Article pubs.acs.org/ac
In Situ Growth of Porous Platinum Nanoparticles on Graphene Oxide for Colorimetric Detection of Cancer Cells Ling-Na Zhang,†,‡ Hao-Hua Deng,†,‡ Feng-Lin Lin,†,‡ Xiong-Wei Xu,§ Shao-Huang Weng,†,‡ Ai-Lin Liu,†,‡ Xin-Hua Lin,†,‡ Xing-Hua Xia,∥ and Wei Chen*,†,‡ †
Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China Nano Medical Technology Research Institute, Fujian Medical University, Fuzhou 350004, China § First Affiliated Hospital, Fujian Medical University, Fuzhou 350004, China ∥ State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China ‡
S Supporting Information *
ABSTRACT: A green approach is proposed for in situ growth of porous platinum nanoparticles on graphene oxide (PtNPs/ GO). The resulting nanocomposite has been proven to function as peroxidase mimetics that can catalyze the reaction of peroxidase substrate in the presence of hydrogen peroxide. On the basis of the peroxidase-like activity, we used the PtNPs/GO as a signal transducer to develop a colorimetric assay for the direct detection of cancer cells. By using folic acid as a recognition element, a total of 125 cancer cells (MCF-7) can be distinguished by naked-eye observation. We envision that this nanomaterial could be used as a power tool for a wide range of potential applications in biotechnology and medicine.
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realize the combination of the respective properties of each component or to achieve cooperatively enhanced performances.11,18−24 Colloidal noble metal nanoparticles have been receiving a great deal of attention in areas from chemistry to physics, material sciences, biology, and medicine.25,26 Conventionally, colloidal noble metal nanoparticles should be stabilized by addition of chemical or biomolecular stabilizers to prevent aggregation. However, their catalytic activity could be severely limited by these stabilizers capped around nanoparticles.13 A solution of this problem could be offered by anchoring of metal particles on specific supports. As a representative derivate of graphene, graphene oxide (GO) has been widely utilized as fascinating support for preparation of graphene-based nanoparticle composites.27−30 The rigid two-dimensional structure of GO can enable most surface of the attached nanoparticles to be exposed to the environment.27 Furthermore, the presence of abundant carbonyl and carboxyl groups makes GO sheets strongly hydrophilic, allowing them to readily disperse in water and polar organic solvents. In this paper, we demonstrated that clean and well-dispersed porous platinum nanoparticles (PtNPs) can be facilely prepared in situ on a GO surface. In contrast to previous studies,31−33 no
atural enzymes have been attracting great interest and widely applied because of their immense catalytic power and high substrate specificity under mild reaction conditions. However, natural enzymes suffer from some serious disadvantages such as sensitivity of catalytic activity to environmental conditions, low operational stability due to denaturation and digestion, as well as time-consuming and expensive preparation and purification, which restrict their widespread applications. Therefore, construction and discovery of novel enzyme mimetics is under intensive investigation. Recently, Fe3O4 nanoparticles, which were generally considered to be biologically inert, have been found to possess intrinsic enzyme mimetic activity similar to that of natural peroxidase, thus opening a new door for the application of nanoscaled materials in the biochemical field.1 In comparison with natural enzymes which are susceptible to the reaction conditions, nanomaterials are considerably more stable over a wide range of pH and temperature. They possess additional advantages of controlled preparation in mass yield and at relatively low cost, flexibility in structure and composition design, and tunable catalytic activities. In light of such advantages, a series of inorganic nanomaterials have emerged as peroxidase mimics and applied in the environmental chemistry and biomedicine fields.2−17 Among the enzyme nanomimics, a new generation of hybrid nanomaterials is particularly impressive and takes one of the most pivotal steps toward enzyme mimicry. Hybrid composite materials, with well-defined structures, have been explored to © 2014 American Chemical Society
Received: December 18, 2013 Accepted: February 13, 2014 Published: February 13, 2014 2711
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Figure 1. (a,b) AFM image of GO and the PtNPs/GO hybrid, (c,d) TEM images of the PtNPs/GO hybrid under different magnifications, (e) EDS spectrum of the PtNPs/GO hybrid, and (f) XPS spectrum of the PtNPs/GO hybrid.
Tetramethylbenzidine, N-hydroxysulfosuccinimide sodium (sulfo-NHS), and folic acid were purchased from Aladdin (Shanghai, China). Poly(allyl amine hydrochloride) (PAH, MW = 58 000) and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were obtained from Sigma-Aldrich. H2PtCl6·6H2O and H2O2 came from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Horseradish peroxidase (HRP, 300 IU/mg) was purchased from Dingguochangsheng Biotech Co. Ltd. (Beijing, China). All chemicals were of analytical reagent grade. Deionized water was used in all experiments. Atomic-force microscopy (AFM) measurements were performed using a Bruker Nanoscope V multimode atomic
additional reductant or surfactant was needed in our synthesis. GO acted as both the stabilizer and the reductant to obtain stable and clean Pt nanoparticles. The resulting PtNPs-loaded GO (PtNPs/GO) revealed excellent peroxidase-like activity that can be used to catalyze the oxidation of 3,3′,5,5′tetramethylbenzidine (TMB) by hydrogen peroxide. For further application, folic acid (FA) functionalized PtNPs/GO was successfully used for the colorimetric detection of cancer cells.
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EXPERIMENTAL SECTION Reagents and Apparatus. Graphite flake (325 mesh) was purchased from XF NANO (Nanjing, China). 3,3′,5,5′2712
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force microscope. Intelligent mode was used to acquire the images under ambient conditions. Transmission electron microscopy (TEM) images and an energy-dispersive X-ray spectroscopy (EDS) spectrum were recorded using a FEI TECNAI G2 20 high-resolution transmission electron microscope operating at 200 kV. An X-ray photoelectron spectrum (XPS) was recorded by a Thermo ESCALAB 250XI X-ray photoelectron spectrometer with nonmonochromatized Al Kα radiation as the excitation source. UV−vis absorbance spectra were performed on a Shimadzu UV-2450 spectrophotometer. Images of cells were viewed with an Olympus IX-70 inverted fluorescence microscope. Synthesis of PtNPs/GO. GO was synthesized from flake graphite by an improved Hummers method.34 PtNPs/GO was fabricated by mixed 0.5 mg/mL GO aqueous solution with 7.9 mM H2PtCl6 at a ratio of 10:1 (v/v). The mixture was continually reacted under vigorous stirring at 80 °C for 24 h to obtain the products. Synthesis of FA-PtNPs/GO. In order to keep the excellent catalytic activity of FA-PtNPs/GO, we prefer to deposit PtNPs on FA-modified GO rather than modify FA on presynthesized PtNPs/GO. The PAH functionalized GO was prepared by vigorously stirring a solution of 40 mg of the graphene oxides, 160 mg of PAH, and 160 mg of KOH in 80 mL of H2O at 70 °C for 24 h. After that, the PAH functionalized GO was collected and purified by centrifugation and adequately washed with water several times to remove the impurities and the excess of PAH by physical absorption. To conjugate the amino functionalized GO with folic acid, folic acid (40 mg, 0.0227 mmol) was first dissolved in 16 mL of phosphate buffer solution (pH 5.0, 20 mM). The solution of folic acid was then mixed with 8 mL of aqueous solution of EDC (0.068 mmol) and sulfo-NHS (0.068 mmol). After agitating overnight at room temperature in the dark, the solution of PAH functionalized GO (0.5 mg/mL, 80 mL) was added to the mixture and stirred at room temperature for 24 h. The folic acid functionalized GO was collected by centrifugation and extensively washed with water to remove the physisorbed folic acid. Finally, 8 mL of H2PtCl6 solution (7.9 mM) was added to 80 mL of folic acid functionalized GO solution (0.5 mg/mL). The resulting mixture was stirred at 80 °C for 24 h to obtain FA-PtNPs/GO. Cell Culture and Treatment. The human breast cancer cells (MCF-7), human gastric cancer cells (SGC-7901), and human umbilical vein endothelial cells (HUVEC) were grown in RPMI 1640 medium (Hyclone) supplemented with 6% fetal bovine serum in a humidified 37 °C incubator with 5% CO2. For the colorimetric assay, cells were plated in 96-well plate for adherence and then allowed to incubate with 5 μg of FAPtNPs/GO in RPMI 1640 medium at 37 °C for 1.5 h. For one of the control experiment, adherent cells in 96-well plate were incubated with 5 μg of PtNPs/GO for 1.5 h. For the other control experiment, cells were first treated with 40 μg of FA in RPMI-1640 medium for 30 min and then incubate with 5 μg of FA-PtNPs/GO for 1.5 h. Then every cell well was washed three times with phosphate buffer solution (10 mM, pH 7.4) to remove unattached FA-PtNPs/GO. After that, 200 μL of phosphate buffer solution (20 mM, pH 5.0) containing 0.8 mM TMB and 1 M H2O2 was added to each cell well and incubated for 15 min at room temperature. Finally, the reaction was terminated by H2SO4 (2 M) and quantitatively monitored by a microplate reader (Thermo Multiskan MK3) at the wavelength of 450 nm.
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RESULTS AND DISCUSSION
Characterization of PtNPs/GO. PtNPs/GO was fabricated by direct incubation with GO with H2PtCl6 at 80 °C for 24 h. The color of the mixture changed to dark brown after incubation (Figure S1 in the Supporting Information). No red shift of the absorption peak of GO in the UV−vis spectrum was observed (Figure S2 in the Supporting Information), indicating that GO was not evolved into reduced GO in the reaction. To verify the successful growth of Pt nanoparticles on graphene oxide sheets, AFM was used to characterize the morphologies of the as-prepared composites. The typical AFM images of GO and PtNPs/GO reveal that the thickness of GO sheets maintained about 1 nm before and after the reaction (Figure 1a,b). The nanoparticles were observed on the GO but not outside the sheet, indicating that there is almost no particle formation in free solution. The H2PtCl6 precursor was directly reduced to PtNPs and anchored to the graphene oxide surface mainly through the defects and oxygen functional groups.30 It was worth to note that the resultant PtNPs/GO hybrid could form well-dispersed aqueous colloids owing to the residual oxygen-containing groups on GO, providing advantages in the chemical modification and biological applications. Figure 1c,d shows representative TEM images of the synthesized PtNPs/ GO composites. A large number of Pt nanoparticles with a size of about 50 nm were well-dispersed on the surface of the graphene oxide nanosheets (Figure S3 in the Supporting Information), which should be attributed to the hydroxyl, epoxide, and carboxylic groups uniformly existing on the GO.35 H2PtCl6 was reduced to Pt nanoparticles without adding any reducing agent in our synthesis. Therefore, it is rationally deduced that the spontaneous deposition of PtNPs on GO sheets is ascribed to the redox reaction between GO and PtCl62−‑, which is similar to the reaction mechanism between metal ions and single-walled carbon nanotubes proposed by Dai et al.36 It should be pointed out that the growth of PtNPs after nucleation might involve a galvanic-reaction-like process in which the reduction of Pt occurs on Pt nuclei by the electrons transferred from GO.30 The selected area electron diffraction (SAED) pattern of PtNPs/GO (inset of Figure 1c) indicates the highly crystalline nature of the PtNPs. The enlarged TEM image reveals the porous structure of PtNPs (Figure 1d), which might endue them more active sites to obtain the extent of catalytic properties. An energy-dispersive X-ray spectroscopy (EDS) spectrum was also used to analyze the elemental distribution of the nanohybrid. The significant Pt signals suggest that the nanoparticles loaded on the surface of graphene oxide were platinum (Figure 1e). Meanwhile, from the X-ray photon spectroscopy (XPS) result demonstrated in Figure 1f, Pt 4f peaks were observed at 71.5 and 74.9 eV,37 verifying again the successful formation of PtNPs on the GO surface. Peroxidase-Like Activity of PtNPs/GO. TMB, one of the most common chromogenic substrates of peroxidase, was used to investigate the peroxidase-like activity of PtNPs/GO. As shown in Figure 2, PtNPs/GO could catalyze the oxidation of TMB in the presence of H2O2 to produce a blue color with maximum absorbance at 652 and 370 nm. Like enzymatic peroxidase activity, such as observed for the commonly used enzyme HRP, this catalytic reaction could even be quenched by H2SO4. The blue product was further oxidized to a yellow diimine with a maximum absorption wavelength of 450 nm. In contrast, no obvious reaction occurred in the absence of 2713
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when the temperature was above 35 °C. To ensure accuracy, the other chromogenic experiments were carried out at 30 °C. As noted, in contrast to that found for natural peroxidase, Fe3O4 nanoparticles, graphene oxide, and porous platinum nanotubes,1,3,16 no inhibition effect on the catalytic activity of PtNPs/GO was observed at high H2O2 concentration (Figure 3c). Figure 3d provides the relationship between the absorbance of the oxidized TMB product at 652 nm and the concentration of PtNPs/GO. We found that the catalytic rate increased with increasing catalyst concentration. With excess reactants, the reaction speed depends on the catalyst concentration over a given range. Therefore, the relationship between the concentration of PtNPs/GO and catalytic activity is nearly linear when the catalyst concentration remained in a relative low range (0.17−6.7 μg/mL). To better understand the peroxidase-like activity of the PtNPs/GO and compare with that of HRP, we analyzed steadystate kinetics for the TMB oxidation reaction. Typical Michaelis−Menten curves were obtained in a certain range of H2O2 or TMB concentrations (Figure 4). The dots are the experimental data and the solid curves are the fittings to the Michaelis−Menten model for enzyme kinetics. From the Lineweaver−Burk plot, the Michaelis constant (Km) and maximal reaction velocity (νmax) were obtained and listed in Table 1. Km is an important parameter to measure binding affinity of the enzyme to the substrate and can be applied similarly here to study enzyme mimic-substrate interaction. The apparent Km value of PtNPs/GO with TMB as the substrate was lower than the natural enzyme, suggesting that the PtNPs/ GO have a higher affinity for TMB than HRP. Because of the high surface-to-volume ratios as well as high affinity of GO for hydrophobic molecules, TMB could be absorbed onto GO
Figure 2. UV−vis spectra of the reaction solution containing TMB and H2O2 (a) in the absence of PtNPs/GO, (b) in the presence of GO, (c) in the presence of PtNPs/GO, and (d) after quenched by H2SO4. Inset: photographs of different solutions corresponding to parts a−d.
PtNPs/GO or in the presence of GO, suggesting the peroxidase-like activity of the hybrid. Like peroxidase and other nanomaterials-based peroxidase mimics, the catalytic activity of PtNPs/GO was also dependent on pH, temperature, and the concentration of substrates and catalyst. To investigate how the acidity of the buffer solution affects the TMB-H2O2 catalytic reaction, we performed the catalytic experiments in phosphate buffer with varying pH. As shown in Figure 3a, the effect of the buffer pH on the catalytic activity of PtNPs/GO is similar to that on HRP. The highest catalytic activity of PtNPs/ GO exhibited over the pH range 4.5−5.5. Although the catalytic activity of PtNPs/GO increased with temperature gradually (Figure 3b), the reaction solution contained flocs
Figure 3. (a−c) Dependency of the relative activity of PtNPs/GO and HRP on (a) pH, (b) temperature, and (c) concentration of H2O2. (d) Relationship between absorbance value at 652 nm with the concentration of PtNPs/GO. 2714
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Figure 4. Steady state kinetic assay of PtNPs/GO hybrid and HRP. Experiments were carried out in 20 mM phosphate buffer (pH 5.0) at 30 °C. (a,c) TMB concentration was fixed at 0.8 mM, and the H2O2 concentration was varied. (b,d) H2O2 concentration was fixed at 400 mM for PtNPs/ GO hybrid or 5 mM for HRP, and the TMB concentration was varied.
higher than that for HRP, consistent with the observation that a higher H2O2 concentration was required to achieve maximal activity for PtNPs/GO nanocomposites. Colorimetric Detection of Cancer Cells. Rapid, economical, and sensitive diagnostic methods for the early and accurate detection of cancer are of great importance for clinical applications. Current clinical methods often require timeconsuming techniques and expensive instrumentation. Therefore, a more cost-effective method requiring simple or no instrumentation yet still providing great sensitivity and accuracy would be ideal for point of care diagnosis.39 To address these issues, we used the PtNPs/GO as a signal transducer to develop the colorimetric assay for the direct detection of cancer cells. As a demonstration, folic acid (FA) was chosen as a recognition element to functionalize PtNPs/GO. FA can effectively target
Table 1. Comparison of the Kinetic Parameters of PtNPs/ GO Hybrid and HRPa PtNPs/GO PtNPs/GO HRP HRP
substrate
Km (mM)
vmax (10−8 M/s)
H2O2 TMB H2O2 TMB
221.4 0.1864 0.2832 0.2343
12.45 10.20 3.053 5.246
a Km is the Michaelis constant, and vmax is the maximal reaction velocity.
efficiently.3 Similar to natural enzymes, the ability to bring substrates into proximity with their active sites leads to high catalytic efficiency.38 On the other hand, the apparent Km value of PtNPs/GO with H2O2 as the substrate was significantly
Scheme 1. Schematic Representation of Colorimetric Direction of Cancer Cells by Using Folic Acid Functionalized PtNPs/GO
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presence of H2O2. In addition, because of the different amounts of folate receptor expression on different types of cancer cells, more FA-PtNPs/GO nanocomposites are bound to MCF-7 cells than SGC-7901 cells. To verify that the PtNPs/GO nanocomposites were assembled on the surface of the target cells, we used diazoaminobenzene (DAB) to locate the binding sites. DAB is a widely used chromogen for immunohistochemical staining and immunoblotting. In the presence of peroxidase enzyme, DAB loses electrons and produces a brown precipitate. The 2000 MCF-7 cells were plated in a 96-well plate and incubated with 5 μg of FA-PtNPs/GO at 37 °C for 1.5 h. After being rinsed three times with phosphate buffer (10 mM, pH 7.4), DAB solution and H2O2 were added into the well. After 10 min, reddish-brown product on the cell surfaces could be observed by a microscope, indicating that FA-PtNPs/GO hybrids could combine with folate receptors effectively (Figure 6). With the initial concept and development of the assay demonstrated, the next step was to verify that the assay could calorimetrically differentiate between different amounts of cells. To accomplish this, 5 μg of FA-PtNPs/GO was incubated with increasing amounts of MCF-7 cells. This was repeated with the same amounts of control cells (HUVECs) for comparison. The image of both cell types is shown in Figure 7a. In the control
many tumor cells that overexpress folate receptor on the cell membrane.40 On the basis of the peroxides-like activity, selective binding PtNPs/GO on the tumor cells can convert the recognition process into quantitative colorimetric signal. The working principle is illustrated in Scheme 1. To demonstrate the principle behind the assay, it was first determined whether the FA functionalized PtNPs/GO (FAPtNPs/GO) could differentiate between target cells and control cells. For these experiments, MCF-7 human breast cancer cell and SGC-7901 human gastric cancer cell were used as target cells while human umbilical vein endothelial cell (HUVEC) was used as a negative control. In these three cell lines, MCF-7 and SGC-7901 cells overexpress folate receptors while HUVECs do not. The six samples are as follow: 5 μg of FA-PtNPs/GO with no cells, 5 μg of FA-PtNPs/GO with 2000 MCF-7 cells, 5 μg of FA-PtNPs/GO with 2000 SGC-7901 cells, 5 μg of FA-PtNPs/ GO with 2000 HUVECs, 40 μg of FA and 5 μg of FA-PtNPs/ GO with 2000 MCF-7 cells, and 5 μg of PtNPs/GO with 2000 MCF-7 cells. In the presence of TMB and H2O2, the PtNPs/ GO conjugated cells will catalyze a color change reaction that can be judged by the naked eye and easily be quantitatively monitored by a microplate reader. Figure 5 shows that the
Figure 5. Colorimetric response of (a) 5 μg of FA-PtNPs/GO with no cells, (b) 5 μg of FA-PtNPs/GO with 2000 MCF-7 cells, (c) 5 μg of FA-PtNPs/GO with 2000 SGC-7901 cells, (d) 5 μg of FA-PtNPs/GO with 2000 HUVECs, (e) 40 μg of FA and 5 μg of FA-PtNPs/GO with 2000 MCF-7 cells, and (f) 5 μg of PtNPs/GO with 2000 MCF-7 cells.
absorbance of FA-PtNPs/GO with 2000 target cells (MCF-7 or SGC-7901) is significantly higher than the same amount of FAPtNPs/GO with 2000 control cells, the same amount of target cells with FA and FA-PtNPs/GO, the same amount of target cells with PtNPs/GO, or FA-PtNPs/GO with no cells. These results indicate that the FA-PtNPs/GO nanocomposites bound selectively to the target cells through the interaction between FA and folate receptor and that the assembly of the PtNPs/GO around the target cells catalyzed the oxidation of TMB in the
Figure 7. (a) Photographs for cell detection with the colorimetric method developed using FA-PtNPs/GO and (b) absorption values at 450 nm depend on the number of cells.
cells, the samples remain almost colorless and there is no significant difference between the samples regardless of the
Figure 6. Photographs of MCF-7 cells (a) before and (b) after DAB staining. 2716
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amount of cells present. However, the results clearly show that the samples with more MCF-7 cells have a darker color. Thus, the assay allows for the detection of MCF-7 cells with the naked eye. The assay also proved to be quite sensitive as 125 cells could be readily detected by the naked eye. In order for a more sensitive detection than allowed by the naked eye, the samples were also analyzed using a microplate reader. The absorbance at 450 nm was recorded for different amounts of control and target cells and plotted in Figure 7b. The absorbance correlates well with the colorimetric results in that the samples with an increasing amount of target cells absorb light more intensely. There is little change in the absorbance of the control cell samples regardless of the amount of cells present. The assay of MCF-7 showed an excellent dynamic range with standard deviations ranging from 4 to 10%. On the basis of three times the standard deviation of the blank measurement, the limit of detection of MCF-7 cells was calculated to be 30 cells. In addition, this experiment was repeated with control cells to measure their response to the assay. The control cells had no response at the lower cell concentrations as these samples had signals comparable to the blank. At the higher cell concentrations, the control cells had a small response although it was still significantly lower than even the smallest concentration of target cells that were evaluated. In order to verify the cytotoxicity of FA-PtNPs/GO, we tested the cell viability by MTT assay. After incubation with 5 μg of FAPtNPs/GO for 1.5 h, the relative survival rate of MCF-7, SGC7901, and HUVEC is 99.58%, 96.33%, and 97.94%, respectively, proving that the cytotoxicity of FA-PtNPs/GO could be ignored in the detection of cancer cells. On the basis of these results, the assay for direct cell detection has demonstrated excellent sensitivity and selectivity.
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Corresponding Author
*Phone/fax: +86 591 22862016. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (Grant 21175023), the Program for New Century Excellent Talents in University (Grant NCET-12-0618), and the Natural Science Foundation of Fujian Province (Grant 2012J06019).
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REFERENCES
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CONCLUSION In summary, by a simple and green in situ reduction process, we have obtained the synergistic PtNPs/GO hybrid that exhibits excellent peroxidase-like activity. As compared to natural enzymes, PtNPs/GO shows several advantages such as low-cost, high-stability, green synthesis, and ease of modification. On the basis of the peroxidase-like activity, we used the PtNPs/GO as a signal transducer to develop a colorimetric assay for the direct detection of cancer cells. As a demonstration, folic acid was chosen as a recognition element to functionalize PtNPs/GO. Since folate receptors are overexpressed on the cell membranes of different types of cancer cells, including ovarian, endometrial, colorectal, breast, lung, renal cell carcinomas, brain metastases derived from epithelial cancers, and neuroendocrine carcinomas, folic acid functionalized PtNPs/GO can effectively target many tumor cells. The selective binding PtNPs/GO can convert the recognition process into a quantitative colorimetric signal. Even by naked-eye observation, 125 cancer cells (MCF-7) can be distinguished. Taking advantage of the attractive properties, we expect that this engineered material has the potential to provide a wide range of approaches for medical diagnosis and biotechnology through utilization of different recognition elements such as aptamers, antibody, peptide, and nucleic acid.
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AUTHOR INFORMATION
ASSOCIATED CONTENT
S Supporting Information *
Photographs of GO + H2PtCl6 before and after reaction, UV− vis spectra of GO and PtNPs/GO, and particle size distribution histogram of the Pt nanoparticles obtained from TEM images. 2717
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In Situ Growth of Porous Platinum Nanoparticles on Graphene Oxide for Colorimetric Detection of Cancer Cells Ling-Na Zhang,†,‡ Hao-Hua Deng,†,‡ Feng-Lin Lin,†,‡ Xiong-Wei Xu,§ Shao-Huang Weng,†,‡ Ai-Lin Liu,†,‡ Xin-Hua Lin,†,‡ Xing-Hua Xia,∥ and Wei Chen*,†,‡ †
Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China Nano Medical Technology Research Institute, Fujian Medical University, Fuzhou 350004, China § First Affiliated Hospital, Fujian Medical University, Fuzhou 350004, China ∥ State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China ‡
S Supporting Information *
ABSTRACT: A green approach is proposed for in situ growth of porous platinum nanoparticles on graphene oxide (PtNPs/ GO). The resulting nanocomposite has been proven to function as peroxidase mimetics that can catalyze the reaction of peroxidase substrate in the presence of hydrogen peroxide. On the basis of the peroxidase-like activity, we used the PtNPs/GO as a signal transducer to develop a colorimetric assay for the direct detection of cancer cells. By using folic acid as a recognition element, a total of 125 cancer cells (MCF-7) can be distinguished by naked-eye observation. We envision that this nanomaterial could be used as a power tool for a wide range of potential applications in biotechnology and medicine.
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realize the combination of the respective properties of each component or to achieve cooperatively enhanced performances.11,18−24 Colloidal noble metal nanoparticles have been receiving a great deal of attention in areas from chemistry to physics, material sciences, biology, and medicine.25,26 Conventionally, colloidal noble metal nanoparticles should be stabilized by addition of chemical or biomolecular stabilizers to prevent aggregation. However, their catalytic activity could be severely limited by these stabilizers capped around nanoparticles.13 A solution of this problem could be offered by anchoring of metal particles on specific supports. As a representative derivate of graphene, graphene oxide (GO) has been widely utilized as fascinating support for preparation of graphene-based nanoparticle composites.27−30 The rigid two-dimensional structure of GO can enable most surface of the attached nanoparticles to be exposed to the environment.27 Furthermore, the presence of abundant carbonyl and carboxyl groups makes GO sheets strongly hydrophilic, allowing them to readily disperse in water and polar organic solvents. In this paper, we demonstrated that clean and well-dispersed porous platinum nanoparticles (PtNPs) can be facilely prepared in situ on a GO surface. In contrast to previous studies,31−33 no
atural enzymes have been attracting great interest and widely applied because of their immense catalytic power and high substrate specificity under mild reaction conditions. However, natural enzymes suffer from some serious disadvantages such as sensitivity of catalytic activity to environmental conditions, low operational stability due to denaturation and digestion, as well as time-consuming and expensive preparation and purification, which restrict their widespread applications. Therefore, construction and discovery of novel enzyme mimetics is under intensive investigation. Recently, Fe3O4 nanoparticles, which were generally considered to be biologically inert, have been found to possess intrinsic enzyme mimetic activity similar to that of natural peroxidase, thus opening a new door for the application of nanoscaled materials in the biochemical field.1 In comparison with natural enzymes which are susceptible to the reaction conditions, nanomaterials are considerably more stable over a wide range of pH and temperature. They possess additional advantages of controlled preparation in mass yield and at relatively low cost, flexibility in structure and composition design, and tunable catalytic activities. In light of such advantages, a series of inorganic nanomaterials have emerged as peroxidase mimics and applied in the environmental chemistry and biomedicine fields.2−17 Among the enzyme nanomimics, a new generation of hybrid nanomaterials is particularly impressive and takes one of the most pivotal steps toward enzyme mimicry. Hybrid composite materials, with well-defined structures, have been explored to © 2014 American Chemical Society
Received: December 18, 2013 Accepted: February 13, 2014 Published: February 13, 2014 2711
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Figure 1. (a,b) AFM image of GO and the PtNPs/GO hybrid, (c,d) TEM images of the PtNPs/GO hybrid under different magnifications, (e) EDS spectrum of the PtNPs/GO hybrid, and (f) XPS spectrum of the PtNPs/GO hybrid.
Tetramethylbenzidine, N-hydroxysulfosuccinimide sodium (sulfo-NHS), and folic acid were purchased from Aladdin (Shanghai, China). Poly(allyl amine hydrochloride) (PAH, MW = 58 000) and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were obtained from Sigma-Aldrich. H2PtCl6·6H2O and H2O2 came from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Horseradish peroxidase (HRP, 300 IU/mg) was purchased from Dingguochangsheng Biotech Co. Ltd. (Beijing, China). All chemicals were of analytical reagent grade. Deionized water was used in all experiments. Atomic-force microscopy (AFM) measurements were performed using a Bruker Nanoscope V multimode atomic
additional reductant or surfactant was needed in our synthesis. GO acted as both the stabilizer and the reductant to obtain stable and clean Pt nanoparticles. The resulting PtNPs-loaded GO (PtNPs/GO) revealed excellent peroxidase-like activity that can be used to catalyze the oxidation of 3,3′,5,5′tetramethylbenzidine (TMB) by hydrogen peroxide. For further application, folic acid (FA) functionalized PtNPs/GO was successfully used for the colorimetric detection of cancer cells.
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EXPERIMENTAL SECTION Reagents and Apparatus. Graphite flake (325 mesh) was purchased from XF NANO (Nanjing, China). 3,3′,5,5′2712
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force microscope. Intelligent mode was used to acquire the images under ambient conditions. Transmission electron microscopy (TEM) images and an energy-dispersive X-ray spectroscopy (EDS) spectrum were recorded using a FEI TECNAI G2 20 high-resolution transmission electron microscope operating at 200 kV. An X-ray photoelectron spectrum (XPS) was recorded by a Thermo ESCALAB 250XI X-ray photoelectron spectrometer with nonmonochromatized Al Kα radiation as the excitation source. UV−vis absorbance spectra were performed on a Shimadzu UV-2450 spectrophotometer. Images of cells were viewed with an Olympus IX-70 inverted fluorescence microscope. Synthesis of PtNPs/GO. GO was synthesized from flake graphite by an improved Hummers method.34 PtNPs/GO was fabricated by mixed 0.5 mg/mL GO aqueous solution with 7.9 mM H2PtCl6 at a ratio of 10:1 (v/v). The mixture was continually reacted under vigorous stirring at 80 °C for 24 h to obtain the products. Synthesis of FA-PtNPs/GO. In order to keep the excellent catalytic activity of FA-PtNPs/GO, we prefer to deposit PtNPs on FA-modified GO rather than modify FA on presynthesized PtNPs/GO. The PAH functionalized GO was prepared by vigorously stirring a solution of 40 mg of the graphene oxides, 160 mg of PAH, and 160 mg of KOH in 80 mL of H2O at 70 °C for 24 h. After that, the PAH functionalized GO was collected and purified by centrifugation and adequately washed with water several times to remove the impurities and the excess of PAH by physical absorption. To conjugate the amino functionalized GO with folic acid, folic acid (40 mg, 0.0227 mmol) was first dissolved in 16 mL of phosphate buffer solution (pH 5.0, 20 mM). The solution of folic acid was then mixed with 8 mL of aqueous solution of EDC (0.068 mmol) and sulfo-NHS (0.068 mmol). After agitating overnight at room temperature in the dark, the solution of PAH functionalized GO (0.5 mg/mL, 80 mL) was added to the mixture and stirred at room temperature for 24 h. The folic acid functionalized GO was collected by centrifugation and extensively washed with water to remove the physisorbed folic acid. Finally, 8 mL of H2PtCl6 solution (7.9 mM) was added to 80 mL of folic acid functionalized GO solution (0.5 mg/mL). The resulting mixture was stirred at 80 °C for 24 h to obtain FA-PtNPs/GO. Cell Culture and Treatment. The human breast cancer cells (MCF-7), human gastric cancer cells (SGC-7901), and human umbilical vein endothelial cells (HUVEC) were grown in RPMI 1640 medium (Hyclone) supplemented with 6% fetal bovine serum in a humidified 37 °C incubator with 5% CO2. For the colorimetric assay, cells were plated in 96-well plate for adherence and then allowed to incubate with 5 μg of FAPtNPs/GO in RPMI 1640 medium at 37 °C for 1.5 h. For one of the control experiment, adherent cells in 96-well plate were incubated with 5 μg of PtNPs/GO for 1.5 h. For the other control experiment, cells were first treated with 40 μg of FA in RPMI-1640 medium for 30 min and then incubate with 5 μg of FA-PtNPs/GO for 1.5 h. Then every cell well was washed three times with phosphate buffer solution (10 mM, pH 7.4) to remove unattached FA-PtNPs/GO. After that, 200 μL of phosphate buffer solution (20 mM, pH 5.0) containing 0.8 mM TMB and 1 M H2O2 was added to each cell well and incubated for 15 min at room temperature. Finally, the reaction was terminated by H2SO4 (2 M) and quantitatively monitored by a microplate reader (Thermo Multiskan MK3) at the wavelength of 450 nm.
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RESULTS AND DISCUSSION
Characterization of PtNPs/GO. PtNPs/GO was fabricated by direct incubation with GO with H2PtCl6 at 80 °C for 24 h. The color of the mixture changed to dark brown after incubation (Figure S1 in the Supporting Information). No red shift of the absorption peak of GO in the UV−vis spectrum was observed (Figure S2 in the Supporting Information), indicating that GO was not evolved into reduced GO in the reaction. To verify the successful growth of Pt nanoparticles on graphene oxide sheets, AFM was used to characterize the morphologies of the as-prepared composites. The typical AFM images of GO and PtNPs/GO reveal that the thickness of GO sheets maintained about 1 nm before and after the reaction (Figure 1a,b). The nanoparticles were observed on the GO but not outside the sheet, indicating that there is almost no particle formation in free solution. The H2PtCl6 precursor was directly reduced to PtNPs and anchored to the graphene oxide surface mainly through the defects and oxygen functional groups.30 It was worth to note that the resultant PtNPs/GO hybrid could form well-dispersed aqueous colloids owing to the residual oxygen-containing groups on GO, providing advantages in the chemical modification and biological applications. Figure 1c,d shows representative TEM images of the synthesized PtNPs/ GO composites. A large number of Pt nanoparticles with a size of about 50 nm were well-dispersed on the surface of the graphene oxide nanosheets (Figure S3 in the Supporting Information), which should be attributed to the hydroxyl, epoxide, and carboxylic groups uniformly existing on the GO.35 H2PtCl6 was reduced to Pt nanoparticles without adding any reducing agent in our synthesis. Therefore, it is rationally deduced that the spontaneous deposition of PtNPs on GO sheets is ascribed to the redox reaction between GO and PtCl62−‑, which is similar to the reaction mechanism between metal ions and single-walled carbon nanotubes proposed by Dai et al.36 It should be pointed out that the growth of PtNPs after nucleation might involve a galvanic-reaction-like process in which the reduction of Pt occurs on Pt nuclei by the electrons transferred from GO.30 The selected area electron diffraction (SAED) pattern of PtNPs/GO (inset of Figure 1c) indicates the highly crystalline nature of the PtNPs. The enlarged TEM image reveals the porous structure of PtNPs (Figure 1d), which might endue them more active sites to obtain the extent of catalytic properties. An energy-dispersive X-ray spectroscopy (EDS) spectrum was also used to analyze the elemental distribution of the nanohybrid. The significant Pt signals suggest that the nanoparticles loaded on the surface of graphene oxide were platinum (Figure 1e). Meanwhile, from the X-ray photon spectroscopy (XPS) result demonstrated in Figure 1f, Pt 4f peaks were observed at 71.5 and 74.9 eV,37 verifying again the successful formation of PtNPs on the GO surface. Peroxidase-Like Activity of PtNPs/GO. TMB, one of the most common chromogenic substrates of peroxidase, was used to investigate the peroxidase-like activity of PtNPs/GO. As shown in Figure 2, PtNPs/GO could catalyze the oxidation of TMB in the presence of H2O2 to produce a blue color with maximum absorbance at 652 and 370 nm. Like enzymatic peroxidase activity, such as observed for the commonly used enzyme HRP, this catalytic reaction could even be quenched by H2SO4. The blue product was further oxidized to a yellow diimine with a maximum absorption wavelength of 450 nm. In contrast, no obvious reaction occurred in the absence of 2713
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when the temperature was above 35 °C. To ensure accuracy, the other chromogenic experiments were carried out at 30 °C. As noted, in contrast to that found for natural peroxidase, Fe3O4 nanoparticles, graphene oxide, and porous platinum nanotubes,1,3,16 no inhibition effect on the catalytic activity of PtNPs/GO was observed at high H2O2 concentration (Figure 3c). Figure 3d provides the relationship between the absorbance of the oxidized TMB product at 652 nm and the concentration of PtNPs/GO. We found that the catalytic rate increased with increasing catalyst concentration. With excess reactants, the reaction speed depends on the catalyst concentration over a given range. Therefore, the relationship between the concentration of PtNPs/GO and catalytic activity is nearly linear when the catalyst concentration remained in a relative low range (0.17−6.7 μg/mL). To better understand the peroxidase-like activity of the PtNPs/GO and compare with that of HRP, we analyzed steadystate kinetics for the TMB oxidation reaction. Typical Michaelis−Menten curves were obtained in a certain range of H2O2 or TMB concentrations (Figure 4). The dots are the experimental data and the solid curves are the fittings to the Michaelis−Menten model for enzyme kinetics. From the Lineweaver−Burk plot, the Michaelis constant (Km) and maximal reaction velocity (νmax) were obtained and listed in Table 1. Km is an important parameter to measure binding affinity of the enzyme to the substrate and can be applied similarly here to study enzyme mimic-substrate interaction. The apparent Km value of PtNPs/GO with TMB as the substrate was lower than the natural enzyme, suggesting that the PtNPs/ GO have a higher affinity for TMB than HRP. Because of the high surface-to-volume ratios as well as high affinity of GO for hydrophobic molecules, TMB could be absorbed onto GO
Figure 2. UV−vis spectra of the reaction solution containing TMB and H2O2 (a) in the absence of PtNPs/GO, (b) in the presence of GO, (c) in the presence of PtNPs/GO, and (d) after quenched by H2SO4. Inset: photographs of different solutions corresponding to parts a−d.
PtNPs/GO or in the presence of GO, suggesting the peroxidase-like activity of the hybrid. Like peroxidase and other nanomaterials-based peroxidase mimics, the catalytic activity of PtNPs/GO was also dependent on pH, temperature, and the concentration of substrates and catalyst. To investigate how the acidity of the buffer solution affects the TMB-H2O2 catalytic reaction, we performed the catalytic experiments in phosphate buffer with varying pH. As shown in Figure 3a, the effect of the buffer pH on the catalytic activity of PtNPs/GO is similar to that on HRP. The highest catalytic activity of PtNPs/ GO exhibited over the pH range 4.5−5.5. Although the catalytic activity of PtNPs/GO increased with temperature gradually (Figure 3b), the reaction solution contained flocs
Figure 3. (a−c) Dependency of the relative activity of PtNPs/GO and HRP on (a) pH, (b) temperature, and (c) concentration of H2O2. (d) Relationship between absorbance value at 652 nm with the concentration of PtNPs/GO. 2714
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Figure 4. Steady state kinetic assay of PtNPs/GO hybrid and HRP. Experiments were carried out in 20 mM phosphate buffer (pH 5.0) at 30 °C. (a,c) TMB concentration was fixed at 0.8 mM, and the H2O2 concentration was varied. (b,d) H2O2 concentration was fixed at 400 mM for PtNPs/ GO hybrid or 5 mM for HRP, and the TMB concentration was varied.
higher than that for HRP, consistent with the observation that a higher H2O2 concentration was required to achieve maximal activity for PtNPs/GO nanocomposites. Colorimetric Detection of Cancer Cells. Rapid, economical, and sensitive diagnostic methods for the early and accurate detection of cancer are of great importance for clinical applications. Current clinical methods often require timeconsuming techniques and expensive instrumentation. Therefore, a more cost-effective method requiring simple or no instrumentation yet still providing great sensitivity and accuracy would be ideal for point of care diagnosis.39 To address these issues, we used the PtNPs/GO as a signal transducer to develop the colorimetric assay for the direct detection of cancer cells. As a demonstration, folic acid (FA) was chosen as a recognition element to functionalize PtNPs/GO. FA can effectively target
Table 1. Comparison of the Kinetic Parameters of PtNPs/ GO Hybrid and HRPa PtNPs/GO PtNPs/GO HRP HRP
substrate
Km (mM)
vmax (10−8 M/s)
H2O2 TMB H2O2 TMB
221.4 0.1864 0.2832 0.2343
12.45 10.20 3.053 5.246
a Km is the Michaelis constant, and vmax is the maximal reaction velocity.
efficiently.3 Similar to natural enzymes, the ability to bring substrates into proximity with their active sites leads to high catalytic efficiency.38 On the other hand, the apparent Km value of PtNPs/GO with H2O2 as the substrate was significantly
Scheme 1. Schematic Representation of Colorimetric Direction of Cancer Cells by Using Folic Acid Functionalized PtNPs/GO
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presence of H2O2. In addition, because of the different amounts of folate receptor expression on different types of cancer cells, more FA-PtNPs/GO nanocomposites are bound to MCF-7 cells than SGC-7901 cells. To verify that the PtNPs/GO nanocomposites were assembled on the surface of the target cells, we used diazoaminobenzene (DAB) to locate the binding sites. DAB is a widely used chromogen for immunohistochemical staining and immunoblotting. In the presence of peroxidase enzyme, DAB loses electrons and produces a brown precipitate. The 2000 MCF-7 cells were plated in a 96-well plate and incubated with 5 μg of FA-PtNPs/GO at 37 °C for 1.5 h. After being rinsed three times with phosphate buffer (10 mM, pH 7.4), DAB solution and H2O2 were added into the well. After 10 min, reddish-brown product on the cell surfaces could be observed by a microscope, indicating that FA-PtNPs/GO hybrids could combine with folate receptors effectively (Figure 6). With the initial concept and development of the assay demonstrated, the next step was to verify that the assay could calorimetrically differentiate between different amounts of cells. To accomplish this, 5 μg of FA-PtNPs/GO was incubated with increasing amounts of MCF-7 cells. This was repeated with the same amounts of control cells (HUVECs) for comparison. The image of both cell types is shown in Figure 7a. In the control
many tumor cells that overexpress folate receptor on the cell membrane.40 On the basis of the peroxides-like activity, selective binding PtNPs/GO on the tumor cells can convert the recognition process into quantitative colorimetric signal. The working principle is illustrated in Scheme 1. To demonstrate the principle behind the assay, it was first determined whether the FA functionalized PtNPs/GO (FAPtNPs/GO) could differentiate between target cells and control cells. For these experiments, MCF-7 human breast cancer cell and SGC-7901 human gastric cancer cell were used as target cells while human umbilical vein endothelial cell (HUVEC) was used as a negative control. In these three cell lines, MCF-7 and SGC-7901 cells overexpress folate receptors while HUVECs do not. The six samples are as follow: 5 μg of FA-PtNPs/GO with no cells, 5 μg of FA-PtNPs/GO with 2000 MCF-7 cells, 5 μg of FA-PtNPs/GO with 2000 SGC-7901 cells, 5 μg of FA-PtNPs/ GO with 2000 HUVECs, 40 μg of FA and 5 μg of FA-PtNPs/ GO with 2000 MCF-7 cells, and 5 μg of PtNPs/GO with 2000 MCF-7 cells. In the presence of TMB and H2O2, the PtNPs/ GO conjugated cells will catalyze a color change reaction that can be judged by the naked eye and easily be quantitatively monitored by a microplate reader. Figure 5 shows that the
Figure 5. Colorimetric response of (a) 5 μg of FA-PtNPs/GO with no cells, (b) 5 μg of FA-PtNPs/GO with 2000 MCF-7 cells, (c) 5 μg of FA-PtNPs/GO with 2000 SGC-7901 cells, (d) 5 μg of FA-PtNPs/GO with 2000 HUVECs, (e) 40 μg of FA and 5 μg of FA-PtNPs/GO with 2000 MCF-7 cells, and (f) 5 μg of PtNPs/GO with 2000 MCF-7 cells.
absorbance of FA-PtNPs/GO with 2000 target cells (MCF-7 or SGC-7901) is significantly higher than the same amount of FAPtNPs/GO with 2000 control cells, the same amount of target cells with FA and FA-PtNPs/GO, the same amount of target cells with PtNPs/GO, or FA-PtNPs/GO with no cells. These results indicate that the FA-PtNPs/GO nanocomposites bound selectively to the target cells through the interaction between FA and folate receptor and that the assembly of the PtNPs/GO around the target cells catalyzed the oxidation of TMB in the
Figure 7. (a) Photographs for cell detection with the colorimetric method developed using FA-PtNPs/GO and (b) absorption values at 450 nm depend on the number of cells.
cells, the samples remain almost colorless and there is no significant difference between the samples regardless of the
Figure 6. Photographs of MCF-7 cells (a) before and (b) after DAB staining. 2716
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amount of cells present. However, the results clearly show that the samples with more MCF-7 cells have a darker color. Thus, the assay allows for the detection of MCF-7 cells with the naked eye. The assay also proved to be quite sensitive as 125 cells could be readily detected by the naked eye. In order for a more sensitive detection than allowed by the naked eye, the samples were also analyzed using a microplate reader. The absorbance at 450 nm was recorded for different amounts of control and target cells and plotted in Figure 7b. The absorbance correlates well with the colorimetric results in that the samples with an increasing amount of target cells absorb light more intensely. There is little change in the absorbance of the control cell samples regardless of the amount of cells present. The assay of MCF-7 showed an excellent dynamic range with standard deviations ranging from 4 to 10%. On the basis of three times the standard deviation of the blank measurement, the limit of detection of MCF-7 cells was calculated to be 30 cells. In addition, this experiment was repeated with control cells to measure their response to the assay. The control cells had no response at the lower cell concentrations as these samples had signals comparable to the blank. At the higher cell concentrations, the control cells had a small response although it was still significantly lower than even the smallest concentration of target cells that were evaluated. In order to verify the cytotoxicity of FA-PtNPs/GO, we tested the cell viability by MTT assay. After incubation with 5 μg of FAPtNPs/GO for 1.5 h, the relative survival rate of MCF-7, SGC7901, and HUVEC is 99.58%, 96.33%, and 97.94%, respectively, proving that the cytotoxicity of FA-PtNPs/GO could be ignored in the detection of cancer cells. On the basis of these results, the assay for direct cell detection has demonstrated excellent sensitivity and selectivity.
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Corresponding Author
*Phone/fax: +86 591 22862016. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (Grant 21175023), the Program for New Century Excellent Talents in University (Grant NCET-12-0618), and the Natural Science Foundation of Fujian Province (Grant 2012J06019).
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CONCLUSION In summary, by a simple and green in situ reduction process, we have obtained the synergistic PtNPs/GO hybrid that exhibits excellent peroxidase-like activity. As compared to natural enzymes, PtNPs/GO shows several advantages such as low-cost, high-stability, green synthesis, and ease of modification. On the basis of the peroxidase-like activity, we used the PtNPs/GO as a signal transducer to develop a colorimetric assay for the direct detection of cancer cells. As a demonstration, folic acid was chosen as a recognition element to functionalize PtNPs/GO. Since folate receptors are overexpressed on the cell membranes of different types of cancer cells, including ovarian, endometrial, colorectal, breast, lung, renal cell carcinomas, brain metastases derived from epithelial cancers, and neuroendocrine carcinomas, folic acid functionalized PtNPs/GO can effectively target many tumor cells. The selective binding PtNPs/GO can convert the recognition process into a quantitative colorimetric signal. Even by naked-eye observation, 125 cancer cells (MCF-7) can be distinguished. Taking advantage of the attractive properties, we expect that this engineered material has the potential to provide a wide range of approaches for medical diagnosis and biotechnology through utilization of different recognition elements such as aptamers, antibody, peptide, and nucleic acid.
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AUTHOR INFORMATION
ASSOCIATED CONTENT
S Supporting Information *
Photographs of GO + H2PtCl6 before and after reaction, UV− vis spectra of GO and PtNPs/GO, and particle size distribution histogram of the Pt nanoparticles obtained from TEM images. 2717
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