Sensitized Luminescent Terbium Nanoparticles: Preparation and Time

Then 1 mL of 0.1 mol/L fresh NaOH solution was added to the microbeads ... The SEM images further show that almost all NPs are very uniform and ...
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Anal. Chem. 2007, 79, 960-965

Sensitized Luminescent Terbium Nanoparticles: Preparation and Time-Resolved Fluorescence Assay for DNA Yang Chen,*,† Yumei Chi,‡ Hongmei Wen,‡ and Zuhong Lu†

State Key Laboratory of Bioelectronics, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, P. R. China, and College of Medicine, Nanjing University of Traditional Chinese Medicine, Nanjing 210029, P. R. China

A highly luminescent terbium nanoparticle as the biolabel based on the sensitization of a dye molecule was prepared. The luminescent complexes included in the particles were composed of a quinolone-based dye molecule as the lightenergy transfer donor and a polyaminocarboxylate-based chelator with excellent water-solubility and a high binding constant for lanthanides. The structure of two functional entities in the single molecule made the complex highly luminescent in aqueous solution. Silica nanoparticles containing terbium complexes were prepared by the reverse microemulsion method. Such a terbium nanoparticle is as bright as about 340 free terbium complexes, and it has a 1.5-ms fluorescence lifetime that enables it to be used in the time-resolved fluorescence assays. The conjugate of the nanoparticle with oligonucleotide was prepared and used to carry out a DNA sandwich hybridization assay based on magnetic microbeads as solidphase carrier. The experimental results showed that the detection sensitivity with the nanoparticles is more than 100-fold as high as that with dye Fluorescein isothiocyanate (FITC) molecules. The fluorescence labeling and detection techniques have been used extensively in bioscience, medical diagnosis, and other fields. However, the traditional fluorescence assays suffer from the interferences of various nonspecific fluorescence. These interferences arise from the background fluorescence of the sample and from Rayleigh, Raman scattering from the instrument’s optics, the cuvettes, and the sample matrix.1 The waveband of interferences is usually located between 350 and 600 nm and overlaps extensively with the emission spectra of many conventional fluorophores. In addition, in the traditional fluorescence assays, the organic dyes as the labels are easily photobleached and quenched by the environment and inter filter effect, resulting in irreproducible signals in ultratrace bioanalysis. In time-resolved fluorescence (TRF) assays, the labels with long-lived fluorescence, usually lanthanide chelates, were used, and the labels’ signals were detected after all short-lived fluorescence completely decayed. This * Corresponding author. E-mail: [email protected] † Southeast University. ‡ Nanjing University of Traditional Chinese Medicine. (1) Diamandis, E. P.; Christopoulos, T. K. Anal. Chem. 1990, 62, 1149A-1157A. (2) Soini, E.; Lovgren, T. CRC Crit. Rev. Anal. Chem. 1987, 18, 105-154.

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Figure 1. Schematic illustration of luminescent nanoparticles based on the sensitization of dye molecules.

allows only special signals from the analytes to be measured with very high sensitivity.2 In some assays with lanthanide chelate labels, a comparable sensitivity with radioisotopes has been obtained.2,3 In the time-resolved fluorescence imaging of cells, the contrast is improved about 400-fold.4,5 The development of nanotechnology is bringing new interest in bioanalysis. Quantum dots, dye-doped nanoparticles, and so forth have been used as biolabels and show more attractive advantages than traditional fluorophores.6-8 The main benefits of particle labels are stronger luminescence and better anti-photobleaching due to the fact that each particle contains a lot of luminescent molecules and was usually protected with a coating layer. But there are also drawbacks. The fluorescent latex particles (e.g., polystyrene particles) have the problems of too large size, tendency to agglomerate, swelling, and dye leaking.9 Meanwhile, (3) Solni, E.; Kojola, H. Clin. Chem. 1983, 29, 65-68. (4) Seveus, L.; Vaisala, M.; Syrjanen, S.; Sandberg, M.; Kuusisto, A.; Harju, R.; Salo, J.; Hemmila, I.; Kojola, H.; Soini, E. Cytometry 1992, 13, 329-338. (5) Marriott, G.; Clegg, R. M.; Arndt-Jovin, D. J.; Jovin, T. M. Biophys. J. 1991, 60, 1374-1387. (6) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (7) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016-2018. (8) Tan, W.; Wang, K.; He, X.; Zhao, X.; Drake, T.; Wang, L.; Bagwe, R. P. Med. Res. Rev. 2004, 24, 621-638. (9) Seydack, M. Biosens. Bioelectron. 2005, 20, 2454-2469. 10.1021/ac061477h CCC: $37.00

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the poor solubility in water, difficult surface conjugation chemistry, and possible toxicity in vivo of quantum dots are still under investigation.10,11 The organic dye-doped nanoparticles have a broad emission and small Stokes shift resulting in cross-talk between excitation and emission signals. More defectively, all particles mentioned do not avoid the interferences of background and scattering fluorescence. Also, the scattering fluorescence is more serious for the nanoparticle labels due to their larger scale than for fluorophores according to the Rayleigh scattering equation.12 The nanosized lanthanide labels enabling the elimination of the background and scattering fluorescence have been presented and discussed recently.13-15 Their unusual spectral characteristics of spiked emission (150 nm), and long fluorescence lifetime make them particularly advantageous for use as biolabels. Due to the water molecules quenching the fluorescence, the luminescence of lanthanide chelates is usually weak in aqueous solution. Despite thorough research, the properties of high quantum yield, long fluorescence lifetime, good watersolubility, and photochemical stability are rarely all present in a single molecule. Attempts to improve aqueous luminescence characteristics of lanthanide chelates have remained largely unsuccessful.16,17 Few applicable chelates have been made,18-20 and the synthesis is quite complicated. In this work, we designed and prepared a sort of luminescent lanthanide nanoparticle label based on the sensitization of dye molecule. The particle is made up of SiO2-coated lanthanide complexes (Figure 1). The lanthanide complexes are comprised of a quinolone-based dye molecule as the light-absorption antenna and a polyaminocarboxylate-based chelator binding lanthanide ions. The dye molecule acts to capture the excitation light and sensitize the lanthanide ion luminescent by fluorescence energy transfer. Polyaminocarboxylates have excellent solubility and a high binding constant for lanthanides (K ) 1016-1022 21), while 7-amino-4-methyl-2-quinolone (Carbostyril 124, Cs124), one of most efficient energy-transfer donors yet synthesized for terbium and europium,22 can make the chelate highly luminescent. The structure of two functional entities easily allowed having both good aqueous solubility and luminescence by optimizing the dye molecule structure and chelator separately. To obtain strong (10) Cottingham, K. Anal. Chem. 2005, 77, 354A-357A. (11) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (12) Hiemenz, P. C.; Rajagopalan, R. Principles of Colloid and Surface Chemistry, 3rd ed., revised and expanded; Marcel Dekker: New York, 1997; pp 193241. (13) Feng, J.; Shan, G.; Maquieira, A.; Koivunen, M. E.; Guo, B.; Hammock, B. D.; Kennedy, I. M. Anal. Chem. 2003, 75, 5282-5286. (14) Ye, Z.; Tan, M.; Wang, G.; Yuan, J. Anal. Chem. 2004, 76, 513-518. (15) Huhtinen, P.; Kivelal, M.; Kuronen, O.; Hagren, V.; Takalo, H.; Tenhu, H.; Lolvgren, T.; Halrmal, H. Anal. Chem. 2005, 77, 2643-2648. (16) Dickson, E. F. G.; Pollak, A.; Diamandis, E. P. Pharmacol. Ther. 1995, 66, 207-235. (17) Alpha, B.; Balzani, V.; Lehn, J. M.; Mathis, G. Angew. Chem., Int. Ed. Engl. 1987, 26, 266-267. (18) Saha, A. K.; Kross, K.; Kloszewski, E. D.; Upson, D. A.; Toner, J. L.; Snow, R. A.; Black, C. D. V.; Desai, V. C. J. Am. Chem. Soc. 1993, 115, 1103211033. (19) Horiguchi, D.; Katayama, Y.; Sasamoto, K.; Terasawa, H.; Sato, N.; Mochizuki, H.; Ohkura, Y. Chem. Pharm. Bull. 1992, 40, 3334-3337. (20) Evangelista, R. A.; Polak, A.; Allore, B.; Templeton, E. F.; Morton, R. C.; Diamandis, E. P. Clin. Biochem. 1988, 21, 173-178. (21) Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum Press: New York, 1989. (22) Li, M.; Selvin, P. R. J. Am. Chem. Soc. 1995, 117, 8132-8138.

Figure 2. TEM (upper) and SEM (lower) images of Cs124-DTPATb luminescent nanoparticles.

luminescence intensity, SiO2 nanoparticles (NPs) containing a lot of these luminescent complexes were prepared by the reverse microemulsion method. The SiO2 surface is easy to conjugate with various biomolecules and very water-soluble and biocompatible; also the size of this kind of NPs is controllable from the range of 5-200 nm.23 Thus, such a configuration of NP can satisfy the desired criteria of high luminescence as well as long-life, easy conjugation, water-solubility, and photostability in development of a biolabel. As an example, we prepared and used terbium nanoparticles to label oligonucleotide for DNA detection and compared the detection results with those of the conventional dye FITC. EXPERIMENTAL SECTION Chemicals. Diethylenetriaminepentaacetic acid dianhydride (98%, DTPA dianhydride); 7-amino-4-methyl-2(1H)-quinolinone (99%, carbostyril 124, Cs124); anhydrous N,N-dimethylformamide (99.8%, DMF); 3-aminopropyltriethoxysilane (99%, APTES); fluorescamine (98%) from Aldrich; succinic anhydride (>97.0%) from Fluka; N-hydroxysuccinimide (NHS); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) from Pierce; streptavidin (SA) from Promega; terbium nitrate (99.99%) from Baotou Rewin Rare Earth Metal Materials Co.; triethylamine (99%); tetraethyl orthosilicate (99%); 1-hexanol (99%); cyclohexane (99.5%) from Sinopharm Chemical Reagent Co.; 2-(N-morpholino)ethanesulfonic acid (MES); Triton X-100 from Amresco. Oligonucleotides from Invitrogen (Shanghai): 5′-biotin-(T)10-TG CGG CAG GTG CGA CGC GGT-3′ for the nanoparticles as probe oligo; 5′-GTC TAC CAG GCA TTC GCT (T)10-3′-biotin for the magnetic microbeads as capture oligo; 3′-C GCC GTC CAC GCT GCG CCA CAG ATG GTC CGT AAG CGA-5′ for the target DNA. 5′-FITC-(T)10TG CGG CAG GTG CGA CGC GGT-3′ as the control probe; streptavidin-coated magnetic microbeads (Dynalbeads Myone (23) Santra, S.; Wang, K.; Tapec, R.; Tan, W. J. Biomed. Opt. 2001, 6 (2), 160166.

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Figure 4. Lifetime at 546-nm emission of Cs124-DTPA-Tb nanoparticles.

Figure 3. Fluorescence (red line) and time-resolved fluorescence (black line) excitation and emission spectra of Cs124-DTPA-Tb complexes and their nanoparticles (diameter 50 nm). (A) 1 × 10-6 mol/L free Cs124-DTPA-Tb; (B) 2.5 × 10-9 mol/L Cs124-DTPA-Tb NPs; (C) 0.1 mol/L Tb3+ water solution.

Streptavidin C1, 1-µm diameter) from Dynal Ltd. Distilled and deionized water (18 MΩ cm-1) was used throughout. Synthesis and Purification of Cs124-DTPA. The synthesis of Cs124-DTPA complex was carried out according to the reported method.22 Typically, 12 mg of Cs124 and 25 mg of DTPA dianhydride were dissolved in 200 µL and 1400 µL of anhydrous DMF, respectively; then 100 µL of dry triethylamine (dried by activated molecular sieves before use) was added to a DMF 962 Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

solution of DTPA, and the solution of Cs124 in DMF was added dropwise to this mixture. The reaction was kept for 2 h at room temperature under stirring. The reaction mixture was purified and analyzed by reversed-phase HPLC (Waters 660), with a yield of about 65%. ESI-MS: m/e ) 548 (M-H+). HPLC was performed on a C18 column (19 × 300 mm, 15 µm, Waters Bondapak) with the flow rate of 16 mL/min at 30 °C column temperature. A 40min gradient, from 20 to 30% solvent B [solvent A, 0.1 M triethylammonium acetate (pH ) 6.5) and 0.5% acetic acid (1:1 v/v); solvent B, methanol] was used. UV detection wavelength was at 328 nm. Synthesis of Cs124-DTPA-Tb Complex and Its Nanoparticles. Tb(NO3)3 aqueous solution was added in 1:1 molar rate to the Cs124-DTPA solution. The solution was used for the preparation of its nanoparticles. The nanoparticles were synthesized by a modified microemulsion method24 as follows. Typically, 4 mL of cyclohexane, 1 mL of n-hexanol, and 1 mL of Triton X-100 were mixed under stirring. Then, 295 µL of an aqueous solution of about 4 mmol/L Cs124-DTPA-Tb complex was added to the mixture. After mixing for 30 min, 85 µL of TEOS and 25 µL of ammonia solution (28%) were added. The reaction was allowed to continue for 24 h. An equal volume of acetone was added to stop the reaction; the particles were centrifuged out of the solution and washed thoroughly with acetone/ethanol (1:1 v/v), ethanol, 0.5% Tween-20 aqueous solution, and water, respectively, for several times by ultrasonic dispersion and centrifugation. For the amination of the surface of nanoparticles, the clean nanoparticles solution was added to the microemulsion solution with the same components, and 20 µL of silanization reagent APTES was introduced to the mixture; the reaction was allowed to continue for 2 h. Finally the reaction was stopped, and the NPs were washed by the same steps as above. Measurements. Fluorescence spectra and emission lifetime were recorded on the LS55 luminescence spectrometer (PerkinElmer). The detection solution was placed in a quartz micro cuvette with 100 µL capacity. The 328-nm excitation wavelength was used for the emission spectra; the 546-nm emission wavelength was used for gathering the excitation spectra. For the timeresolved fluorescence spectra, a delay time of 0.05 ms and a gate (24) Santra, S.; Zhang, P.; Wang, K.; Tapec, R.; Tan, W. Anal. Chem. 2001, 73, 4988-4993.

Scheme 1. Structure and Synthesis of Cs124-DTPA-Tb

Scheme 2. Cs124-DTPA-Tb Nanoparticles Conjugated with Oligonucleotide

Scheme 3. DNA Sandwich Hybridization Assay Based on the Magnetic Microbeads

time of 2 ms were used. The images of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were acquired using a JEM-2000EX (JEOL, Japan) and LEO1530VP (LEO, Germany), respectively. Surface Modification of Nanoparticles with Oligonucleotide. A quantity of 2 mg of nanoparticles silanized with APTES was suspended in 3 mL of 0.5 mol/L phosphate buffer (pH ) 7.0) by ultrasonication. An amount of 20 mg of succinic anhydride was added to this solution, and the resulting mixture was stirred for 2 h. After centrifuging and washing with 0.5% Tween-20, the particles were dispersed in 3 mL of 0.1 mol/L MES buffer (pH ) 6.0, 0.1 mol/L MES, 0.5 mol/L NaCl). Quantities of 1.2 mg of EDC and 1.8 mg of NHS were added to the solution to react for 15 min under stirring, 5 µL mercaptoethanol and 1 mg streptavidin (SA) were added, respectively, and the reaction was continued for 2 h. After centrifuging and washing, the particles was resuspended in 1 mL of PBS buffer (pH ) 7.0, 0.15 mol/L NaCl,

10 mmol/L sodium citrate). A quantity of 200 µL of 100 µmol/L biotin-oligo (probe oligo) was added to the above SA-coated NPs to react for 30 min. The particles were removed from the solution by centrifugal precipitation, washed with 0.5% Tween-20 solution, and resuspended in 0.5% Tween-20 aqueous solution for use. DNA Assay Based on Magnetic Microbeads. Excess 3′terminal biotinylated capture oligo was incubated with 1 mL of 1× B&W buffer (pH ) 7.5, 1.0 mol/L NaCl, 0.5 mmol/L EDTA, 5 mmol/L tris-HCl) of washed 200 µg of streptavidin-coated magnetic microbeads for 15 min. After magnetic separation and washing to remove unbound oligonucleotide, the microbeads were resuspended in 1 mL of 1×hybridization buffer (pH ) 7.0, 0.75 mol/L NaCl, 10 mmol/L sodium citrate). Excess NPs labeled probe oligo and different amounts of the target oligo were added to the above microbeads solution and slow-tilt rotated for 1 h. The microbeads were separated magnetically and washed three times with 1×hybridization buffer. Then 1 mL of 0.1 mol/L fresh NaOH solution was added to the microbeads precipitate and stirred for 3 min, and the supernatant was removed in the magnetic stand for the time-resolved fluorescence assay. RESULTS AND DISCUSSION The synthesis of the luminescent terbium complex is presented in Scheme 1. Carbostyril 124 first was covalently coupled with the polyaminocarboxylates by its dianhydride, and the product was chelated with terbium ions.22 The terbium complexes were integrated into nanoparticles (NPs) by the hydrolysis and condensation of tetraethyl orthosilicate (TEOS) in water-in-oil emulsions.24 Figure 2 is the TEM and SEM images of the Cs124-DTPATb NPs. Under typical conditions, (50 ( 3)-nm size and spherical NPs were obtained. The SEM images further show that almost all NPs are very uniform and round (Figure 2 lower). Figure 3 shows the fluorescence and time-resolved fluorescence spectra of Cs124-DTPA-Tb complexes and their nanoparticles. The excitation spectra (λmax ) 328 nm) of Cs124-DTPA-Tb were identical to the sensitizer Cs124 absorption spectra (data Analytical Chemistry, Vol. 79, No. 3, February 1, 2007


Figure 5. Different concentrations of target DNA versus response of Cs124-DTPA-Tb NPs and FITC in 0.1 mol/L NaOH uncoupling solution. FITC: general fluorescence assay, Cs124-DTPA-Tb NP: time-resolved fluorescence assay. Other determining conditions are the same.

not shown), indicating that the absorption of Cs124-DTPA-Tb is due to Cs124 attached via an amide linkage to the chelate, and the interaction between the Cs124 and terbium does not significantly perturb the Cs124 absorption. The highly spiked emission peaks at 490 nm (full width at half-maximum, fwhm ) 8 nm), 546 nm (fwhm ) 10 nm), 585nm, and 621 nm are the special peaks of terbium, which arise from its 5D4 state to its ground-state 7F6, 7F , 7F , 7F , respectively.25 The free Cs124 or DTPA-Cs124 has 5 4 3 no emission peaks at these positions (data not shown); however, free Tb3+ has the same peaks but the luminescent intensity is only about 10-5 fold as strong as these complexes (Figure 3C), which is undetectable at the micromole concentration. This showed that the light emission of terbium in the complex is from the fluorescence energy transfer of Cs124, and the emission was enhanced about 175000-fold by the sensitization of Cs124. The excited-state lifetime of Cs124-DTPA-Tb NPs in aqueous solution is about 1.53 ms, as shown in Figure 4, almost identical with that of its complex (τ )1.51 ms), which is long enough for the entire decay of the various nonspecific fluorescence. In timeresolved fluorescence spectra, the strong scattering peaks appearing in the conventional fluorescence were eliminated cleanly and there is at least a 160-nm Stokes shift from 328-nm excitation to longer than 490-nm emission (Figure 3). The leakage of Cs124-DTPA-Tb complex from the NPs was checked after the suspension of washed NPs was allowed to stand for 24 h. The suspension of NPs was ultrasonically dispersed and centrifuged down. The fluorescence intensity of the supernatant was close to that of the background signal, while that of the redispersed precipitate is close to that of the suspension, showing no Cs124-DTPA-Tb complex leaking. We estimated the number of molecules inside the particles. According to the concentrations of free complexes and nanoparticles (from the size, the weight of dry nanoparticles, and density of silica NPs, here 1.96 g/cm3 24) and comparing their emission intensity of the same peak, the (25) Richardson, F. S. Chem. Rev. 1982, 82, 541-552.


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fluorescence of about 340 complex molecules is as bright as that of one Cs124-DTPA-Tb NP. Unlike dye fluorophores, the selfquenching effects of lanthanide complexes from multiple labeling are usually negligible,26-28 so it is educed that one Cs124-DTPATb particle included about 340 free terbium complexes. In order for conjugation to take place with various biomolecules, the surface of the nanoparticles was modified chemically (Scheme 2). The homobifunctional cross reagents often create a broad range of poorly defined conjugates.29 For the higher yield of desired conjugate, we used a heterobifunctional reagent for the cross-linking. The amino groups on the particle surface were converted into carboxylic groups by succinic anhydride. The amount of amine groups on the particles surface was determined by the fluorescamine method.30,31 The generated fluorescent intensity corresponds to about 6400 amino groups per particle (50nm diameter) according to the work curve of known concentrations of APTES. After treatment with succinic anhydride, the particles showed a negative fluorescamine assay indicating that the carboxyl groups were formed via amino groups. The particles with -COOH were further cross-linked with streptavidin for conjugating with the biotinylated probe oligonucleotide by the EDC and NHS on the basis of the protocol of the manufacturer. As an example of the applications of these particle labels, DNA sandwich hybridization assay based on magnetic microbeads as the solid-phase carrier was carried out (Scheme 3). Such an assay can easily detect these particle labels just by general fluorescence spectrometers and need not use expensive time-resolved laser confocal microscope equipment. Cs124-DTPA-Tb NPs labeled probe oligos were immobilized on the magnetic microbeads with capture oligos via the hybridization of target DNA complementary to both of them. After magnetic separation and washing to remove unbound oligos, the bound target and NPs were released by uncoupling alkali solution for the time-resolved fluorescence detection. Figure 5 shows the luminescent response of labeling with Cs124-DTPA-Tb NPs and dye FITC versus different amounts of target DNA. The particles have a detection limit about 8 × 10-11 mol/L, compared with that of 1 × 10-8 mol/L for FITC; the detection sensitivity was improved slightly more than 100-fold by general solution spectra measurement. If using more sensitive measuring equipment or increasing the number of luminescent molecules inside the particle, the detection limit should be improved significantly. CONCLUSIONS In summary, we have for the first time synthesized lanthanide nanoparticle labels on the basis of the principle of dye sensitization. The nanoparticles are strongly luminescent in aqueous solution, have a long fluorescence lifetime, are easily conjugated with biomolecules, and are excellent in shape. The luminescent complexes inside the particles have two functional entities which allow optimizing their luminescence and aqueous solubility (26) Morton, R. C.; Diamandis, E. P. Anal. Chem. 1990, 62, 1841-1845. (27) Scorilas, A.; Diamandis, E. P. Clin. Biochem. 2000, 33, 345-350. (28) Takalo, H.; Mukkala, V. M.; Mikola, H.; Liitti, P.; Hemmila, I. Bioconjugate Chem. 1994, 5, 278-282. (29) Hermanson, G. T. Bioconjugate Techniques; Academic Press: New York, 1996; pp 187-188. (30) Udenfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.; Leimgruber, W.; Weigele, M. Science 1972, 178, 871-872. (31) Polak, T. B.; Kassai, M.; Grant, K. B. Anal. Biochem. 2001, 297, 128-136.

separately. Such a constitution is more flexible, permitting us to make a new type of luminescent complexes by suitable combination of different sensitizers and lanthanide chelates. The synthetic materials are commercially available. A simple coupling reaction between them is easy to do by the nonprofessional, avoiding the very complicated synthesis of ligands by conventional methods. As an example, preliminary DNA assay labeled with terbium nanoparticles showed at least 100-fold improvement of detection sensitivity over general solution spectra measurement. We expect that this preparation method of luminescent nanoparticle labels based on the sensitization of dye molecules will provide a new

way for applying various lanthanide compounds to ultrasensitive time-resolved bioanalysis and bioimaging. ACKNOWLEDGMENT We acknowledge the financial support from the NSFC (Grants 90206027, 60671014) and the SRF for ROCS, State Education Ministry. Received for review August 9, 2006. Accepted November 9, 2006. AC061477H

Analytical Chemistry, Vol. 79, No. 3, February 1, 2007