Article pubs.acs.org/ac
Two-Photon Graphene Oxide/Aptamer Nanosensing Conjugate for In Vitro or In Vivo Molecular Probing Mei Yi,†,§ Sheng Yang,†,§ Zanying Peng,† Changhui Liu,† Jishan Li,*,† Wenwan Zhong,‡ Ronghua Yang,† and Weihong Tan† †
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China ‡ Department of Chemistry, University of California-Riverside, Riverside, California 92521, United States S Supporting Information *
ABSTRACT: Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source have the unique properties of lower tissue autofluorescence and self-absorption, reduced photodamage and photobleaching, higher spatial resolution, and deeper penetration depth (>500 μm). Carbon nanomaterials, for example, graphene oxide (GO), have the advantages of good biocompatibility, efficient transporters into cells, protecting the carried DNA or peptides from enzymatic cleavage, and super fluorescence quenching efficiency. By combination of the nanostructured carbon materials with the TPE technique, herein we have designed an aptamer-twophoton dye (TPdye)/GO TPE fluorescent nanosensing conjugate for molecular probing in biological fluids, living cells, and zebrafish. This approach takes advantage of the exceptional quenching capability of GO for the proximate TP dyes and the higher affinity of single-stranded DNA on GO than the aptamer− target complex. Successful in vitro and in vivo detection of ATP was demonstrated with this sensing strategy. Our results reveal that the GO/Aptamer−TPdye system not only is a robust, sensitive, and selective sensor for quantitative detection of ATP in the complex biological environment but also can be efficiently delivered into live cells or tissues and act as a “signal-on” in vivo sensor for specific, high-contrast imaging of target biomolecules. Our design provides a methodology model scheme for development of future carbon nanomaterial-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species.
T
These limitations in OPE motivated us to design the twophoton dye-embedded nanoprobes for molecular detection in complicated biological samples or in vivo imaging through twophoton excitation (TPE).9 TPE with NIR photons as the excitation source have the advantages of lower tissue autofluorescence and self-absorption, reduced photodamage and photobleaching, and deeper penetration depth (>500 μm), etc. Together with the development of two-photon microscopy (TPM), TPE has become a powerful tool for research in life science.10,11 In recent years, although a variety of organic molecule-based two-photon probes have been reported for various biotargets such as anions,12 metal ions,13,14 reactive oxygen species,15,16 and other biomolecules,17−19 the twophoton dye (TPdye)/nanostructure conjugates as TP nanoprobes for in vitro or in vivo assays have not been reported so far. Therefore, considering the unique properties of the nanomaterials and the TPE technique, development of carbon nanostructure-based TPE nanoprobes with good biocompati-
he development of hybrid biological-synthetic nanomaterials for sensing and delivery is of crucial importance for clinical diagnostics and therapeutics.1 Carbon nanomaterials, as the popular star nanomaterial with the properties of good biocompatibility, high internalization efficiency, simple functionalization, excellent quenching ability to the adjacent fluorophores and the protecting effect on the carried DNA or peptides from enzymatic cleavage, have gained great attentions in biomedical and bioanalytical fields.2−5 However, most of the fluorescent dyes used in previous carbon nanomaterials-based sensing systems are the traditional one-photon excited (OPE) fluorophores, for example, fluorescein, rhodamine, Cy3, etc.6 Such dyes require rather short excitation wavelengths (18.3 MΩ. All experiments were carried out at room temperature. UV−vis absorption spectra were measured on a Hitachi U4100 spectrophotometer (Kyoto, Japan). One-photon fluorescence and anisotropy measurements were performed on a PTI QM4 Fluorescence System (Photo Technology International, Birmingham, NJ). Two-photon fluorescence spectra were obtained with a mode-locked Ti:sapphire pulsed laser (Chameleon Ultra II, Coherent Inc.) and then recording with a DCS200PC single photon counting (Beijing Zolix Instruments Co., Ltd.). TPM imaging of HeLa cells and zebrafish were carried out by using an Olympus FV1000-MPE multiphoton laser scanning confocal microscope (Japan). Atomic force microscopy (AFM) measurements were performed by using a Nanoscope Vmultimode atomic force microscope (Veeco Instruments). Energy-dispersive X-ray (EDX) analysis was carried out using a HITACHI S-4500 instrument. pH was measured by a model 868 pH meter (Orion). Preparation of GO/Aptamer−TPdye Conjugate. According to the previous reported methods for preparation of GO/DNA complexes,35 the TPdye-labeled ATP aptamer/GO conjugates were prepared as the following procedures. Briefly, the GO was sonicated in doubly deionized water for 2 h and then the centrifugation step was carried out at 3000 rpm to get rid of the larger aggregates of GO, leading to a homogeneous black solution (the concentration of GO is ∼2 mg/mL). After the pretreatment, enough GO was introduced into 500 μL of the HEPES buffer (20 mM, pH 7.4) containing 100 nM of Aptamer−TPdye (the final concentration of GO is ∼40 μg/ mL), and the mixture was incubated for several minutes at room temperature to form the GO/Aptamer−TPdye conjugate. Then the obtained GO/Aptamer−TPdye conjugate was stored in a refrigerator at 4 °C before further usage. The GO/ R-TPdye conjugate was also prepared as the above descriptions. Both GO and GO/Aptamer conjugate were characterized on an atomic force microscopy using tapping mode to simultaneously collect height and phase data. EDX analysis of GO and GO/ Aptamer conjugate was carried out using a HITACHI S-4500 instrument. In Vitro Detection of ATP by the GO/Aptamer−TPdye Nanosensing Conjugate. In a typical assay, an aliquot of 200 μL of GO/Aptamer−TPdye conjugate suspension was first added in 200 μL of HEPES buffer or cell media (the final
bility and efficient transporting property, lower biological autofluorescence and self-absorption, higher spatial resolution and deeper penetration depth for in vitro or in vivo assay will have a fascinating prospect. Herein, we report a TPdye labeled aptamer/graphene oxide (GO)-conjugate for in vitro or in vivo detection of adenosine triphosphate (ATP) (Scheme 1). Aptamers are single-stranded Scheme 1. Schematic Illustration of Two-Photon Graphene Oxide/Aptamer-Based Nanosensing Conjugatea
Assembly of aptamer−TPdye with GO led to the fluorescence emission in the “off” state due to the fluorescence resonance energy transfer (FRET) effect between TPdye and GO. After the GO/ Aptamer−TPdye conjugate was incubated with ATP-containing sample, the nanoprobe would be in the “on” state as a result of the release of TP fluorophores from the GO surface, thus providing greatly enhanced fluorescence emission intensity. a
DNA molecules that can specifically bind to non-nucleic acid targets.20 They have been widely used for detection of a broad range of molecules including ions, small biomolecules, and proteins.21,22 GO, a water-soluble derivative of one-atom-thick two-dimensional carbon material, has recently attracted great interest for biomedical applications.23 The unique capacity of GO in adsorbing biomolecules, such as nucleic acids and peptides, with super fluorescence quenching efficiency creates a robust platform for the development of biosensors.24 ATP, as a multifunctional intracellular signaling molecule, plays significant roles in many biological processes, such as universal energy flow in living cells, metabolic reactions, material trafficking, and membrane transport.25−28 Because of its great importance, many methods have been developed for in situ detection and imaging of ATP.29−34 However, efficient TPE nanoprobes for in vitro or in vivo assay of ATP appear to be rare. Thus we prepared a GO/ATP aptamer−TPdye conjugate as proof-ofconcept of the sensing platform for in vitro or in vivo measurement of ATP. When the analyte is not presented, the nanoprobe was initially in “off” state due to the efficient quenching of GO for fluorophores adjacent to its surface. After the GO−ATP aptamer conjugate was incubated with targetcontaining sample or transported into cells followed by binding of the aptamer by intracellular ATP, the nanoprobe would be in the “on” state as a result of the release of TP fluorophores from the GO surface, thus providing greatly enhanced fluorescence emission intensity.
■
EXPERIMENTAL SECTION Chemicals and Instruments. The TPdye used in this work was synthesized as described in the Supporting Information, and the TPdye-labeled ATP aptamer (Aptamer3549
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
Analytical Chemistry
Article
concentration of Aptamer−TPdye is 50 nM and GO is 20 μg/ mL), then ATP (final concentrations ranging from 0 to 3 mM) or other biomolecules was added and incubated at roomtemperature for 30 min. After reaction, the resulting solution was subjected to fluorescence measurements using the OPE or TPE method. For the OPE measurement, the fluorescence spectra were recorded in a quartz cuvette on PTI QM4 Fluorescence System with the excitation wavelength of 370 nm and the emission wavelengths in the range from 430 to 580 nm with both excitation and emission slits of 10 nm. For the TPE measurement, the two-photon emission fluorescence spectra in the range from 430 to 550 nm were obtained by exciting all samples at 750 nm with a mode-locked Ti:sapphire pulsed laser (Output laser pulses were centered at 750 nm and an average power of 100 mW was as the excitation source. The laser pulses have pulse duration of 120 fs and repetition rate of 80 MHz.), followed by recording with a DCS200PC single photon counting. Cytotoxicity Assays and Live Cell Imaging with the GO/Aptamer−TPdye Conjugate. HeLa cells were grown in RPMI 1640 medium (Thermo Scientific HyClone) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/mL penicillin, and 100 U/mL gentamicin at 37 °C in a humidified atmosphere containing 5% CO2. The cytotoxic effects of GO were assessed using the MTT assay.36 For cell imaging experiments, cells were seeded in 24-well culture plate and grown overnight on glass coverslips at the bottom of the plate. When the cells were ∼90% confluent, the coverslips were washed three times with phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). Before incubation with probes, the HeLa cells were incubated with/without 10 μM oligomycin or 5 mM Ca2+ for 30 min. Then, 500 μL of fresh cell growth medium supplemented with the GO/Aptamer−TPdye conjugate ([Aptamer−TPdye] = 50 nM, [GO] = 20 μg/mL), the GO/R−TPdye mixture ([R−TPdye] = 50 nM, [GO] = 20 μg/ mL), the GO (∼20 μg/mL) only, or the aptamer−TPdye (50 nM) was added in each well, respectively. After incubation for 8 h, the cells were washed with Dulbecco’s phosphate buffered saline (DPBS) three times. Two-photon confocal fluorescence imaging of HeLa cells was observed under an Olympus FV1000-MPE multiphoton laser scanning confocal microscope, with a mode-locked titanium-sapphire laser source (120 fs pulse width, 80 MHz repetition rate) set at wavelength 750 nm. Three dimensional images were taken every 2 μm by scanning the samples across a defined section along the z-axis. Taking into account the possible photothermal effect of GO under NIR irradiation, the laser power was set at 2 mW to prevent potential thermal damage to cells during imaging.37 TPM Images of Zebrafish by Using GO/Aptamer− TPdye Nanosensing Conjugate. Zebrafishes were maintained in E3 embryo media (15 mM NaCl, 0.5 mM KCl, 1.0 mM MgSO4, 1.0 mM CaCl2, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3, 5−10% methylene blue, pH 7.5). In fluorescence imaging experiments, two-day-old zebrafishes were, respectively, incubated with GO/Aptamer−TPdye conjugate ([Aptamer−TPdye] = 100 nM, [GO] = 40 μg/ mL) or GO/R−TPdye conjugate ([R−TPdye] = 100 nM, [GO] = 40 μg/mL) in E3 embryo media for 15 h at 28 °C. After washing with PBS (pH 7.4) to remove the remaining probe, two-photon confocal fluorescence imaging and the 3D two-photon confocal fluorescence images (accumulated along the z direction at depth of 0−270 μm with 60× magnification
of these treated zebrafishes) were observed under an Olympus FV1000-MPE multiphoton laser scanning confocal microscope using a mode-locked titanium-sapphire laser source (120 fs pulse width, 80 MHz repetition rate) set at wavelength 750 nm with laser power of 2 mW.
■
RESULTS AND DISCUSSION Characterization of the Two-Photon Fluorescent Dye. As a newly synthesized fluorescent label, its optical properties should be carefully examined before biomedical applications. As is exhibited in Figure 1A, this TPdye displays a strong
Figure 1. (A) Absorption (dashed lines) and one-photon emission spectra (solid lines) of 10 μM TPdye in HEPES buffer solution (pH 7.4), λex = 370 nm. Inset: photographs of the corresponding absorption and fluorescent species. (B) Two-photon absorption action cross sections of TPdye in HEPES buffer solution. All error bars were obtained through the detection of three parallel samples. The inset shows TP excited fluorescence intensity of TPdye (10 μM) in HEPES buffer solution, λex = 750 nm.
electronic transition at λmax,abs = 370 nm (ε = 2.2 × 104 M−1 cm−1) and its OPE emission spectrum shows a peak maximum at 460 nm with an obvious cyan fluorescence under UV lamp excitation. The measurement of luminescence quantum yield (Φ) with quinine sulfate dihydrate as the reference shows that the quantum yield of the TPdye is 0.47.38 The luminescent brightness of this TPdye (defined as ε × Φ) is estimated to be 1.03 × 104 M−1 cm−1, suggesting a remarkable signal response capability in bioanalysis. We also measured the two-photon absorption (TPA) action cross section (δ × Φ, δ is the TPA cross section) and the TPE emission spectrum of the TPdye. As shown in Figure 1B, the maximal TPA action cross section of the TPdye in aqueous solution was measured to be 50 Goeppert-Mayer (GM) at room temperature (λex = 750 nm, 1 GM = 10−50 cm4 s photon−1 molecule−1) with rhodamine B as the reference,39 which is approximate to that of the similar compound reported previously.40 The TPE emission spectrum of the TPdye is shown in the inset of Figure 1B. The sample was excited with femtosecond laser pulses with a central wavelength at 750 nm and a pulse duration of 40 fs. As can be seen, the corresponding maximal emission peak of this TPE emission spectrum is also located at 460 nm and the profile of this TPE emission spectrum matches exactly to that of the OPE emission spectrum, indicating that the fluorescence emission excited at the near-infrared region is purely the two-photoninduced fluorescence. Long-term stability of the two-photon dyes is crucial to their practical applications in biolabeling or bioimaging. So the stability of this TPdye under various environmental conditions was investigated through testing its luminescence intensity. The photostability of the TPdye was first measured by using a 150 W xenon lamp as an excitation source. The emission intensity 3550
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
Analytical Chemistry
Article
decreased by only 1% after irradiation for 1 h, indicating the good photostability of this TPdye (see Figure S1 in the Supporting Information). We then explored the effect of pH on the TPdye at pH values ranging from 4.0 to 10.0 and observed little changes in the fluorescence intensity which demonstrated that this new two-photon dye was stable at physiological pH conditions (Figure S2, Supporting Information). Additionally, there was no significant variation in fluorescence intensity of the TPdye with increasing concentrations of the physiological relevant metal ions, including Na+, K+, Ca2+, and Mg2+ (Figure S3, Supporting Information), indicating that this TPdye is stable under the physiological ion strength, even with extremely high ion concentrations. Furthermore, the stability of this TPdye in human serum or human cervix carcinoma (HeLa) cell lysate was also investigated through the fluorescence intensity measurements. The results show that the TPdye molecules is still stable after tens of hours of incubation (Figure S4, Supporting Information). The higher δ × Φ value, good photostability, and long-term stability in various environmental conditions support that this TPdye is a promising luminescence emitter for TP imaging applications in complex biological samples. Formation and Characterization of the GO/Aptamer− TPdye Conjugate. Because single-stranded DNA sequences (ssDNA) have been proven to be assembled on GO surfaces with strong affinity,41 a noncovalent complex of GO/Aptamer− TPdye was prepared by simple self-assembly of Aptamer− TPdye with GO. We can see from Figure S5 (Supporting Information) that the fluorescence intensity of the Aptamer− TPdye solution decreases proportionally with the addition of increasing amounts of GO, indicating the growing number of Aptamer−TPdye adsorbed onto the surface of GO, thus resulting in the quenching of the TPdye’s fluorescence by way of fluorescence resonance energy transfer (FRET) processes.42 When the concentration of GO added reached about 20 μg/ mL, the fluorescence of 50 nM Aptamer−TPdye was nearly completely quenched. The GO/Aptamer−TPdye conjugate was then characterized by atomic force microscopy (AFM) and energy-dispersive X-ray (EDX) spectroscopy to obtain its morphological profile and structural information. The GO sheets mostly have a lateral width of less than 200 nm with a topological height of about 1 nm (Figure S6A in the Supporting Information), which would increase the cell uptake effciency of GO and be beneficial for bioimaging.43 After conjugation of the aptamer−TPdye with GO, the GO/Aptamer−TPdye conjugate had a topological height of over 2 nm across the lateral dimension (Figure S6B in the Supporting Information) and an appearance of the P element which belongs to oligonucleotides in the EDX pattern of GO/Aptamer−TPdye conjugate. The increase in the ratio of O and C compared with that of GO (Figure S7 in the Supporting Information) implied coverage of Aptamer−TPdye on the GO surfaces and the successful formation of GO/Aptamer−TPdye nanosensing conjugate. Validation of the Design Scheme. Scheme 1 illustrates the signaling mechanism of the GO/Aptamer−TPdye nanosensing conjugate. To verify this design scheme, we investigated the real-time records of OPE fluorescence intensity and the anisotropy changes of Aptamer−TPdye in the HEPES buffer solution upon addition of GO and subsequently ATP (Figure 2A). In aqueous solution, Aptamer−TPdye has a strong fluorescence emission, while the fluorescence emission was quenched greatly after the addition of GO (curve a), indicating the interaction of the TPdye with GO, allowing the energy
Figure 2. (A) Real-time recordings of the one-photon fluorescence emission intensity (a) and anisotropy (b) changes of Aptamer−TPdye as a function of time upon additions of GO and subsequently ATP. (B) Relative fluorescence responses of GO/Aptamer−TPdye to CTP, GTP, UTP, and ATP. All error bars were obtained through the detection of three parallel samples. [Aptamer−TPdye] = 50 nM, [GO] = 20 μg/mL, [NTP] = 1 mM. λex = 370 nm.
transfer process to occur. Distinct increase of fluorescence emission after ATP addition verified the feasibility of the sensing scheme. Curve b shows the fluorescence anisotropy (FA) value change of the Aptamer−TPdye under the same conditions. The FA value of Aptamer−TPdye at the free state in the buffer is very low (0.047). However, it underwent a remarkable enhancement upon addition of GO. This result indicates that binding of GO to the ssDNA strand created a larger mass complex, which hindered the rotation diffusion rate of the labeled TPdye.44 The significant difference in the FA values of GO and the GO/Aptamer−TPdye further confirms the formation of GO/Aptamer−TPdye conjugate. The FA value was reduced from 0.228 to 0.113 with addition of ATP, meaning that formation of the Aptamer−TPdye/ATP complexes freed the Aptamer−TPdye from the GO surface. It is worth noting that the fluorescence intensity and anisotropy changes of GO/Aptamer−TPdye nanosensing conjugate reached equilibrium within a few minutes. This indicates the potential of our assay for rapid and real time monitoring of the target in homogeneous solutions. Further inspection of the GO/Aptamer−TPdye nanosensing conjugate was also performed by using gel electrophoresis (Figure S8 in the Supporting Information). The results show that there is no significant ATP aptamer band on the gel image for the supernatant of GO/Aptamer−TPdye conjugates (lane b), while for the supernatant of GO/Aptamer−TPdye conjugates with addition of ATP, there is a clear aptamer band on the gel image (lane c), indicating that the formed aptamer−target complex fell off from the GO surface and stayed in the supernatant after centrifugation. These results agree well with the above fluorescence emission experiments. To evaluate the selectivity of our assay, the TPdye fluorescence intensity changes with the addition of different targets (CTP, UTP, GTP, and ATP) were studied. As shown in Figure 2B, when CTP, UTP, and GTP were at a concentration of 1.0 mM, the system only gives a small TPdye fluorescence response, while a significant TPdye fluorescence enhancement occurs after the introduction of 1.0 mM ATP to the solution under the same conditions. This selectivity is similar to that of the original ATP aptamer, confirming that the conjugation of GO with the aptamer does not affect the selectivity of the aptamer. GO/Aptamer−TPdye Nanosensing Conjugate for ATP Probing in Cell Media by OPE and TPE Technique. Measurement in complex biological samples often suffers from the high fluorescence background produced by the ubiquitous 3551
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
Analytical Chemistry
Article
endogenous components in the sample matrix,45 failing the commonly used biosensors without sample pretreatment. To address this limitation, NIR fluorophores, time-resolved fluorescence method, or TP technique are usually employed to realize the target assay in complex biological samples. The TPE-based fluorescence strategies might be the best choice due to the key advantages of the TP technique, namely, lower selfabsorption and lower biological autofluorescence, reduced photodamage, greater penetration depth (>500 μm), and higher spatial resolution. To evaluate the performance of this GO/Aptamer−TPdye nanosensing conjugate in complex conditions, we carried out ATP measurements in cell media using the OPE and TPE techniques. Figure 3A,B shows the
obtained when the ATP concentration is increased, thus failing to measure the target successfully. However, Figure 3D showed that the TPE fluorescence emission was very weak when the target ATP was not presented, while remarkable enhancements were observed with increasing ATP concentrations. The dosedependent intensity of the 460 nm band showed the fluorescence signal dynamically increased with the addition of ATP in the concentration range of 10 μM to 3 mM, and a satisfactory detection limit of 0.5 μM ATP that was taken to be 3 times the standard derivation in blank solution was achieved (inset of Figure 3D). These results suggest that the TPE method is more suitable for biological assays compared with the OPE-based methods, owing to the low background fluorescence. GO/Aptamer−TPdye Nanosensing Conjugate for ATP Probing in Living Cells. After demonstrating in vitro sensing of ATP by the GO/Aptamer−TPdye conjugate in the complex biological sample, we then explored its potential for live-cell imaging of ATP. First, the cytotoxicities of GO on HeLa cells were evaluated using the standard cell viability assay, the MTT assay. After the HeLa cells were treated with GO concentrations up to 50 μg/mL for 24 h, high cell viability was observed (survival rate was higher than 90% in 1.0 × 104 cells/ well) (Figure S9, Supporting Information). Throughout the present study for cell imaging, we used GO of less than 20 μg/ mL, which ensures high viability of all the cells we tested. The TPE confocal fluorescence imaging in HeLa cells was then carried out. As shown in Figure 4, a significant TPE
Figure 3. OPE (A) and TPE (B) emission spectra of the only cell growth media (a), Aptamer−TPdye in HEPES buffer solution (b), and Aptamer−TPdye in cell growth media (c). OPE (C) and TPE (D) fluorescence emission changes of GO/Aptamer−TPdye nanosensing conjugate in the presence of different concentrations of ATP in cell growth media. Inset: Signal to background ratios (S/B) of GO/ Aptamer−TPdye conjugate as a function of ATP concentrations. All error bars were obtained through the detection of three parallel samples. [Aptamer−TPdye] = 50 nM, [GO] = 20 μg/mL, [ATP] = 0−3 mM. λex = 370 nm/750 nm.
Figure 4. TP confocal microscopy fluorescence images (up), brightfield images (middle), and the overlay of fluorescence and bright-field images (down) of HeLa cells after incubation with GO/Aptamer− TPdye conjugates (A), Aptamer−TPdye (B), GO/R−TPdye (C), and treated with 10 μg/mL oligomycin (D) or with 5 mM Ca2+ (E) followed by incubation with GO/Aptamer−TPdye nanosensing conjugate. Scale bar: 20 μm. [Aptamer−TPdye] = 50 nM, [R− TPdye] = 50 nM, [GO] = 20 μg/mL.
OPE and TPE emission spectra of the Aptamer−TPdye in the HEPES buffer and the 1640 cell growth media, respectively. One can see from Figure 3A that the cell growth media had a high autofluorescence and dominated the fluorescence spectra from 430 to 580 nm under the OPE. Furthermore, the fluorescence emission intensity inversely decreased when the aptamer−TPdye was dispersed into the cell growth media, which might be a result from the higher self-absorption of the cell growth media. However, in contrast to the OPE measurements, one can find that the TPE fluorescence emission intensity and its emission spectrum in the cell growth media were almost the same as in the HEPES buffer (Figure 3B). Figure 3C,D shows the fluorescence emission spectra of this nanosensing conjugate in cell media with various concentrations of ATP by using the OPE and TPE method, respectively. For the OPE method (Figure 3C), the background fluorescence emission was very strong even without target ATP and a significant fluorescence emission increase cannot be
fluorescence image was obtained for the cells incubated with the GO/Aptamer−TPdye conjugates (Figure 4A), indicating successful intracellular aptamer delivery and ATP measurement in living cells. As controls, HeLa cells were also cultured either with or without the GO/R−TPdye conjugates as well as with only the ATP aptamer−TPdye, and almost no TPE fluorescence signals were observed (Figure 4B,C). In order to verify the internalization of GO/Aptamer−TPdye conjugate in HeLa cells, Z-scanning confocal imaging was performed (Figure S10 in the Supporting Information) by two-photon confocal microscopy. It was clear that bright TPE fluorescence was 3552
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
Analytical Chemistry
Article
biological conditions. TP confocal fluorescence microscopy experiments with HeLa cells and zebrafish suggested that the GO/Aptamer−TPdye conjugate was efficiently delivered into live cells or tissues and acted as a “signal-on” in vivo sensor for specific, high-contrast imaging of target biomolecules. To the best of our knowledge, it is the first time that a carbon nanomaterials/aptamer-based two-photon fluorescent probe has been successfully used for in vitro or in vivo assays. Considering the unique properties of nanostructured materials and the TPM technique, this new GO/Aptamer−TPdye conjugate-based strategy for developing robust biomolecules sensors is expected to hold great potential for in vitro and in vivo applications in medical research and clinical diagnostics.
present throughout the whole cells, which suggested efficient delivery of this nanosensing conjugate to the cytosol. To further confirm that the fluorescence signal resulted from the endogenously produced ATP of the HeLa cell, we also designed an assay for in situ ATP semi quantification, and the results were shown in Figure 4D,E. From these images, one can see that the TPE fluorescence decreased dramatically upon treatment with oligomycin (Figure 4D), a well-known inhibitor of ATP.46 In the meanwhile, a significant enhancement was observed when the cells were preincubated with Ca2+ (Figure 4E), a commonly used ATP inducer.47 All these results demonstrate that the GO/Aptamer−TPdye conjugate can afford a robust intracellular biomolecule sensor for highcontrast TPA imaging of biomolecules in live cells. GO/Aptamer−TPdye Nanosensing Conjugate for ATP Probing in Zebrafish. On the basis of successful cell imaging with the GO/Aptamer−TPdye conjugate, we further determined whether it could be used for in vivo ATP imaging in zebrafish. As shown in Figure 5, a 2-day-old zebrafish treated
■
ASSOCIATED CONTENT
S Supporting Information *
Experimental details including synthesis of the TPdye and its characterizations as well as additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +86-731-88822587. Author Contributions §
M.Y. and S.Y. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
Figure 5. TP confocal microscopy bright-field images (up) and fluorescence images (down) of zebrafishes after incubation with GO/ R−TPdye conjugates (A) and GO/Aptamer−TPdye conjugates (B). (C) 3D TP confocal fluorescence images accumulated along the z direction at depth of 0−270 μm with 60× magnification. [Aptamer− TPdye] = 100 nM, [R−TPdye] = 100 nM, [GO] = 40 μg/mL.
ACKNOWLEDGMENTS This work was supported by NSFC (Grants 21135001, 21205143, 21305036, and J1103312), the Program for New Century Excellent Talents in University (Grant NCET-130188), and the ‘‘973’’National Key Basic Research Program (Grant 2011CB91100-0). The authors also thank Professor Zhihong Liu of Wuhan University for the TPE spectra measurements.
with GO/R−TPdye conjugates gave virtually no fluorescence signal under the TPE method (Figure 5A), whereas the zebrafish treated with the GO/ATP aptamer−TPdye conjugates showed a strong TPE fluorescence (Figure 5B), clearly demonstrating the high ATP concentration level of the growing zebrafish. In addition, the 3D two-photon confocal fluorescence imaging along the z-direction (inset of Figure 5B) at 0−270 μm tissue depth demonstrates that the GO/Aptamer−TPdye nanosensing conjugate is capable of monitoring target biomolecules at a larger depth in living tissues using twophoton microscopy.
■
REFERENCES
(1) Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A. Angew. Chem. 2010, 122, 3352−3366; Angew. Chem., Int. Ed. 2010, 49, 3280−3294. (2) Stankovich, S.; Dikin, D. A.; Dommett, G. H.; Kohlhaas, K. M.; Zimney, E. J.; Stach, E. A.; Piner, R. D.; Nguyen, S. T.; Ruoff, R. S. Nature 2006, 442, 282−286. (3) Liu, Y. X.; Dong, X. C.; Chen, P. Chem. Soc. Rev. 2012, 41, 2283− 2307. (4) Wu, Y. R.; Phillips, J. A.; Liu, H. P.; Yang, R. H.; Tan, W. H. ACS Nano 2008, 2, 2023−2028. (5) Lu, C. H.; Li, J.; Lin, M. H.; Wang, Y. W.; Yang, H. H.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2010, 49, 8454−8457. (6) (a) Wang, H. B.; Zhang, Q.; Chu, X.; Chen, T. T.; Ge, J.; Yu, R. Q. Angew. Chem., Int. Ed. 2011, 50, 7065−7069. (b) Ryoo, S. R.; Lee, J.; Yeo, J.; Na, H. K.; Kim, Y. K.; Jang, H. J.; Lee, J. H.; Han, S. W.; Lee, Y.; Kim, V. N.; Min, D. H. ACS Nano 2013, 7, 5882−5891. (7) Chen, Z.; Chen, H.; Hu, H.; Yu, M.; Li, F.; Zhang, Q.; Zhou, Z.; Yi, T.; Huang, C. J. Am. Chem. Soc. 2008, 130, 3023−3029. (8) Peng, X.; Song, F.; Lu, E.; Wang, Y.; Zhou, W.; Fan, J.; Gao, Y. J. Am. Chem. Soc. 2005, 127, 4170−4171. (9) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic: New York, 1999. (10) Helmchen, F.; Denk, W. Nat. Methods 2005, 2, 932−940. (11) Dong, X. H.; Lian, W. L.; Peng, X. N.; Liu, Z. H.; He, Z. K.; Wang, Q. Q. Anal. Chem. 2010, 82, 1381−1388.
■
CONCLUSION In summary, we have developed a GO/aptamer-based twophoton fluorescent nanoprobe for in vitro or in vivo assay of biomolecules with high sensitivity and selectivity. Compared with the nowadays prevailing strategy of carbon nanomaterialsbased fluorescent biosensor construction, introduction of a twophoton dye as the signal reporter effectively eliminates the selfabsorption and autofluorescence of the biological molecules in biological matrixes, reduces the photodamage resulted from the high-energy excitation, and enhances the penetration depth and spatial resolution, thus substantially improves the performance of carbon nanomaterials-based sensors in complex biological conditions. In vitro assays revealed that the GO/Aptamer− TPdye conjugate provided a robust, sensitive, and selective sensor for quantitative detection of ATP even if under complex 3553
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
Analytical Chemistry
Article
(12) Kim, D.; Singha, S.; Wang, T. J.; Seo, E.; Lee, J. H.; Lee, S. J.; Kim, K. H.; Ahn, K. H. Chem. Commun. 2012, 48, 10243−10245. (13) Kim, H. M.; Cho, B. R. Chem. Asian. J. 2011, 6, 58−69. (14) Dong, X. H.; Han, J. H.; Heo, C. H.; Kim, H. M.; Liu, Z. H.; Cho, B. R. Anal. Chem. 2012, 84, 8110−8113. (15) Ahn, H. Y.; Fairfull-Smith, K. E.; Morrow, B. J.; Lussini, V.; Kim, B.; Bondar, M. V.; Bottle, S. E.; Belfield, K. D. J. Am. Chem. Soc. 2012, 134, 4721−4730. (16) Chung, C.; Srikun, D.; Lim, C. S.; Chang, C. J.; Cho, B. R. Chem. Commun. 2011, 47, 9618−9620. (17) Liu, L. Z.; Shao, M.; Dong, X. H.; Yu, X. F.; Liu, Z. H.; He, Z. K.; Wang, Q. Q. Anal. Chem. 2008, 80, 7735−7741. (18) Andrade, C. D.; Yanez, C. O.; Ahn, H.-Y.; Urakami, T.; Bondar, M. V.; Komatsu, M.; Belfield, K. D. Bioconjugate Chem. 2011, 22, 2060−2071. (19) Li, L.; Shen, X. Q.; Xu, Q. H.; Yao, S. Q. Angew. Chem., Int. Ed. 2013, 52, 424−428. (20) Wilson, D. S.; Szostak, J. W. Annu. Rev. Biochem. 1999, 68, 611− 647. (21) Cho, E. J.; Lee, J. W.; Ellington, A. D. Annu. Rev. Anal. Chem. 2009, 2, 241−264. (22) Iliuk, A. B.; Hu, L. H.; Tao, W. A. Anal. Chem. 2011, 83, 4440− 4452. (23) Chung, C.; Kim, Y. K.; Shin, D.; Rood, S. R.; Hong, B. H.; Min, D. H. Acc. Chem. Res. 2013, 46, 2211−2224. (24) (a) Liu, Y. X.; Dong, X. C.; Chen, P. Chem. Soc. Rev. 2012, 41, 2283−2307. (b) Cui, L.; Song, Y. L.; Ke, G. L.; Guan, Z. C.; Zhang, H. M.; Lin, Y.; Huang, Y. S.; Zhu, Z.; Yang, C.Y. J. Chem.Eur. J. 2013, 19, 10442−10451. (25) Palleros, D. R.; Reid, K. L.; Shi, L.; Welch, W. J.; Fink, A. L. Nature 1993, 365, 664−666. (26) Finger, T. E.; Danilova, V.; Barrows, J.; Bartel, D. L.; Vigers, A. J.; Stone, L.; Hellekant, G.; Kinnamon, S. C. Science 2005, 310, 1495− 1499. (27) Nutiu, R.; Li, Y. F. J. Am. Chem. Soc. 2003, 125, 4771−4778. (28) Li, C.; Numata, M.; Takeuchi, M.; Shinkai, S. Angew. Chem., Int. Ed. 2005, 44, 6371−6374. (29) Zheng, D.; Seferos, D. S.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Nano Lett. 2009, 3258−3261. (30) Xu, Z. C.; Singh, N. J.; Lim, J.; Pan, J.; Kim, H. N.; Park, S.; Kim, K. S.; Yoon, J. J. Am. Chem. Soc. 2009, 131, 15528−15533. (31) Wang, Y.; Li, Z. H.; Hu, D. H.; Lin, C. T.; Li, J. H.; Lin, Y. H. J. Am. Chem. Soc. 2010, 132, 9274−9296. (32) Rao, A. S.; Kim, D.; Nam, H.; Jo, H.; Kim, K. H.; Ban, C.; Ahn, K. H. Chem. Commun. 2012, 48, 3206−3208. (33) Wu, C. C.; Chen, T.; Han, D.; You, M. X.; Peng, L.; Cansiz, S.; Zhu, G. Z.; Li, C. M.; Xiong, X. L.; Jimenez, E.; Yang, C. J.; Tan, W. H. ACS Nano 2013, 7, 5724−5731. (34) Liu, J. H.; Wang, C. Y.; Jiang, Y.; Hu, Y. P.; Li, J. S.; Yang, S.; Li, Y. H.; Yang, R. H.; Tan, W. H.; Huang, C. Z. Anal. Chem. 2013, 85, 1424−1430. (35) Wang, Y.; Li, Z. H.; Weber, T. J.; Hu, D. H; Lin, C. T.; Li, J. H.; Lin, Y. H. Anal. Chem. 2013, 85, 6775−6782. (36) Hu, H. B.; Zhang, X. F.; Xiao, Y.; Zhou, W.; Wang, L. P.; Jin, L. J. Anal. Chem. 2013, 85, 7076−7084. (37) (a) Yang, K.; Zhang, S.; Zhang, G. X.; Sun, X. M.; Lee, S. T.; Liu, Z. Nano Lett. 2013, 85, 7076−7084. (b) Li, J. L; Bao, H. C.; Hou, X. L.; Sun, L.; Wang, X. G.; Gu, M. Angew. Chem., Int. Ed. 2012, 51, 1830−1834. (38) Parker, C. A.; Rees, W. T. Analyst 1960, 85, 587−600. (39) Nikolay, S.; Makarov, N. K.; Drobizhev, M.; Rebane, A. Opt. Exp. 2008, 16, 4029−4047. (40) Son, J. H.; Lim, C. S.; Han, J. H.; Danish, I. A.; Kim, H. M.; Cho, B. R. J. Org. Chem. 2011, 76, 8113−8116. (41) Lu, C. H.; Yang, H. H.; Zhu, C. L.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2009, 48, 4785−4787. (42) (a) Morales-Narváez, E.; Merkoçi, A. Adv. Mater. 2012, 24, 3298−3308. (b) Kim, J. Y.; Cote, L. J.; Kim, F.; Huang, J. X. J. Am. Chem. Soc. 2010, 134, 260−267.
(43) Lu, C. H.; Zhu, C. L.; Li, J.; Liu, J. J.; Chen, X.; Yang, H. H. Chem. Commun. 2010, 46, 3116−3118. (44) Schröder, G. F.; Alexiev, U.; Helmut Grubmüller, H. Biophys. J. 2005, 89, 3757−3770. (45) Fu, C. C.; Lee, H. S.; Chen, K.; Lim, T. S.; Wu, H. Y.; lin, P. K.; Wei, P. K.; Tsao, P. H.; Chang, H. C.; Fann, W. S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 727−732. (46) Gong, Y.; Sohn, H.; Xue, L.; Firestone, G. L.; Bjeldanes, L. F. Cancer Res. 2006, 66, 4880−4887. (47) Ainscow, E. K.; Rutter, G. A. Diabetes 2002, 51 (Suppl.1), S162−170.
3554
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
Two-Photon Graphene Oxide/Aptamer Nanosensing Conjugate for In Vitro or In Vivo Molecular Probing Mei Yi,†,§ Sheng Yang,†,§ Zanying Peng,† Changhui Liu,† Jishan Li,*,† Wenwan Zhong,‡ Ronghua Yang,† and Weihong Tan† †
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China ‡ Department of Chemistry, University of California-Riverside, Riverside, California 92521, United States S Supporting Information *
ABSTRACT: Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source have the unique properties of lower tissue autofluorescence and self-absorption, reduced photodamage and photobleaching, higher spatial resolution, and deeper penetration depth (>500 μm). Carbon nanomaterials, for example, graphene oxide (GO), have the advantages of good biocompatibility, efficient transporters into cells, protecting the carried DNA or peptides from enzymatic cleavage, and super fluorescence quenching efficiency. By combination of the nanostructured carbon materials with the TPE technique, herein we have designed an aptamer-twophoton dye (TPdye)/GO TPE fluorescent nanosensing conjugate for molecular probing in biological fluids, living cells, and zebrafish. This approach takes advantage of the exceptional quenching capability of GO for the proximate TP dyes and the higher affinity of single-stranded DNA on GO than the aptamer− target complex. Successful in vitro and in vivo detection of ATP was demonstrated with this sensing strategy. Our results reveal that the GO/Aptamer−TPdye system not only is a robust, sensitive, and selective sensor for quantitative detection of ATP in the complex biological environment but also can be efficiently delivered into live cells or tissues and act as a “signal-on” in vivo sensor for specific, high-contrast imaging of target biomolecules. Our design provides a methodology model scheme for development of future carbon nanomaterial-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species.
T
These limitations in OPE motivated us to design the twophoton dye-embedded nanoprobes for molecular detection in complicated biological samples or in vivo imaging through twophoton excitation (TPE).9 TPE with NIR photons as the excitation source have the advantages of lower tissue autofluorescence and self-absorption, reduced photodamage and photobleaching, and deeper penetration depth (>500 μm), etc. Together with the development of two-photon microscopy (TPM), TPE has become a powerful tool for research in life science.10,11 In recent years, although a variety of organic molecule-based two-photon probes have been reported for various biotargets such as anions,12 metal ions,13,14 reactive oxygen species,15,16 and other biomolecules,17−19 the twophoton dye (TPdye)/nanostructure conjugates as TP nanoprobes for in vitro or in vivo assays have not been reported so far. Therefore, considering the unique properties of the nanomaterials and the TPE technique, development of carbon nanostructure-based TPE nanoprobes with good biocompati-
he development of hybrid biological-synthetic nanomaterials for sensing and delivery is of crucial importance for clinical diagnostics and therapeutics.1 Carbon nanomaterials, as the popular star nanomaterial with the properties of good biocompatibility, high internalization efficiency, simple functionalization, excellent quenching ability to the adjacent fluorophores and the protecting effect on the carried DNA or peptides from enzymatic cleavage, have gained great attentions in biomedical and bioanalytical fields.2−5 However, most of the fluorescent dyes used in previous carbon nanomaterials-based sensing systems are the traditional one-photon excited (OPE) fluorophores, for example, fluorescein, rhodamine, Cy3, etc.6 Such dyes require rather short excitation wavelengths (18.3 MΩ. All experiments were carried out at room temperature. UV−vis absorption spectra were measured on a Hitachi U4100 spectrophotometer (Kyoto, Japan). One-photon fluorescence and anisotropy measurements were performed on a PTI QM4 Fluorescence System (Photo Technology International, Birmingham, NJ). Two-photon fluorescence spectra were obtained with a mode-locked Ti:sapphire pulsed laser (Chameleon Ultra II, Coherent Inc.) and then recording with a DCS200PC single photon counting (Beijing Zolix Instruments Co., Ltd.). TPM imaging of HeLa cells and zebrafish were carried out by using an Olympus FV1000-MPE multiphoton laser scanning confocal microscope (Japan). Atomic force microscopy (AFM) measurements were performed by using a Nanoscope Vmultimode atomic force microscope (Veeco Instruments). Energy-dispersive X-ray (EDX) analysis was carried out using a HITACHI S-4500 instrument. pH was measured by a model 868 pH meter (Orion). Preparation of GO/Aptamer−TPdye Conjugate. According to the previous reported methods for preparation of GO/DNA complexes,35 the TPdye-labeled ATP aptamer/GO conjugates were prepared as the following procedures. Briefly, the GO was sonicated in doubly deionized water for 2 h and then the centrifugation step was carried out at 3000 rpm to get rid of the larger aggregates of GO, leading to a homogeneous black solution (the concentration of GO is ∼2 mg/mL). After the pretreatment, enough GO was introduced into 500 μL of the HEPES buffer (20 mM, pH 7.4) containing 100 nM of Aptamer−TPdye (the final concentration of GO is ∼40 μg/ mL), and the mixture was incubated for several minutes at room temperature to form the GO/Aptamer−TPdye conjugate. Then the obtained GO/Aptamer−TPdye conjugate was stored in a refrigerator at 4 °C before further usage. The GO/ R-TPdye conjugate was also prepared as the above descriptions. Both GO and GO/Aptamer conjugate were characterized on an atomic force microscopy using tapping mode to simultaneously collect height and phase data. EDX analysis of GO and GO/ Aptamer conjugate was carried out using a HITACHI S-4500 instrument. In Vitro Detection of ATP by the GO/Aptamer−TPdye Nanosensing Conjugate. In a typical assay, an aliquot of 200 μL of GO/Aptamer−TPdye conjugate suspension was first added in 200 μL of HEPES buffer or cell media (the final
bility and efficient transporting property, lower biological autofluorescence and self-absorption, higher spatial resolution and deeper penetration depth for in vitro or in vivo assay will have a fascinating prospect. Herein, we report a TPdye labeled aptamer/graphene oxide (GO)-conjugate for in vitro or in vivo detection of adenosine triphosphate (ATP) (Scheme 1). Aptamers are single-stranded Scheme 1. Schematic Illustration of Two-Photon Graphene Oxide/Aptamer-Based Nanosensing Conjugatea
Assembly of aptamer−TPdye with GO led to the fluorescence emission in the “off” state due to the fluorescence resonance energy transfer (FRET) effect between TPdye and GO. After the GO/ Aptamer−TPdye conjugate was incubated with ATP-containing sample, the nanoprobe would be in the “on” state as a result of the release of TP fluorophores from the GO surface, thus providing greatly enhanced fluorescence emission intensity. a
DNA molecules that can specifically bind to non-nucleic acid targets.20 They have been widely used for detection of a broad range of molecules including ions, small biomolecules, and proteins.21,22 GO, a water-soluble derivative of one-atom-thick two-dimensional carbon material, has recently attracted great interest for biomedical applications.23 The unique capacity of GO in adsorbing biomolecules, such as nucleic acids and peptides, with super fluorescence quenching efficiency creates a robust platform for the development of biosensors.24 ATP, as a multifunctional intracellular signaling molecule, plays significant roles in many biological processes, such as universal energy flow in living cells, metabolic reactions, material trafficking, and membrane transport.25−28 Because of its great importance, many methods have been developed for in situ detection and imaging of ATP.29−34 However, efficient TPE nanoprobes for in vitro or in vivo assay of ATP appear to be rare. Thus we prepared a GO/ATP aptamer−TPdye conjugate as proof-ofconcept of the sensing platform for in vitro or in vivo measurement of ATP. When the analyte is not presented, the nanoprobe was initially in “off” state due to the efficient quenching of GO for fluorophores adjacent to its surface. After the GO−ATP aptamer conjugate was incubated with targetcontaining sample or transported into cells followed by binding of the aptamer by intracellular ATP, the nanoprobe would be in the “on” state as a result of the release of TP fluorophores from the GO surface, thus providing greatly enhanced fluorescence emission intensity.
■
EXPERIMENTAL SECTION Chemicals and Instruments. The TPdye used in this work was synthesized as described in the Supporting Information, and the TPdye-labeled ATP aptamer (Aptamer3549
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
Analytical Chemistry
Article
concentration of Aptamer−TPdye is 50 nM and GO is 20 μg/ mL), then ATP (final concentrations ranging from 0 to 3 mM) or other biomolecules was added and incubated at roomtemperature for 30 min. After reaction, the resulting solution was subjected to fluorescence measurements using the OPE or TPE method. For the OPE measurement, the fluorescence spectra were recorded in a quartz cuvette on PTI QM4 Fluorescence System with the excitation wavelength of 370 nm and the emission wavelengths in the range from 430 to 580 nm with both excitation and emission slits of 10 nm. For the TPE measurement, the two-photon emission fluorescence spectra in the range from 430 to 550 nm were obtained by exciting all samples at 750 nm with a mode-locked Ti:sapphire pulsed laser (Output laser pulses were centered at 750 nm and an average power of 100 mW was as the excitation source. The laser pulses have pulse duration of 120 fs and repetition rate of 80 MHz.), followed by recording with a DCS200PC single photon counting. Cytotoxicity Assays and Live Cell Imaging with the GO/Aptamer−TPdye Conjugate. HeLa cells were grown in RPMI 1640 medium (Thermo Scientific HyClone) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/mL penicillin, and 100 U/mL gentamicin at 37 °C in a humidified atmosphere containing 5% CO2. The cytotoxic effects of GO were assessed using the MTT assay.36 For cell imaging experiments, cells were seeded in 24-well culture plate and grown overnight on glass coverslips at the bottom of the plate. When the cells were ∼90% confluent, the coverslips were washed three times with phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). Before incubation with probes, the HeLa cells were incubated with/without 10 μM oligomycin or 5 mM Ca2+ for 30 min. Then, 500 μL of fresh cell growth medium supplemented with the GO/Aptamer−TPdye conjugate ([Aptamer−TPdye] = 50 nM, [GO] = 20 μg/mL), the GO/R−TPdye mixture ([R−TPdye] = 50 nM, [GO] = 20 μg/ mL), the GO (∼20 μg/mL) only, or the aptamer−TPdye (50 nM) was added in each well, respectively. After incubation for 8 h, the cells were washed with Dulbecco’s phosphate buffered saline (DPBS) three times. Two-photon confocal fluorescence imaging of HeLa cells was observed under an Olympus FV1000-MPE multiphoton laser scanning confocal microscope, with a mode-locked titanium-sapphire laser source (120 fs pulse width, 80 MHz repetition rate) set at wavelength 750 nm. Three dimensional images were taken every 2 μm by scanning the samples across a defined section along the z-axis. Taking into account the possible photothermal effect of GO under NIR irradiation, the laser power was set at 2 mW to prevent potential thermal damage to cells during imaging.37 TPM Images of Zebrafish by Using GO/Aptamer− TPdye Nanosensing Conjugate. Zebrafishes were maintained in E3 embryo media (15 mM NaCl, 0.5 mM KCl, 1.0 mM MgSO4, 1.0 mM CaCl2, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3, 5−10% methylene blue, pH 7.5). In fluorescence imaging experiments, two-day-old zebrafishes were, respectively, incubated with GO/Aptamer−TPdye conjugate ([Aptamer−TPdye] = 100 nM, [GO] = 40 μg/ mL) or GO/R−TPdye conjugate ([R−TPdye] = 100 nM, [GO] = 40 μg/mL) in E3 embryo media for 15 h at 28 °C. After washing with PBS (pH 7.4) to remove the remaining probe, two-photon confocal fluorescence imaging and the 3D two-photon confocal fluorescence images (accumulated along the z direction at depth of 0−270 μm with 60× magnification
of these treated zebrafishes) were observed under an Olympus FV1000-MPE multiphoton laser scanning confocal microscope using a mode-locked titanium-sapphire laser source (120 fs pulse width, 80 MHz repetition rate) set at wavelength 750 nm with laser power of 2 mW.
■
RESULTS AND DISCUSSION Characterization of the Two-Photon Fluorescent Dye. As a newly synthesized fluorescent label, its optical properties should be carefully examined before biomedical applications. As is exhibited in Figure 1A, this TPdye displays a strong
Figure 1. (A) Absorption (dashed lines) and one-photon emission spectra (solid lines) of 10 μM TPdye in HEPES buffer solution (pH 7.4), λex = 370 nm. Inset: photographs of the corresponding absorption and fluorescent species. (B) Two-photon absorption action cross sections of TPdye in HEPES buffer solution. All error bars were obtained through the detection of three parallel samples. The inset shows TP excited fluorescence intensity of TPdye (10 μM) in HEPES buffer solution, λex = 750 nm.
electronic transition at λmax,abs = 370 nm (ε = 2.2 × 104 M−1 cm−1) and its OPE emission spectrum shows a peak maximum at 460 nm with an obvious cyan fluorescence under UV lamp excitation. The measurement of luminescence quantum yield (Φ) with quinine sulfate dihydrate as the reference shows that the quantum yield of the TPdye is 0.47.38 The luminescent brightness of this TPdye (defined as ε × Φ) is estimated to be 1.03 × 104 M−1 cm−1, suggesting a remarkable signal response capability in bioanalysis. We also measured the two-photon absorption (TPA) action cross section (δ × Φ, δ is the TPA cross section) and the TPE emission spectrum of the TPdye. As shown in Figure 1B, the maximal TPA action cross section of the TPdye in aqueous solution was measured to be 50 Goeppert-Mayer (GM) at room temperature (λex = 750 nm, 1 GM = 10−50 cm4 s photon−1 molecule−1) with rhodamine B as the reference,39 which is approximate to that of the similar compound reported previously.40 The TPE emission spectrum of the TPdye is shown in the inset of Figure 1B. The sample was excited with femtosecond laser pulses with a central wavelength at 750 nm and a pulse duration of 40 fs. As can be seen, the corresponding maximal emission peak of this TPE emission spectrum is also located at 460 nm and the profile of this TPE emission spectrum matches exactly to that of the OPE emission spectrum, indicating that the fluorescence emission excited at the near-infrared region is purely the two-photoninduced fluorescence. Long-term stability of the two-photon dyes is crucial to their practical applications in biolabeling or bioimaging. So the stability of this TPdye under various environmental conditions was investigated through testing its luminescence intensity. The photostability of the TPdye was first measured by using a 150 W xenon lamp as an excitation source. The emission intensity 3550
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
Analytical Chemistry
Article
decreased by only 1% after irradiation for 1 h, indicating the good photostability of this TPdye (see Figure S1 in the Supporting Information). We then explored the effect of pH on the TPdye at pH values ranging from 4.0 to 10.0 and observed little changes in the fluorescence intensity which demonstrated that this new two-photon dye was stable at physiological pH conditions (Figure S2, Supporting Information). Additionally, there was no significant variation in fluorescence intensity of the TPdye with increasing concentrations of the physiological relevant metal ions, including Na+, K+, Ca2+, and Mg2+ (Figure S3, Supporting Information), indicating that this TPdye is stable under the physiological ion strength, even with extremely high ion concentrations. Furthermore, the stability of this TPdye in human serum or human cervix carcinoma (HeLa) cell lysate was also investigated through the fluorescence intensity measurements. The results show that the TPdye molecules is still stable after tens of hours of incubation (Figure S4, Supporting Information). The higher δ × Φ value, good photostability, and long-term stability in various environmental conditions support that this TPdye is a promising luminescence emitter for TP imaging applications in complex biological samples. Formation and Characterization of the GO/Aptamer− TPdye Conjugate. Because single-stranded DNA sequences (ssDNA) have been proven to be assembled on GO surfaces with strong affinity,41 a noncovalent complex of GO/Aptamer− TPdye was prepared by simple self-assembly of Aptamer− TPdye with GO. We can see from Figure S5 (Supporting Information) that the fluorescence intensity of the Aptamer− TPdye solution decreases proportionally with the addition of increasing amounts of GO, indicating the growing number of Aptamer−TPdye adsorbed onto the surface of GO, thus resulting in the quenching of the TPdye’s fluorescence by way of fluorescence resonance energy transfer (FRET) processes.42 When the concentration of GO added reached about 20 μg/ mL, the fluorescence of 50 nM Aptamer−TPdye was nearly completely quenched. The GO/Aptamer−TPdye conjugate was then characterized by atomic force microscopy (AFM) and energy-dispersive X-ray (EDX) spectroscopy to obtain its morphological profile and structural information. The GO sheets mostly have a lateral width of less than 200 nm with a topological height of about 1 nm (Figure S6A in the Supporting Information), which would increase the cell uptake effciency of GO and be beneficial for bioimaging.43 After conjugation of the aptamer−TPdye with GO, the GO/Aptamer−TPdye conjugate had a topological height of over 2 nm across the lateral dimension (Figure S6B in the Supporting Information) and an appearance of the P element which belongs to oligonucleotides in the EDX pattern of GO/Aptamer−TPdye conjugate. The increase in the ratio of O and C compared with that of GO (Figure S7 in the Supporting Information) implied coverage of Aptamer−TPdye on the GO surfaces and the successful formation of GO/Aptamer−TPdye nanosensing conjugate. Validation of the Design Scheme. Scheme 1 illustrates the signaling mechanism of the GO/Aptamer−TPdye nanosensing conjugate. To verify this design scheme, we investigated the real-time records of OPE fluorescence intensity and the anisotropy changes of Aptamer−TPdye in the HEPES buffer solution upon addition of GO and subsequently ATP (Figure 2A). In aqueous solution, Aptamer−TPdye has a strong fluorescence emission, while the fluorescence emission was quenched greatly after the addition of GO (curve a), indicating the interaction of the TPdye with GO, allowing the energy
Figure 2. (A) Real-time recordings of the one-photon fluorescence emission intensity (a) and anisotropy (b) changes of Aptamer−TPdye as a function of time upon additions of GO and subsequently ATP. (B) Relative fluorescence responses of GO/Aptamer−TPdye to CTP, GTP, UTP, and ATP. All error bars were obtained through the detection of three parallel samples. [Aptamer−TPdye] = 50 nM, [GO] = 20 μg/mL, [NTP] = 1 mM. λex = 370 nm.
transfer process to occur. Distinct increase of fluorescence emission after ATP addition verified the feasibility of the sensing scheme. Curve b shows the fluorescence anisotropy (FA) value change of the Aptamer−TPdye under the same conditions. The FA value of Aptamer−TPdye at the free state in the buffer is very low (0.047). However, it underwent a remarkable enhancement upon addition of GO. This result indicates that binding of GO to the ssDNA strand created a larger mass complex, which hindered the rotation diffusion rate of the labeled TPdye.44 The significant difference in the FA values of GO and the GO/Aptamer−TPdye further confirms the formation of GO/Aptamer−TPdye conjugate. The FA value was reduced from 0.228 to 0.113 with addition of ATP, meaning that formation of the Aptamer−TPdye/ATP complexes freed the Aptamer−TPdye from the GO surface. It is worth noting that the fluorescence intensity and anisotropy changes of GO/Aptamer−TPdye nanosensing conjugate reached equilibrium within a few minutes. This indicates the potential of our assay for rapid and real time monitoring of the target in homogeneous solutions. Further inspection of the GO/Aptamer−TPdye nanosensing conjugate was also performed by using gel electrophoresis (Figure S8 in the Supporting Information). The results show that there is no significant ATP aptamer band on the gel image for the supernatant of GO/Aptamer−TPdye conjugates (lane b), while for the supernatant of GO/Aptamer−TPdye conjugates with addition of ATP, there is a clear aptamer band on the gel image (lane c), indicating that the formed aptamer−target complex fell off from the GO surface and stayed in the supernatant after centrifugation. These results agree well with the above fluorescence emission experiments. To evaluate the selectivity of our assay, the TPdye fluorescence intensity changes with the addition of different targets (CTP, UTP, GTP, and ATP) were studied. As shown in Figure 2B, when CTP, UTP, and GTP were at a concentration of 1.0 mM, the system only gives a small TPdye fluorescence response, while a significant TPdye fluorescence enhancement occurs after the introduction of 1.0 mM ATP to the solution under the same conditions. This selectivity is similar to that of the original ATP aptamer, confirming that the conjugation of GO with the aptamer does not affect the selectivity of the aptamer. GO/Aptamer−TPdye Nanosensing Conjugate for ATP Probing in Cell Media by OPE and TPE Technique. Measurement in complex biological samples often suffers from the high fluorescence background produced by the ubiquitous 3551
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
Analytical Chemistry
Article
endogenous components in the sample matrix,45 failing the commonly used biosensors without sample pretreatment. To address this limitation, NIR fluorophores, time-resolved fluorescence method, or TP technique are usually employed to realize the target assay in complex biological samples. The TPE-based fluorescence strategies might be the best choice due to the key advantages of the TP technique, namely, lower selfabsorption and lower biological autofluorescence, reduced photodamage, greater penetration depth (>500 μm), and higher spatial resolution. To evaluate the performance of this GO/Aptamer−TPdye nanosensing conjugate in complex conditions, we carried out ATP measurements in cell media using the OPE and TPE techniques. Figure 3A,B shows the
obtained when the ATP concentration is increased, thus failing to measure the target successfully. However, Figure 3D showed that the TPE fluorescence emission was very weak when the target ATP was not presented, while remarkable enhancements were observed with increasing ATP concentrations. The dosedependent intensity of the 460 nm band showed the fluorescence signal dynamically increased with the addition of ATP in the concentration range of 10 μM to 3 mM, and a satisfactory detection limit of 0.5 μM ATP that was taken to be 3 times the standard derivation in blank solution was achieved (inset of Figure 3D). These results suggest that the TPE method is more suitable for biological assays compared with the OPE-based methods, owing to the low background fluorescence. GO/Aptamer−TPdye Nanosensing Conjugate for ATP Probing in Living Cells. After demonstrating in vitro sensing of ATP by the GO/Aptamer−TPdye conjugate in the complex biological sample, we then explored its potential for live-cell imaging of ATP. First, the cytotoxicities of GO on HeLa cells were evaluated using the standard cell viability assay, the MTT assay. After the HeLa cells were treated with GO concentrations up to 50 μg/mL for 24 h, high cell viability was observed (survival rate was higher than 90% in 1.0 × 104 cells/ well) (Figure S9, Supporting Information). Throughout the present study for cell imaging, we used GO of less than 20 μg/ mL, which ensures high viability of all the cells we tested. The TPE confocal fluorescence imaging in HeLa cells was then carried out. As shown in Figure 4, a significant TPE
Figure 3. OPE (A) and TPE (B) emission spectra of the only cell growth media (a), Aptamer−TPdye in HEPES buffer solution (b), and Aptamer−TPdye in cell growth media (c). OPE (C) and TPE (D) fluorescence emission changes of GO/Aptamer−TPdye nanosensing conjugate in the presence of different concentrations of ATP in cell growth media. Inset: Signal to background ratios (S/B) of GO/ Aptamer−TPdye conjugate as a function of ATP concentrations. All error bars were obtained through the detection of three parallel samples. [Aptamer−TPdye] = 50 nM, [GO] = 20 μg/mL, [ATP] = 0−3 mM. λex = 370 nm/750 nm.
Figure 4. TP confocal microscopy fluorescence images (up), brightfield images (middle), and the overlay of fluorescence and bright-field images (down) of HeLa cells after incubation with GO/Aptamer− TPdye conjugates (A), Aptamer−TPdye (B), GO/R−TPdye (C), and treated with 10 μg/mL oligomycin (D) or with 5 mM Ca2+ (E) followed by incubation with GO/Aptamer−TPdye nanosensing conjugate. Scale bar: 20 μm. [Aptamer−TPdye] = 50 nM, [R− TPdye] = 50 nM, [GO] = 20 μg/mL.
OPE and TPE emission spectra of the Aptamer−TPdye in the HEPES buffer and the 1640 cell growth media, respectively. One can see from Figure 3A that the cell growth media had a high autofluorescence and dominated the fluorescence spectra from 430 to 580 nm under the OPE. Furthermore, the fluorescence emission intensity inversely decreased when the aptamer−TPdye was dispersed into the cell growth media, which might be a result from the higher self-absorption of the cell growth media. However, in contrast to the OPE measurements, one can find that the TPE fluorescence emission intensity and its emission spectrum in the cell growth media were almost the same as in the HEPES buffer (Figure 3B). Figure 3C,D shows the fluorescence emission spectra of this nanosensing conjugate in cell media with various concentrations of ATP by using the OPE and TPE method, respectively. For the OPE method (Figure 3C), the background fluorescence emission was very strong even without target ATP and a significant fluorescence emission increase cannot be
fluorescence image was obtained for the cells incubated with the GO/Aptamer−TPdye conjugates (Figure 4A), indicating successful intracellular aptamer delivery and ATP measurement in living cells. As controls, HeLa cells were also cultured either with or without the GO/R−TPdye conjugates as well as with only the ATP aptamer−TPdye, and almost no TPE fluorescence signals were observed (Figure 4B,C). In order to verify the internalization of GO/Aptamer−TPdye conjugate in HeLa cells, Z-scanning confocal imaging was performed (Figure S10 in the Supporting Information) by two-photon confocal microscopy. It was clear that bright TPE fluorescence was 3552
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
Analytical Chemistry
Article
biological conditions. TP confocal fluorescence microscopy experiments with HeLa cells and zebrafish suggested that the GO/Aptamer−TPdye conjugate was efficiently delivered into live cells or tissues and acted as a “signal-on” in vivo sensor for specific, high-contrast imaging of target biomolecules. To the best of our knowledge, it is the first time that a carbon nanomaterials/aptamer-based two-photon fluorescent probe has been successfully used for in vitro or in vivo assays. Considering the unique properties of nanostructured materials and the TPM technique, this new GO/Aptamer−TPdye conjugate-based strategy for developing robust biomolecules sensors is expected to hold great potential for in vitro and in vivo applications in medical research and clinical diagnostics.
present throughout the whole cells, which suggested efficient delivery of this nanosensing conjugate to the cytosol. To further confirm that the fluorescence signal resulted from the endogenously produced ATP of the HeLa cell, we also designed an assay for in situ ATP semi quantification, and the results were shown in Figure 4D,E. From these images, one can see that the TPE fluorescence decreased dramatically upon treatment with oligomycin (Figure 4D), a well-known inhibitor of ATP.46 In the meanwhile, a significant enhancement was observed when the cells were preincubated with Ca2+ (Figure 4E), a commonly used ATP inducer.47 All these results demonstrate that the GO/Aptamer−TPdye conjugate can afford a robust intracellular biomolecule sensor for highcontrast TPA imaging of biomolecules in live cells. GO/Aptamer−TPdye Nanosensing Conjugate for ATP Probing in Zebrafish. On the basis of successful cell imaging with the GO/Aptamer−TPdye conjugate, we further determined whether it could be used for in vivo ATP imaging in zebrafish. As shown in Figure 5, a 2-day-old zebrafish treated
■
ASSOCIATED CONTENT
S Supporting Information *
Experimental details including synthesis of the TPdye and its characterizations as well as additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +86-731-88822587. Author Contributions §
M.Y. and S.Y. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
Figure 5. TP confocal microscopy bright-field images (up) and fluorescence images (down) of zebrafishes after incubation with GO/ R−TPdye conjugates (A) and GO/Aptamer−TPdye conjugates (B). (C) 3D TP confocal fluorescence images accumulated along the z direction at depth of 0−270 μm with 60× magnification. [Aptamer− TPdye] = 100 nM, [R−TPdye] = 100 nM, [GO] = 40 μg/mL.
ACKNOWLEDGMENTS This work was supported by NSFC (Grants 21135001, 21205143, 21305036, and J1103312), the Program for New Century Excellent Talents in University (Grant NCET-130188), and the ‘‘973’’National Key Basic Research Program (Grant 2011CB91100-0). The authors also thank Professor Zhihong Liu of Wuhan University for the TPE spectra measurements.
with GO/R−TPdye conjugates gave virtually no fluorescence signal under the TPE method (Figure 5A), whereas the zebrafish treated with the GO/ATP aptamer−TPdye conjugates showed a strong TPE fluorescence (Figure 5B), clearly demonstrating the high ATP concentration level of the growing zebrafish. In addition, the 3D two-photon confocal fluorescence imaging along the z-direction (inset of Figure 5B) at 0−270 μm tissue depth demonstrates that the GO/Aptamer−TPdye nanosensing conjugate is capable of monitoring target biomolecules at a larger depth in living tissues using twophoton microscopy.
■
REFERENCES
(1) Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A. Angew. Chem. 2010, 122, 3352−3366; Angew. Chem., Int. Ed. 2010, 49, 3280−3294. (2) Stankovich, S.; Dikin, D. A.; Dommett, G. H.; Kohlhaas, K. M.; Zimney, E. J.; Stach, E. A.; Piner, R. D.; Nguyen, S. T.; Ruoff, R. S. Nature 2006, 442, 282−286. (3) Liu, Y. X.; Dong, X. C.; Chen, P. Chem. Soc. Rev. 2012, 41, 2283− 2307. (4) Wu, Y. R.; Phillips, J. A.; Liu, H. P.; Yang, R. H.; Tan, W. H. ACS Nano 2008, 2, 2023−2028. (5) Lu, C. H.; Li, J.; Lin, M. H.; Wang, Y. W.; Yang, H. H.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2010, 49, 8454−8457. (6) (a) Wang, H. B.; Zhang, Q.; Chu, X.; Chen, T. T.; Ge, J.; Yu, R. Q. Angew. Chem., Int. Ed. 2011, 50, 7065−7069. (b) Ryoo, S. R.; Lee, J.; Yeo, J.; Na, H. K.; Kim, Y. K.; Jang, H. J.; Lee, J. H.; Han, S. W.; Lee, Y.; Kim, V. N.; Min, D. H. ACS Nano 2013, 7, 5882−5891. (7) Chen, Z.; Chen, H.; Hu, H.; Yu, M.; Li, F.; Zhang, Q.; Zhou, Z.; Yi, T.; Huang, C. J. Am. Chem. Soc. 2008, 130, 3023−3029. (8) Peng, X.; Song, F.; Lu, E.; Wang, Y.; Zhou, W.; Fan, J.; Gao, Y. J. Am. Chem. Soc. 2005, 127, 4170−4171. (9) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic: New York, 1999. (10) Helmchen, F.; Denk, W. Nat. Methods 2005, 2, 932−940. (11) Dong, X. H.; Lian, W. L.; Peng, X. N.; Liu, Z. H.; He, Z. K.; Wang, Q. Q. Anal. Chem. 2010, 82, 1381−1388.
■
CONCLUSION In summary, we have developed a GO/aptamer-based twophoton fluorescent nanoprobe for in vitro or in vivo assay of biomolecules with high sensitivity and selectivity. Compared with the nowadays prevailing strategy of carbon nanomaterialsbased fluorescent biosensor construction, introduction of a twophoton dye as the signal reporter effectively eliminates the selfabsorption and autofluorescence of the biological molecules in biological matrixes, reduces the photodamage resulted from the high-energy excitation, and enhances the penetration depth and spatial resolution, thus substantially improves the performance of carbon nanomaterials-based sensors in complex biological conditions. In vitro assays revealed that the GO/Aptamer− TPdye conjugate provided a robust, sensitive, and selective sensor for quantitative detection of ATP even if under complex 3553
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
Analytical Chemistry
Article
(12) Kim, D.; Singha, S.; Wang, T. J.; Seo, E.; Lee, J. H.; Lee, S. J.; Kim, K. H.; Ahn, K. H. Chem. Commun. 2012, 48, 10243−10245. (13) Kim, H. M.; Cho, B. R. Chem. Asian. J. 2011, 6, 58−69. (14) Dong, X. H.; Han, J. H.; Heo, C. H.; Kim, H. M.; Liu, Z. H.; Cho, B. R. Anal. Chem. 2012, 84, 8110−8113. (15) Ahn, H. Y.; Fairfull-Smith, K. E.; Morrow, B. J.; Lussini, V.; Kim, B.; Bondar, M. V.; Bottle, S. E.; Belfield, K. D. J. Am. Chem. Soc. 2012, 134, 4721−4730. (16) Chung, C.; Srikun, D.; Lim, C. S.; Chang, C. J.; Cho, B. R. Chem. Commun. 2011, 47, 9618−9620. (17) Liu, L. Z.; Shao, M.; Dong, X. H.; Yu, X. F.; Liu, Z. H.; He, Z. K.; Wang, Q. Q. Anal. Chem. 2008, 80, 7735−7741. (18) Andrade, C. D.; Yanez, C. O.; Ahn, H.-Y.; Urakami, T.; Bondar, M. V.; Komatsu, M.; Belfield, K. D. Bioconjugate Chem. 2011, 22, 2060−2071. (19) Li, L.; Shen, X. Q.; Xu, Q. H.; Yao, S. Q. Angew. Chem., Int. Ed. 2013, 52, 424−428. (20) Wilson, D. S.; Szostak, J. W. Annu. Rev. Biochem. 1999, 68, 611− 647. (21) Cho, E. J.; Lee, J. W.; Ellington, A. D. Annu. Rev. Anal. Chem. 2009, 2, 241−264. (22) Iliuk, A. B.; Hu, L. H.; Tao, W. A. Anal. Chem. 2011, 83, 4440− 4452. (23) Chung, C.; Kim, Y. K.; Shin, D.; Rood, S. R.; Hong, B. H.; Min, D. H. Acc. Chem. Res. 2013, 46, 2211−2224. (24) (a) Liu, Y. X.; Dong, X. C.; Chen, P. Chem. Soc. Rev. 2012, 41, 2283−2307. (b) Cui, L.; Song, Y. L.; Ke, G. L.; Guan, Z. C.; Zhang, H. M.; Lin, Y.; Huang, Y. S.; Zhu, Z.; Yang, C.Y. J. Chem.Eur. J. 2013, 19, 10442−10451. (25) Palleros, D. R.; Reid, K. L.; Shi, L.; Welch, W. J.; Fink, A. L. Nature 1993, 365, 664−666. (26) Finger, T. E.; Danilova, V.; Barrows, J.; Bartel, D. L.; Vigers, A. J.; Stone, L.; Hellekant, G.; Kinnamon, S. C. Science 2005, 310, 1495− 1499. (27) Nutiu, R.; Li, Y. F. J. Am. Chem. Soc. 2003, 125, 4771−4778. (28) Li, C.; Numata, M.; Takeuchi, M.; Shinkai, S. Angew. Chem., Int. Ed. 2005, 44, 6371−6374. (29) Zheng, D.; Seferos, D. S.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Nano Lett. 2009, 3258−3261. (30) Xu, Z. C.; Singh, N. J.; Lim, J.; Pan, J.; Kim, H. N.; Park, S.; Kim, K. S.; Yoon, J. J. Am. Chem. Soc. 2009, 131, 15528−15533. (31) Wang, Y.; Li, Z. H.; Hu, D. H.; Lin, C. T.; Li, J. H.; Lin, Y. H. J. Am. Chem. Soc. 2010, 132, 9274−9296. (32) Rao, A. S.; Kim, D.; Nam, H.; Jo, H.; Kim, K. H.; Ban, C.; Ahn, K. H. Chem. Commun. 2012, 48, 3206−3208. (33) Wu, C. C.; Chen, T.; Han, D.; You, M. X.; Peng, L.; Cansiz, S.; Zhu, G. Z.; Li, C. M.; Xiong, X. L.; Jimenez, E.; Yang, C. J.; Tan, W. H. ACS Nano 2013, 7, 5724−5731. (34) Liu, J. H.; Wang, C. Y.; Jiang, Y.; Hu, Y. P.; Li, J. S.; Yang, S.; Li, Y. H.; Yang, R. H.; Tan, W. H.; Huang, C. Z. Anal. Chem. 2013, 85, 1424−1430. (35) Wang, Y.; Li, Z. H.; Weber, T. J.; Hu, D. H; Lin, C. T.; Li, J. H.; Lin, Y. H. Anal. Chem. 2013, 85, 6775−6782. (36) Hu, H. B.; Zhang, X. F.; Xiao, Y.; Zhou, W.; Wang, L. P.; Jin, L. J. Anal. Chem. 2013, 85, 7076−7084. (37) (a) Yang, K.; Zhang, S.; Zhang, G. X.; Sun, X. M.; Lee, S. T.; Liu, Z. Nano Lett. 2013, 85, 7076−7084. (b) Li, J. L; Bao, H. C.; Hou, X. L.; Sun, L.; Wang, X. G.; Gu, M. Angew. Chem., Int. Ed. 2012, 51, 1830−1834. (38) Parker, C. A.; Rees, W. T. Analyst 1960, 85, 587−600. (39) Nikolay, S.; Makarov, N. K.; Drobizhev, M.; Rebane, A. Opt. Exp. 2008, 16, 4029−4047. (40) Son, J. H.; Lim, C. S.; Han, J. H.; Danish, I. A.; Kim, H. M.; Cho, B. R. J. Org. Chem. 2011, 76, 8113−8116. (41) Lu, C. H.; Yang, H. H.; Zhu, C. L.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2009, 48, 4785−4787. (42) (a) Morales-Narváez, E.; Merkoçi, A. Adv. Mater. 2012, 24, 3298−3308. (b) Kim, J. Y.; Cote, L. J.; Kim, F.; Huang, J. X. J. Am. Chem. Soc. 2010, 134, 260−267.
(43) Lu, C. H.; Zhu, C. L.; Li, J.; Liu, J. J.; Chen, X.; Yang, H. H. Chem. Commun. 2010, 46, 3116−3118. (44) Schröder, G. F.; Alexiev, U.; Helmut Grubmüller, H. Biophys. J. 2005, 89, 3757−3770. (45) Fu, C. C.; Lee, H. S.; Chen, K.; Lim, T. S.; Wu, H. Y.; lin, P. K.; Wei, P. K.; Tsao, P. H.; Chang, H. C.; Fann, W. S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 727−732. (46) Gong, Y.; Sohn, H.; Xue, L.; Firestone, G. L.; Bjeldanes, L. F. Cancer Res. 2006, 66, 4880−4887. (47) Ainscow, E. K.; Rutter, G. A. Diabetes 2002, 51 (Suppl.1), S162−170.
3554
dx.doi.org/10.1021/ac5000015 | Anal. Chem. 2014, 86, 3548−3554
