Two-Photon Excitation Fluorescence Resonance Energy Transfer

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Bioconjugate Chem. 2008, 19, 574–579

Two-Photon Excitation Fluorescence Resonance Energy Transfer with Small Organic Molecule as Energy Donor for Bioassay Lingzhi Liu,† Genghui Wei,† Zhihong Liu,*,† Zhike He,† Si Xiao,‡ and Ququan Wang*,‡ College of Chemistry and Molecular Sciences and Key Laboratory of Acoustic and Photonic Materials and Devices of Ministry of Education, Wuhan University, Wuhan 430072, P. R. China. Received October 4, 2007; Revised Manuscript Received November 7, 2007

A fluorescence resonance energy transfer (FRET) model using two-photon excitable small organic molecule DMAHAS as energy donor has been constructed and tried in an assay for avidin. In the FRET model, biotin was conjugated to the FRET donor, and avidin was labeled with a dark quencher DABS-Cl. Binding of DABS-Cl labeled avidin to biotinylated DMAHAS resulted in the quenching of fluorescence emission of the donor, based on which a competitive assay for free avidin was established. With using such donors that are excited in IR region, it is capable of overcoming some primary shortcomings of conventional one-photon FRET methods, especially in bioassays, such as the interference from background fluorescence or scattering light, the coexcitation of the energy acceptor with the donor. And such small molecules also show advantages over inorganic upconverting particles that also give anti-Stokes photoluminescence and have been applied as FRET donor recently. The results of this work suggest that two-photon excitable small molecules could be a promising energy donor for FRET-based bioassays.

INTRODUCTION 1

Fluorescence resonance energy transfer (FRET) is defined as the nonradiative transfer of the excited-state energy from a fluorescent donor to an acceptor, which may be fluorescent or not. FRET is considered as a sensitive and reliable “ruler” over distances of 10—100 Å (1) and has broad applications in studying interactions of biological macromolecules (2), in immunoassay (3), and so on. However, it is often seen that the energy acceptor is coexcited with the energy donor because of the overlap of their excitation spectra (4, 5). Meanwhile, autofluorescence or scattering light always arises from biomolecules upon the excitation of energy donor. Therefore, new pairs of energy donor–acceptor are desired for FRET methods, especially in bioassays. Recently, two-photon (TP) excitation has attracted a lot of attention in biological applications. In such a nonlinear optical process, two low-energy photons are simultaneously absorbed to reach the excited state (6). TP excitable materials can be excited in IR region to give emission in the visible region. Under TP excitation with IR light, the autofluorescence of biomolecules and the scattered excitation light could be eliminated, thus the sensitivity is significantly improved. Besides, the IR light causes * Corresponding authors. Zhihong Liu, E-mail: [email protected], Tel: 86-27-87218754, Fax: 86-27-68754067, Address: College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China. Ququan Wang, E-mail: [email protected], Tel: 86-27-68752989-8445, Fax: 86-27-6875-2569, Address: Key Laboratory of Acoustic and Photonic Materials and Devices of Ministry of Education, Wuhan University, Wuhan 430072, China. † College of Chemistry and Molecular Sciences. ‡ Key Laboratory of Acoustic and Photonic Materials and Devices of Ministry of Education. 1 Abbreviations: DABS-Av, DABS-Cl conjugated avidin; DABSBSA, DABS-Cl labeled bovine serum albumin; DABS-Cl, 4-(Dimethylamino)azobenzene-4′-sulfonyl chloride; DMAHAS, trans-4-(N2-hydroxyethyl-N-ethyl amino)-4′-(dimethyl amino)stilbene; FRET, fluorescence resonance energy transfer; QDs, quantum dots; TP, twophoton; TP-FRET, two-photon excitation fluorescence resonance energy transfer; UCPs, up-converting phosphors.

much less photobleaching off the focal plane and less photodamage to biological samples (7, 8). The above unique features have led to the wide use of TP excitation, notably in microscopic imaging (9, 10). Because of the anti-Stokes photoluminescence nature of TP excitation which avoids the direct excitation of energy acceptor, one can expect two-photon excitable materials to be promising energy donors for FRET microscopy. TP-FRET microscopy has been used to characterize the intranuclear dimerization of protein molecules in living cells (11) and to illuminate protein interactions in tissue (12). In biological imaging applications combined with microscopy, TP-FRET has shown higher energy transfer efficiency, less background, and deeper imaging depth than confocal FRET. TP-FRET also has advantages over wide-field FRET as there is less photodamage above and below the focal plane (13). While efforts are being made to apply the anti-Stokes photoluminescence in biological imaging, TP-FRET based quantitative bioassays in homogeneous solutions have been studied much less. So far there have been several reports on FRET bioassays using up-converting phosphors (UCPs) as donors. UCPs are kinds of inorganic particles that give antiStokes photoluminescence via the sequential absorption of two or more low-energy photons, which is similar to TP excitation. Kuningas et al. (14, 15) and Li et al. (16) have developed bioassays based on upconversion fluorescence resonance energy transfer (UC-FRET). Most recently, luminescent quantum dots (QDs)-based FRET driven by a two-photon process, which eliminates the direct excitation of the acceptor and thus results in a near-zero background, has also been reported (17). The significance of using the IR light excitable UCPs as energy donor in bioassays has been proved in these papers. Nevertheless, the relatively large size of UCPs has caused some negative effects (15, 18), such as signal variation, reabsorptive energy transfer of ions in the core, instability of the phosphor bioconjugates, and nonspecific binding interactions. These have reduced the sensitivity of UC-FRET and restricted its applications. Therefore, TP excitable small organic molecules would be a better alternative to UCPs in some aspects as the FRET donor, especially when the nonspecific binding/adsorbing of target

10.1021/bc700369q CCC: $40.75  2008 American Chemical Society Published on Web 01/16/2008

Technical Notes

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Figure 1. Schematic representation of the model FRET system with biotinylated DMAHAS as donor and DABS-Cl conjugated avidin as acceptor. FRET occurred upon the binding of DABS-Av to biotinylated DMAHAS, resulting in the quenching of the donor. With the addition of free avidin, the labeled avidin was partially replaced due to the competition towards biotin, which led to partial recovery of the fluorescence of donor.

molecules on the surface of particles, which always causes false positive results, is considered. Small molecules are capable of overcoming the problems resulted from the size effect of UCPs and thus improve the sensitivity and specificity of quantitative assays. What’s more, organic molecules are structurally more flexible so that it is much easier to configure the molecules and motifs for the purpose of improving FRET efficiency and conjugating with biomolecules. Herein in this work, we have constructed a model assay for avidin (see Figure 1) to demonstrate the merits of TP excitation in FRET-based bioassays. Organic molecule trans-4-(N-2-hydroxyethyl-N-ethyl amino)4′-(dimethyl amino)stilbene (DMAHAS) was employed as energy donor since it is convenient to conjugate with biotin through the reactive hydroxyl group. 4-(Dimethylamino)azobenzene-4′-sulfonyl chloride (DABS-Cl) is a nonfluorescent quencher with high molar extinction coefficient, which makes it a good acceptor in FRET. It was chosen as the acceptor in this work because it has a maximum absorption around 470 nm (19), which matches well with the emission spectrum of DMAHAS. To the best of our knowledge, such TP excitation FRET model for bioassay using small organic molecule as energy donor has not yet been reported in literature.

EXPERIMENTAL PROCEDURES Synthesis of DMAHAS and Biotinylated DMAHAS. DMAHAS was synthesized according to a reported pathway (20), and then biotinylated DMAHAS was prepared with a normal labeling method (21). Biotin (29.3 mg, 0.12 mmol), DMAHAS (31 mg, 0.1 mmol), N,N′-dicyclohexylcarbodiimide (24.8 mg, 0.12 mmol), and 4-dimethylaminopyridine (1.23 mg, 0.01 mmol) were dissolved in anhydrous dichloromethane (10 mL). The mixture was stirred at room temperature for 5 days under argon atmosphere, after which the solution was filtered and the filtrate was evaporated. The residue was purified by column chromatography on silica gel using a 20:1 (v/v) ethyl acetate/methanol as eluent, and a yellow crystal was obtained (structures shown in Figure 2).1H NMR (CDCl3), δ: 7.35 (m, 4H), 6.83 (s, 2H), 6.71 (m, 4H), 5.20 (s, 1 H, NH), 4.87 (s, 1 H, NH), 4.44 (m, 1H), 4.25 (m, 3H), 3.56 (m, 2H), 3.41 (m, 2H), 3.08 (m, 1H), 2.96 (s, 6H), 2.90—2.67 (m, 2H), 2.31 (t, 2H), 1.63 (m, 4H), 1.38 (m, 2H), 1.17 (t, 3H). HRMS (ESITOF) calcd for C30H40N4O3S [M + H]+ 537.2899, found 537.2901. Preparation of DABS-Cl Conjugated Avidin. DABS-Av conjugate was prepared using a method analogous to others (22). Avidin was dissolved in 0.1 M sodium carbonate-sodium bicarbonate buffer, pH 9.1, at a concentration of 1 mg/mL. DABS-Cl was dissolved in DMF at a concentration of 1 mg/ mL. The DABS-Cl solution was protected from light and used immediately. With gentle shaking, 30 µL of the DABS-Cl solution was slowly added to the avidin solution. After reacting

Figure 2. Structures of (A) DMAHAS, (B) biotinylated DMAHAS.

at room temperature for 2 h in the dark, the unbound DABS-Cl molecules were removed by exhaustive dialysis against the above buffer overnight at 4 °C. The dye to protein ratio was estimated as follows: the concentration of DABS-Cl was determined by the absorbance at 474 nm, using the molar extinction coefficient of 3.3 × 104 M-1 cm-1. Since DABS-Cl has a contribution to the absorbance at 280 nm, the concentration of avidin was determined by the absorbance at 280 nm after deducting the contribution of DABSCl at this wavelength, using the molar extinction coefficient of 8.88 × 104 M-1 cm-1 (23). Preparation of DABS-BSA Conjugate. Similar to the process for making DABS-Av, BSA was dissolved in 0.1 M sodium carbonate-sodium bicarbonate buffer, pH 9.1, at a concentration of 1 mg/mL. And DABS-Cl was also dissolved in DMF at a concentration of 1 mg/mL, which was protected from light and used immediately. With gentle shaking, 100 µL of the DABS-Cl solution was slowly added to the BSA solution. After reacting at room temperature for 2 h in the dark, the unbound DABS-Cl molecules were removed by exhaustive dialysis against the above buffer overnight at 4 °C. The dye-to-protein ratio was determined with the same manner as DABS-to-avidin ratio described above. Measurement of TP Excited Fluorescence. Two-photon excited fluorescence detection system included two parts. The excitation light was provided by a mode-locked pulsed laser (Mira 900, Coherent) to provide high instantaneous photon flux density. The photoluminescence was recorded on a liquidnitrogen cooled CCD detector (SPEC-10, Princeton) through a monochromator (Spectrapro 2500i, Acton). Two-photon fluorescence was excited at 800 nm with ∼3 ps pulse width and 76 MHz repetition rate. The fluorescence intensity was given in normalized form, i.e., the maximum emission of each group of data was set as 1, and others were calculated proportionally. A LS 55 fluorometer (Perkin-Elmer) equipped with 1 cm cell was used for one-photon fluorescence measurements. In a typical FRET experiment, 1 × 10-4 M biotinylated DMAHAS in DMF was diluted to 1 × 10-6 M with phosphate buffer, thus the solvent was a mixture of aqueous buffer (0.1 M phosphate buffer, pH 7.4) and 1% DMF (v/v). Under room temperature, DABS-Cl labeled avidin (0.145 µM avidin with the dye-to-protein ratio as 1.6) was added to the solution, which was then vortexed and incubated for 15 min. Then the fluorescence emission of biotinylated DMAHAS was recorded under excitation at 800 nm. For the fluorescence recovery, 1 ×

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Figure 3. Linear absorption spectrum of DMAHAS (1 × 10-5 M in DMF). -6

10 M free avidin was added to another tube containing the same amount of biotinylated DMAHAS and DABS-Cl labeled avidin, which had been allowed to react for 15 min. After thoroughly mixing and incubating for another 15 min, the mixture was subjected to fluorescence measurement. In the control experiments which were performed to exclude nonspecific interactions of biomolecules, 1 × 10-6 M biotinylated DMAHAS was incubated, also for 15 min under room temperature, with DABS-Cl labeled BSA (0.122 µM BSA with the dye-to-protein ratio as 1.6) and 0.18 µM free avidin, respectively. And the fluorescence of the donor was detected likewise. For competitive assay of avidin, varying concentrations of free avidin (0, 0.1 µM, 0.4 µM, 0.8 µM, 1.3 µM, 1.9 µM, and 2.6 µM) were incubated with 1 × 10-5 M biotinylated DMAHAS and DABS-Cl labeled avidin (concentration fixed at 2.4 µM) for 15 min, and then the same fluorescence detecting procedure as above was followed for each sample.

Liu et al.

Figure 4. Two-photon excitation fluorescence spectra. (—) DMAHAS (1 × 10-2 M in DMF), (0) biotinylated DMAHAS (1 × 10-2M in DMF). Excitation wavelength ) 800 nm.

Figure 5. UV–vis absorption spectra for the calculation of DABS-toavidin ratio. (—) the DABS-Av conjugate, (0) DABS-Cl (1 × 10-5 M in acetonitrile).

RESULTS Spectral Properties of the Donor. In order to confirm the TP excitation luminescence of the donor, the linear absorption spectrum of DMAHAS was examined first. From Figure 3, it is seen that its maximum absorption locates at 372 nm and there is no linear absorption in the range from 430 to 900 nm, which indicates that the photoluminescence of DMAHAS excited with 800 nm is purely the two-photon induced fluorescence. The TP excited fluorescence of the organic molecule is structurally originated from its symmetrical D-π-D conjugate motif. The biotinylation on the terminal hydroxyl group does not destroy the symmetrical charge transfer within the conjugated chain or the rigid plane of the molecule. Therefore, the biotinylation did not alter the nonlinear photoluminescence of DMAHAS. The fluorescence emission of both DMAHAS and biotin-conjugated DMAHAS under TP excitation model at 800 nm are presented in Figure 4, which shows that the TP fluorescence emission of biotinylated DMAHAS did not differ much from that of DMAHAS. Optimization of the DABS-to-Avidin Ratio. The FRET efficiency can be adjusted by altering the dye-to-protein ratio. Generally speaking, increasing the dye-to-protein ratio will enhance the overall FRET efficiency due to the increased overlap integral (24). However, high dye-to-protein ratio does not always work well because labeling with more than one fluorophore may also result in problems, like concentration-dependent quenching, which will limit the detection sensitivity. Besides, the solubility and activity of the complex may be decreased in cases when coupling water-insoluble molecules to the surface of proteins owing to the hydrophobicity of the conjugate (22). In our experiments, we also found that adding excessive DABS-Cl to avidin solution would cause severe aggregation of the protein.

Figure 6. Two-photon excitation fluorescence spectra of the FRET model. (—) biotinylated DMAHAS (1 × 10-6 M), (. . .) biotinylated DMAHAS + DABS-Av (0.145 µM avidin with the dye-to-protein ratio as 1.6), (0) biotinylated DMAHAS + DABS-Av + unlabeled avidin (1 × 10-6 M). Excitation wavelength ) 800 nm.

In this case, the dye-to-protein ratio should be scaled back. The UV–vis absorption spectra of pure DABS-Cl and the DABSAv complex are illustrated in Figure 5, based on which the DABS-to-avidin ratio was calculated as 1.6. Construction of the TP Excitation FRET Model. FRET occurred upon the addition of DABS-Cl conjugated avidin to the biotinylated DMAHAS solution, since DMAHAS and DABS-Cl were in proximity due to the specific binding of biotin and avidin. As is shown in Figure 6, when avidin conjugate was present, the two-photon excited fluorescence of biotinylated DMAHAS, the donor, was quenched by the acceptor. With adding free avidin to the above system, the fluorescence intensity

Technical Notes

Figure 7. Two-photon excitation fluorescence spectra of control experiments. (A) (—) biotinylated DMAHAS (1 × 10-6 M), (0) biotinylated DMAHAS + DABS-Cl labeled BSA (0.122 µM BSA with the dye-to-protein ratio as 1.6). (B) (—) biotinylated DMAHAS (1 × 10-6 M), (0) biotinylated DMAHAS + unlabeled avidin (0.18 µM). Excitation wavelength ) 800 nm.

recovered partially, which resulted from the competition between the labeled and unlabeled avidin toward biotin. The replacement of labeled avidin with free avidin is due to the stronger biotin binding ability of the latter (25). Examination on the Specificity of the TP-FRET Model. Two control experiments were performed to exclude nonspecific interactions of biomolecules, in which biotinylated DMAHAS was incubated with DABS-Cl labeled BSA or with free avidin. DABS-BSA conjugate was prepared with the same method as DABS-Av. The dye-to-protein ratio was also determined to be 1.6. The fluorescence of biotinylated DMAHAS was not quenched by DABS-Cl labeled BSA which had almost the same concentration as avidin used to quench the fluorescence of biotinylated DMAHAS in the FRET model, i.e., 0.122 µM BSA versus 0.145 µM avidin (Figure 7A). And in another experiment, the fluorescence of biotinylated DMAHAS was not directly enhanced with 0.18 µM free avidin either (Figure 7B). These results indicated that the quenching and the recovery of the fluorescence of the donor were not induced by any nonspecific interactions of macromolecules, which is always a troublesome problem in large-size-particle-based biosensors (nanometer to micrometer magnitude), such as the UCPs described above. In order to illustrate the ability of TP excitation FRET to exclude interferences from autofluorescence or scattering light, the background signal of biomolecules under one-photon and two-photon excitation was compared. Figure 8A indicates that in the case of one-photon excitation, when exciting avidin and DABS-Cl labeled avidin at the optimum excitation wavelength of DMAHAS at 372 nm, the background light around 420–480 nm could not be ignored, which would interfere the luminescence of DMAHAS and thus reduce the sensitivity. While under two-photon excitation at 800 nm there was only noise signal from the instrument, which could be negligible (Figure 8B). Competitive Assay for Avidin. Finally, the TP-FRET model was tried to investigate the dependence of the relative fluorescence intensity (FL/FL0) of the donor on the concentration of free avidin (where FL0 represents the fluorescence intensity in

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Figure 8. Comparison on the background signal under one-photon and two-photon excitation. (A) one-photon excitation. (—) DABS-Cl labeled avidin (0.145 µM avidin with the dye-to-protein ratio as 1.6), (0) avidin (1 × 10-6 M). Excitation wavelength ) 372 nm, Excitation slit ) 5 nm, Emission slit ) 15 nm. (B) two-photon excitation. (—) DABS-Cl labeled avidin (0.145 µM avidin with the dye-to-protein ratio as 1.6), (0) avidin (1 × 10-6 M). Excitation wavelength ) 800 nm.

Figure 9. Competitive assay for avidin. (A) The dependence of FL/FL0 of biotinylated DMAHAS on the concentration of free avidin. (B) The linear calibration curve of FL/ FL0 to the concentration of free avidin within the range 0.4-2.6 µM.

the absence of free avidin and FL represents the fluorescence intensity under different concentrations of free avidin). As shown in Figure 9, the FL/FL0 of the donor is dependent on avidin concentration and is linear to the concentration in a certain range (0.4-2.6 µM) with a correlation coefficient of 0.9947, which suggests that the two-photon FRET model could be a candidate for quantitative bioassay.

DISCUSSION The results show that TP-FRET bioassays can overcome the shortcomings of conventional one-photon-FRET and that small organic molecules have advantages over the newly reported inorganic particles as the energy donor in bioassays. They have supported the hypothesis that TP excitable small organic molecules could be a kind of competent energy donor for FRET in homogeneous bioassays. However, it must be pointed out that, as a start of investigating the applicability of TP-FRET

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with small molecule as energy donor in bioassay, the biocompatibility of the donor molecule used here and the analytical performance of the model have not been considered intensively. As a quantitative bioassay, the linear range for avidin is somewhat narrow, and the sensitivity is not as good as the reported UCPs-based biosensors. This might be mainly caused by the rather poor solubility of biotinylated DMAHAS in aqueous solution, which results in aggregation or precipitation out of water at high concentrations. Besides, the moderate TP absorption cross section of DMAHAS also restricted the detection sensitivity. An ideal TP-FRET donor molecule is expected to have large TP absorption cross section, high fluorescence quantum yield as well as good biological compatibility such as water solubility. Although the investigations on the relationship between molecular structure and TP absorption property over the past few years have enabled people to design and synthesize molecules with large TP absorption cross section (26, 27), most of them have poor solubility in water preventing them from biological applications. Whereas many of the water soluble fluorophores commonly used as fluorescent tags in biological samples, if TP excitable, only have a small TP absorption cross section (28, 29), for example, the value for fluorescein in water at pH 13 is only 36 GM (30) (1 GM ) 1 × 10-50 cm4 s photon-1). Therefore for further research concerning TP-FRET methods in quantitative bioassay, emphasis should be put on development of donor molecules with both high water solubility and larger TP absorption cross section. It seems there is a conflict between the two aspects because, generally speaking, a larger absorption cross section requires a larger hydrophobic conjugated motif within the molecule, which always leads to the decrease of water solubility. Nevertheless, great efforts are being made and some exciting results have already been obtained in most recent years. For instance, by encapsulating the hydrophobic TP excitable molecules into the hydrophobic interior of micelles, large TP absorption cross section in water is achieved (31–33). Recently, a new type of fluorescent chromophores with efficient TP absorption cross sections and high fluorescence quantum yield in aqueous medium as well as in organic medium have been successfully developed (34). As such examples are so limited, it remains a challenge for scientists to investigate the effect of solvent especially water on the properties of two-photon excited molecules and to develop molecules with large TP absorption cross section in aqueous solutions.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 20675059) and the Science Fund for Creative Research Groups (No. 20621502), NSFC and The National Key Scientific Program-Nanoscience and Nanotechnology (No. 2006CB933103). Authors thank Dr. Song Wu and Dr. Bao-Ping Zhai for helping in synthetic work.

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