Silicon Nanowire-Based Fluorescent Nanosensor for Complexed

Letter pubs.acs.org/NanoLett

Silicon Nanowire-Based Fluorescent Nanosensor for Complexed Cu2+ and its Bioapplications Rong Miao,†,‡ Lixuan Mu,† Hongyan Zhang,† Guangwei She,† Bingjiang Zhou,†,‡ Haitao Xu,† Pengfei Wang,† and Wensheng Shi*,† †

Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190 China ‡ University of Chinese Academy of Sciences, Beijing, 100039 China S Supporting Information *

ABSTRACT: A silicon nanowires (SiNWs)-based fluorescent sensor for complexed Cu2+ was realized. High sensitivity and selectivity of the present sensor facilitate its bioapplications. The sensor was successfully used to detect the Cu2+ in liver extract. Meanwhile, real-time and in situ monitoring of Cu2+ released from apoptotic HeLa cell was performed using the as-prepared SiNW arrays-based sensor. These results indicate that the present SiNWs-based sensor would be of potential applications in revealing the physiological and pathological roles of Cu2+. KEYWORDS: SiNWs, SiNW arrays, fluorescent sensor, Cu2+, complexed Cu2+

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u2+ is one of the most abundant metal ions in the human body and the total concentration of Cu2+ is generally as high as around 100 μM in healthy cells.1,2 It is known that most of the Cu2+ exist as a complexed form in which the Cu2+ are tightly bonded into specific proteins and a small proportion is chelated by numerous small nutrient molecules, such as SOD1, ceruloplasmin, tyrosinase, and so forth.1−3 What’s more, it is the complexed Cu2+ that plays a critical role in fundamental physiological processes, such as biocatalysis, oxidative stress, and disease.4−7 Therefore, the ability of the sensor to monitor the complexed Cu2+ in living cells would be of great significance for understanding the roles of Cu2+ in biological systems. So far, although various methods have been developed to detect Cu2+, most of them worked well for free Cu2+ but not for the complexed one.8−12 Only high-performance liquid chromatography with online inductively coupled plasma mass spectrometric (HPLC-ICPMS) has been reported to be able to realize the detection of the complexed Cu2+ (SOD1).13,14 It is known that the HPLC-ICPMS often suffers from obvious disadvantages, especially the requirement of complicated instruments. In order to conveniently investigate the roles of Cu2+ in organisms, it is vital to establish a rational method to selectively and sensitively detect the complexed Cu2+.15 During the past decade with the development of nanomaterials, various fluorescent nanosensors for Cu2+ have been fabricated and © XXXX American Chemical Society

many of them exhibit enhanced properties. Unfortunately, most of these fluorescent nanosensors possess satisfactory sensitivity and selectivity only for free Cu2+, therefore the capability to detect the complexed Cu2+ is still quite limited. One rational approach is to develop a fluorescent sensor that owns remarkable binding affinity with Cu2+. When such a sensor works in a complexed Cu2+ containing system, it could easily snatch the Cu2+ from the complexation and then exhibit a change in its fluorescence signal. Meanwhile, the sensor should be selective enough to avoid the interference from other metal ions and biomolecules during practical application. Recently some experimental results demonstrated that the sensitivity and selectivity of fluorescent sensor could be highly improved by immobilizing the highly specific recognition ligands (organic molecules or biomolecules) onto the one-dimensional (1D) nanostructures.16−19 Among these 1D nanostructures, Si nanowires (SiNWs) are good candidates for bioapplications due to their nontoxicity, biocompatibility, and convenience for integration with IC.20−22 Moreover, the SiNW arrays could also enhance the adhesion force between cell and substrate and restrict cell spreading, which would appreciably offer the Received: January 23, 2014 Revised: April 3, 2014

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Scheme 1. Structure of 3-AD-SiNWs and Procedure of the Modification of SiNWs

convenience to the investigation of the cells.23−25 On the basis of these advantages of the SiNWs, it has been utilized as excellent biomedical materials in cellular studies, such as gene transduction, intracellular biomolecules delivery, and biochemical activity measurement.26−29 Considering the importance of complexed Cu2+ detection and the advantages of the SiNWs-based fluorescent sensor, in this study the combination of 3-[2-(2-aminoethylamino)ethylamino] propyl-trimethoxysilane (3-A, receptor) and dansyl group (D, fluorophores) was covalently immobilized onto the surface of SiNWs and a fluorescent sensor (3-AD-SiNWs) is realized for complexed Cu2+ (SOD1 and liver extract) with high selectivity and sensitivity. Furthermore, 3-AD-SiNW arrays have been successfully used in real time and in situ monitoring of the Cu2+ released from apoptotic HeLa cells. Present sensors show potential application in further investigation of the physiological and pathological roles of the Cu2+ in biosystems and it could be used in studying of the interaction between the nanosensor and cellular interfaces.30,31 Meanwhile, the reported method used to construct the sensor for complexed Cu2+ could be extended to develop other metalloenzyme sensors for bioapplications. The 3-[2-(2-aminoethylamino)ethylamino] propyl-trimethoxysilane (3-A) and dansyl chloride (D) were purchased from Alfa Aesar. Other reagents were purchased from Beijing Chemical Regent Co. All reagents and chemicals were AR grade and used without further purifications unless otherwise noted. Water used for measurement is of chromatographic grade, and was purified by Millipore filtration system. The HeLa cell line was obtained from Peking Union Medical College Hospital. The cell line was maintained in DMEM medium (containing 10% heat-inactivated FBS). The fluorescence spectra were measured with the F-4600 spectrophotometers. The transmission electron microscopy

(TEM) images were recorded by the JEOL JEM-2100 at the acceleration voltage of 200 kV. The scanning electron microscopy (SEM) images were recorded by Hitachi S4300FEG. The fluorescence images were taken with the laser scanning confocal microscope (LSCM, Nikon- Ti + UltraVIEWVoX) under excitation of 405 nm argon laser. SiNWs were prepared through simple thermal evaporation method.32 The as-prepared SiNWs own a crystalline Si core of 6−9 nm in diameter and a silicon oxide sheath of 3−5 nm in thickness determined by TEM (Figure S1, Supporting Information). High-quality SiNW arrays were fabricated by chemical etching33 and SEM images show that the diameters of the SiNWs are in the range of 100−250 nm, while the wire length is around 25 μm (Figure S2, Supporting Information). Both SiNWs and SiNW arrays were modified by the procedure showed in Scheme 1 and the acquired SiNWs- and SiNW arrays-based sensor were defined as 3-AD-SiNWs and 3-ADSiNW arrays, respectively (preparation of SiNWs and their modification are described in Supporting Information). The modification of SiNWs was characterized by X-ray photoelectron spectroscopy (XPS) and Fourier transform IR (FTIR) techniques. Figure S3 (Supporting Information) shows the XPS spectra of the bare SiNW arrays and 3-AD-SiNW arrays. It could be found that a small amount of nitrogen (Supporting Information Figure S3a) or sulfur (Supporting Information Figure S3b) was detected from the surface of the bare SiNW arrays, while a large quantity of nitrogen and sulfur was observed from the surface of the 3-AD-SiNW arrays. Furthermore, compared with the bare SiNWs, the 3-AD-SiNWs (Figure S4, Supporting Information) showed an additional peak in its FTIR spectra at around 2930 cm−1 that corresponded to the −CH vibration.32 Combining XPS and FTIR results, it can be confirmed that the organic molecules have been covalently immobilized onto the surface of SiNWs. B

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Figure 1. Fluorescence spectra of 3-AD-SiNWs with addition of Cu2+ (a) and its corresponding titration curve (b).The inset plot in (b) shows the linear relationship between the FI and the concentration of Cu2+, λex = 350 nm and λem = 505 nm. Conditions: 15 μg/mL 3-AD-SiNWs in pH 7.4 buffer (10 mM HEPES). Experiment has been repeated 5 times with a RSD of 0.85%.

The fluorescence quenching of 3-AD-SiNWs by Cu2+ was investigated and the results are shown in Figure 1. It is found that the fluorescence intensity of 3-AD-SiNWs gradually decreased with increase of Cu2+ concentration and finally reached at a plateau at about 1300 nM (Figure 1a). The dependence of fluorescence intensity of 3-AD-SiNWs on the Cu2+ concentration was obtained (Figure 1b). The plot inserted in Figure 1b exhibits a good linear relationship between fluorescence intensities and Cu2+ concentrations (50−400 nM) and the detection limit is about 31.0 nM. To check the selectivity of 3-AD-SiNWs for Cu2+ over other metal ions, competition experiments were performed in the presence of 100 equiv of K+, Ca2+, Na+, or Mg2+ and 5 equiv of Mn2+, Zn2+, Fe3+, Fe2+, Hg2+, Pb2+, Cd2+, Co2+, Ni2+, Cr2+, Ba2+, or Li+ with the subsequent addition of 1 equiv of Cu2+. As shown in Figure 2, the emission from the complex of 3-AD-

avoid the interference from various metal ions including cobalt and nickel ions. The 3-A moieties on the surface of SiNWs could better serve as Cu2+ receptors to snatch the Cu2+ and form a secure complex with 3-AD-SiNWs due to the suitable radius and electronic structure of the Cu2+. As a result, the selectivity of the 3-AD-SiNWs was greatly improved. Usually, Cu2+ is easily bound to some amino acids, such as Cys, His, Asp, Glu, and Arg as well as some other molecules.2 So a Cu2+ sensor that can be used in biosystems must bear the interference from these coexist biomolecules. Therefore, the interferences of Asp, Cys, Arg, Glu, His, GSH, vitamin C, vitamin H, and DPA on the detection of Cu2+ by 3-AD-SiNWs were examined.8 As shown in Figure 3, little influence on the

Figure 3. Fluorescence response of 3-AD-SiNWs to 10 μM amino acid (Asp, Cys, Arg, Glu, and His) and other coexisting biomolecules bearing potential complexation to Cu2+ (GSH, vitamin C, vitamin H, and DPA). Ten micromolar of each interference was added into the system (black bars) and then 400 nM Cu2+ was added into the system immediately (gray bars). λex = 350 nm, λem = 505 nm. Conditions: 15 μg/mL 3-AD-SiNWs in pH 7.4 buffer (10 mM HEPES).

Figure 2. Relative fluorescence intensity of 3-AD-SiNWs: gray bars, in the presence of metal ions alone (1, Mn2+; 2, Zn2+; 3, Fe3+; 4, Fe2+; 5, Hg2+; 6, Pb2+; 7, Cd2+; 8, Co2+; 9, Ni2+; 10, Cr2+; 11, Ba2+; 12, Li+; 13, K+; 14, Ca2+; 15, Na+; 16, Mg2+; 17, none); black bars, after subsequent addition of Cu2+ (1 equiv). K+, Ca2+, Na+, and Mg2+ ions are 100 equiv. Other metal ions are 5 equiv. Chloride salts were used. Conditions: 15 μg/mL 3-AD-SiNWs in pH 7.4 buffer (10 mM HEPES), λex = 350 nm, λem = 505 nm.

fluorescence intensity of the sensor was observed after the sensor was exposed to a solution containing 10 μM of each interference (black bars in Figure 3). However, the fluorescence of the sensor was immediately quenched when 400 nM Cu2+ was subsequently added into the system (gray bars in Figure 3). These results reveal that the present 3-AD-SiNWs could bear the interference from these coexist biomolecules and be further used to detect the Cu2+ in biosystem. A sensor that was employed to detect the complexed Cu2+ in the biosystem has to own high binding affinity with Cu2+. In order to check the binding ability of 3-AD-SiNWs to Cu2+, a widely used complexing agent EDTA was selected. It is well-

SiNWs and Cu2+ is unperturbed in the presence of these metal ions, indicating that the present sensor, 3-AD-SiNWs, owns favorable selectivity for Cu2+. Montalti et al.34 used the combination of 3-A and D in hydrolysis−condensation process of tetraethoxysilane to form silica nanosensor. However, the fluorescence intensity of the silica nanosensor was quenched by not only free copper ions but also cobalt or nickel ions. In our work, the combination of 3-A and D was covalently immobilized onto the surface of SiNWs and the results showed that the present SiNWs-based sensor (3-AD-SiNWs) could C

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Figure 4. Fluorescence spectra of 3-AD-SiNWs with addition of SOD1 unit (a) and its corresponding titration curve (b). λex = 350 nm, λem = 505 nm. Conditions: 15 μg/mL 3-AD-SiNWs in pH 7.4 buffer (10 mM HEPES).

Figure 5. Fluorescence spectra of 3-AD-SiNWs by increasing the amount of liver extract (a) and the linear relationship between fluorescence intensity and the volume of liver extract (b). λex = 350 nm, λem = 505 nm. Conditions: 15 μg/mL 3-AD-SiNWs in pH 7.4 buffer (10 mM HEPES). Experiment has been repeated 4 times with a RSD of 1.0%.

known that EDTA can easily react with Cu2+ and produce EDTA-Cu2+ complex that owns a high stability constant.35 Figure S5 (Supporting Information) shows the influence of EDTA on the binding behavior between 3-AD-SiNWs and Cu2+. The fluorescence of 3-AD-SiNWs changed little when excess EDTA was added, but it was quenched immediately by the subsequent addition of Cu2+ (Supporting Information Figure S5a). Meanwhile, we can see that fluorescence of 3-ADSiNWs decreased obviously when Cu2+ was added and the subsequent addition of excessive EDTA could not recover the fluorescence (Supporting Information Figure S5b). These results demonstrate that the 3-AD-SiNWs own high binding affinity with Cu2+ and could be further utilized to detect complexed Cu2+. Meanwhile, the binding affinity of the combination of 3-A and D molecules (3-AD molecules) with Cu2+ was also checked (Figure S6, Supporting Information). It was found that the 3-AD molecules also exhibit a high binding affinity with Cu2+. On the basis of these results, we could attribute the high binding affinity between the sensor and Cu2+ to the inherent properties of the organic molecules (3-AD molecules). In order to investigate the capability of 3-AD-SiNWs to detect the complexed Cu2+, a typical copper-binding enzyme, Cu−Zn-superoxide dismutase (SOD1), was chosen as analyte. SOD1 is a typical copper-binding enzyme and plays a critical role in defense against oxygen radicals.1 It is found that the fluorescence intensity of 3-AD-SiNWs gradually decreased when the SOD1 was added into the system (Figure 4a) and the dependence of the fluorescence intensity of the 3-AD-SiNWs on the unit of SOD1 was obtained (Figure 4b). On the basis of

Figure 1b, it can be determined that the quenching efficiency of every 1.11 U/L SOD1 is equal to that of 1.0 nM Cu2+. It has been reported that overload of copper usually happens in the liver.1 So we tried to use 3-AD-SiNWs to detect the Cu2+ (both free and complexed) in freshly prepared mouse liver extract. The liver extract was prepared according to previous work30 (preparation process is provided in Supporting Information). As shown in Figure 5a, fluorescence intensity of the sensor is gradually quenched with the addition of liver extract and a linear relationship between the fluorescence intensity and the amount of the liver extract was achieved (Figure 5b). Compared with the linear relationship presented in Figure 1b, it can be determined that the concentration of Cu2+-equiv in the liver extract is about 13.5 μM, which is in accordance with the concentration of Cu2+ that normally existed in the liver.1 In another experiment, the FBS was used as a control sample. It was found that the fluorescence intensity of the sensor changed little when 0−10% of FBS was added into the system (Figure S7, Supporting Information). Accordingly, it can be determined that the quenching of the fluorescence of the sensor indeed resulted from the specific binding between the sensor and Cu2+. In order to explore the application of 3-AD-SiNW arrays for complexed Cu2+ in the biosystem, the fluorescence images of 3AD-SiNW arrays were recorded before and after the 3-ADSiNW arrays were immersed into SOD1 solution (6000 mU/ mL) and the results are shown in Supporting Information Figure S8. It is observed that the fluorescence of the 3-ADSiNW arrays was quenched obviously in the system consisting of SOD1. This phenomenon implies the potential application D

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Figure 6. Fluorescence images of cell-captured 3-AD-SiNW arrays culturing in trypsin solution. (a−c) Fluorescence images of the cell nucleus (DAPI) under different incubation time (0, 10, and 20 min); (d−f) Fluorescence images of the 3-AD-SiNW arrays under different incubation time; (g) the fluorescence intensities of cell nucleus indicated in panels (a−c) under different time; (h) the fluorescence intensities of the plots in (d−f) under different time.

small amount (Figure S9, Supporting Information) even though 1 mL trypsin was added during this process. These results demonstrated that the 3-AD-SiNW array sensor was capable of real-time and in situ monitoring of the released Cu2+ during the apoptosis of cells. In conclusion, a SiNW-based fluorescent sensor that can be used to detect the complexed Cu2+ was realized by covalently immobilizing the combination of 3-[2-(2-aminoethylamino) ethylamino] propyl-trimethoxysilane and dansyl group onto the surface of the SiNWs. With high sensitivity and enhanced selectivity, the sensor was utilized to detect the Cu2+ in liver extract and a good linear relationship between its fluorescence intensity and the amount of liver extract was exhibited. Moreover, a sensor chip was fabricated for real-time and in situ monitoring of both free and complexed Cu2+ released from apoptotic HeLa cell. It was observed that the fluorescence of the chip was quenched gradually during apoptosis of the captured HeLa cells. The results indicated that the Cu2+ released from the apoptotic HeLa cells can be real-time and in situ detected by the present sensor chip. Moreover, this new method used to detect complexed Cu2+ could be extended to develop more powerful sensors for other metalloenzymes, which would facilitate further study of biocatalysis.

of 3-AD-SiNW arrays in real-time and in situ monitoring of the complexed Cu2+ released from biosystems. It is known that trypsin can induce digestion and apoptosis of cells and the Cu2+ (both free and complexed) will be released during these processes.1,36 To inspect the ability of 3-AD-SiNW array sensor to monitor the Cu2+ released from the apoptotic cells a cell-captured 3-AD-SiNW array sensor chip was prepared (preparation details are in Supporting Information). First, the HeLa cell suspension was loaded onto the 3-AD-SiNW arrays. After being incubated for 24 h, the sensor chip (3-AD-SiNW arrays) was rinsed with PBS to remove the inadherent HeLa cells from the arrays. In order to facilitate the visualization of the adherent HeLa cells under the LSCM observation, the cells were further stained with 4′, 6-diamidino-2-phenylindole (DAPI), a fluorescent DNA-binding agent.37−39 Finally, the chip was placed upside down in a specific dish containing 1 mL of trypsin for the LSCM investigation. Under the excitation of 405 nm, two fluorescence emissions were observed. One is blue fluorescence that comes from the DAPI-stained cell nucleus and the other green fluorescence was from the sensor chip. From the change of the blue fluorescence image, it can be determined that the HeLa cells captured on the chip were gradually ruptured (Figure 6a−c). Meanwhile, the green fluorescence emission from the chip is quenched simultaneously (Figure 6d−f). Accordingly, it can be revealed that the fluorescence of the chip was weakened by the Cu2+ released from the apoptosis of the captured HeLa cells. The fluorescence emissions (both blue and green channels) from seven cell locations (as indicated in Figure 6a−f) are recorded for quantifying the apoptosis of the HeLa cells and the fluorescence quenching of the sensor arrays, and the development of fluorescence intensity of both emissions from these seven points are shown in Figure 6g,h, respectively. In accordance with the observation from the fluorescence images, the fluorescence intensities (both blue and green emissions) of the seven points are gradually decreased during the experiments. However, if no cell is captured onto the sensor arrays, the fluorescence of the bare 3-AD-SiNW arrays only changed a

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ASSOCIATED CONTENT

S Supporting Information *

Details of the experiment section and additional figures (TEM and SEM images, XPS, FTIR, and fluorescence spectra). This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-10-82543513. Fax: +86-10-82543513. Notes

The authors declare no competing financial interest. E

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Gertner, R. S.; Amit, I.; Brown, R. J.; Hacohen, N.; Regev, A.; Wu, C. J.; Park, H. Nano Lett. 2012, 12, 6498−6504. (29) Robinson, J. T.; Jorgolli, M.; Shalek, A. K.; Yoon, M.-H.; Gertner, R. S.; Park, H. Nat. Nanotechnol. 2012, 7, 180−184. (30) Wu, Y.-L.; Putcha, N.; Ng, K. W.; Leong, D.; Lim, C. T.; Loo, S. C.; Chen, X. Acc. Chem. Res. 2013, 46, 782−791. (31) Tay, C. Y.; Cai, P.; Setyawati, M. I.; Fang, W.; Tan, L. P.; Hong, C.; Chen, X.; Leong, D. T. Nano Lett. 2014, 14, 83−888. (32) Mu, L. X.; Shi, W. S.; She, G. W.; Chang, J. C.; Lee, S. T. Angew. Chem., Int. Ed. 2009, 48, 3469−3472. (33) Miao, R.; Mu, L. X.; Zhang, H. Y.; Xu, H. T.; She, G. W.; Wang, P. F.; Shi, W. S. J. Mater. Chem. 2012, 22, 3348−3353. (34) Montalti, M.; Prodi, L.; Zaccheroni, N. J. Mater. Chem. 2005, 15, 2810−2814. (35) Yang, J.-K.; Davis, A. D. J. Colloid Interface Sci. 1999, 216, 77− 85. (36) Richard, J. P.; Melikov, K.; Vives, E.; Ramos, C.; Verbeure, B.; Gait, M. J.; Chernomordik, L. V.; Lebleu, B. J. Biol. Chem. 2003, 278, 585−590. (37) Coleman, A. W.; Maguire, M. J.; Coleman, J. R. J. Histochem. Cytochem. 1981, 29, 959−968. (38) Willingham, M. C. J. Histochem. Cytochem. 1999, 47, 1101− 1109. (39) Li, C.; Liu, X. L.; Bin, S.; Jing, L.; Lei, J.; Dong, H.; Wang, S. T. Adv. Mater. 2011, 23, 4376−4380.

ACKNOWLEDGMENTS This work was supported by Chinese Academy of Sciences (Grant KGZD-EW-T02), NSFC (Grants 61025003, 51272302, 21103211, 51272258, 91333119, and 61204128) and National Basic Research Program of China (973 Program) (Grant 2012CB932400).

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