6589
2008, 112, 6589-6593 Published on Web 04/04/2008
Resonance Energy Transfer in Steady-State and Time-Decay Fluoro-Immunoassays for Lanthanide Nanoparticles Based on Biotin and Avidin Affinity Jian-Qin Gu, Jie Shen, Ling-Dong Sun,* and Chun-Hua Yan* Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Rare Earth Materials Chemistry and Applications and PKU-HKU Joint Laboratory in Rare Earth Materials and Bioinorganic Chemistry, Peking UniVersity, Beijing 100871, China ReceiVed: February 10, 2008; In Final Form: March 5, 2008
Compared with conventional organic fluorophores, lanthanide-doped nanoparticles as donor and gold nanoparticles as quencher-like acceptor in energy transfer studies offer many technological advantages; in particular, the long-lifetime donor endows the fluorescence resonance energy transfer (FRET) assay with great signal-to-noise ratio, and the quencher acceptor allows monitoring of the donor channel alone. The LaPO4:Ce,Tb nanoparticles with high quantum yield in aqueous solution are readily paired with differently sized gold nanoparticles through the well-established binding affinity of biotin and avidin. The spectra overlap between the emission of lanthanide nanoparticles and absorption of gold nanoparticles meets the prerequisite for energy transfer, and the resonance energy transfer process is characterized by both steady-state and timedecay luminescent measurements. With the emission contributions from Tb3+ ions located at the surface and inside the lanthanide nanoparticles taken into account, the comparison of the donor emission in the presence and absence of acceptors in combination with lifetime changes are used to determine luminescence resonance energy transfer (LRET) efficiency more accurately. The results indicate that the Tb3+ ions located at/near the surface of the nanoparticles play a more important role than Tb3+ ions inside the nanoparticles in the LRET process. The present study introduces the possibility of multiplexing donor/acceptor pairs if sufficient spectral separation is achieved.
Fluorescence resonance energy transfer (FRET) is a nonradiative process whereby an excited-state donor (D) transfers energy to a proximal ground-state acceptor (A), through longrange dipole-dipole interactions.1 Over the past years, FRET has been proven to be a powerful technique in bioanalysis, especially in the detection of molecular binding events2 and changes in protein conformation,3 because of its intrinsic sensitivity (proportional to R-6) to D-A separation distance (R) changes in nanoscale.4 Although organic fluorophores widely used in FRET studies5 have been demonstrated down to the single-pair level,6 the small Stokes shifts, broad emission spectra, crosstalk between multicomponents, and the photobleaching behaviors severely influence the detection sensitivity, thus limiting the potential practical application of FRET. Lanthanides complexes, such as Eu or Tb chelates, show advantages for use as donors for resonance energy transfer studies due to their large Stokes shifts and narrow emission lines arising from 4f-4f transitions, which are technically called luminescence to distinguish from fluorescence.7 Lanthanide-chelate-based resonance energy transfer is also called luminescence resonance energy transfer (LRET), which is an extension of FRET.8 The narrow donor emission, especially the atomic-like emission, can be wellresolved from the emission of the acceptor,8b and the donor emission can be excited at wavelengths much shorter than the acceptor absorption, thus minimizing direct acceptor excitation. * Correspondingauthor.Fax: +86-10-6275-4179;E-mail:
[email protected].
10.1021/jp801203w CCC: $40.75
In comparison with organic fluorophores, a long lifetime of a few milliseconds of the excited state of the lanthanide donors makes the donor lifetime measurements facile and precise.8b Accompanied with the resonance energy transfer, sensitized emission from the acceptor as well as the decreased emission and shortened lifetime of the donor are observed. The lifetime shortening of the donor is insensitive to incomplete pairing, and thus allows measurements of small signals and consequently large distances.9 Lanthanide nanoparticles, such as LaPO4:Ce,Tb and YVO4: Eu, with unique photophysical properties similar to the lanthanide chelates but higher photo- and chemical-stability,10 are also very attractive for use as donors in LRET analysis11 like quantum dots, as has recently been demonstrated.12-14 The nanoparticles endowed with functional groups during preparation or well-established surface chemistry for bioimmobilization are effective in applications as biosensors.10c,d In comparison with more traditional organic fluorophores, gold nanoparticle acceptors have been utilized to follow dynamics in biological systems.15 The surface plasmonic behavior related to electric field density is easy to tune just by simply changing the size or shape of the gold nanoparticles. Furthermore, this surface plasmon resonance effect can be characterized with extinction spectroscopy, which makes operation and characterization easy in the FRET studies with gold nanoparticles as a quencher. The quencher-like acceptor used in FRET studies also makes the donor emission behavior measurements simple and accurate.16 © 2008 American Chemical Society
6590 J. Phys. Chem. C, Vol. 112, No. 17, 2008 Here, we report a simple sensing platform based on LRET studies of luminescent biotinylated LaPO4:Ce,Tb nanoparticles and avidin-coated gold nanoparticles with the specific biotinavidin binding events (Scheme 1) in steady-state and time-decay. The naturally strong binding of avidin with the small molecule biotin is one of the most popular methods of noncovalent conjugation; the strength of the biotin-avidin interaction has made it useful in specific targeting applications and assay designs.17,18 The LaPO4:Ce,Tb nanoparticles with high quantum yield and long lifetime of excited states were synthesized directly in water, and bioconjugation was carried out with the established method19 through the carboxylate groups of the nanoparticles and the hydrazide groups of biotin hydrazides. Gold nanoparticles were chosen as acceptors mainly because of their exceptional quenching ability,20 and the quenching modes were described as the Fo¨rster dipole-dipole interaction and nanosurface energy transfer.16 Gold nanoparticles with an average diameter of ca. 13 nm labeled with avidin through the high affinity to protein19 are used as acceptor (quencher) in the LRET event. The spectral overlap between the donor emission and the acceptor absorption satisfies the basic requirement of resonance energy transfer. Gold nanoparticles used as acceptors also have the advantage of being labeled with multiple biologically active groups and reducing the background fluorescence originating from direct acceptor excitation or re-emission, thus efficiently improving the detection sensitivity.21 The resonance energy transfer efficiency was studied by the comparison of the donor emission and the variation of excited lifetime of Tb3+ in the presence and absence of acceptors. Considering the contributions from Tb3+ ions located at/near the surface and inside of the lanthanide nanoparticles, the comparison of the donor emission gives a more accurate determination in the FRET efficiency. Experimental Section Synthesis of Biotinylated LaPO4:Ce,Tb Nanoparticles. 8 mL of 0.10 mol‚L-1 phosphorus-containing poly(acrylic acid) and 2 mL of 0.10 mol‚L-1 of Ln(NO3)3 (Ln ) La 40%, Ce 45%, Tb 15%) solutions were mixed with magnetic stirring, and 2 mL of 0.10 mol‚L-1 Na2HPO4 aqueous solution was added dropwise. After adjusting pH to 10, 0.75 mL of 0.10 mol‚L-1 Na2S2O3 was added. The solution was adjusted to 15 mL in volume and transferred into a 25 mL autoclave; then, it was maintained at 180 °C for 24 h. A transparent solution of LaPO4:Ce,Tb nanoparticles was obtained after further dialysis for 48 h using a membrane of molecular weight cutoff of 12 000 Da (pore size of ca. 2.5 nm). The powders of LaPO4:Ce,Tb nanoparticles were obtained through lyophilizing the colloidal solution. 2 mg of the as-prepared LaPO4:Ce,Tb nanoparticles and 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1 mg of N-hydroxysulfosuccinimide (SulfoNHS) were added to 2 mL of 0.10 mol‚L-1 phosphate buffer (pH 7.1) containing 1 mg of biotin hydrazide. After magnetic stirring for 2 h at room temperature, the solution was incubated at 4 °C for 12 h. Synthesis of Avidin-Coated Gold Nanoparticles. 1.0 mL of 1% chloroauric acid (HAuCl4) solution was added to 100 mL of deionized water and heated to boiling while mixing. Then, 2 mL of 1% sodium citrate solution was added rapidly to the boiling solution. The solution was refluxed for 15 min. The color of the suspension would change from dark blue to red as the monodisperse colloidal gold particles with an average diameter of ca. 13 nm (Figure S1, Supporting Information) were formed,
Letters SCHEME 1: Schematic Illustration of the LRET Process from the Biotinylated LaPO4:Ce,Tb Nanoparticles to Avidin-Coated Gold Nanoparticles Based on the High Affinity of Biotin and Avidin
then cooled to room temperature. Colloidal gold nanoparticles with larger diameters could be obtained by reducing the amount of sodium citrate solution; for example, gold nanoparticles with an average diameter of 70 nm were synthesized through a similar process but with 0.7 mL of 1% sodium citrate solution in the reaction. 5 mL of 1 mg‚mL-1 succinylated avidin solution was formed by dissolving the protein in 50 mmol‚L-1 sodium phosphate with stirring, and the succinylated avidin solution was added to 200 µL colloidal gold suspension at room temperature and reacted for 30 min with constant mixing. LRET Experiments. 20 µL as-prepared biotinylated LaPO4: Ce,Tb nanoparticles solution was diluted into 3000 mL PBS buffer, and a 20 µL solution of avidin-coated gold nanoparticles was added in aliquots until the emission intensity and lifetime measurements reached a constant. Instrumentation. Absorption spectra were recorded on a Hitachi U-3010 spectrophotometer with colloidal nanoparticles. Fluorescence spectra were recorded on a Hitachi F-4500 spectrophotometer equipped with a 150 W Xe-arc lamp at room temperature. The spectra were measured at a fixed bandpass of 0.2 nm with the same instrument parameters as 5.0 nm for excitation slit and 2.5 nm for emission slit. The lifetime measurements were performed with an Edinburgh Instruments FLS920 transient/steady-state luminescence spectrometer at room temperature. Results and Discussion The as-prepared LaPO4:Ce,Tb nanoparticles with a hydrodynamic radius of ca. 20 nm (Figure S2, Supporting Information) could be well-dispersed in water, and the biotinylated LaPO4:Ce,Tb nanoparticles also possess unique luminescent properties with a quantum yield of 68% and an average lifetime of 3.08 ms. The plasmonic behavior of gold nanoparticles is size- and shape-dependent; therefore, both ca. 13 and 70 nm gold nanoparticles were synthesized through the reduction of
Letters
Figure 1. Extinction spectrum of colloidal gold nanoparticles with a diameter of ca. 13 nm (red line) and emission spectrum of LaPO4:Ce,Tb nanoparticles (blue line).
J. Phys. Chem. C, Vol. 112, No. 17, 2008 6591
Figure 3. Luminescence decay curves of biotinylated LaPO4:Ce,Tb nanoparticles and the conjugates of biotinylated LaPO4:Ce,Tb-avidin coated gold nanoparticles.
TABLE 1: Comparison of Luminescent Lifetimes of Biotinylated LaPO4:Ce,Tb Nanoparticles in the LRET Process with Different Donor to Acceptor Ratios
Figure 2. Evolution of emission intensity of biotinylated LaPO4:Ce,Tb nanoparticles with the addition of avidin coated gold nanoparticles.
chloroauric acid (HAuCl4) by sodium citrate, whose extinction overlapped with the emission of LaPO4:Ce,Tb nanoparticles.22 Figure 1 shows the extinction spectra of ca. 13 nm gold nanoparticles and emission spectra of LaPO4:Ce,Tb nanoparticles, respectively. The emission of LaPO4:Ce,Tb nanoparticles overlaps with the absorption of gold nanoparticles, which satisfies the prerequisite for efficient LRET. The luminescence resonance energy transfer experiment as shown in Scheme 1 was done in aqueous solution in order to match biological conditions. It was carried out by stepwise addition of avidinlabeled gold acceptors into a PBS buffer, which contained a fixed amount of biotinylated LaPO4:Ce,Tb nanoparticles as donors, like a titration experiment. After each addition of aliquots, the steady-state luminescence of LaPO4:Ce,Tb nanoparticles was measured. Figure 2 shows the evolution of the emission spectra of LaPO4:Ce,Tb nanoparticles in this process. As expected, the luminescence from the donor is obviously quenched by gold nanoparticles Via energy transfer through the specific binding of avidin and biotin. As gold nanoparticles do not emit as a sensitizer acceptor in this energy transfer process, only the examination of LaPO4:Ce,Tb nanoparticles luminescence is necessary. Increasing the volume of the donor to more than six times that of the acceptor, the emission spectra of LaPO4:Ce,Tb nanoparticles did not change anymore, and this phenomenon indicated that the equilibrium was reached. The status of the gold nanoparticle acceptor is also inspected with the extinction spectra. There is no shift in the absorption as a result of the large distance between gold nanoparticles; thus, no influence of the dipole interactions was observed. The phenomenon of quenching provides a valuable understanding of the excited-state lifetime in luminescence measurements. The resonance energy transfer process is further confirmed by the luminescence decay measurements for the LRETinduced shortening lifetime of the donor, which is insensitive to incomplete pairing and environment. Figure 3 shows the
Au/NPa
I1b (%)
τ1b (ms)
I2b (%)
0 1 2 3 4 5 6
28.79 24.75 21.26 18.71 18.69 16.90 15.58
2.02 1.88 1.74 1.41 1.31 1.28 1.24
71.20 75.25 78.74 81.29 81.31 83.10 84.42
τ2b τb (ms) (ms) Φτ1c
Φτ2c
Φτc
ΦIc
3.91 3.86 3.75 3.48 3.41 3.37 3.37
0 0.01 0.041 0.11 0.13 0.14 0.14
0 0.01 0.02 0.11 0.15 0.14 0.14
0 0.07 0.14 0.31 0.35 0.39 0.39
3.08 3.06 3.01 2.73 2.62 2.64 2.64
0 0.07 0.14 0.30 0.35 0.37 0.39
a Au/NP is the acceptor to donor volume ratio in the LRET process, with the primordial concentration of 0.1 mg‚mL-1 for Au and 1 mg‚mL-1 for LaPO4:Ce,Tb nanoparticles, respectively. b τ1 and τ2 are the lifetimes of Tb3+ fitting with double exponential function; I1 and I2 are the contribution fractions of τ1 and τ2, respectively; τ is the average lifetime calculated according to ref 23. c Φ is the LRET efficiency, calculated with ΦI ) 1 - Iconj/I and Φτ )1 - τconj/τ, respectively, where Iconj and I represent the luminescent intensity in the presence and absence of the acceptor, respectively; τconj and τ are the luminescent lifetimes in the presence and absence of the acceptor.
decay curves of 5D4-7F5 transition from the biotinylated LaPO4: Ce,Tb nanoparticles and the conjugates of biotinylated LaPO4: Ce,Tb-avidin labeled gold nanoparticles. Both of the curves can be fitted well with a double exponential function of I ) I1 exp(-t/τ1) + I2 exp(-t/τ2) (τ1 and τ2 correspond to the lifetimes of Tb3+). The biotinylated LaPO4:Ce,Tb nanoparticles have a τ1 of 2.02 ms, τ2 of 3.91 ms, and an average τ of 3.08 ms,23 and the conjugates have a τ1 of 1.24 ms, τ2 of 3.37 ms, and an average τ of 2.64 ms. This analytical result indicated that both τ1 and τ2 decreased in the LRET process, and Table 1 further shows the decreasing tendency of both τ1 and τ2 with an increasing amount of avidin-labeled gold acceptors. The emission of LaPO4:Ce,Tb nanoparticles comes from a large number of individual dopant ions as isolated luminescent centers, and luminescence can behave differently in the transition probability for luminescent centers located at the surface or inside the nanoparticles.24 Consequently, the short lifetime τ1 should come from the relatively faster decay process of the Tb3+ located at/ near the surface of the nanoparticles, and the relative long lifetime τ2 should come from the slower decay process of Tb3+ inside the nanoparticles.25 In addition, the fitted lifetime curve indicated that the luminescence is dominated by the slow process, and this is consistent with the relatively large particle size of the lanthanide nanoparticles. The LRET efficiencies, Φ, can be obtained from either the change of the emission intensity of LaPO4:Ce,Tb nanoparticles or lifetime τ1 and τ2 and average lifetime τ values by the equation of ΦI ) 1 - Iconj/I and Φτ ) 1 - τconj/τ, respectively,
6592 J. Phys. Chem. C, Vol. 112, No. 17, 2008
Figure 4. The LRET efficiencies of different acceptor/donor ratios calculated by the emission intensities (squares) and lifetimes (spheres, triangles, and stars correspond to Φτ1, Φτ2, and Φτ, respectively) of LaPO4:Ce,Tb nanoparticles, respectively.
where Iconj and I represent the luminescent intensity in the presence and absence of the acceptor, respectively; τconj and τ are the luminescent lifetime in the presence and absence of the acceptor, respectively. As shown in Figure 4, the LRET efficiency is improved with the increasing acceptor to donor ratio in the conjugates, consistent with the scheme model in which a single avidin-coated gold nanoparticle is conjugated with multiple biotinylated LaPO4:Ce,Tb nanoparticles. The LRET efficiencies calculated with the emission intensities are in good agreement with the efficiencies calculated with lifetime τ1; however, an apparent discrepancy is observed with the efficiencies calculated with lifetime τ2 and average lifetime τ. This behavior should also arise from the intrinsic emission behavior of lanthanide-doped nanoparticles. On the basis of the lifetime studies and previous reports,24,25 the luminescence of LaPO4:Ce,Tb nanoparticles involves that from Tb3+ located at/ near the surface of the nanoparticles or inside the nanoparticles. These two parts behave differently in the LRET process. The distance to the gold nanoparticle acceptor accessed by Tb3+ ions at the surface of the LaPO4:Ce,Tb nanoparticles is relatively short; the LRET efficiencies are thus practically high in this case. Although the Tb3+ at/near the surface of the nanoparticles contributes less to the luminescence (as indicated in Table 1), it benefits the LRET process. On the contrary, the Tb3+ ions inside the nanoparticles induce a relatively low LRET efficiency. With the contributions from Tb3+ ions at/near the surface and inside taken into account, the comparison of the donor emission in the presence and absence of acceptors, on the other hand, should give a more accurate determination of the LRET efficiency. It is known that the efficiency of energy transfer is highly dependent on the extent of spectral overlap, the relative orientation of the transition dipoles, and the distance between the donor and the acceptor molecules. In our case, the spectral overlap between the emission of LaPO4:Ce,Tb and the extinction of ca. 13 nm gold nanoparticles is calculated as ∼1.2 × 1015 nm4‚M-1. This is the upper limit, since 5D4-7F5 of Tb3+ may arise from the magnetic dipole transition as well as the electric dipole transition, and the former does not contribute to LRET.9 The extent of electric dipole transition to magnetic dipole transition is dependent on local symmetry and can be calculated theoretically, but it is rather complicated for lanthanide nanoparticles with a prominent surface contribution.24a The LRETbased detection limit is still constrained on the order of
