Article pubs.acs.org/ac
Gold Nanocluster-Assisted Fluorescent Detection for Hydrogen Peroxide and Cholesterol Based on the Inner Filter Effect of Gold Nanoparticles Heng-Chia Chang† and Ja-an Annie Ho*,†,‡ †
Department of Chemistry, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan BioAnalytical Chemistry and Nanobiomedicine Laboratory, Department of Biochemical Science and Technology, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan
‡
S Supporting Information *
ABSTRACT: We developed a simple, sensitive inner filter effect (IFE)-based fluorescent assay for sensing H2O2 and cholesterol. In the process, poly(vinylpyrrolidone)-protected gold nanoparticles (PVP-AuNPs) and fluorescent BSA-protected gold nanoclusters (BSA-AuNCs) were used as an IFE absorber/ fluorophore pair. PVP-AuNPs can be a powerful absorber to influence the emission of the fluorophore, BSA-AuNCs, in the IFE-based fluorescent assays. That is due to the high extinction coefficient of AuNPs and the complementary overlap between the surface plasmon resonance (SPR) absorption of PVP-AuNPs and the excitation of BSA-AuNCs. The PVPAu seeds, produced by directly mixing PVP with HAuCl4, were able to catalyze H2O2 to enlarge AuNPs. The SPR absorption of PVP-AuNPs was enhanced with an increased concentration of H2O2 and, subsequently, induced significant fluorescence quenching of BSA-AuNCs. The IFE-based fluorescent assay enabled the detection of H2O2 and generation of H2O2 in the presence of O2/cholesterol and cholesterol oxidase (ChOx) by the fluorescence response of BSA-AuNCs. The present IFE-based approach can detect H2O2 ranging from 1 to 100 μM with a detection limit of 0.8 μM and cholesterol ranging from 1 to 100 μM with a detection limit of 1.4 μM.
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glucose/glucose oxidase10 or cholesterol/cholesterol oxidase.11 Moreover, AuNPs have been reported to be involved in the design of the fluorescent assays. For instance, AuNPs can be used as an efficient fluorescence quencher through energy or electron transfer processes for the determination of target analytes.12−14 More recently, several studies have described the detection of environmentally or biologically important analytes, such as CN−, H2O2, aminothiols, and immunoglobulin,15−17 via the inner filter effect of gold nanoparticles on the fluorescence of fluorophores or metal nanoparticles. This is ascribed to the unique characteristics of the AuNPs that can absorb a significant percentage of the excitation and/or emission light of the fluorophore. In most AuNPs-based fluorescence quenching assays, the quenching effects are associated with the distance between the quencher (AuNPs) and the fluorophore. Besides, a linker element is not usually necessary for the IFE-based fluorescence assays, which provide a comparatively simple and facile approach for the analytical detection of target analytes. However, some restrictions still exist in the design and development of IFE-based fluorescent assays due to the limited choices of suitable absorber and fluorophore, because IFE only occurs effectively if the absorption band of the absorber possesses the complementary
he detection of hydrogen peroxide (H2O2) is of practical importance in chemical, biological, clinical, environmental, and other fields. For instance, H2O2 can be harmful to biological systems and appears to be involved in the neuropathology of central nervous system diseases.1 The H2O2 level in urine is also considered as a potential biomarker for oxidation stress in patients with a malignancy.2 Additionally, H2O2 is an end-product in oxidation reactions, catalyzed by glucose oxidase, uricase, and cholesterol oxidase, etc.3−5 Cholesterol and its fatty acid esters are important compounds of nerve and brain cells; they are precursors of other biological materials, such as bile acid and steroid hormones. An excessive concentration of cholesterol in blood is often associated with the occurrence of atherosclerosis;6 however, a low concentration of cholesterol results in hypocholesterolemia, which may cause hemorrhagic stroke.7 Without cholesterol, brain cells cannot send messages that power every organ in our body. Therefore, being able to estimate cholesterol level is of interest to the biological science and food industries. Recent studies have revealed that a larger dimension of gold nanoparticles (AuNPs) can be formed with the presence of Au seeds, cetyltrimethylammonium chloride (CTAC) as a surfactant, and a reducing agent (e.g., ascorbic acid, phenolic acid) as a reductant. The spectroscopic features of AuNPs with larger dimensions depend on the concentrations of the reducing agents used, such as neurotransmitters,8 nicotinamide adenine dinucleotide (NADH),9 or H2O2 that are produced by © 2015 American Chemical Society
Received: July 1, 2015 Accepted: September 17, 2015 Published: September 17, 2015 10362
DOI: 10.1021/acs.analchem.5b02452 Anal. Chem. 2015, 87, 10362−10367
Article
Analytical Chemistry
Figure 1. SEM images of (A) PVP-Au seeds, (B) catalytic growth of AuNPs mediated by 50 μM H2O2, and (C) catalytic growth of AuNPs mediated by 100 μM H2O2.
Preparation of the BSA-AuNCs. The fluorescent BSAAuNCs synthesized through BSA-mediated reduction of HAuCl4 was based on a modification of a previously reported method.18 Briefly, 10 mM HAuCl4 solution (5 mL) was mixed with 50 mg/mL BSA solution (5 mL) under vigorous stirring for 2 min at ambient temperature. Then, 1.0 M NaOH solution (0.5 mL) was introduced, and the reaction was allowed to proceed under vigorous stirring overnight at 37 °C. The asprepared BSA-AuNCs were stored at 4 °C for further use. The concentration of BSA-AuNCs was estimated as ∼0.02 M, and detailed calculation was shown in Supporting Information. IFE-Based Fluorescent Detection of H2O2 and Cholesterol. The stock solution of H2O2 was freshly diluted from a 30% solution by 0.1 M phosphate buffer (pH 8.0). The cholesterol solution was prepared in 0.1 M phosphate buffer (pH 8.0) containing 1.0% Triton X-100. The Au seeds were prepared by mixing 1.5 mM PVP in phosphate buffer (100 μL, pH 8.0) with 10 mM HAuCl4 solution (50 μL) for 3 min. Concurrently, 1.0 mL of various concentrations of H2O2 or cholesterol solutions (containing 5.0 units of cholesterol oxidase) were added to the PVP-Au seeds solution and then allowed to react for 1 h. The Au seeds and the enlarged AuNPs were characterized by a JEOL JSM-7000F thermal-type field emission scanning electron microscope (Akishima, Tokyo, Japan). The absorption spectra of the H2O2-mediated enlargement of the AuNP solution were recorded by a Cary 300 bio UV−visible spectrophotometer (Varian, Mulgrave, Victoria, Australia). Subsequently, the fluorescent BSA-AuNCs (10 μL) were mixed homogeneously with the resulting AuNP solution (1.0 mL). The fluorescence spectra of BSA-AuNCs were obtained by a Varian Cary Eclipse fluorescence spectrophotometer (Varian) in the wavelength range of 550−850 nm with an excitation at 540 nm and maximum emission at 657 nm. The slit widths of the excitation and emission were both 5.0 nm.
overlap with the excitation and/or emission bands of the fluorophore. Water-soluble gold nanocluster (AuNC)-based fluorescent probes have garnered great interest in the past decade due to their exceptional optical properties. The fluorescent AuNCs, especially protein-protected AuNCs that possess excellent biocompatibility and photostability,18,19 have been employed extensively in bioimaging,19,20 sensing of metal ions,21 and fabrication of biosensors.22,23 In the present study, we attempt to demonstrate a new IFEbased approach using poly(vinylpyrrolidone)-protected AuNPs as an IFE absorber and bovine serum albumin-protected AuNCs (BSA-AuNCs) as an IFE fluorophore for sensing H2O2 and cholesterol, respectively. The catalytic growth of PVPAuNCs by H2O2 influences the emission of BSA-AuNCs in a delicate yet significant way, which may be due to the overlap between the SPR band of the AuNPs and the excitation band of AuNCs. The current fluorescent assay allows the quantification of H2O2 and cholesterol to levels as low as 0.8 and 1.4 μM, respectively. Moreover, to the best of our knowledge, this is the first application of fluorescent AuNCs for fabricating an IFEbased fluorescent assay.
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EXPERIMENTAL SECTION Reagents and Materials. All chemicals were of reagent grade or better. Bovine serum albumin (BSA), cholesterol, cholesterol oxidase, hydrogen peroxide, poly(vinylpyrrolidone), sodium hydroxide, and sodium phosphate dibasic were ordered from Sigma-Aldrich (St. Louis, MO, USA). Potassium phosphate monobasic was purchased from J. T. Baker (Phillipsburg, NJ, USA). Hydrogen tetrachloroaurate was acquired from Alfa Aesar (Ward Hill, MA, USA). Triton X100 was obtained from Amersham Bioscience (Uppsala, Sweden). 10363
DOI: 10.1021/acs.analchem.5b02452 Anal. Chem. 2015, 87, 10362−10367
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Figure 2. (A) Absorbance spectra of AuNP seeds after growth in the presence of different concentrations of H2O2 from 0 to 1000 μM. (B) Calibration curve that reveals the reducing effect of H2O2 on the absorbance of PVP-AuNPs at 540 nm. The inset shows the linear range of 1 to 100 μM.
Figure 3. (A) UV−vis absorbance (black line) of PVP-AuNPs enlarged by 1 mM H2O2, fluorescent emission spectrum (gray line) of BSA-AuNCs with a maximum at 657 nm, and fluorescent excitation spectrum (dashed line) of BSA-AuNCs with a maximum at 522 nm. (B) Fluorescence intensity of 10 μL BSA-AuNCs in 1.0 mL of 0.1 M phosphate buffer (pH 8) at 657 nm (black line) was quenched significantly about 43% upon addition of 10 μM H2O2-mediated produced PVP-AuNPs under an excitation wavelength of 540 nm (dashed line). The gray line illustrates the recovered fluorescence of the supernatant containing BSA-AuNCs that was obtained from the same solution after being centrifuged at 16000 rpm for 10 min to remove PVP-AuNPs. (C) The UV−vis absorbance spectra for a mixture of BSA-AuNCs solution and PVP-AuNPs solution synthesized by 0.1 mM H2O2 (black line), PVP-AuNPs solution synthesized by 0.1 mM H2O2 (gray line), and BSA-AuNCs solution (dashed line).
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DOI: 10.1021/acs.analchem.5b02452 Anal. Chem. 2015, 87, 10362−10367
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showed the recovered fluorescence. Moreover, in the absorption spectra (Figure 3C), the SPR band of AuNPs did not shift in the presence of BSA-AuNCs, suggesting that no interaction between AuNPs and BSA-AuNCs occurred. In addition, it is also confirmed that the reduced fluorescence of BSA-AuNCs was not ascribed to the AuNPs aggregationinduced fluorescence quenching. The IFE of AuNPs on the fluorescent BSA-AuNCs is understood, therefore, to be significant. Analytical Performance of the IFE Assay for the Determination of H2O2. The fluorescence intensity of BSAAuNCs was decreased exponentially with the addition of AuNPs enlarged by various concentrations of H2O2 (Figure 4A). After the growth of AuNPs by H2O2, the absorbance of
RESULTS AND DISCUSSION H2O2-Mediated Enlargement of AuNPs. The dimension of the AuNPs can be easily enlarged in the presence of PVP-Au seeds and H2O2. PVP can not only be used readily as a mild reductant for the formation of Au seeds, but it served also as a capping agent for both Au seeds and the larger dimension AuNPs.24 PVP-Au seeds could be prepared simply by mixing PVP and Au3+. SEM images (Figure 1A) revealed that the average diameters of PVP-Au seeds were 17.3 ± 3.6 nm. Previous studies have demonstrated that Au seeds were able to function as catalysts for the reduction of Au3+ by H2O2, resulting in the enlargement of the AuNPs, and enhanced absorbance features.10 The PVP-Au seeds can be enlarged to AuNPs in the presence of 50 μM of H2O2 (Figure 1B). The diameters of enlarged AuNPs appear in a wide range of sizes, 24.0−95.8 nm, and with an average of 52.9 ± 18 nm. Interestingly, the higher concentration (100 μM) of H2O2 in the tested solution resulted in the production of AuNPs of smaller average diameter (35.8 ± 22.4 nm), but a broader range in size (16.6−101.8 nm) (see Figure 1C). From the behavior of catalytic growth of AuNPs, we suggest that the smaller AuNPs can be generated on the surface of enlarged AuNPs in the presence of H2O2, and they can be detached subsequently from the surface. The formation mechanism of AuNPs via H2O2 was investigated previously by Willner’s group.10 The absorption spectra imply the formation of heterogeneous sizes of AuNPs with the existence of H2O2. Figure 2A shows the spectra of the AuNPs formed upon addition of various concentrations of H2O2 to PVP-Au seeds after 1 h of incubation. As the concentration of H2O2 increased, the characteristic SPR absorbance of the enlarged AuNPs was intensified and the absorbance maxima were slightly blueshifted. The results of absorption spectra were consistent with the SEM images, which showed the formation of smaller AuNPs at higher concentrations of H2O2. Inner Filter Effect of AuNPs on the Fluorescent AuNCs. We have also developed a fluorescent assay for sensing H2 O 2 based on H2 O2 -induced change in the absorbance of the absorber (AuNPs), which then influences the fluorescence intensity of our fluorophore, BSA-AuNCs, due to the IFE effect. In our study, the fluorescent BSA-AuNCs synthesized through BSA-mediated reduction of HAuCl4 were based on a previously reported method.18 Strong emission of the BSA-AuNCs was observed at 657 nm with an excitation maximum at 522 nm (Figure 3A). Fluorescent BSA-AuNCs were chosen as IFE fluorophores in the study due to the following advantages: (i) a good overlap between excitation spectrum of fluorescent AuNCs and the plasmonic absorption band of AuNPs (as shown in Figure 3A); (ii) a large Stoke shift (135 nm) between excitation and emission spectra that reduced the reabsorption of emitted light; (iii) greater photostability and facile preparation of BSA-AuNCs than organic dyes;19 (iv) environmental friendliness. To demonstrate further the generality of our design using H2O2-mediated growth of AuNPs as an IFE-absorber and BSA-AuNCs as an IFEfluorophore, we then recorded the fluorescence spectra of BSAAuNCs mixed with AuNPs. The fluorescence intensity of BSAAuNCs at 657 nm under an excitation wavelength of 540 nm was quenched significantly, about 43% upon an addition of formed AuNPs mediated by 10 μM H2O2 (Figure 3B). When the mixed solution was centrifuged at 16000 rpm for 10 min to remove AuNPs, the supernatant containing BSA-AuNCs
Figure 4. (A) Fluorescent spectra of BSA-AuNCs solutions synthesized from PVP-AuNPs solutions with addition of various concentrations of H2O2 at excitation wavelength of 540 nm (concentrations from a to h: 0 M, 1 μM, 5 μM, 10 μM, 50 μM, 0.1 mM, 0.5 mM, and 1 mM). (B) Calibration curve of points depicted from panel A at 657 nm and the inset illustrating the linear range for detection of H2O2 from 1 to 100 μM.
the absorber (AuNPs) increased, which led to less light available to excite the fluorophore (BSA-AuNCs). Thus, the fluorescence of BSA-AuNCs was decreased significantly. This IFE-based assay allowed the detection of H2O2 in a range of 1− 100 μM. (Figure 4B). The linear fitting could be expressed as I/ I0 = −0.1386 log[H2O2] + 0.9684 (R2 = 0.982) with a detection limit as low as 0.8 μM (defined as 3 × standard deviation of the blank), which is much lower than the level (∼35 μM) of H2O2 in human blood plasma.2 The coefficients of variation (CV) among all of the measurements were 0.95−1.73%. It is noteworthy that our IFE assay provided a lower detection limit than the previously reported methods for H 2 O 2 detection.25−32 Analytical Performance of the IFE Assay for the Determination of Cholesterol. Numerous oxidases generate H2O2 after the oxidation of their corresponding substrates by molecular O2. Our IFE-based assay was further extended to the determination of cholesterol using cholesterol oxidase (ChOx) and O2/cholesterol as the H2O2-generating system. The spectral changes of the PVP-Au seeds solution were ascribed to the presence of H2O2 that were produced from various concentrations of cholesterol with ChOx (Figure 5A). The characteristic SPR peaks of enlarged AuNPs mediated by H2O2 were intensified as the concentration of cholesterol increased (Figure 5B). Figure 5C shows that the fluorescence intensity decreased after mixing BSA-AuNCs with the AuNPs, which were enlarged by H2O2 generated from various concentrations of cholesterol in the presence of ChOx. Our assay allows the detection of cholesterol in a range of 1−100 μM, the linear fitting can be expressed as I/I0 = −0.15721 log[cholesterol] + 10365
DOI: 10.1021/acs.analchem.5b02452 Anal. Chem. 2015, 87, 10362−10367
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Analytical Chemistry
Figure 5. (A) Absorbance spectra of AuNP seeds upon growth in the presence of different concentrations of cholesterol from 0 to 100 μM. (B) Dose−response curve revealing the reducing effect of cholesterol on the absorbance of PVP-AuNPs at 540 nm, ranging from 1 to 100 μM. (C) Fluorescent spectra of PVP-AuNPs solutions synthesized from BSA-AuNCs solutions with addition of various concentrations of cholesterol in the presence of catalyst ChOx at excitation wavelength of 540 nm (concentrations from a to f: 0 M, 1 μM, 5 μM, 10 μM, 50 μM, and 0.1 mM). (D) Calibration curve of points depicted from panel C at 657 nm indicating the linear range for detection of cholesterol from 1 to 100 μM.
saves time, because modification of the fluorophore is no longer required. Furthermore, it is our ultimate goal to utilize this sensing platform for other biologically important molecules with different H2O2-producing oxidases. The detection of glucose, lactate, choline, and uric acid seems to be feasible using our approach.
0.98178 (R2 = 0.991), and LOD is determined to be as low as 1.4 μM (defined as 3 × standard deviation of the blank), which is lower than the total cholesterol level found in human blood (200 mg/dL that is equivalent to 5 mM) (Figure 5D). The performance of our assay is comparable to or relatively better than methods reported previously for the detection of cholesterol.33−38 The coefficients of variations (CV%) among all of the measurements were 0.41−3.97%, which suggested that our IFE-based assay is reproducible and possesses potential for further use in the detection of cholesterol in a clinical setting. In comparison with the previously reported Au NPs−βCDs−FL system by Tang et al.,39 our IFE method is a simpler process for sensing cholesterol without the need for further modification to AuNPs and fluorescent BSA-AuNCs.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02452. Calculation for BSA-AuNC concentration (PDF)
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CONCLUSIONS We have demonstrated the feasibility of a new IFE-based fluorescent assay using AuNPs as an IFE absorber and BSAAuNCs as an IFE fluorophore. H2O2-mediated enlargement of AuNPs influenced the fluorescent intensity of the BSA-AuNCs, which can be used to detect H2O2 and cholesterol sensitively. To the best of our knowledge, this is the first work using fluorescent BSA-AuNCs in the fabrication of an IFE-based assay. The present IFE-based strategy allows the design of fluorescent assays in a cost-effective way that is simpler and
AUTHOR INFORMATION
Corresponding Author
*Tel.: 886-2-33664438. Fax: 886-2-33662271. E-mail: jaho@ ntu.edu.tw. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the Taiwan Ministry of Science and Technology under Grants Nos. 101-2113-M-002-003-MY3 10366
DOI: 10.1021/acs.analchem.5b02452 Anal. Chem. 2015, 87, 10362−10367
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N.; Illingworth, D. R.; Sadin, H. C.; Schonfeld, G.; Cleeman, J. I.; Brewer, H. B., Jr; Ernst, N.; Friedewald, W.; Hoeg, J. M.; Rifkind, B.; Gordon, D. Arch. Intern. Med. 1988, 148 (1), 36−69. (33) Pineiro-Avila, G. Analyst 1998, 123, 999−1003. (34) Pineiro-Avila, G.; Salvador, A.; de la Guardia, M. Analyst 1998, 123, 2291−2295. (35) Gilmartin, M. A. T.; Hart, J. P. Analyst 1994, 119, 2331−2336. (36) Kaminikado, K.; Ikeda, R.; Idegami, K.; Nagatani, N.; Vestergaard, M. d. C.; Saito, M.; Tamiya, E. Analyst 2011, 136, 1826−1830. (37) Arya, S. K.; Pandey, P.; Singh, S. P.; Datta, M.; Malhotra, B. D. Analyst 2007, 132, 1005−1009. (38) Wu, C.; Ifa, D. R.; Manicke, N. E.; Cooks, R. G. Anal. Chem. 2009, 81, 7618−7624. (39) Zhang, N.; Liu, Y.; Tong, L.; Xu, K.; Zhuo, L.; Tang, B. Analyst 2008, 133, 1176−1181.
and 102-2628-M-002-004-MY4 and the Taiwan Ministry of Education “Aim for the Top University Plan”. We thank Ms. Amily Fang-Ju Jou for valuable discussion and her help in formulating the paper and preparing the TOC.
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REFERENCES
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DOI: 10.1021/acs.analchem.5b02452 Anal. Chem. 2015, 87, 10362−10367
Gold Nanocluster-Assisted Fluorescent Detection for Hydrogen Peroxide and Cholesterol Based on the Inner Filter Effect of Gold Nanoparticles Heng-Chia Chang† and Ja-an Annie Ho*,†,‡ †
Department of Chemistry, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan BioAnalytical Chemistry and Nanobiomedicine Laboratory, Department of Biochemical Science and Technology, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan
‡
S Supporting Information *
ABSTRACT: We developed a simple, sensitive inner filter effect (IFE)-based fluorescent assay for sensing H2O2 and cholesterol. In the process, poly(vinylpyrrolidone)-protected gold nanoparticles (PVP-AuNPs) and fluorescent BSA-protected gold nanoclusters (BSA-AuNCs) were used as an IFE absorber/ fluorophore pair. PVP-AuNPs can be a powerful absorber to influence the emission of the fluorophore, BSA-AuNCs, in the IFE-based fluorescent assays. That is due to the high extinction coefficient of AuNPs and the complementary overlap between the surface plasmon resonance (SPR) absorption of PVP-AuNPs and the excitation of BSA-AuNCs. The PVPAu seeds, produced by directly mixing PVP with HAuCl4, were able to catalyze H2O2 to enlarge AuNPs. The SPR absorption of PVP-AuNPs was enhanced with an increased concentration of H2O2 and, subsequently, induced significant fluorescence quenching of BSA-AuNCs. The IFE-based fluorescent assay enabled the detection of H2O2 and generation of H2O2 in the presence of O2/cholesterol and cholesterol oxidase (ChOx) by the fluorescence response of BSA-AuNCs. The present IFE-based approach can detect H2O2 ranging from 1 to 100 μM with a detection limit of 0.8 μM and cholesterol ranging from 1 to 100 μM with a detection limit of 1.4 μM.
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glucose/glucose oxidase10 or cholesterol/cholesterol oxidase.11 Moreover, AuNPs have been reported to be involved in the design of the fluorescent assays. For instance, AuNPs can be used as an efficient fluorescence quencher through energy or electron transfer processes for the determination of target analytes.12−14 More recently, several studies have described the detection of environmentally or biologically important analytes, such as CN−, H2O2, aminothiols, and immunoglobulin,15−17 via the inner filter effect of gold nanoparticles on the fluorescence of fluorophores or metal nanoparticles. This is ascribed to the unique characteristics of the AuNPs that can absorb a significant percentage of the excitation and/or emission light of the fluorophore. In most AuNPs-based fluorescence quenching assays, the quenching effects are associated with the distance between the quencher (AuNPs) and the fluorophore. Besides, a linker element is not usually necessary for the IFE-based fluorescence assays, which provide a comparatively simple and facile approach for the analytical detection of target analytes. However, some restrictions still exist in the design and development of IFE-based fluorescent assays due to the limited choices of suitable absorber and fluorophore, because IFE only occurs effectively if the absorption band of the absorber possesses the complementary
he detection of hydrogen peroxide (H2O2) is of practical importance in chemical, biological, clinical, environmental, and other fields. For instance, H2O2 can be harmful to biological systems and appears to be involved in the neuropathology of central nervous system diseases.1 The H2O2 level in urine is also considered as a potential biomarker for oxidation stress in patients with a malignancy.2 Additionally, H2O2 is an end-product in oxidation reactions, catalyzed by glucose oxidase, uricase, and cholesterol oxidase, etc.3−5 Cholesterol and its fatty acid esters are important compounds of nerve and brain cells; they are precursors of other biological materials, such as bile acid and steroid hormones. An excessive concentration of cholesterol in blood is often associated with the occurrence of atherosclerosis;6 however, a low concentration of cholesterol results in hypocholesterolemia, which may cause hemorrhagic stroke.7 Without cholesterol, brain cells cannot send messages that power every organ in our body. Therefore, being able to estimate cholesterol level is of interest to the biological science and food industries. Recent studies have revealed that a larger dimension of gold nanoparticles (AuNPs) can be formed with the presence of Au seeds, cetyltrimethylammonium chloride (CTAC) as a surfactant, and a reducing agent (e.g., ascorbic acid, phenolic acid) as a reductant. The spectroscopic features of AuNPs with larger dimensions depend on the concentrations of the reducing agents used, such as neurotransmitters,8 nicotinamide adenine dinucleotide (NADH),9 or H2O2 that are produced by © 2015 American Chemical Society
Received: July 1, 2015 Accepted: September 17, 2015 Published: September 17, 2015 10362
DOI: 10.1021/acs.analchem.5b02452 Anal. Chem. 2015, 87, 10362−10367
Article
Analytical Chemistry
Figure 1. SEM images of (A) PVP-Au seeds, (B) catalytic growth of AuNPs mediated by 50 μM H2O2, and (C) catalytic growth of AuNPs mediated by 100 μM H2O2.
Preparation of the BSA-AuNCs. The fluorescent BSAAuNCs synthesized through BSA-mediated reduction of HAuCl4 was based on a modification of a previously reported method.18 Briefly, 10 mM HAuCl4 solution (5 mL) was mixed with 50 mg/mL BSA solution (5 mL) under vigorous stirring for 2 min at ambient temperature. Then, 1.0 M NaOH solution (0.5 mL) was introduced, and the reaction was allowed to proceed under vigorous stirring overnight at 37 °C. The asprepared BSA-AuNCs were stored at 4 °C for further use. The concentration of BSA-AuNCs was estimated as ∼0.02 M, and detailed calculation was shown in Supporting Information. IFE-Based Fluorescent Detection of H2O2 and Cholesterol. The stock solution of H2O2 was freshly diluted from a 30% solution by 0.1 M phosphate buffer (pH 8.0). The cholesterol solution was prepared in 0.1 M phosphate buffer (pH 8.0) containing 1.0% Triton X-100. The Au seeds were prepared by mixing 1.5 mM PVP in phosphate buffer (100 μL, pH 8.0) with 10 mM HAuCl4 solution (50 μL) for 3 min. Concurrently, 1.0 mL of various concentrations of H2O2 or cholesterol solutions (containing 5.0 units of cholesterol oxidase) were added to the PVP-Au seeds solution and then allowed to react for 1 h. The Au seeds and the enlarged AuNPs were characterized by a JEOL JSM-7000F thermal-type field emission scanning electron microscope (Akishima, Tokyo, Japan). The absorption spectra of the H2O2-mediated enlargement of the AuNP solution were recorded by a Cary 300 bio UV−visible spectrophotometer (Varian, Mulgrave, Victoria, Australia). Subsequently, the fluorescent BSA-AuNCs (10 μL) were mixed homogeneously with the resulting AuNP solution (1.0 mL). The fluorescence spectra of BSA-AuNCs were obtained by a Varian Cary Eclipse fluorescence spectrophotometer (Varian) in the wavelength range of 550−850 nm with an excitation at 540 nm and maximum emission at 657 nm. The slit widths of the excitation and emission were both 5.0 nm.
overlap with the excitation and/or emission bands of the fluorophore. Water-soluble gold nanocluster (AuNC)-based fluorescent probes have garnered great interest in the past decade due to their exceptional optical properties. The fluorescent AuNCs, especially protein-protected AuNCs that possess excellent biocompatibility and photostability,18,19 have been employed extensively in bioimaging,19,20 sensing of metal ions,21 and fabrication of biosensors.22,23 In the present study, we attempt to demonstrate a new IFEbased approach using poly(vinylpyrrolidone)-protected AuNPs as an IFE absorber and bovine serum albumin-protected AuNCs (BSA-AuNCs) as an IFE fluorophore for sensing H2O2 and cholesterol, respectively. The catalytic growth of PVPAuNCs by H2O2 influences the emission of BSA-AuNCs in a delicate yet significant way, which may be due to the overlap between the SPR band of the AuNPs and the excitation band of AuNCs. The current fluorescent assay allows the quantification of H2O2 and cholesterol to levels as low as 0.8 and 1.4 μM, respectively. Moreover, to the best of our knowledge, this is the first application of fluorescent AuNCs for fabricating an IFEbased fluorescent assay.
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EXPERIMENTAL SECTION Reagents and Materials. All chemicals were of reagent grade or better. Bovine serum albumin (BSA), cholesterol, cholesterol oxidase, hydrogen peroxide, poly(vinylpyrrolidone), sodium hydroxide, and sodium phosphate dibasic were ordered from Sigma-Aldrich (St. Louis, MO, USA). Potassium phosphate monobasic was purchased from J. T. Baker (Phillipsburg, NJ, USA). Hydrogen tetrachloroaurate was acquired from Alfa Aesar (Ward Hill, MA, USA). Triton X100 was obtained from Amersham Bioscience (Uppsala, Sweden). 10363
DOI: 10.1021/acs.analchem.5b02452 Anal. Chem. 2015, 87, 10362−10367
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Figure 2. (A) Absorbance spectra of AuNP seeds after growth in the presence of different concentrations of H2O2 from 0 to 1000 μM. (B) Calibration curve that reveals the reducing effect of H2O2 on the absorbance of PVP-AuNPs at 540 nm. The inset shows the linear range of 1 to 100 μM.
Figure 3. (A) UV−vis absorbance (black line) of PVP-AuNPs enlarged by 1 mM H2O2, fluorescent emission spectrum (gray line) of BSA-AuNCs with a maximum at 657 nm, and fluorescent excitation spectrum (dashed line) of BSA-AuNCs with a maximum at 522 nm. (B) Fluorescence intensity of 10 μL BSA-AuNCs in 1.0 mL of 0.1 M phosphate buffer (pH 8) at 657 nm (black line) was quenched significantly about 43% upon addition of 10 μM H2O2-mediated produced PVP-AuNPs under an excitation wavelength of 540 nm (dashed line). The gray line illustrates the recovered fluorescence of the supernatant containing BSA-AuNCs that was obtained from the same solution after being centrifuged at 16000 rpm for 10 min to remove PVP-AuNPs. (C) The UV−vis absorbance spectra for a mixture of BSA-AuNCs solution and PVP-AuNPs solution synthesized by 0.1 mM H2O2 (black line), PVP-AuNPs solution synthesized by 0.1 mM H2O2 (gray line), and BSA-AuNCs solution (dashed line).
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DOI: 10.1021/acs.analchem.5b02452 Anal. Chem. 2015, 87, 10362−10367
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showed the recovered fluorescence. Moreover, in the absorption spectra (Figure 3C), the SPR band of AuNPs did not shift in the presence of BSA-AuNCs, suggesting that no interaction between AuNPs and BSA-AuNCs occurred. In addition, it is also confirmed that the reduced fluorescence of BSA-AuNCs was not ascribed to the AuNPs aggregationinduced fluorescence quenching. The IFE of AuNPs on the fluorescent BSA-AuNCs is understood, therefore, to be significant. Analytical Performance of the IFE Assay for the Determination of H2O2. The fluorescence intensity of BSAAuNCs was decreased exponentially with the addition of AuNPs enlarged by various concentrations of H2O2 (Figure 4A). After the growth of AuNPs by H2O2, the absorbance of
RESULTS AND DISCUSSION H2O2-Mediated Enlargement of AuNPs. The dimension of the AuNPs can be easily enlarged in the presence of PVP-Au seeds and H2O2. PVP can not only be used readily as a mild reductant for the formation of Au seeds, but it served also as a capping agent for both Au seeds and the larger dimension AuNPs.24 PVP-Au seeds could be prepared simply by mixing PVP and Au3+. SEM images (Figure 1A) revealed that the average diameters of PVP-Au seeds were 17.3 ± 3.6 nm. Previous studies have demonstrated that Au seeds were able to function as catalysts for the reduction of Au3+ by H2O2, resulting in the enlargement of the AuNPs, and enhanced absorbance features.10 The PVP-Au seeds can be enlarged to AuNPs in the presence of 50 μM of H2O2 (Figure 1B). The diameters of enlarged AuNPs appear in a wide range of sizes, 24.0−95.8 nm, and with an average of 52.9 ± 18 nm. Interestingly, the higher concentration (100 μM) of H2O2 in the tested solution resulted in the production of AuNPs of smaller average diameter (35.8 ± 22.4 nm), but a broader range in size (16.6−101.8 nm) (see Figure 1C). From the behavior of catalytic growth of AuNPs, we suggest that the smaller AuNPs can be generated on the surface of enlarged AuNPs in the presence of H2O2, and they can be detached subsequently from the surface. The formation mechanism of AuNPs via H2O2 was investigated previously by Willner’s group.10 The absorption spectra imply the formation of heterogeneous sizes of AuNPs with the existence of H2O2. Figure 2A shows the spectra of the AuNPs formed upon addition of various concentrations of H2O2 to PVP-Au seeds after 1 h of incubation. As the concentration of H2O2 increased, the characteristic SPR absorbance of the enlarged AuNPs was intensified and the absorbance maxima were slightly blueshifted. The results of absorption spectra were consistent with the SEM images, which showed the formation of smaller AuNPs at higher concentrations of H2O2. Inner Filter Effect of AuNPs on the Fluorescent AuNCs. We have also developed a fluorescent assay for sensing H2 O 2 based on H2 O2 -induced change in the absorbance of the absorber (AuNPs), which then influences the fluorescence intensity of our fluorophore, BSA-AuNCs, due to the IFE effect. In our study, the fluorescent BSA-AuNCs synthesized through BSA-mediated reduction of HAuCl4 were based on a previously reported method.18 Strong emission of the BSA-AuNCs was observed at 657 nm with an excitation maximum at 522 nm (Figure 3A). Fluorescent BSA-AuNCs were chosen as IFE fluorophores in the study due to the following advantages: (i) a good overlap between excitation spectrum of fluorescent AuNCs and the plasmonic absorption band of AuNPs (as shown in Figure 3A); (ii) a large Stoke shift (135 nm) between excitation and emission spectra that reduced the reabsorption of emitted light; (iii) greater photostability and facile preparation of BSA-AuNCs than organic dyes;19 (iv) environmental friendliness. To demonstrate further the generality of our design using H2O2-mediated growth of AuNPs as an IFE-absorber and BSA-AuNCs as an IFEfluorophore, we then recorded the fluorescence spectra of BSAAuNCs mixed with AuNPs. The fluorescence intensity of BSAAuNCs at 657 nm under an excitation wavelength of 540 nm was quenched significantly, about 43% upon an addition of formed AuNPs mediated by 10 μM H2O2 (Figure 3B). When the mixed solution was centrifuged at 16000 rpm for 10 min to remove AuNPs, the supernatant containing BSA-AuNCs
Figure 4. (A) Fluorescent spectra of BSA-AuNCs solutions synthesized from PVP-AuNPs solutions with addition of various concentrations of H2O2 at excitation wavelength of 540 nm (concentrations from a to h: 0 M, 1 μM, 5 μM, 10 μM, 50 μM, 0.1 mM, 0.5 mM, and 1 mM). (B) Calibration curve of points depicted from panel A at 657 nm and the inset illustrating the linear range for detection of H2O2 from 1 to 100 μM.
the absorber (AuNPs) increased, which led to less light available to excite the fluorophore (BSA-AuNCs). Thus, the fluorescence of BSA-AuNCs was decreased significantly. This IFE-based assay allowed the detection of H2O2 in a range of 1− 100 μM. (Figure 4B). The linear fitting could be expressed as I/ I0 = −0.1386 log[H2O2] + 0.9684 (R2 = 0.982) with a detection limit as low as 0.8 μM (defined as 3 × standard deviation of the blank), which is much lower than the level (∼35 μM) of H2O2 in human blood plasma.2 The coefficients of variation (CV) among all of the measurements were 0.95−1.73%. It is noteworthy that our IFE assay provided a lower detection limit than the previously reported methods for H 2 O 2 detection.25−32 Analytical Performance of the IFE Assay for the Determination of Cholesterol. Numerous oxidases generate H2O2 after the oxidation of their corresponding substrates by molecular O2. Our IFE-based assay was further extended to the determination of cholesterol using cholesterol oxidase (ChOx) and O2/cholesterol as the H2O2-generating system. The spectral changes of the PVP-Au seeds solution were ascribed to the presence of H2O2 that were produced from various concentrations of cholesterol with ChOx (Figure 5A). The characteristic SPR peaks of enlarged AuNPs mediated by H2O2 were intensified as the concentration of cholesterol increased (Figure 5B). Figure 5C shows that the fluorescence intensity decreased after mixing BSA-AuNCs with the AuNPs, which were enlarged by H2O2 generated from various concentrations of cholesterol in the presence of ChOx. Our assay allows the detection of cholesterol in a range of 1−100 μM, the linear fitting can be expressed as I/I0 = −0.15721 log[cholesterol] + 10365
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Figure 5. (A) Absorbance spectra of AuNP seeds upon growth in the presence of different concentrations of cholesterol from 0 to 100 μM. (B) Dose−response curve revealing the reducing effect of cholesterol on the absorbance of PVP-AuNPs at 540 nm, ranging from 1 to 100 μM. (C) Fluorescent spectra of PVP-AuNPs solutions synthesized from BSA-AuNCs solutions with addition of various concentrations of cholesterol in the presence of catalyst ChOx at excitation wavelength of 540 nm (concentrations from a to f: 0 M, 1 μM, 5 μM, 10 μM, 50 μM, and 0.1 mM). (D) Calibration curve of points depicted from panel C at 657 nm indicating the linear range for detection of cholesterol from 1 to 100 μM.
saves time, because modification of the fluorophore is no longer required. Furthermore, it is our ultimate goal to utilize this sensing platform for other biologically important molecules with different H2O2-producing oxidases. The detection of glucose, lactate, choline, and uric acid seems to be feasible using our approach.
0.98178 (R2 = 0.991), and LOD is determined to be as low as 1.4 μM (defined as 3 × standard deviation of the blank), which is lower than the total cholesterol level found in human blood (200 mg/dL that is equivalent to 5 mM) (Figure 5D). The performance of our assay is comparable to or relatively better than methods reported previously for the detection of cholesterol.33−38 The coefficients of variations (CV%) among all of the measurements were 0.41−3.97%, which suggested that our IFE-based assay is reproducible and possesses potential for further use in the detection of cholesterol in a clinical setting. In comparison with the previously reported Au NPs−βCDs−FL system by Tang et al.,39 our IFE method is a simpler process for sensing cholesterol without the need for further modification to AuNPs and fluorescent BSA-AuNCs.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02452. Calculation for BSA-AuNC concentration (PDF)
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CONCLUSIONS We have demonstrated the feasibility of a new IFE-based fluorescent assay using AuNPs as an IFE absorber and BSAAuNCs as an IFE fluorophore. H2O2-mediated enlargement of AuNPs influenced the fluorescent intensity of the BSA-AuNCs, which can be used to detect H2O2 and cholesterol sensitively. To the best of our knowledge, this is the first work using fluorescent BSA-AuNCs in the fabrication of an IFE-based assay. The present IFE-based strategy allows the design of fluorescent assays in a cost-effective way that is simpler and
AUTHOR INFORMATION
Corresponding Author
*Tel.: 886-2-33664438. Fax: 886-2-33662271. E-mail: jaho@ ntu.edu.tw. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the Taiwan Ministry of Science and Technology under Grants Nos. 101-2113-M-002-003-MY3 10366
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N.; Illingworth, D. R.; Sadin, H. C.; Schonfeld, G.; Cleeman, J. I.; Brewer, H. B., Jr; Ernst, N.; Friedewald, W.; Hoeg, J. M.; Rifkind, B.; Gordon, D. Arch. Intern. Med. 1988, 148 (1), 36−69. (33) Pineiro-Avila, G. Analyst 1998, 123, 999−1003. (34) Pineiro-Avila, G.; Salvador, A.; de la Guardia, M. Analyst 1998, 123, 2291−2295. (35) Gilmartin, M. A. T.; Hart, J. P. Analyst 1994, 119, 2331−2336. (36) Kaminikado, K.; Ikeda, R.; Idegami, K.; Nagatani, N.; Vestergaard, M. d. C.; Saito, M.; Tamiya, E. Analyst 2011, 136, 1826−1830. (37) Arya, S. K.; Pandey, P.; Singh, S. P.; Datta, M.; Malhotra, B. D. Analyst 2007, 132, 1005−1009. (38) Wu, C.; Ifa, D. R.; Manicke, N. E.; Cooks, R. G. Anal. Chem. 2009, 81, 7618−7624. (39) Zhang, N.; Liu, Y.; Tong, L.; Xu, K.; Zhuo, L.; Tang, B. Analyst 2008, 133, 1176−1181.
and 102-2628-M-002-004-MY4 and the Taiwan Ministry of Education “Aim for the Top University Plan”. We thank Ms. Amily Fang-Ju Jou for valuable discussion and her help in formulating the paper and preparing the TOC.
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