Nonbleaching Fluorescence of Gold Nanoparticles and Its

Anal. Chem. 2008, 80, 5951–5957

Nonbleaching Fluorescence of Gold Nanoparticles and Its Applications in Cancer Cell Imaging Hua He, Chao Xie, and Jicun Ren* College of Chemistry and Chemical Engineering, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, 200240, P. R. China In this paper, we investigated the fluorescent properties of gold nanoparticles (GNPs) with several tens of nanometers by ensemble fluorescence spectrometry, fluorescence correlation spectroscopy (FCS), and fluorescence microscopy. We observed that GNPs synthesized by the citrate reduction of chloroauric acid possessed certain fluorescence, narrow full width at half-maximum (17 nm), and with an increase of particle sizes, the emission intensity showed a gradual increase while the emission wavelength remained almost constant (at 610 nm). Especially, the fluorescence of GNPs possessed the excellent behavior of antiphotobleaching under strong light illumination. Despite their low quantum yields, GNPs exhibited strong native fluorescence under relatively high excitation power. The fluorescence of GNPs could be characterized by fluorescence imaging and FCS at the single particle level. On the basis of this excellent antiphotobleaching of GNPs and easy photobleaching of cellular autofluorescence, we developed a new method for imaging of cells using GNPs as fluorescent probes. The principle of this method is that after cells stained with GNPs or GNPs bioconjugates are illuminated by strong light, the cellular autofluorescence are photobleached and the fluorescence of GNPs on cell membrane or inside cells can be collected for cell imaging. On the basis of this principle, we imaged living HeLa cells using GNPs as fluorescent probes and obtained good cell images by photobleaching of cellular autofluorescence. Furthermore, anti-EGFR/GNPs were successfully used as targeted probes for fluorescence imaging of cancer cells. Our preliminary results demonstrated that GNPs possessed excellent behaviors of antiphotobleaching and were good fluorescent probes in cell imaging. Our cellular imaging method described has potential applications in cancer diagnostics, studies, and immunoassays. Optical imaging techniques provide great potential for understanding biological processes at the molecular level and for sensitive cancer diagnosis, particularly at the early stage of cancer development. Biological imaging with an optical technique however greatly relies upon the use of sensitive and stable optical labels. So far, organic fluorescent dyes are the most commonly * To whom correspondence should be addressed. Prof. and Dr. Jicun Ren, College of Chemistry and Chemical Engineering, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, P. R. China. Phone: +86-21-54746001. Fax: +86-21-54741297. E-mail: [email protected]. 10.1021/ac8005796 CCC: $40.75  2008 American Chemical Society Published on Web 07/01/2008

used optical labels. Their major drawback, however, is the limited observation time due to rapid photobleaching. In recent years, semiconductor quantum dots (QDs) have been used as optical markers.1–3 Although QDs offer greatly improved photostability, they still suffer a certain degree of photodecomposition and they are difficult to functionalize in a controlled way. Additionally, the most well studied QDs are composed of heavy metals that are cytotoxic, making them unsuitable for in vitro and in vivo applications.4,5 Colloidal gold nanoparticles (GNPs) have become an alternative consideration due to ease of synthesis, relative inertia, simplicity of conjugation chemistry, and excellent biocompatibility.6–14 To date, GNPs are finding increased applications in certain biological fields, such as DNA hybridization detection, immunoassay, single particle tracking, drug delivery, and cancer diagnostics and therapy.15–24Among these applications, optical signal is mostly based on the fact that GNPs resonantly scatter (1) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016–2018. (2) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013–2016. (3) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538–544. (4) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2004, 4, 11–18. (5) Kirchner, C.; Liedl, T.; Kudera, S.; Pellegrino, T.; Javier, A. M.; Gaub, H. E.; Stolzle, S.; Fertig, N.; Parak, W. J. Nano Lett. 2005, 5, 331–338. (6) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959–1964. (7) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757–1760. (8) Han, M. S.; Lytton-Jean, A. K. R.; Oh, B. K.; Heo, J.; Mirkin, C. A. Angew. Chem., Int. Ed. 2006, 45, 1807–1810. (9) Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K. R.; Han, M. S.; Mirkin, C. A. Science 2006, 312, 1027–1030. (10) Medley, C. D.; Smith, J. E.; Tang, Z.; Wu, Y.; Bamrungsap, S.; Tan, W. Anal. Chem. 2008, 80, 1067–1072. (11) Shi, X. G.; Wang, S. H.; Meshinchi, S.; Van Antwerp, M. E.; Bi, X. D.; Lee, I. H.; Baker, J. R. Small 2007, 3, 1245–1252. (12) Tong, L.; Zhao, Y.; Huff, T. B.; Hansen, M. N.; Wei, A.; Cheng, J. X. Adv. Mater. 2007, 19, 3136–3141. (13) Lee, K. J.; Nallathamby, P. D.; Browning, L. M.; Osgood, C. J.; Xu, X. H. N. ACS Nano 2007, 1, 133–143. (14) Curry, A. C.; Crow, M.; Wax, A. J. Biomed. Opt. 2008, 13, 014022. (15) Schultz, S.; Smith, D. R.; Mock, J. J.; Schultz, D. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 996–1001. (16) Sonnichsen, C.; Reinhard, B. M.; Liphardt, J.; Alivisatos, A. P. Nat. Biotechnol. 2005, 23, 741–745. (17) Paciotti, G. F.; Myer, L.; Weinreich, D.; Goia, D.; Pavel, N.; McLaughlin, R. E.; Tamarkin, L. Drug Deliv. 2004, 11, 169–183. (18) Kusumi, A.; Sako, Y.; Yamamoto, M. Biophys. J. 1993, 65, 2021–2040. (19) Meier, J.; Vannier, C.; Serge, A.; Triller, A.; Choquet, D. Nat. Neurosci. 2001, 4, 253–260. (20) Borgdorff, A. J.; Choquet, D. Nature 2002, 417, 649–653. (21) Sokolov, K.; Follen, M.; Aaron, J.; Pavlova, I.; Malpica, A.; Lotan, R.; RichardsKortum, R. Cancer Res. 2003, 63, 1999–2004.

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light upon excitation of the surface plasmon, and the scattering signal must be discriminated from a strong background, especially in a cell or in a scattering tissue. In a certain sense, one hopes that GNPs possess fluorescence emission similar to semiconductor QDs. However, different from semiconductor materials, metallic materials such as silver and gold do not have a discrete band gap and quantum-mechanical properties are only observed in atomic clusters at sizes comparable to the Fermi wavelength of the electron (∼1 nm). As a result, fluorescent gold particles that show discrete and size-tunable electronic transitions consist of only several to tens of metal atoms, and they are currently synthesized in the presence of stabilizing ligands, such as triphenylphosphine and thiols,25–29 or they are encapsulated and stabilized by polyamidoamine (PAMAM) dendrimers or polyethylenimine (PEI).30–34Although these few-atom gold clusters possessed high fluorescence efficiency (up to 10%), problems associated with their chemical and fluorescence instability limited their application to cells or tissues imaging. Recently, Geddes et al. observed fluorescent blinking from spin-coated gold colloids with an average diameter of 50 nm on glass slides upon laser-light illumination.35–37 Surprisingly, based on intrinsic gold fluorescence, these colloidal GNPs remained immune to photobleaching under continuous illumination. However, mainly because of their low quantum yields (QYs), these fluorescent GNPs do not attract people’s attention, and to date there have been no further investigation reports on such fluorescent GNPs and their applications as fluorescence probes. In fact, experimentally, colloidal GNPs in the range of several tens of nanometers are readily synthesized and they are very stable and biocompatible. Therefore, it is of great significance to further investigate the fluorescent behaviors of GNPs in solution and explore their applications in chemical or biological fields. In this work, we first synthesized colloidal GNPs with sizes varying between 16 and 55 nm by the citrate reduction of chloroauric acid and then investigated the optical properties of the as-prepared GNPs by various optical techniques including UV-visible absorption spectroscopy, fluorescence spectroscopy, fluorescence correlation spectroscopy (FCS), and fluorescence microscopy. We observed that the (22) El-Sayed, I. H.; Huang, X. H.; El-Sayed, M. A. Nano Lett. 2005, 5, 829– 834. (23) Huang, X. H.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am. Chem. Soc. 2006, 128, 2115–2120. (24) Kumar, S.; Harrison, N.; Richards-Kortum, R.; Sokolov, K. Nano Lett. 2007, 7, 1338–1343. (25) Huang, C. C.; Yang, Z.; Lee, K. H.; Chang, H. T. Angew. Chem., Int. Ed. 2007, 46, 6824–6828. (26) Negishi, Y.; Nobusada, K.; Tsukuda, T. J. Am. Chem. Soc. 2005, 127, 5261– 5270. (27) Negishi, Y.; Tsukuda, T. Chem. Phys. Lett. 2004, 383, 161–165. (28) Huang, T.; Murray, R. W. J. Phys. Chem. B 2001, 105, 12498–12502. (29) Schaaff, T. G.; Whetten, R. L. J. Phys. Chem. B 2000, 104, 2630–2641. (30) Zheng, J.; Zhang, C. W.; Dickson, R. M. Phys. Rev. Lett. 2004, 93, 077402. (31) Zheng, J.; Petty, J. T.; Dickson, R. M. J. Am. Chem. Soc. 2003, 125, 7780– 7781. (32) Tran, M. L.; Zvyagin, A. V.; Plakhotnik, T. Chem. Commun. 2006, 2400– 2401. (33) Shi, X. Y.; Ganser, T. R.; Sun, K.; Balogh, L. P.; Baker, J. R. Nanotechnology 2006, 17, 1072–1078. (34) Duan, H. W.; Nie, S. M. J. Am. Chem. Soc. 2007, 129, 2412–2413. (35) Geddes, C. D.; Parfenov, A.; Gryczynski, I.; Lakowicz, J. R. Chem. Phys. Lett. 2003, 380, 269–272. (36) Geddes, C. D.; Parfenov, A.; Lakowicz, J. R. J. Fluoresc. 2003, 13, 297– 299. (37) Geddes, C. D.; Gryczynski, I.; Parfenov, A.; Aslan, K.; Lakowicz, J. R. Proc. SPIE-Int. Soc. Opt. Eng. 2004, 5329, 276–286.

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fluorescence of GNPs possessed the excellent behavior of antiphotobleaching under strong light illumination. On the basis of this excellent antiphotobleaching of GNPs and easy photobleaching of cellular autofluorescence, we developed a new method for imaging of cells using GNPs as fluorescent probes. After photobleaching of cellular autofluorescence by illumination of strong light, we clearly observed GNPs in living HeLa cells, which entered cells via the endocytic pathway during cell differentiation and proliferation processes. Furthermore, we conjugated GNPs to antiepidermal growth factor receptor (anti-EGFR) antibodies and successfully used the anti-EGFR/GNPs conjugates as targeted probes for fluorescent imaging of cancer cells. To the best of our knowledge, this may be first report on fluorescent imaging of cells using GNPs as probes via photobleaching of cellular autofluorescence by illumination with strong light. EXPERIMENTAL SECTION Preparation and Characterization of GNPs. Variable sizes of GNPs were synthesized using a reported method with a minor modification.6 In brief, aqueous solutions of HAuCl4 (0.01%, w/w) and sodium citrate (1%, w/w) were prepared. A solution of HAuCl4 (100 mL) was heated to boiling, a given amount of sodium citrate solution (volume depended on the requirement size of GNPs) was added rapidly to the boiling solution. Heating was continued for 30 min after the solution color remained unchanged. After cooling to room temperature, the GNPs stock solution was placed in a refrigerator for further use. All solutions were prepared with ultrapure water (18.2 MΩ) purified on Millipore Simplicity (Millipore). Transmission electron micrograph (TEM) images were taken with a JEM-100CX transmission electron microscope. UV-vis absorption spectra of GNPs were obtained by using a UV3501 spectrophotometer (Tianjin Gangdong Sci. & Tech. Development Co. Ltd., China). Fluorescence spectra were recorded on a WGY-10 fluorescence spectrophotometer (Tianjin Gangdong Sci. & Tech. Development Co. Ltd., China). Laser-induced fluorescence spectra were recorded on a Jobin Yvon HR 800 instrument with an Ar+ laser (514.5 nm) as the excitation source. FCS measurements were performed on a home-built FCS system with a 532 nm laser. The details of the experimental setup can be found elsewhere.38 In brief, the expanded laser line was focused with a water immersion objective (UplanApo, 60× NA 1.2, Olympus, Japan) to a small volume within the diluted sample. The resulting excitation volume is on the order of 1 fL. The excited fluorescence signal collected by the objective passed through the dichroic mirror (570DRLP, Omega Optical) and then was filtered by a band-pass filter (605DF50, Omega Optical) to block scattering laser light. Finally, the fluorescence was coupled into a 30 µm pinhole at the image plane in front of the single-photon counting module (SPCM-AQR16, Perkin-Elmer EG&G, Canada). The fluorescence fluctuations were correlated with a correlator card (ALV-5000/EPP, ALV-GmbH, Germany). To prepare anti-EGFR/GNPs conjugates, colloidal GNPs were diluted with 20 mM HEPES buffer (pH 7.4), and anti-EGFR antibodies (1 mg mL-1, Beijing CellChip Biotechnology Co. Ltd., China) were reconstituted in the same buffer at 0.1 mg mL-1. Then, the solutions were mixed at a 1:1 volume ratio and allowed to interact for 20 min at room temperature. Poly(ethyleneglycol) (38) Dong, C. Q.; Bi, R.; Qian, H. F.; Li, L.; Ren, J. C. Small 2006, 2, 534–538.

Figure 1. (a) The normalized optical absorption spectra of colloidal GNPs; (b) fluorescence spectra of colloidal GNPs upon 532 nm excitation; (c) fluorescence spectrum of colloidal GNPs with an average diameter of 38 nm upon 514.5 nm laser excitation; and (d) the normalized FCS curves for a variable size of GNPs.

(MW 20 000) was added to the mixture up to a final concentration of 0.5 mg mL-1, and the solution was centrifuged at 2000 rpm for 2 h to wash unbound antibodies. Then the anti-EGFR/GNPs pellet was redispersed in PBS buffer (pH 7.4) and stored at 4 °C. Cell Culture. Cervical cancer HeLa cells were cultured with high Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum (Hyclone) in a humidified incubator (MCO15AC, Sanyo) at 37 °C in which the CO2 level was kept constant at 5%. The cell culture conditions remain the same if not mentioned in following tests. GNPs are introduced into cells via the endocytic pathway during cell differentiation and proliferation processes. In our experiments, 1 mL of solution with approximately 8000 cells was seeded onto the coverslip and then GNPs were added into the medium grown for 48 h at 37 °C under 5% CO2. The cells on the coverslip were rinsed with PBS buffer (pH 7.4) and then directly observed with fluorescence microscopy. For the incubation of conjugated nanoparticles, the cell suspensions were labeled with anti-EGFR/GNPs conjugates in the incubator at 37 °C for about 20 min. The cells were then centrifuged at 2000 rpm for 5 min to remove unbound particles, and the cells were resuspended in PBS buffer (pH 7.4). A drop of the above solution was placed on top of a microscope slide and then sealed with another coverslip for observation under total internal reflection fluorescence (TIR) microscopy. Imaging Setup. GNPs and cell imaging were accomplished by an Olympus IX71 inverted fluorescence microscope (Olympus Optical Co., Tokyo, Japan) equipped with a 100 W mercury lamp for epi-illumination and a Nd:YVO4 laser (532 nm) for the total internal reflection illumination system (IX2-RFAEVA-2, Olympus).

The emission from the sample was collected by the Olympus objective (PlanApo N 60×/1.45 oil), separated from Rayleigh and Raman-shifted light by a combination of filters, an excitation filter 530DF35, a dichroic mirror 570DRLP, and an emission filter 605DF50 (Omega Optical, Inc.) and then focused into a cooled 16-bit CCD camera (Cascade 650, Photometrics, Tucson, AZ). Image acquisition and processing were performed with the MetaMorph imaging software (Universal Imaging, Downingtown, PA). Coverslips were thoroughly cleaned prior to sample preparation by sonication in sulfuric acid/potassium dichromate solution, NaOH solution (0.1 M), ethanol, and ultrapure water (Millipore), each for 30 min, and subsequently dried in a vacuum oven. All measurements were performed at room temperature. RESULTS AND DISCUSSION Fluorescence of Gold Nanoparticles. Parts a and b of Figure 1 present the UV-visible absorption and fluorescence spectra of colloidal GNPs with average diameters of 16, 20, 28, 38, 43, and 55 nm (as determined by TEM, see Supporting Information Figure S-1 for TEM micrographs). As the particle sizes increase, the absorption spectra show a gradual red-shift in the plasmon resonance absorption peak while the fluorescent emission remains almost constant at around 610 nm along with a gradual increase in the emission intensity, suggesting intrinsic gold fluorescence. This emission wavelength is also consistent with the report from Geddes et al.35 As a confirmation, we recorded the laser-induced fluorescence spectrum of GNPs in solution by a Jobin Yvon HR 800 spectrometer. The laser-induced fluorescence spectrum exhibits a better peak shape with a half-peak width of about 17 Analytical Chemistry, Vol. 80, No. 15, August 1, 2008

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Figure 2. The fluorescence images of 38 nm GNPs taken in the same field view by epi-illumination fluorescence microscopy using a 100 W mercury lamp (a) before and (b) after 32 min continuous illumination. Exposure time: 10 s. (c) Cross sections marked by solid lines in parts a and b, respectively. (d) The same cross sections only after subtracting the background, showing that no photobleaching took place.

Figure 3. Bright-field images (a) and autofluorescence images of HeLa cells without nanoparticles (b) before and (c) after 5 min photobleaching. The other two images in the same row for each imaging condition are shown to test reproducibility. Exposure time: 5 s.

nm (Figure 1c) compared with that obtained by a conventional fluorescence spectrophotometer. It should be noted that, due to low fluorescence efficiency and enhanced surface-plasmon resonance absorption phenomenon, it is difficult for us to accurately estimate the quantum yields (QYs, generally using rhodamine or other highly fluorescent dyes as a reference standard) of these GNPs. Nevertheless, experimentally GNPs have the fluorescence 5954


strong enough to be detected by the fluorescence instruments, and even the emission intensity can be further improved by increasing excitation power. Subsequently, fluorescence correlation spectroscopy experiments have been performed to characterize GNPs in solution. FCS is an ultrasensitive and noninvasive single molecule technique using statistical analysis of the fluctuations of fluorescence emitted from a small, optically well-defined

Figure 4. Bright-field images (a) and fluorescence images of HeLa cells incubated with 38 nm GNPs (b) before and (c) after 5 min photobleaching. The other two images in the same row for each imaging condition are shown to test reproducibility. Exposure time: 5 s.

Figure 5. TIR fluorescence images of HeLa cells incubated (a) in pure medium, (b) in the medium with free GNPs, (c) in the medium with BSA conjugated GNPs, and (d) in the medium with anti-EGFR antibody conjugated GNPs. Exposure time: 5 s. Here, 38 nm GNPs were employed.

open volume element. Its autocorrelation curve can provide us some important information about luminescent particles such as the average number of particles in the defined volume, diffusion coefficient, and the brightness per particle (BPP) obtained by the count rate and the number of particles indirectly.38–40 In experiments, we obtained good FCS curves of GNPs in solution shown Figure 1d (see Supporting Information Figure S-2 for the corresponding count-rate traces). As expected, the normalized auto-

correlation curves of GNPs were monotonically displaced to the right with increased particle size, which suggested that the mean “apparent” diffusion times of GNPs monotonically increased with increased particle size. Note that 100 kW cm-2 or larger excitation power was required to obtain FCS curves from the 20 nm GNPs solution. Meanwhile, in the FCS measurements, we also calculated the brightness per particle (BPP) of GNPs in solution based on the acquired average count rate and mean number of GNPs (N)

(39) Dong, C. Q.; Qian, H. F.; Fang, N. H.; Ren, J. C. J. Phys. Chem. B 2006, 110, 11069–11075.

(40) Dong, C. Q.; Qian, H. H.; Fang, N. H.; Ren, J. C. J. Phys. Chem. C 2007, 111, 7918–7923.


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in the focused volume (BPP ) count rate/N) and found that the brightness of singe gold particle was considerably high under high excitation power. For example, at 600 kW cm-2 excitation power, the BPP of 38 nm GNPs was 9 kHz and, as a comparison, BPP of rhodamine B was 36 kHz at the same laser power by our FCS system. The satisfactory BPP of GNPs was mainly attributed to the extremely high molar absorptivity of GNPs. Although GNPs were low in quantum yield, the absorbance of single GNP was several orders of magnitude more than a single rhodamine B molecule. More importantly, despite high laser excitation power used in this case, the count rate trace from GNPs in the FCS measurements, which monitored the fluctuation of ensemble luminescence intensity in real time, showed that GNPs did not suffer from any degradation and displayed antibleaching luminescence behavior (as shown in Figure S-2). Furthermore, the antibleaching property of GNPs was examined by epi-illumination fluorescence microscopy equipped with a 100 W mercury lamp through a 535DF35 filter for excitation. The actual excitation intensity on the sample was about 15 W cm-2. Figure 2 presents the typical fluorescence images of GNPs with an average diameter of 38 nm before and after continuous illumination for 32 min. It can be seen that even though the long-time continuous illumination led to a great decrease of the background intensity (Figure 2c), GNPs still had the same (or even higher) signal-background ratios(Figure2d),furtherconfirmingtheirantibleachingfluorescence. Cancer Cell Imaging via Photobleaching of Cellular Autofluorescence. Because of a high numerical-aperture objective and a highly sensitive CCD camera used in our experiments, GNPs (or GNP aggregates) can be clearly imaged with a typical exposure time of 1∼10 s under a several watt per centimeter squared mercury lamp illumination. Compared to the laserillumination imaging system, such excitation intensity is not high for many applications in the biological imaging field. However, the major problem confronted was the serious interference of autofluorescence from cells or tissues under the same imaging condition. In Figure 3b, we present the autofluorescence images of HeLa cancer cells adhering to the coverslips. Obviously, such strong autofluorescence will seriously confuse cell images using GNPs as fluorescent probes. Fortunately, the endogenous autofluorescence can decay quickly with time under illumination. As shown in Figure 3c, the cellular autofluorescence was almost eliminated after illumination for 5 min. Meanwhile, we compared bright-field images of cells before and after 10 min continuous illumination and found that such illumination had no discernible adverse effects on the cell morphology (data not shown). This result suggests to us that GNPs can be efficiently used as fluorescent probes in cell imaging due to their antiphotobleaching characteristic. In subsequent experiments, we chose GNPs with an average diameter of 38 nm as imaging probes considering the particle uptake efficiency and luminescent intensities. Here, GNPs were introduced into cells by the endocytosis process during cell differentiation and proliferation processes. When incubated in the presence of GNPs, the cells grew at a normal rate and the GNPs entered and accumulated inside the cells. From the bright-field images of these cells (Figure 4a), we can see that GNPs did not enter the nucleus and they predominantly accumulated inside the cytoplasm of the cells. Figure 4b shows the corresponding fluorescence images of cells before photobleaching. Although, on 5956


the whole, these cells exhibit stronger luminescence and have more bright spots inside the cytoplasm than those without nanoparticles, it is difficult to resolve which of these spots come from fluorescent GNPs due to the interference of autofluorescence from the cellular organelles. Then, after photobleaching of cellular autofluorescence for 5 min, the cell images became very clear and displayed extremely improved contrast as shown in Figure 4c, which allowed observing only fluorescent GNPs inside the cells. In all above figures, two other images in the same row for each imaging condition are shown to test the reproducibility of our method. We also imaged cells incubated with 16 nm GNPs with extremely weak fluorescence (as shown in Figure S-3). Experimentally, small GNPs crossed the cytoplasmic membrane more easily, and the larger amount of GNPs accumulated and aggregated inside the cells. Thus, on the basis of the photobleaching method, the cells incubated with 16 nm GNPs could still be clearly observed but exhibited weaker signal than those incubated with 38 nm GNPs. Cancer Cell Imaging Using Targeted GNPs as Probes. In the experiments, we conjugated GNPs to antiepidermal growth factor receptor (anti-EGFR) antibodies and then targeted the resulting conjugates to cancer cells for specific fluorescence imaging. HeLa cells are derived from a human epithelial carcinoma, and it has been reported that they exhibit significantly elevated EGFR expression.41,42 The high level of EGFR expression is often associated with enhanced aggressiveness of epithelial cancers and poor prognosis.43,44 Here, 38 nm GNPs were still employed as a probe. HeLa cell suspensions were labeled with anti-EGFR/GNPs conjugates in the incubator at 37 °C for about 20 min. After centrifugation, the labeled cells were resuspended in PBS buffer. A droplet of this solution was placed on top of a microscope slide and then sealed with another coverslip for observation under total internal reflection (TIR) fluorescence microscopy. Figure 5 shows the fluorescence images of the cells in each incubation condition. Since these cells were not cultured on the coverslip, they have a near circular shape without cell spreading. Meanwhile, because of the limited excitation depth of TIR, only the structures in the immediate proximity of the basal membrane were illuminated and then observed. Therefore, as shown in Figure 5a, the unlabeled cells that were incubated in pure medium exhibited very weak autofluorescence, not requiring the photobleaching procedure. Figure 5d shows the TIR images of HeLa cancer cells that were incubated with anti-EGFR/GNPs conjugates. From this figure, we can see that the large amount of GNPs were bound on the cell surface due to the specific binding of overexpressed EGFR on the cancer cells with the anti-EGFR antibodies on the GNPs surface. Meanwhile, to further confirm anti-EGFR/GNPs conjugates specifically bound on the cell membrane, the contrast experiments were conducted using GNPs and BSA/GNPs (BSA, bovine serum albumin) conjugates as probes at the same conditions. Parts b and c of Figure 5 show the typical images of HeLa cells using GNPs and BSA/GNPs as probes. As expected, only a little unspecific binding of GNPs or BSA/GNPs (41) Hu, G. Y.; Liu, W. B.; Mendelsohn, J.; Ellis, L. M.; Radinsky, R.; Andreeff, M.; Deisseroth, A. B. J. Natl. Cancer Inst. 1997, 89, 1271–1276. (42) Hemminki, A.; Dmitriev, I.; Liu, B.; Desmond, R. A.; Alemany, R.; Curiel, D. T. Cancer Res. 2001, 61, 6377–6381. (43) Pfeiffer, D.; Stellwag, B.; Pfeiffer, A.; Borlinghaus, P.; Meier, W.; Scheidel, P. Gynecol. Oncol. 1989, 33, 146–150. (44) Todd, R.; Wong, D. T. W. Histol. Histopath. 1999, 14, 491–500.

to cell membranes was observed. These preliminary results suggest that the targeted GNPs may be potentially useful as fluorescent probes in cancer diagnostics and research. CONCLUSIONS In this paper, the fluorescent properties of colloidal GNPs with sizes between 16 and 55 nm were investigated by various optical techniques including UV-visible absorption spectroscopy, fluorescence spectroscopy, fluorescence correlation spectroscopy, and fluorescence microscopy. Our results demonstrated that GNPs possessed the excellent behaviors of antiphotobleaching under strong light illumination. Although they had low quantum yields, the fluorescence of GNPs was strong enough to be detected by the fluorescence instruments such as fluorescence microscopy and FCS at the single particle level, and even their intensity could be further improved by increasing excitation power. On the basis of this antiphotobleaching of GNPs and easy photobleaching of cellular autofluorescence, we developed a new method for imaging of cells using GNPs as fluorescent probes. Our method was successfully used for imaging of living HeLa cells using free GNPs and anti-EGFR/GNPs conjugates as fluorescent probes. In particular, under some specific illumination such as total internal reflection fluorescence microscopy, GNPs can also be directly used, not requiring the photobleaching procedure due to the extremely weak interference of autofluorescence from cell mem-

brane. Compared to scattering imaging techniques such as darkfield imaging using GNPs as probes, the fluorescent imaging method presented here possessed high selectivity and should have wide application fields. Compared to currently used fluorescent probes such as fluorescent dyes and quantum dots, GNPs as fluorescent probes have unique properties such as ease of synthesis, good chemical and photostability, simplicity of conjugation chemistry, and excellent biocompatibility. We believe that our imaging method will become a very useful tool for cell imaging, targeted drug delivery, and cancer diagnostics and studies using GNPs as fluorescent probes. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (Grant Numbers 20675052, 20727005) and the National High-Tech R&D Program (Grant 2006AA03Z324). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review March 21, 2008. Accepted May 30, 2008. AC8005796


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Nonbleaching Fluorescence of Gold Nanoparticles and Its

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