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Preparation of optical functional composite films and their application in protein detection
Colloids and Surfaces A 535 (2017) 69–74
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Research paper
Preparation of optical functional composite films and their application in protein detection
MARK
⁎
Xiaoyu Wang1, Xiaofeng Jiang1, Shuxian Zhu, Lu Liu, Junhan Xia, Lidong Li
State Key Laboratory for Advanced Metals and Materials, School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, PR China
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Fluorescence Composite film Self-assembly Protein detection
In this work, an optical functional composite film was prepared through self-assembly technique for specific protein detection. The Au nanostructures with metal enhanced fluorescence (MEF) effect were prepared via the in situ reduction of Au ions in a filter paper. Then, a multilayer film was prepared on the Au nanostructures by layer-by-layer adsorption of poly-(ethylenimine), poly-(glycolic acid) and biotinylated poly(L-lysine)-graft-poly (ethylene glycol) (PLL-g-PEG-biotin). Through adjusting the structure of multilayer film, an optimum interaction distance between Au nanostructures and fluorophores for the MEF effect was achieved. Meanwhile, the surface PLL-g-PEG-biotin layer allows the film to capture specific streptavidin through biotin-streptavidin interaction. Owing to the MEF effect of Au nanostructures, a significant enhancement in the fluorescence of fluorescein isothiocyanate (FITC) that labeled streptavidin was successfully obtained. This optical functional composite film with enhanced fluorescence could be used to recognize specific protein in a facile and efficient way.
1. Introduction Fluorescence detection technology is a dominant tool in biological and medical research, mainly due to its high sensitivity, high efficiency, and ease of use [1–5]. It has become massively popular in analytical sciences, particularly in the protein detection based on antibody-antigen specific recognition [6–9]. With excellent optical properties, organic fluorophores [10–12], conjugated polymers [13–17], quantum dots [18–20], metal nanostructures [21–23] and upconversion nanoparticles [24–26] have been used as fluorescent probes in fluorescence detection. Currently, to meet the increasing demands of high⁎
1
throughput assay, solid-phase detection system begins to be developed, such as paper-based sensors, gene chips and microarrays [27–31]. However, the applications of fluorescent probes in solid-phase system are often limited by aggregation-induced fluorescence quenching, low fluorescence quantum yield, and low resistance to photobleaching. Introducing metal nanoparticles into solid matrices will provide a means to bypass the limitation [32–34]. Metal nanoparticles including gold (Au), silver, aluminum and copper nanoparticles are known as plasmonic nanoparticles [35–38]. They exhibit an extraordinary capability to enhance the fluorescence intensity of nearby fluorophores. The strong interaction of excitation
Corresponding author. E-mail address: [email protected] (L. Li). These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.colsurfa.2017.09.026 Received 24 August 2017; Received in revised form 14 September 2017; Accepted 15 September 2017 Available online 18 September 2017 0927-7757/ © 2017 Elsevier B.V. All rights reserved.
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2. Experimental
light with metallic nanostructures leads to the collective oscillations of conduction-band electrons and then an amplified near-field. The amplified near-field results in significant improvements in the optical properties of excited fluorophores. This phenomenon is named as metal-enhanced fluorescence (MEF) effect [39]. Direct synthesis of nanoparticles in solid matrixes is an efficient way for preparing metallic nanostructures that induces MEF effect [40–42]. Cellulose fibers are composed of microfibrils which form three-dimensional network structure and exhibit porous structures [43]. Such structural features can provide a unique microreactor for synthesizing nanoparticles in an in situ way and make cellulose fibers as ideal matrixes. More importantly, MEF effect has a distance-dependent nature [44], only affecting the fluorophores which reside in the region of the amplified near-field produced by metal nanoparticles. However, if the fluorophores directly contact with metallic nanostructures, energy and/ or charge transfer occurs that result in significant quenching of fluorophores by metallic nanostructures [45]. To avoid fluorescence quenching, nanometer-thick film is usually desired to separate fluorophores and metal nanostructures. Layer-by-layer (LbL) multilayer films [46–50], whose thickness can be accurately controlled at the nanometer scale, can be used as interlayer between fluorophores and metal nanostructures to achieve the optimal MEF effect. Moreover, in the preparation of LbL multilayer films, it is easy to introduce biological functional groups to the film system [51–53], which facilitates their further application in biological analysis and detection. In this work, we prepared an optical functional composite film based on LbL self-assembly for protein detection. The film structures and its detection mechanism for special protein are illustrated in Scheme 1. The Au nanoparticles performing MEF effect were prepared via the in situ reduction of Au ions in a filter paper. Through self-assembly technology, a multilayer film containing poly-(ethylenimine) (PEI), poly-(glycolic acid) (PGA) and biotinylated poly(L-lysine)-graftpoly(ethylene glycol) (PLL-g-PEG-biotin) was covered on the Au nanostructures. Adjusting the structure of multilayer film can effectively optimize the interaction distance of the MEF effect. Taking advantage of biotin-streptavidin (SA) coupling [54], assembled PLL-g-PEG-biotin layer specifically captured fluorescein isothiocyanate-labled SA (FITCSA). With the MEF effect in our composite film, the excited FITC produced an enhanced fluorescent signal in response to SA binding allowing specific protein detection. The easily prepared optical functional composite film is expected to serve as novel detection system for sensitive protein detection.
2.1. Materials Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O), PEI (Mn = 60,000), PGA, FITC-labeled lectin protein concanavalin A (FITCCon A), FITC-SA, FITC-labeled goat anti-mouse IgG (FITC-IgG) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Whatman brand filter paper (grade 2, ϕ70 mm) and HyClone Phosphate Buffered Saline (PBS) were purchased from GE Healthcare Life Sciences. Ascorbic acid was purchased from Beijing ABOXING Bio-Tech Co., Ltd. PLL-g-PEG-biotin was purchased from SoSuS AG (Dübendorf, Switzerland). FITC-labeled bovine serum albumin (FITC-BSA) was purchased from Sangon Biotech. Ultrapure Millipore water (18.6 MΩ cm) was used throughout the experiments. All reagents were used without further purification unless otherwise stated. 2.2. Measurements The morphologies of samples were characterized by scanning electron micro-scope (SEM, Carl Zeiss Jena, SUPRA 55 SAPPHIRE). The prepared samples were sputter-coated with carbon to enhance the contrast of the SEM images. Ultraviolet visible (UV–vis) near-infrared absorption spectra were recorded with the Varian Cary 5000 UV-vis near-infrared spectrophotometer. UV–vis absorption spectra were characterized with a Hitachi U3900 spectrophotometer. Fluorescence spectra were recorded with a Hitachi F-7000 fluorescence spectrometer equipped with a Xenon lamp excitation source at room temperature. Time-domain lifetime measurements were performed by an Edinburgh Instruments F900 spectrometer with excitation at 490 nm. Photographs of samples were captured by a Nikon D-7000 camera with excitation at 365 nm provided by the portable UV lamp ZF-7A. 2.3. Preparation of optical functional composite films Filter paper was cut into 1 cm × 3 cm size. Ascorbic acid and HAuCl4 were respectively dissolved to form 30 mM ascorbic solution and 30 mM HAuCl4 solution. The above solutions were stored at 4 °C for further use. Quartz slides were immersed in piranha solution (H2O2/ H2SO4 = 1:3 v/v) for 30 min, washed three times with deionized water and ethanol, and then dried with a gentle stream of nitrogen gas. (CAUTION: “Piranha” solution reacts violently with organic materials; it must be handled with extreme care.) The cleaned slides were placed Scheme 1. Schematic illustration of the optical functional composite film for protein detection and molecule structures of PEI, PGA and PLL-g-PEGbiotin.
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in a glass dish. Then, 200 μL of 30 mM HAuCl4 was dropped to the slide, and filter paper was placed on the solution for 1 min. After adsorption, the filter paper was soaked into 30 mM ascorbic acid solution to form Au nanoparticles. After reacting for 10 min, the filter paper was placed on glass dish and nature drying for 12 h. The prepared filter paper@Au substrates were alternatively immersed in charged 5 mg/ml PEI and 0.5 mg/ml PGA for 15 min under the same conditions. In this experiment, 0–3 bilayers were added to control the thickness of the interlayer films. The final layer was PGA. The prepared substrates were then immersed in 0.5 mg/ml PLL-g-PEG-biotin solution for 15 min to prepare the filter paper@Au/(PEI/PGA)n/PLL-g-PEG-biotin (n = 0–3) films. As a control group, filter papers without Au nanoparticles were covered with the same interlayers following the procedures described as above. 5 μL 0.1 mg/ml FITC-SA, were added dropwise to each sample, respectively. After incubation for 45 min, the samples were rinsed with PBS buffer and dried by nitrogen. The MEF effect was studied by comparing the fluorescence intensities of FITC on the different samples. 2.4. Protein detection on the optical functional composite films Four groups of filter paper@Au/PEI/PGA/PLL-g-PEG-biotin were prepared as described. 5 μL 0.1 mg/ml FITC-SA, FITC-BSA, FITC-IgG and FITCeCon A were added dropwise to each samples, respectively. After incubation for 45 min, the samples were rinsed with PBS buffer and dried by nitrogen. Filter paper/PEI/PGA/PLL-g-PEG-biotin was used as a control experiment. Finally, the fluorescence intensities of FITC on the different samples were measured. 3. Results and discussion Fig. 2. (a) Photographs and (b) UV–vis absorption spectra of filter paper and filter paper@Au, respectively.
3.1. Preparation and characterization of the optical functional composite films
nanoparticles located in the visible region, absorbing visible light will cause plasma resonance of Au nanoparticles and produce MEF effects. Therefore, the prepared Au nanostructures are suitable for MEF studies. In order to efficiently utilize MEF effect of Au nanostructures and avoid fluorescence quenching, we built a multilayer composed of PEI, PGA and PLL-g-PEG-biotin on the Au nanostructures by electrostatic self-assembly. The Au nanoparticles were protected by ascorbic acid and displayed negative charges. Meanwhile, the electron-rich oxygen atoms of hydroxyl and ether groups of cellulose make the matrix negatively charged [56]. Hence, the cationic PEI could be deposited via electrostatic interactions. Then, the anionic PGA layer reversed the charge of the surface. Finally, positively charged PLL-g-PEG-biotin with functional biotin was deposited on the nanocomposite film to offer an application of protein detection. Meanwhile, the stretched PEG brushes of PLL-g-PEG-biotin had a synergistic effect to prevent nonspecific protein adsorption and further improve the detection sensitivity [32]. As revealed by SEM image in Fig. 1b, the Au nanoparticles display a clear and rough surface. Compared Fig. 1b with c, it can be concluded that the Au nanoparticles in Fig. 1c were coated by polymer layers with a high surface coverage. The structure difference confirmed that this interlayer was successfully assembled.
A filter paper was used in our work as the source of cellulose fibers to fabricate Au nanoparticles. As the SEM image shown in Fig. 1a, cellulose fibers in the filter paper three-dimensionally connect with each other and form porous structures. After soaked in HAuCl4 solution, AuCl4− ions were adsorbed in the paper. Immersing Au ion-loaded papers into ascorbic acid solution, Au ions were reduced in situ to form Au nanoparticles. As shown in Fig. 1b, irregular Au nanoparticles anchor to cellulose fibers and randomly distribute throughout the paper matrix. Almost no changes were observed by SEM image in the morphologies of cellulose fibers. Accordingly, the original colorless filter paper turned light pink (Fig. 2a). The results indicated the formation of Au nanoparticles, and Au nanoparticles had no effect on the structure of cellulose fibers. The light pink color probably resulted from the plasmon absorption of Au nanoparticles [55]. We then measured the plasmon absorption of Au nanostructures which strongly depends on the morphology of the Au nanoparticles. UV–vis absorption spectra in Fig. 2b shows that the single absorption band of Au nanoparticles centered at about 534 nm, which is consistent with the plasmon absorption band of spherical Au nanoparticles. While pure filter paper has no absorption peak in the wavelength range of 400–800 nm, which further demonstrate that the Au nanoparticles were successfully prepared in filter paper. As the plasmon absorption of the prepared Au
Fig. 1. SEM image of (a) filter paper, (b) filter paper@Au and (c) filter paper@Au/PEI/PGA/PLL-gPEG-biotin films, respectively. The scale bar represents 1 μm.
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Fig. 3. Normalized UV–vis absorption spectra of filter paper@Au and FITC; normalized fluorescence emission spectrum of FITC in aqueous solution. The excitation wavelength is 490 nm.
3.2. MEF effects of the optical functional composite films Then, the MEF effect of the prepared composite film was determined. As shown in Fig. 3, fluorophore FITC exhibits an absorption peak at 490 nm and a strong emission peak at 519 nm. It was selected as a fluorophore in our system to label proteins as its absorption/emission overlaps well with the plasmon absorption of Au nanostructures. When the plasmon absorption peak matches the FITC excitation/emission wavelength, exciting FITC will cause plasma resonance of Au nanoparticle and produce corresponding MEF effects [57]. The MEF effect of metal nanostructure depends not only on the spectral overlap between metal nanostructure and fluorophores, but also on the separation distance between them [58]. Four different (PEI/ PGA)n/PLL-g-PEG-biotin (n = 0–3) interlayers were prepared by electrostatic self-assembly on the Au nanostructure. The obtained interlayers are named as biotin, 1BL-biotin, 2BL-biotin and 3BL-biotin, respectively. Then, FITC-labeled SA was used as the analyte which can bind to immobilized biotin in our system. Changing the thickness of (PEI/PGA)n/PLL-g-PEG-biotin will vary the distance between FITC and the Au nanostructure. We then examined the fluorescence intensity of FITC at different distances from the Au nanoparticle by varying interlayer structures. As shown in Fig. 4a, in the presence of Au nanoparticles, the fluorescence intensity of FITC changed markedly with the interlayer. When the interlayer was 1BL-biotin, the fluorescence intensity of FITC reached its maximum. Whether increasing or decreasing the layer number of the interlayer resulted in a decrease in fluorescence of FITC. Conversely, in the absence of Au nanoparticles, the fluorescence intensity of FITC was not significantly increased as the interlayer changed. The fluorescence intensity of FITC-SA on the filter paper@Au/ 1BL-biotin film was about 3.5 times as high as that of on the filter paper/1BL-biotin film. These results indicate that MEF depends strongly on the distance between FITC and the Au nanoparticles. The sample with 1BL-biotin exhibited best MEF effect. Thus, 1BL-biotin was selected as the optimal interlayer to separate Au nanoparticles and FITC. To further investigate the enhancement in fluorescence intensity of FITC, we investigated the fluorescence intensity decays of FITC-SA on filter paper@Au/1BL-biotin and filter paper/1BL-biotin. As displayed in Fig. 5, the radiative decay rate of FITC-SA on filter paper@Au/1BL-biotin is obviously faster than that detected on filter paper/1BL-biotin. The intensities of FITC-SA show multiexponential decays. The fitted parameters for intensity decays were displayed in Table 1. B values are the pre-exponential factors and τ values are characteristic lifetimes. The calculated average lifetimes (Table 1) of FITC-SA from filter paper@Au/1BL-biotin and filter paper/1BL-biotin were 2.49 and 6.79 ns, respectively. The shortened lifetime suggested that the fluorescence enhancement of FITC on filter paper@Au/1BL-biotin derived from the increased radiation decay affected by Au nanostructure [59,60]. Therefore, the excitation light of
Fig. 4. (a) The emission intensity of FITC-SA on filter paper@Au film and filter paper film with biotin, 1BL-biotin, 2BL-biotin and 3BL-biotin interlayers, respectively. (b) Emission spectra of FITC-SA on filter paper@Au/1BL-biotin film and filter paper/1BL-biotin film. The excitation wavelength is 490 nm.
Fig. 5. Fluorescence lifetime measurements of FITC-SA on filter paper@Au/1BL-biotin film and filter paper/1BL-biotin film, respectively.
Table 1 Exponential components analysis of the fluorescence intensity decays of FITC-SA on filter paper@Au/1BL-biotin and filter paper/1BL-biotin with the time-correlated single-photon counting technique, respectively. sample
Bi
τi (ns)
fi
χ2
< τ > (ns)
filter paper/1BL-biotin/FITCSA
0.0298
2.819
63.009%
1.199
6.79
0.0037 0.0411
13.477 1.394
36.991% 61.913%
1.184
2.49
0.0083
4.255
38.087%
filter paper@Au/1BL-biotin/ FITC-SA
FITC will cause plasma resonance of Au nanoparticle. And the plasma resonance of Au nanoparticle produces an amplified near-field. Finally, the amplified near-field results in the increased radiation decay of FITC and enhanced fluorescence of FITC. 72
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Acknowledgments This work was supported by the National Natural Science Foundation of China (51373022), the Fundamental Research Funds for the Central Universities (FRF-TP-16-026A1) and the State Key Laboratory for Advanced Metals and Materials (2017Z-03). References [1] A.B. Chinen, C.M. Guan, J.R. Ferrer, S.N. Barnaby, T.J. Merkel, C.A. Mirkin, Nanoparticle probes for the detection of cancer biomarkers cells, and tissues by fluorescence, Chem. Rev. 115 (2015) 10530–10574. [2] M.E. Tanenbaum, L.A. Gilbert, L.S. Qi, J.S. Weissman, R.D. Vale, A protein-tagging system for signal amplification in gene expression and fluorescence imaging, Cell 159 (2014) 635–646. [3] C. Zhu, L. Liu, Q. Yang, F. Lv, S. Wang, Water-soluble conjugated polymers for imaging diagnosis, and therapy, Chem. Rev. 112 (2012) 4687–4735. [4] Q.T. Nguyen, E.S. Olson, T.A. Aguilera, T. Jiang, M. Scadeng, L.G. Ellies, R.Y. 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Fig. 6. (a) The emission intensity of FITC on the optical functional composite films after incubation with FITC-BSA, FITC-IgG, FITC-Con A and FITC-SA, respectively. The excitation wavelength is 490 nm. (b) Fluorescence photographs of the optical functional composite films after incubation with FITC-BSA, FITC-IgG, FITC-Con A and FITC-SA under 365-nm UV light.
3.3. Protein detection of the optical functional composite films By taking advantage of the enhanced fluorescence intensity of FITC, we examined the ability of the optical functional composite films to detect specific protein SA. The stretched PEG brushes of outermost PLLg-PEG-biotin show a synergistic effect and can efficiently circumvent nonspecific protein adsorption [32]. Three more non-target proteins such as FITC-BSA, FITC-IgG and FITC-Con A were chosen as the control groups. Fluorescence spectra were measured for FITC on the filter paper@Au/1BL-biotin films after incubation with different proteins. As shown in Fig. 6a, the fluorescence emission of FITC could be obviously obtained with target protein SA, while the fluorescence signal cannot be observed for other proteins. The results indicate that the SA was selectively captured and detected by our optical functional composite films. Under UV light irradiation, a brilliant blue-green fluorescence from the protein FITC-SA appears against the blue background color of the filter paper (Fig. 6b), which is consistent with the result of SA capture. Hence, the developed optical functional composite film can be used to detect special protein.
4. Conclusions In summary, an optical functional composite film based on self-assembly approach was prepared for specific protein detection. The in situ reduction of Au nanoparticles in a filter paper provided the metal nanostructures with MEF effect. Based on the driving force of electrostatic interactions, an interlayer film composed of PEI, PGA and biotinylated PLL-g-PEG-biotin was built between the Au nanostructure and FITC. The structure of interlayer film was simply adjusted to obtain the optimal MEF effect of Au nanostructure. Exploiting the specific biotin–streptavidin interaction, the target protein SA was captured by biotin in our composite film. With MEF effect of the film, fluorescence intensity of FITC that labeled SA was significantly enhanced. It allowed the optical functional composite film to detect SA in a facile and efficient way. We believe that the research provides a new approach to prepare optical functional films for protein detection. 73
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Contents lists available at ScienceDirect
Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Research paper
Preparation of optical functional composite films and their application in protein detection
MARK
⁎
Xiaoyu Wang1, Xiaofeng Jiang1, Shuxian Zhu, Lu Liu, Junhan Xia, Lidong Li
State Key Laboratory for Advanced Metals and Materials, School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, PR China
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Fluorescence Composite film Self-assembly Protein detection
In this work, an optical functional composite film was prepared through self-assembly technique for specific protein detection. The Au nanostructures with metal enhanced fluorescence (MEF) effect were prepared via the in situ reduction of Au ions in a filter paper. Then, a multilayer film was prepared on the Au nanostructures by layer-by-layer adsorption of poly-(ethylenimine), poly-(glycolic acid) and biotinylated poly(L-lysine)-graft-poly (ethylene glycol) (PLL-g-PEG-biotin). Through adjusting the structure of multilayer film, an optimum interaction distance between Au nanostructures and fluorophores for the MEF effect was achieved. Meanwhile, the surface PLL-g-PEG-biotin layer allows the film to capture specific streptavidin through biotin-streptavidin interaction. Owing to the MEF effect of Au nanostructures, a significant enhancement in the fluorescence of fluorescein isothiocyanate (FITC) that labeled streptavidin was successfully obtained. This optical functional composite film with enhanced fluorescence could be used to recognize specific protein in a facile and efficient way.
1. Introduction Fluorescence detection technology is a dominant tool in biological and medical research, mainly due to its high sensitivity, high efficiency, and ease of use [1–5]. It has become massively popular in analytical sciences, particularly in the protein detection based on antibody-antigen specific recognition [6–9]. With excellent optical properties, organic fluorophores [10–12], conjugated polymers [13–17], quantum dots [18–20], metal nanostructures [21–23] and upconversion nanoparticles [24–26] have been used as fluorescent probes in fluorescence detection. Currently, to meet the increasing demands of high⁎
1
throughput assay, solid-phase detection system begins to be developed, such as paper-based sensors, gene chips and microarrays [27–31]. However, the applications of fluorescent probes in solid-phase system are often limited by aggregation-induced fluorescence quenching, low fluorescence quantum yield, and low resistance to photobleaching. Introducing metal nanoparticles into solid matrices will provide a means to bypass the limitation [32–34]. Metal nanoparticles including gold (Au), silver, aluminum and copper nanoparticles are known as plasmonic nanoparticles [35–38]. They exhibit an extraordinary capability to enhance the fluorescence intensity of nearby fluorophores. The strong interaction of excitation
Corresponding author. E-mail address: [email protected] (L. Li). These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.colsurfa.2017.09.026 Received 24 August 2017; Received in revised form 14 September 2017; Accepted 15 September 2017 Available online 18 September 2017 0927-7757/ © 2017 Elsevier B.V. All rights reserved.
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2. Experimental
light with metallic nanostructures leads to the collective oscillations of conduction-band electrons and then an amplified near-field. The amplified near-field results in significant improvements in the optical properties of excited fluorophores. This phenomenon is named as metal-enhanced fluorescence (MEF) effect [39]. Direct synthesis of nanoparticles in solid matrixes is an efficient way for preparing metallic nanostructures that induces MEF effect [40–42]. Cellulose fibers are composed of microfibrils which form three-dimensional network structure and exhibit porous structures [43]. Such structural features can provide a unique microreactor for synthesizing nanoparticles in an in situ way and make cellulose fibers as ideal matrixes. More importantly, MEF effect has a distance-dependent nature [44], only affecting the fluorophores which reside in the region of the amplified near-field produced by metal nanoparticles. However, if the fluorophores directly contact with metallic nanostructures, energy and/ or charge transfer occurs that result in significant quenching of fluorophores by metallic nanostructures [45]. To avoid fluorescence quenching, nanometer-thick film is usually desired to separate fluorophores and metal nanostructures. Layer-by-layer (LbL) multilayer films [46–50], whose thickness can be accurately controlled at the nanometer scale, can be used as interlayer between fluorophores and metal nanostructures to achieve the optimal MEF effect. Moreover, in the preparation of LbL multilayer films, it is easy to introduce biological functional groups to the film system [51–53], which facilitates their further application in biological analysis and detection. In this work, we prepared an optical functional composite film based on LbL self-assembly for protein detection. The film structures and its detection mechanism for special protein are illustrated in Scheme 1. The Au nanoparticles performing MEF effect were prepared via the in situ reduction of Au ions in a filter paper. Through self-assembly technology, a multilayer film containing poly-(ethylenimine) (PEI), poly-(glycolic acid) (PGA) and biotinylated poly(L-lysine)-graftpoly(ethylene glycol) (PLL-g-PEG-biotin) was covered on the Au nanostructures. Adjusting the structure of multilayer film can effectively optimize the interaction distance of the MEF effect. Taking advantage of biotin-streptavidin (SA) coupling [54], assembled PLL-g-PEG-biotin layer specifically captured fluorescein isothiocyanate-labled SA (FITCSA). With the MEF effect in our composite film, the excited FITC produced an enhanced fluorescent signal in response to SA binding allowing specific protein detection. The easily prepared optical functional composite film is expected to serve as novel detection system for sensitive protein detection.
2.1. Materials Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O), PEI (Mn = 60,000), PGA, FITC-labeled lectin protein concanavalin A (FITCCon A), FITC-SA, FITC-labeled goat anti-mouse IgG (FITC-IgG) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Whatman brand filter paper (grade 2, ϕ70 mm) and HyClone Phosphate Buffered Saline (PBS) were purchased from GE Healthcare Life Sciences. Ascorbic acid was purchased from Beijing ABOXING Bio-Tech Co., Ltd. PLL-g-PEG-biotin was purchased from SoSuS AG (Dübendorf, Switzerland). FITC-labeled bovine serum albumin (FITC-BSA) was purchased from Sangon Biotech. Ultrapure Millipore water (18.6 MΩ cm) was used throughout the experiments. All reagents were used without further purification unless otherwise stated. 2.2. Measurements The morphologies of samples were characterized by scanning electron micro-scope (SEM, Carl Zeiss Jena, SUPRA 55 SAPPHIRE). The prepared samples were sputter-coated with carbon to enhance the contrast of the SEM images. Ultraviolet visible (UV–vis) near-infrared absorption spectra were recorded with the Varian Cary 5000 UV-vis near-infrared spectrophotometer. UV–vis absorption spectra were characterized with a Hitachi U3900 spectrophotometer. Fluorescence spectra were recorded with a Hitachi F-7000 fluorescence spectrometer equipped with a Xenon lamp excitation source at room temperature. Time-domain lifetime measurements were performed by an Edinburgh Instruments F900 spectrometer with excitation at 490 nm. Photographs of samples were captured by a Nikon D-7000 camera with excitation at 365 nm provided by the portable UV lamp ZF-7A. 2.3. Preparation of optical functional composite films Filter paper was cut into 1 cm × 3 cm size. Ascorbic acid and HAuCl4 were respectively dissolved to form 30 mM ascorbic solution and 30 mM HAuCl4 solution. The above solutions were stored at 4 °C for further use. Quartz slides were immersed in piranha solution (H2O2/ H2SO4 = 1:3 v/v) for 30 min, washed three times with deionized water and ethanol, and then dried with a gentle stream of nitrogen gas. (CAUTION: “Piranha” solution reacts violently with organic materials; it must be handled with extreme care.) The cleaned slides were placed Scheme 1. Schematic illustration of the optical functional composite film for protein detection and molecule structures of PEI, PGA and PLL-g-PEGbiotin.
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in a glass dish. Then, 200 μL of 30 mM HAuCl4 was dropped to the slide, and filter paper was placed on the solution for 1 min. After adsorption, the filter paper was soaked into 30 mM ascorbic acid solution to form Au nanoparticles. After reacting for 10 min, the filter paper was placed on glass dish and nature drying for 12 h. The prepared filter paper@Au substrates were alternatively immersed in charged 5 mg/ml PEI and 0.5 mg/ml PGA for 15 min under the same conditions. In this experiment, 0–3 bilayers were added to control the thickness of the interlayer films. The final layer was PGA. The prepared substrates were then immersed in 0.5 mg/ml PLL-g-PEG-biotin solution for 15 min to prepare the filter paper@Au/(PEI/PGA)n/PLL-g-PEG-biotin (n = 0–3) films. As a control group, filter papers without Au nanoparticles were covered with the same interlayers following the procedures described as above. 5 μL 0.1 mg/ml FITC-SA, were added dropwise to each sample, respectively. After incubation for 45 min, the samples were rinsed with PBS buffer and dried by nitrogen. The MEF effect was studied by comparing the fluorescence intensities of FITC on the different samples. 2.4. Protein detection on the optical functional composite films Four groups of filter paper@Au/PEI/PGA/PLL-g-PEG-biotin were prepared as described. 5 μL 0.1 mg/ml FITC-SA, FITC-BSA, FITC-IgG and FITCeCon A were added dropwise to each samples, respectively. After incubation for 45 min, the samples were rinsed with PBS buffer and dried by nitrogen. Filter paper/PEI/PGA/PLL-g-PEG-biotin was used as a control experiment. Finally, the fluorescence intensities of FITC on the different samples were measured. 3. Results and discussion Fig. 2. (a) Photographs and (b) UV–vis absorption spectra of filter paper and filter paper@Au, respectively.
3.1. Preparation and characterization of the optical functional composite films
nanoparticles located in the visible region, absorbing visible light will cause plasma resonance of Au nanoparticles and produce MEF effects. Therefore, the prepared Au nanostructures are suitable for MEF studies. In order to efficiently utilize MEF effect of Au nanostructures and avoid fluorescence quenching, we built a multilayer composed of PEI, PGA and PLL-g-PEG-biotin on the Au nanostructures by electrostatic self-assembly. The Au nanoparticles were protected by ascorbic acid and displayed negative charges. Meanwhile, the electron-rich oxygen atoms of hydroxyl and ether groups of cellulose make the matrix negatively charged [56]. Hence, the cationic PEI could be deposited via electrostatic interactions. Then, the anionic PGA layer reversed the charge of the surface. Finally, positively charged PLL-g-PEG-biotin with functional biotin was deposited on the nanocomposite film to offer an application of protein detection. Meanwhile, the stretched PEG brushes of PLL-g-PEG-biotin had a synergistic effect to prevent nonspecific protein adsorption and further improve the detection sensitivity [32]. As revealed by SEM image in Fig. 1b, the Au nanoparticles display a clear and rough surface. Compared Fig. 1b with c, it can be concluded that the Au nanoparticles in Fig. 1c were coated by polymer layers with a high surface coverage. The structure difference confirmed that this interlayer was successfully assembled.
A filter paper was used in our work as the source of cellulose fibers to fabricate Au nanoparticles. As the SEM image shown in Fig. 1a, cellulose fibers in the filter paper three-dimensionally connect with each other and form porous structures. After soaked in HAuCl4 solution, AuCl4− ions were adsorbed in the paper. Immersing Au ion-loaded papers into ascorbic acid solution, Au ions were reduced in situ to form Au nanoparticles. As shown in Fig. 1b, irregular Au nanoparticles anchor to cellulose fibers and randomly distribute throughout the paper matrix. Almost no changes were observed by SEM image in the morphologies of cellulose fibers. Accordingly, the original colorless filter paper turned light pink (Fig. 2a). The results indicated the formation of Au nanoparticles, and Au nanoparticles had no effect on the structure of cellulose fibers. The light pink color probably resulted from the plasmon absorption of Au nanoparticles [55]. We then measured the plasmon absorption of Au nanostructures which strongly depends on the morphology of the Au nanoparticles. UV–vis absorption spectra in Fig. 2b shows that the single absorption band of Au nanoparticles centered at about 534 nm, which is consistent with the plasmon absorption band of spherical Au nanoparticles. While pure filter paper has no absorption peak in the wavelength range of 400–800 nm, which further demonstrate that the Au nanoparticles were successfully prepared in filter paper. As the plasmon absorption of the prepared Au
Fig. 1. SEM image of (a) filter paper, (b) filter paper@Au and (c) filter paper@Au/PEI/PGA/PLL-gPEG-biotin films, respectively. The scale bar represents 1 μm.
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Fig. 3. Normalized UV–vis absorption spectra of filter paper@Au and FITC; normalized fluorescence emission spectrum of FITC in aqueous solution. The excitation wavelength is 490 nm.
3.2. MEF effects of the optical functional composite films Then, the MEF effect of the prepared composite film was determined. As shown in Fig. 3, fluorophore FITC exhibits an absorption peak at 490 nm and a strong emission peak at 519 nm. It was selected as a fluorophore in our system to label proteins as its absorption/emission overlaps well with the plasmon absorption of Au nanostructures. When the plasmon absorption peak matches the FITC excitation/emission wavelength, exciting FITC will cause plasma resonance of Au nanoparticle and produce corresponding MEF effects [57]. The MEF effect of metal nanostructure depends not only on the spectral overlap between metal nanostructure and fluorophores, but also on the separation distance between them [58]. Four different (PEI/ PGA)n/PLL-g-PEG-biotin (n = 0–3) interlayers were prepared by electrostatic self-assembly on the Au nanostructure. The obtained interlayers are named as biotin, 1BL-biotin, 2BL-biotin and 3BL-biotin, respectively. Then, FITC-labeled SA was used as the analyte which can bind to immobilized biotin in our system. Changing the thickness of (PEI/PGA)n/PLL-g-PEG-biotin will vary the distance between FITC and the Au nanostructure. We then examined the fluorescence intensity of FITC at different distances from the Au nanoparticle by varying interlayer structures. As shown in Fig. 4a, in the presence of Au nanoparticles, the fluorescence intensity of FITC changed markedly with the interlayer. When the interlayer was 1BL-biotin, the fluorescence intensity of FITC reached its maximum. Whether increasing or decreasing the layer number of the interlayer resulted in a decrease in fluorescence of FITC. Conversely, in the absence of Au nanoparticles, the fluorescence intensity of FITC was not significantly increased as the interlayer changed. The fluorescence intensity of FITC-SA on the filter paper@Au/ 1BL-biotin film was about 3.5 times as high as that of on the filter paper/1BL-biotin film. These results indicate that MEF depends strongly on the distance between FITC and the Au nanoparticles. The sample with 1BL-biotin exhibited best MEF effect. Thus, 1BL-biotin was selected as the optimal interlayer to separate Au nanoparticles and FITC. To further investigate the enhancement in fluorescence intensity of FITC, we investigated the fluorescence intensity decays of FITC-SA on filter paper@Au/1BL-biotin and filter paper/1BL-biotin. As displayed in Fig. 5, the radiative decay rate of FITC-SA on filter paper@Au/1BL-biotin is obviously faster than that detected on filter paper/1BL-biotin. The intensities of FITC-SA show multiexponential decays. The fitted parameters for intensity decays were displayed in Table 1. B values are the pre-exponential factors and τ values are characteristic lifetimes. The calculated average lifetimes (Table 1) of FITC-SA from filter paper@Au/1BL-biotin and filter paper/1BL-biotin were 2.49 and 6.79 ns, respectively. The shortened lifetime suggested that the fluorescence enhancement of FITC on filter paper@Au/1BL-biotin derived from the increased radiation decay affected by Au nanostructure [59,60]. Therefore, the excitation light of
Fig. 4. (a) The emission intensity of FITC-SA on filter paper@Au film and filter paper film with biotin, 1BL-biotin, 2BL-biotin and 3BL-biotin interlayers, respectively. (b) Emission spectra of FITC-SA on filter paper@Au/1BL-biotin film and filter paper/1BL-biotin film. The excitation wavelength is 490 nm.
Fig. 5. Fluorescence lifetime measurements of FITC-SA on filter paper@Au/1BL-biotin film and filter paper/1BL-biotin film, respectively.
Table 1 Exponential components analysis of the fluorescence intensity decays of FITC-SA on filter paper@Au/1BL-biotin and filter paper/1BL-biotin with the time-correlated single-photon counting technique, respectively. sample
Bi
τi (ns)
fi
χ2
< τ > (ns)
filter paper/1BL-biotin/FITCSA
0.0298
2.819
63.009%
1.199
6.79
0.0037 0.0411
13.477 1.394
36.991% 61.913%
1.184
2.49
0.0083
4.255
38.087%
filter paper@Au/1BL-biotin/ FITC-SA
FITC will cause plasma resonance of Au nanoparticle. And the plasma resonance of Au nanoparticle produces an amplified near-field. Finally, the amplified near-field results in the increased radiation decay of FITC and enhanced fluorescence of FITC. 72
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Fig. 6. (a) The emission intensity of FITC on the optical functional composite films after incubation with FITC-BSA, FITC-IgG, FITC-Con A and FITC-SA, respectively. The excitation wavelength is 490 nm. (b) Fluorescence photographs of the optical functional composite films after incubation with FITC-BSA, FITC-IgG, FITC-Con A and FITC-SA under 365-nm UV light.
3.3. Protein detection of the optical functional composite films By taking advantage of the enhanced fluorescence intensity of FITC, we examined the ability of the optical functional composite films to detect specific protein SA. The stretched PEG brushes of outermost PLLg-PEG-biotin show a synergistic effect and can efficiently circumvent nonspecific protein adsorption [32]. Three more non-target proteins such as FITC-BSA, FITC-IgG and FITC-Con A were chosen as the control groups. Fluorescence spectra were measured for FITC on the filter paper@Au/1BL-biotin films after incubation with different proteins. As shown in Fig. 6a, the fluorescence emission of FITC could be obviously obtained with target protein SA, while the fluorescence signal cannot be observed for other proteins. The results indicate that the SA was selectively captured and detected by our optical functional composite films. Under UV light irradiation, a brilliant blue-green fluorescence from the protein FITC-SA appears against the blue background color of the filter paper (Fig. 6b), which is consistent with the result of SA capture. Hence, the developed optical functional composite film can be used to detect special protein.
4. Conclusions In summary, an optical functional composite film based on self-assembly approach was prepared for specific protein detection. The in situ reduction of Au nanoparticles in a filter paper provided the metal nanostructures with MEF effect. Based on the driving force of electrostatic interactions, an interlayer film composed of PEI, PGA and biotinylated PLL-g-PEG-biotin was built between the Au nanostructure and FITC. The structure of interlayer film was simply adjusted to obtain the optimal MEF effect of Au nanostructure. Exploiting the specific biotin–streptavidin interaction, the target protein SA was captured by biotin in our composite film. With MEF effect of the film, fluorescence intensity of FITC that labeled SA was significantly enhanced. It allowed the optical functional composite film to detect SA in a facile and efficient way. We believe that the research provides a new approach to prepare optical functional films for protein detection. 73
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