys. Chem. B 102, 8360 (1998). 21. H. Zhang, Z. Zhou, B. Yang and M. Y. Gao, J. Phys. Chem. B 107, 8 (2003). 22. J. Georges, N. Arnaud and L. Parise, Appl. Spectrosc. 50, 1505 (1996). 23. Y. J. Guo, Laboratory Technique for Protein Electrophoresis (Jilin University Press, Changchun, 1999), p. 76. 24. O. Schmelz, A. Mews, T. Basché, A. Herrmann and K. Müllen, Langmuir 17, 2861 (2001). 25. C. Landes, C. Burda, M. Braun and M. A. EI-sayed, J. Phys. Chem. B 105, 4973 (2001). 26. H. Mattoussi, J. M. Mauro, E. R. Goldman, G. P. Anderson, V. C. Sundar, F. V. Mikulec and M. G. Bawendi, J. Am. Chem. Soc. 122, 12142 (2000). 27. Y. L. Wang, P. L. Dubin and H. W. Zhang, Langmuir 17, 1670 (2001).