Co-enhancement of fluorescence and singlet oxygen generation by silica-coated gold nanorods core-shell nanoparticle
© Ke et al.; licensee Springer. 2014
Received: 16 October 2014
Accepted: 26 November 2014
Published: 10 December 2014
Metal-enhanced fluorescence (MEF) as a newly recognized technology has been attracting considerable attention and is widely used in fluorescence-based technology. In this paper, we reported a novel distance-dependent MEF and metal-enhanced singlet oxygen generation phenomenon based on silica-coated gold nanorods (AuNRs@SiO2) core-shell structure with tetra-substituted carboxyl aluminum phthalocyanine (AlC4Pc) that serve as both fluorophore and photosensitizer. When the AlC4Pc was linked on the surface of AuNRs@SiO2, the fluorescence intensity and singlet oxygen productivity varied with the thickness difference of silica shell from 2.1 to 28.6 nm. The co-enhancement effect reached the maximum of 7-fold and 2.1-fold, respectively, when the separation distance was 10.6 nm. These unique characteristics make the prepared core-shell nanoparticles promising for MEF-based biological imaging and photodynamics therapy.
Noble metal nanoparticles (NPs) have been widely applied in chemical and biological sensing [1–4], surface-enhanced Raman scattering (SERS) [5, 6], and bioimaging technologies [7, 8] due to their unique shape- and size-dependent localized surface plasmon resonance (LSPR) and local electromagnetic field features. It has also been found that many kinds of metal NPs can dramatically enhance or quench the fluorescence of fluorophores along with the changing of separation distance between them . Such fluorescence enhancement is known as metal-enhanced fluorescence (MEF). Furthermore, when the wavelength of fluorescence emission overlap well with the plasmonic response of metal NPs, the fluorophores can couple with metal NPs, which result in a shortening of lifetime and an increasing of photostability as compared with that of free fluorophores [10, 11]. Therefore, the metal NP-associated fluorophores have illustrated a promising candidate for cell imaging and tracking owing to this fluorophore-metal interaction [11, 12].
Until now, numerous nanodevices have been used to investigate the mechanism of MEF. Most studies of MEF were based on silver island films (SIFs) with the depositing of Ag NPs on a glass substrate [12–14]. However, these structures exhibited irregular morphology and unsuitable size, which would limit their in vivo application research. In some other distance-dependent MEF studies, flexible molecules like DNA [15, 16], polyethylene glycol , and some other polymers [18, 19] were used as spacer layers, while they were not rigid enough to obtain accurate distance control. To overcome these problems, Gerritsen et al.  have developed an AgNPs@SiO2 core-shell structure to achieve well performance in MEF by taking the advantage of the easy and accurate tunable feature of silica shell. Such method introduced a new way to manage the distance between fluorophores and metal core in the study of MEF. In addition to MEF, Geddes et al.  have found that the photon-induce excited states could enhance both fluorescence and phosphorescence emission, like the phosphorescence emission of singlet oxygen (1O2) at 1,270 nm. The production of reactive oxygen species (ROS) could be enhanced by the intensified electromagnetic field when the photosensitizers (PSs) were placed in close proximity to the metal NP.
To achieve the co-enhancement of fluorescence intensity and 1O2 generation, an ideal model system of silica-coated gold nanorod (AuNRs@SiO2) with tetra-substituted carboxyl aluminum phthalocyanine (AlC4Pc) linked on the surface to serve as both fluorophore and photosensitizer was demonstrated in our work. Gold nanorods were chosen as MEF substrates, which offered the possibility of plasmon resonance tuning to match the spectra of AlC4Pc. The distance between AuNRs core and AlC4Pc was controlled by varying the thickness of silica shell spacer layers. By adjusting this spacer layer to an appropriate distance, both of the fluorescence intensity and 1O2 generation of AlC4Pc was obviously enhanced by the AuNRs@SiO2 core-shell structure. Our current results shed a new light on the PSs delivery system for the real-time visualization of photodynamics therapy (PDT) by using metal-enhanced effect.
Chloroauric acid (HAuCl4 · 3H2O), silver nitrate (AgNO3), sodium borohydride (NaBH4), cetyltrimethyl ammonium bromide (CTAB), L-ascorbic acid (AA), tetraethyoxysilane (TEOS), and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China). 3-aminopropyl triethoxysilane (APTMS), 9, 10-anthracenediyl-bis (methylene) dimalonic acid (ABDA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC · HCl), and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich (Sigma-Aldrich St Louis, MO, USA). AlC4Pc was synthesized and purified according to a method in the literature . The other reagents were analytical grade and used without further purification. Milli-Q water (less than 18.2 MΩ) was used throughout the synthesis experiments.
Synthesis of AuNRs
AuNRs were prepared by the well-known seed-mediated growth process . The seed solution was prepared firstly; 9.5 mL of 0.1 M CTAB solution was mixed with 0.25 mL of 0.01 M HAuCl4. After that, a freshly prepared 0.6 mL of 10 mM ice-cold NaBH4 solution was quickly injected into the mixed solution and magnetically stirred for 2 min. The mixture rapidly turned into a brownish-yellow color and was kept at room temperature for 2 h.
To prepare the growth solution, 5 mL of 0.01 M HAuCl4 and 0.35 mL of 10 mM AgNO3 were mixed with 95 mL of 0.1 M CTAB, followed by adding a freshly prepared 0.55 mL of 0.1 M AA solution to reduce the Au3+ to Au0. A seed solution of 120 μL was gently added into the resultant solution and was continuously stirred for 2 min, and then the reaction mixture was stored at 30°C for at least 3 h without agitation.
Synthesis of AuNRs@SiO2 with different shell thickness
The silica-coated AuNRs were achieved by a modified StÖber method , and the silica shells with different thicknesses were obtained by adding varying amounts of TEOS. Briefly, 10 mL of the as-prepared AuNRs solution was centrifuged at 10,000 rpm for 30 min twice to remove the excess CTAB surfactant and was re-dispersed in 10 mL pure water. Subsequently, 100 μL of 0.1 M NaOH solution was added into the AuNRs solution under gentle stirring to adjust the pH value to 10.0. After that, 5, 8, 10, 15, 20, or 25 μL of TEOS/methanol (V/V: 1/4) solution together with 10 μL of APTMS/methanol (V/V: 1/49) solution was injected three times at a 30-min interval to form the silica shell. The reaction was allowed to be gently stirred for 24 h, and the resultant colloidal solution was centrifuged at 7,500 rpm for 30 min twice to remove the unreacted TEOS and APTMS.
Covalent binding of AlC4Pc to AuNRs@SiO2
The covalent binding of AlC4Pc to the surface of amine-capped AuNRs@SiO2 was performed by using EDC and NHS cross-linking procedure. In a typical coupling reaction, 5 μL of 10 μM AlC4Pc was pre-activated by an EDC/NHS solution for 30 min and then added into 2 mL of 0.4 nM AuNRs@SiO2 solution. The mixed solution was incubated with gentle shaking at room temperature for 6 h. The unlinked AlC4Pc molecules were separated from AuNRs@SiO2 NPs after synthesis completion via centrifugation at 7,500 rpm for 30 min three times. By measuring the fluorescence intensity of every supernatant solution, the amount of unlinked AlC4Pc was deduced via a calibration curve plotted with known concentrations.
Transmission electron microscopy (TEM) images were acquired by using a JEM-2100 HRTEM (JEOL, Akishima-shi, Japan) operating with an accelerating voltage of 200 kV. The absorption spectra were measured using a UV-1750 UV-vis spectrophotometer (Shimadzu, Kyoto, Japan). The fluorescence emission spectra and fluorescence lifetime decay curves were obtained by FluoroMax-4 spectrofluorometer (HORIBA Jobin Yvon, Palaiseau, France); fluorescence lifetime decay curves were measured with a pulsed 591-nm nano LED as the excitation source. Detection of singlet oxygen generation was obtained using a FLS 920 spectrofluorimeter (FLS 920 spectrofluorimeter, Edinburgh, UK) with an excitation wavelength at 380 nm in the dark room.
Fluorescence spectroscopic measurements
where I i is the fluorescence intensity of AuNRs@SiO2-AlC4Pc varying with shell thickness, I0 is the fluorescence intensity of free AlC4Pc in the same amount, and Ibackground is the fluorescence intensity of pure water.
With the numerous measurements, the average fluorescence lifetimes in different cases were determined.
Measurement of singlet oxygen (1O2) generation
ABDA, a chemical-sensitive probe of ROS, was used to monitor the generation of 1O2. In the photochemical experiment, 5 μL of 10 μM ABDA in DMSO solution was added to 2 mL of 0.4 nM AuNRs@SiO2-AlC4Pc with different shell thickness and free AlC4Pc solution, respectively. The mixtures were photo-activated by using an LED lamp excitation at 680 nm (10 mW cm−2), and the generation of 1O2 was measured by recording the fluctuation of ABDA fluorescence intensity after different amount of light exposures.
Results and discussion
Synthesis and characterization of AuNRs@SiO2-AlC4Pc
Spectral analysis of AuNRs@SiO2-AlC4Pc
Distance-dependent MEF of AuNRs@SiO2-AlC4Pc
To further confirm whether the observed fluorescence enhancement was attributed to the resonance interaction, a fluorescence lifetime measurement was carried out. As can be observed in the Figure 5b, the fluorescence intensity decays were fitted in terms of the multi-exponential model. The fluorescence lifetime of AlC4Pc was dramatically shortened from 4.78 to 0.515 ns after linking on the AuNRs@SiO2, when the distance between AlC4Pc and AuNRs was 10.6 nm.
Comparing with the free-space radiation, the increase in the radiative rate would directly increase the quantum yield and decrease the lifetime of a fluorophore. The increased radiative rate of fluorophore, together with the concentrated local electromagnetic field of metal substrate, can give a well explanation for the MEF phenomenon of AuNRs@SiO2-AlC4Pc .
Effects of different shell thickness on singlet oxygen generation
In summary, the AuNRs@SiO2 core-shell NPs linked with AlC4Pc were prepared to study the MEF phenomenon and 1O2 generation. As we have shown, the fluorescence of AlC4Pc was significantly enhanced by optimizing the distance between AlC4Pc and AuNRs. Meanwhile, 1O2 generation could also be increased probably by the promotion of intersystem crossing and triplet state yield. Co-enhancement of fluorescence intensity and 1O2 generation reached their maximum effect when the distance between AlC4Pc and AuNRs was 10.6 nm. Although the mechanism of MEF and metal-enhanced 1O2 generation are still under investigation, these results provide a desirable insight towards evaluating and improving the performance of AuNRs-based NPs in fluorescence imaging and PDT.
This work was financially supported by the National Basic Research Program of China (973 Program) (2013CB933703), the National Natural Science Foundation of China (Nos. 31371012, 81171448, 31428007), and the Medical Science and Technology Innovation Program of Nanjing Military Region (11Z022).
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