Biosensors and Bioelectronics 64 (2015) 345–351
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Selectively assaying CEA based on a creative strategy of gold nanoparticles enhancing silver nanoclusters' fluorescence Xiaoming Yang n,1, Yan Zhuo 1, Shanshan Zhu, Yawen Luo, Yuanjiao Feng, Yan Xu College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China
art ic l e i nf o
a b s t r a c t
Article history: Received 26 June 2014 Received in revised form 2 September 2014 Accepted 4 September 2014 Available online 18 September 2014
Herein, we have successfully built up connections between nanoparticles and nanoclusters, and further constructed a surface-enhanced fluorescence (SEF) strategy based on the two types of nanomaterials for selectively assaying carcinoembryonic antigen (CEA). Specifically, silver nanoclusters provided the original fluorescence signal, while gold nanoparticles modified with DNA served as the fluorescence enhancer simultaneously. On the basis of this proposed nano-system, the two nanomaterials were linked by CEA–aptamer, thus facilitating SEF occurring. Nevertheless, more competitive interactions between CEA and CEA–aptamer emerged once CEA added, leading to SEF failed and their fluorescence decreased. Significantly, this creative method was further applied to detect CEA, and showed the linear relationship between the fluorescence intensity and CEA concentrations in the range of 0.01–1 ng mL 1 with a detection limit of 3 pg mL 1 at a signal-to-noise ratio of 3, demonstrating its sensitivity and promising towards multiple applications. On the whole, this approach we established may broaden potential ways of combining nanoparticles and nanoclusters for detecting trace targets in bioanalytical fields. & 2014 Elsevier B.V. All rights reserved.
Keywords: Silver nanoclusters Gold nanoparticles Aptamer CEA Surface-enhanced fluorescence
1. Introduction Tumor markers, as molecules appearing in blood or tissue, have been proved to be associated with carcinogenesis, and their related measurement or identification exhibited great importance for patient diagnosis or clinical management (Miyake et al., 2010; Tang et al., 2007). Carcinoembryonic antigen (CEA), a tumorassociated antigen, has been identified as a critical marker for clinical diagnosis of colon tumors, breast tumors, ovarian carcinoma and cystadenocarcinoma (Eppler et al., 2002; Naghibalhossaini and Ebadi, 2006). Basically, amounts of CEA in colon tissue of adults are reported as a low level of 2.5–5.0 μg L 1 (Zamcheck and Martin, 1981). However, the levels of CEA are claimed to be obviously elevated while tumors arise in endodermal tissue including gastrointestinal tract, respiratory tract, pancreas, and breast (Shen et al., 2005). Accordingly, investigating the amounts of CEA plays an important role towards clinical purpose. Currently, immunoassays and related techniques have been considered as major analytical methods towards detections of CEA mainly including radioimmunoassay, enzyme immunoassay, chemiluminescence immunoassay and fluorescence immunoassay (Lang et al., 2014; Li et al., 2010; Yu et al., 2013; Yuan et al., 2001). n
Corresponding author. Fax: þ 86 23 68251225. E-mail address: [email protected] (X. Yang). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.09.029 0956-5663/& 2014 Elsevier B.V. All rights reserved.
Unfortunately, most of these methods exhibited drawbacks of requiring radiation hazards, time-consuming or sophisticated instrumentation (Darain et al., 2003), resulting in alternative approaches still desirable. Aptamer, emerging as single-stranded oligonucleotides with definite structures, can selectively bind with specific targets (Nielsen et al., 2010; Nutiu and Li, 2003). In particular, nucleic acid aptamer to specifically bind cells, proteins, low-molecularweight inorganic or organic substrates, usually contains unmodified DNA and RNA, which is isolated through a selection process in vitro or systematic evolution of ligands by exponential enrichment (SELEX) (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Simultaneously, aptamer are indeed able to bind their complementary DNA sequence, and form a duplex structure (Jhaveri et al., 2000; Lin and Patel, 1997). Compared with other natural receptors such as antibodies and enzymes, aptamer showed various unapproachable advantages mainly including easy operation of synthesis and readily acquired from commercial sources (Wilson and Szostak, 1999), facilitating their satisfactory chemical stability. Additionally, aptamer generally exhibited high specificity and affinity towards their targets, and their structural flexibility allows for adaptation of undergoing significant conformational changes once aptamer and targets specifically bind with each other (Nutiu and Li, 2005; Sefah et al., 2009). Hence, these attractive superiorities result in potentiating them as ideal biosensing molecules (Jayasena, 1999; Tang and Breaker, 1997), and aptamer have broadened ways as sensors in fields of assaying metal ions, small
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molecules, proteins and cancer cell, etc. (Liu et al., 2009; Sun et al., 2011; Zhou et al., 2010; Zhou et al., 2012). Fluorescent nanomaterials, especially gold nanoclusters (AuNCs), silver nanoclusters (AgNCs) and copper nanoclusters (CuNCs), have appeared as powerful and sensitive probes (Gerion et al., 2003; Guo et al., 2010; Huang et al., 2008) and attracted numerous attentions. Among these nanoclusters, oligonucleotidestabilized AgNCs (DNA-AgNCs) are usually synthesized by NaBH4 mediating reductions of Ag ions in the presence of oligonucleotides, and biocompatible and low toxic (Yu et al., 2009). Besides, most of DNA-AgNCs were obtained by cytosine-rich DNA and exhibited notable spectral and photophysical properties due to the binding interactions between silver and cytosine (Ono et al., 2008; Robichaud et al., 2005), and introducing various DNA sequences provided diverse fluorescence colors of DNA-AgNCs (Gwinn et al., 2008; Richards et al., 2008). Again, DNA not only serves as templates, but also plays roles for molecular recognition. All these advantages of DNA-AgNCs suggested their promising value for analytical applications (Li et al., 2012). Combinations of fluorescent nanomaterials and biochemical molecules have exhibited great prospects for biochemical applications, since both the unique structural and photophysical performance and excellent properties have been effectively integrated (Zhang et al., 2012; Zhang et al.,
2011a; Zhang et al., 2011b). Again, employing nanomaterials may improve the sensitivity and accuracy of biosensors. Moreover, nano-sized devices generally response faster due to mass transport occurring over shorter distances. Surface-enhanced fluorescence (SEF) on metal nanostructures (Lakowicz, 2005), usually happens while a fluorophore locating near the surface of metallic nanoparticles (Antunes et al., 2001; Kulakovich et al., 2002; Parfenov et al., 2003). SEF is believed to occur through the coupling of the fluorophores with radiating plasmons from the metallic particles (Kawasaki and Mine, 2005; Wells et al., 2012; Zhang et al., 2005). Nevertheless, fluorescence is also quenched once the fluorophore is excessively close to the metal core, and maximum enhancement occurs at about 10 nm away from the metal surface (Kulakovich et al., 2002). Additionally, the size of the metal core has also been considered as a critical factor affecting enhancement of the fuorophore on nanoparticles (Gryczynski et al., 2002; Lakowicz, 2001; Malicka et al., 2002). Fortunately, gold nanoparticles functions as one major member of metallic particles for fluorescence enhancement as well as silver nanoclusters playing the role as the fluorophore. In this contribution, we successfully combined AgNCs and AuNPs to build up a SEF-based strategy for detecting CEA, and the assaying mechanism was illustrated in detail in Fig. 1A.
Fig. 1. (A) Schematic illustration of SEF occurring: AuNPs enhanced the fluorescence intensity of AgNCs on the basis of complementary DNA hybridization by introducing CEA–aptamer linking AuNPs with AgNCs together; (B) schematic illustration of the creative strategy for assaying CEA: more competitive interactions emerged once CEA and CEA–aptamer coexisted, leading to SEF failed and their fluorescence decreased.
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Specifically, AuNPs were modified with a half-complementary DNA for CEA–aptamer. Meanwhile, the AgNCs were synthesized by another DNA containing half-complementary sequence towards the aptamer. Once the CEA–aptamer was introduced, AuNPs will be linked with AgNCs together through the hybridization of DNA, thus a SEF-system formed. Crucially, the distance from the core of AuNPs to AgNCs was 25 bases estimated as about 10 nm (Yang et al., 2013; Zhang et al., 2005), providing the possibility for enhanced-fluorescence appearing (Lakowicz, 2001). Conversely, AuNPs and AgNCs showed apart from each other in the absence of CEA–aptamer, leading to scarce variation of the fluorescence signal (Fig. S1). Interestingly, CEA preferred to bind with its corresponding aptamer to form complexes rather than the interactions of between aptamer and DNA, while CEA and its aptamer were simultaneously added into the solution of AuNPs and AgNCs (Fig. 1B). Ultimately, we have established a novel detection approach for CEA based on this competitive strategy. Accordingly, this proposed method was employed by assaying CEA with a linear relationship of 0.01 ng mL 1 to 1 ng mL 1 and a detection limit of 3 pg mL 1, suggesting its satisfactory sensitivity. Importantly, the practicality of the current assay was further validated by supplementing CEA in human blood samples, indicating its application future.
2. Experimental 2.1. Apparatus All fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) with excitation slit set at 10 nm band pass and emission at 10 nm band pass in 1 cm 1 cm quartz cell. Meanwhile, UV/vis absorption spectra were recorded by a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). The high-resolution transmission electron microscopy (HR-TEM) images were obtained by using a TECNAI G2 F20 microscope (Hillsbroro, US) at 200 kV. Cell images were acquired by using an Olympus fluorescence microscope 1-71 (Tokyo, Japan). A Fangzhong pHS-3C digital pH meter (Chengdu, China) was employed to measure pH values of all the aqueous solutions and a vortex mixer QL-901 (Haimen, China) for blending solutions. Zeta potential of AuNPs solutions was performed at 25 °C using dynamic laser light scattering (Malvern, UK). The thermostatic water bath (DF-101s) was purchased from Gongyi Instrument Co., Ltd. (Hennan, China).
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0502-P ultrapure water system (Chongqing, China) was applied during the following experiments. 2.3. Preparation of DNA-templated silver nanoclusters Synthesis of DNA-AgNCs was according to the reported together with slight modification (Lan et al., 2011). In brief, AgNO3 solution (1 mM) was initially added to DNA solution (50 μM,160 μL) prepared in phosphate buffer (20 mM, pH 7.0) to provide an Ag þ -to-DNA molar ratio of 6:1, and this mixture was incubated in the dark ice-bath for 15 min. Subsequently, NaBH4 (2 mM, 15 μL) was added into the mixture under vigorous shaking, and the reactions were kept at 25 °C for 3 h. Following the previous procedures, fluorescent DNA-AgNCs were finally produced with fluorescence emission at 540 nm (excitation at 473 nm). 2.4. Synthesis of AuNPs AuNPs were prepared according to a previously described method as well as the corresponding concentration of 13 nM (Zhao et al., 2006). Specifically, trisodium citrate (2.5 mL, 38.8 mM) was added to a boiling solution of HAuCl4 (25 mL, 1 mM) accompanied by its color varying from pale yellow to deep red within a short while. Then, the mixture was heated to reflux for another 30 min, ensuring completed reductions. After slowly cooling to room temperature, the nanoparticles obtained here were filtered through a 0.22 μm filter membrane. 2.5. Modification of AuNPs To be specific, AuNPs modified with thiolated DNA proceeded on the basis of a published report (Zhao et al., 2007). Particularly, it started by mixing the AuNPs solution (600 μL, 13 nM) with thiolmodified oligonucleotide (280 μL, 6.6 μM), and the mixture was incubated at room temperature for 45 h. Then, Tris-HCl buffer (10 mL, 1 M, pH 7.4) and aqueous NaCl (90 μL, 1 M) were added into the mixture above, and further incubated for another 28 h. Again, additions of Tris-HCl buffer (5 μL, 1 M, pH 7.4) and aqueous NaCl (50 μL, 5 M) were performed for one more time, and the mixture was incubated for 18 h under room temperature. Next, this solution was separated by centrifuging at 20,000 rpm for 15 min, and the precipitated AuNPs were collected followed by centrifuging with wash buffer (2 1 mL; 20 mm Tris-HCl, pH 7.4, 300 mM NaCl) to remove other impurity. Eventually, the AuNPs obtained here were redispersed in wash buffer (600 mL) for following applications.
2.2. Chemicals and materials 2.6. Detection of CEA CEA, AFP, CA125, CA15-3 were purchased from Boaosen Biotech Co., Ltd. (Beijing, China). Thrombin, tyrosinase, dopamine, glucose oxidase, heparin sodium, bovine serum albumin (BSA) glucose, folic acid, norepinephrine (NE), bile acid and GSH were obtained from Sangon Biotechnonlog Co., Ltd. (Shanghai, China). All the DNA were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China), and their corresponding sequences were as follows: 5′-ATACCAGCTTATTCAATT-3′ (CEA–aptamer); 5′-AAGCTGGTATCCCCCCC-SH-3′ (modified on the surfaces of AuNPs); 5′CCCCCCCCCCCCAATTGAAT-3′ (for synthesizing AgNCs). Metal ions (Fe3 þ , Ca2 þ , K þ , Na þ ), disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4), trihydroxymethyl aminomethane (Tris), HCl, sodium citrate (CA), sodium chloride (NaCl), sodium borohydride (NaBH4), hydrogen tetrachloroaurate trihydrate (HAuCl4), silver nitrate (AgNO3) were purchased from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). Ultrapure water, 18.25 MΩ cm, produced with an Aquapro AWL-
At the very beginning, 50 μL AgNCs, 50 μL AuNPs (13 mM) and CEA–aptamer (70 μL, 50 μM) were successively pipetted into a 1.5mL vial. Next, an appropriate volume of CEA working solution or sample solution was introduced, and then diluted to 250 μL with Milli-Q purified water along with thoroughly vortexing. Finally, this mixture remained reacting at 37 °C for 30 min and subjected to fluorescence measurements. 2.7. Preparation of blood samples Human serum samples from three healthy volunteers were originally collected from Southwest University Hospital (Chongqing, China). Towards the recovery experiments, impurities of the blood samples were removed by centrifugation (10,000 rpm, 10 min). Then, the supernatant was collected into a 1.5-mL vial and supplemented with standard CEA solutions (0.3 ng, 0.6 ng,
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1.0 ng). Finally, the serum samples were respectively subjected to this proposed enhanced-fluorescence strategy.
data. Taken together, these results suggested DNA were successfully immobilized around the surface of AuNPs. 3.2. Establishing the assaying strategy
3. Results and discussion 3.1. Characterization of AgNCs and AuNPs To characterize this synthesized AgNCs, their corresponding maximum excitation and emission spectra were explored as 473 nm and 540 nm, respectively (Fig. 2A). Meanwhile, as indicated in Fig. 2A, this AgNCs prepared emitted obvious red fluorescence (photograph||) under UV light (365 nm) while appearing as light brown transparent under daylight (photograph I), suggesting satisfactory AgNCs obtained. Simultaneously, HEp-2 cells were incubated in the absence and presence of AgNCs for 30 min (Fig. 2B and C), and the obviously fluorescent signal was observed compared with that without AgNCs, proving their applicability of this nanoclusters for cell imaging. Accordingly, to evaluate multiple DNA assembly onto the AuNPs surface and their related stability, further characterization were performed. In particular, spectrophotometric analysis revealed only a slight change in AuNPs' peak absorbance, confirming successful retention of AuNPs stability modified by thiolated DNA (Luo et al., 2011) (Fig. S2). Furthermore, HR-TEM was employed to directly observe the morphology of the AuNPs with or without modifications, as clearly shown in Fig. 2D and F, both types of AuNPs obtained here appeared as sphere and well-dispersed. For the size distribution analysis, the diameter of unmodified-AuNPs existed in the range of 13.1 70.5 nm determined by dynamic light scattering (DLS), whereas DNA modified-AuNPs growing to the size of 15.4 70.7 nm (Fig. 2E and G), claiming their efficient modification. Besides, DNA-modified AuNPs showed more negative ζ-potentials ( 37.3 71.5) than that of AuNPs without modification ( 5.39 71.9) obtained from ζ-potential measurement (Table S1), indicating general agreement with both HR-TEM images and DLS
Towards building up a more sensitive method by improving the fluorescence of AgNCs, we were trying to construct a SEF system based on DNA modified-AuNPs and AgNCs linked by CEA–aptamer. As shown in Fig. 3A, the fluorescence intensity of AgNCs together with AuNPs has been obviously enhanced about 3-fold once CEA– aptamer (50 μm L 1) introduced. In striking contrast, their signal varied scarcely while no additions of CEA–aptamer (Fig. 3A), demonstrating that CEA–aptamer effectively linked AgNCs with AuNPs and caused SEF happened. Followed by introducing different concentrations of CEA–aptamer into the solution of AgNCs without AuNPs, there is no obvious fluorescence change observed (Fig. 3B), indicating that the enhanced-fluorescence was induced by efficient SEF rather than CEA–aptamer only. Again, the absorbance signal of DNA modified-AuNPs showed slight change in the presence of CEA–aptamer only, whereas the absorbance intensity of AgNCs was enhanced in the presence of both DNA modifiedAuNPs and CEA–aptamer (Fig. 3C). Taken all above together, these data proved the fluorescence intensity of the hypothetical method was dramatically enhanced by CEA–aptamer linking AgNCs with DNA modified-AuNPs together, and SEF indeed occurring. To achieve the ultimate purpose of detecting CEA, further experiments were designed. As shown in Fig. 3D, the fluorescence intensity of this SEF system decreased to about 50% accompanied by 0.5 ng mL 1 CEA added, suggesting it can be applied for assaying CEA. 3.3. Optimization of detection conditions Considering incubation time of CEA interacting with CEA– aptamer playing a key role, further explorations were provided. As shown in Fig. S3, the enhanced fluorescence of the analytical
Fig. 2. (A) The maximum excitation and emission spectra of DNA-AgNCs (inset: photographs of under visible light (�) and UV light (�) respectively); Hep-2 cells were incubated in the absence (B) and presence (C) of AgNCs for 30 min; HR-TEM images of AuNPs (D) and DNA modified-AuNPs (F); DLS data of AuNPs (E) and DNA modifiedAuNPs (G).
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Fig. 3. (A) FL spectra of AgNCs, AgNCs with CEA–aptamer, AgNCs and AuNPs in the presence and absence of CEA–aptamer; (B) FL spectra of AgNCs by adding various concentrations of CEA–aptamer; (C) UV–vis absorption spectra of AuNPs, AuNPs with CEA–aptamer, AgNCs, the mixture of AgNCs, AuNPs and CEA–aptamer; (D) FL spectra of the mixture including AgNCs, AuNPs and CEA–aptamer in the presence or absence of 0.5 ng mL 1 CEA.
system returned to the original-level signal of AgNCs together with AuNPs, while the incubation time reached 30 min after CEA (0.1 ng mL 1) engaging in the mixture of AgNCs, AuNPs and CEA– aptamer, suggesting 30 min can serving as the optimal incubation time. Additionally, 37 °C were determined as the reaction temperature owing to their biochemical nature and occurring in human bodies. Consequently, 30 min and 37 °C were finally selected as optimized analytical conditions.
3.4. Selectivity and interference from coexisting substance Next, the selectivity of this proposed strategy was evaluated by investigating responses to other eight related proteins including AFP, CA125, CA15-3, thrombin, BSA, tyrosinase, dopamine, Glucose oxidase (5 ng mL 1 for each) upon optimal conditions for the case of 0.8 ng mL 1 CEA. By comparing (F0 F)/F of CEA with that of other proteins (F0 and F correspond to the fluorescence intensity of
Fig. 4. (A) Selectivity of the proposed method by comparing CEA (0.8 ng mL 1) with other related proteins such as AFP, CA125, CA15-3, thrombin, BSA, tyrosinase, dopamine, Glucose oxidase (5 ng mL 1 for each); (B) influence of coexisting compounds and ions on the fluorescence of the system including AgNCs, AuNPs, CEA–aptamer and CEA by introducing glucose, folic acid, heparin sodium, norepinephrine, bile acid, Na þ , K þ , Ca2 þ , Fe3 þ (1 ng mL 1 for each).
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Fig.5. (A) Fluorescence emission spectra of AgNCs and AuNPs with different concentrations of CEA–aptamer; (B) plot of F/F0 versus the logarithm concentrations of CEA– aptamer; (C) fluorescence spectra of AgNCs, AuNPs and CEA–aptamer with various concentrations of CEA; (D) plot of F/F0 versus concentrations of CEA from 0.01 ng mL 1 to 1 ng mL 1.
AgNCs, AuNPs, CEA–aptamer before or after adding CEA, respectively), CEA was clearly distinguished from others (Fig. 4A), indicating that the proposed method implied satisfactory selectivity. To explore the influence originated from compounds and ions in physiological circumstances towards detecting CEA (0.5 ng mL 1), various substance such as bilirubin, bile acid, folic acid, heparin sodium, glucose, norepinephrine, Na þ K þ , Ca2 þ , and Fe3 þ (1 ng mL 1 for each) were separately introduced into current detection procedures. As Fig. 4B indicated, these biological compounds and ions showed scarce effect on the fluorescence intensity of the analytical system, describing its acceptable endurance for interference. 3.5. Detection of CEA–aptamer and CEA Fundamentally, the fluorescence intensity of AgNCs combing with AuNPs increased in accord with the amounts of CEA–aptamer (Fig. 5A), and the relevant signals regularly increased versus concentrations of 12.25 nM–50 μM (R2 ¼0.967) with the detection Table 1 Recoveries of CEA in human serum samples detected by the proposed method (n¼6). Sample
Supplemented (ng) Measured (ng) Recovery (%) RSD (%)
Human serum blood
0.3 0.6 1
0.294 0.615 0.953
98.16 102.5 95.31
0.843 0.685 0.751
limit of 4.8 nM at a signal-to-noise ratio of 3 (Fig. 5B). Accordingly, the final concentration of CEA–aptamer was fixed to 50 μM to obtain the maximal enhanced-fluorescence and improve the sensitivity during the following detections. Based on the established strategy, the standard curve for this method was established by a series concentrations of CEA (0.01, 0.05, 0.1, 0.3, 0.5, 0.7, 0.9, 1.0 ng mL 1). As shown in Fig. 5C and D, the fluorescence intensity decreased proportionally with the amount of CEA introduced. The fluorescence intensity decrease (F/F0) versus CEA concentrations displayed a linear range from 0.01 ng mL 1 to 0.1 ng mL 1 (R2 ¼0.984) together with the detection limit of 3 pg mL 1 at a signal-to-noise ratio of 3, suggesting the sensitivity and promising of this analytical strategy. To our knowledge, the linear range and the detection limit here are obviously lower than that of the previous methods (Table S2), demonstrating that our method can match the requirement of assaying CEA towards various purpose.
3.6. Analysis of blood samples For testing the practicality of this developed approach, standard recovery experiments were performed in human blood samples (Table 1). As listed, the recoveries of all samples were obtained as 98.16%, 102.5%, 95.31% respectively, proving little inference from substrates of the blood. Thus, the proposed method may broaden ways for practical detections of CEA in real samples.
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4. Conclusions In summary, we here successfully built up a creative SEF system by employing AuNPs to enhance the fluorescence intensity of AgNCs on the basis of complementary DNA hybridization, and further applied this system for assaying CEA due to its more competitive interactions with CEA–aptamer by comparison to hybridization between complementary DNA sequences. In particular, the fluorescence signals of AgNCs were first effectively enhanced by AuNPs, and introductions of CEA subsequently competed with the complementary DNA for interacting with CEA–aptamer, leading to SEF destroyed and the enhanced-fluorescence decreased. Importantly, the decrease of fluorescence intensity permitted detecting CEA in a linear range of 0.01 ng mL 1 to 0.1 ng mL 1 together with a detection limit of 3 pg mL 1 at a signal-to-noise ratio of 3, indicating its sensitivity of this proposed strategy. Eventually, a sensitive and selective approach towards CEA has been constructed. Additionally, this innovative strategy was subjected to detect CEA in human blood samples by recovery experiments, demonstrating its potentiality for diverse applications. Overall, the unique combination of AgNCs and AuNPs by virtue of aptamer has provided a valuable model for sensitively assaying other trace targets.
Acknowledgements We gratefully acknowledge financial support by National Natural Science Foundation of China (31100981), Research Fund for the Doctoral Program of Higher Education of China (20110182120014), Natural Science Foundation Project of CQ CSTC (cstc2013jcyjA10117), Fundamental Research Funds for the Central Universities (XDJK2013B038), and Program for Innovative Research Team in University of Chongqing (2013).
Appendix A. Suplementary materials Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.09.029.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Selectively assaying CEA based on a creative strategy of gold nanoparticles enhancing silver nanoclusters' fluorescence Xiaoming Yang n,1, Yan Zhuo 1, Shanshan Zhu, Yawen Luo, Yuanjiao Feng, Yan Xu College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China
art ic l e i nf o
a b s t r a c t
Article history: Received 26 June 2014 Received in revised form 2 September 2014 Accepted 4 September 2014 Available online 18 September 2014
Herein, we have successfully built up connections between nanoparticles and nanoclusters, and further constructed a surface-enhanced fluorescence (SEF) strategy based on the two types of nanomaterials for selectively assaying carcinoembryonic antigen (CEA). Specifically, silver nanoclusters provided the original fluorescence signal, while gold nanoparticles modified with DNA served as the fluorescence enhancer simultaneously. On the basis of this proposed nano-system, the two nanomaterials were linked by CEA–aptamer, thus facilitating SEF occurring. Nevertheless, more competitive interactions between CEA and CEA–aptamer emerged once CEA added, leading to SEF failed and their fluorescence decreased. Significantly, this creative method was further applied to detect CEA, and showed the linear relationship between the fluorescence intensity and CEA concentrations in the range of 0.01–1 ng mL 1 with a detection limit of 3 pg mL 1 at a signal-to-noise ratio of 3, demonstrating its sensitivity and promising towards multiple applications. On the whole, this approach we established may broaden potential ways of combining nanoparticles and nanoclusters for detecting trace targets in bioanalytical fields. & 2014 Elsevier B.V. All rights reserved.
Keywords: Silver nanoclusters Gold nanoparticles Aptamer CEA Surface-enhanced fluorescence
1. Introduction Tumor markers, as molecules appearing in blood or tissue, have been proved to be associated with carcinogenesis, and their related measurement or identification exhibited great importance for patient diagnosis or clinical management (Miyake et al., 2010; Tang et al., 2007). Carcinoembryonic antigen (CEA), a tumorassociated antigen, has been identified as a critical marker for clinical diagnosis of colon tumors, breast tumors, ovarian carcinoma and cystadenocarcinoma (Eppler et al., 2002; Naghibalhossaini and Ebadi, 2006). Basically, amounts of CEA in colon tissue of adults are reported as a low level of 2.5–5.0 μg L 1 (Zamcheck and Martin, 1981). However, the levels of CEA are claimed to be obviously elevated while tumors arise in endodermal tissue including gastrointestinal tract, respiratory tract, pancreas, and breast (Shen et al., 2005). Accordingly, investigating the amounts of CEA plays an important role towards clinical purpose. Currently, immunoassays and related techniques have been considered as major analytical methods towards detections of CEA mainly including radioimmunoassay, enzyme immunoassay, chemiluminescence immunoassay and fluorescence immunoassay (Lang et al., 2014; Li et al., 2010; Yu et al., 2013; Yuan et al., 2001). n
Corresponding author. Fax: þ 86 23 68251225. E-mail address: [email protected] (X. Yang). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.09.029 0956-5663/& 2014 Elsevier B.V. All rights reserved.
Unfortunately, most of these methods exhibited drawbacks of requiring radiation hazards, time-consuming or sophisticated instrumentation (Darain et al., 2003), resulting in alternative approaches still desirable. Aptamer, emerging as single-stranded oligonucleotides with definite structures, can selectively bind with specific targets (Nielsen et al., 2010; Nutiu and Li, 2003). In particular, nucleic acid aptamer to specifically bind cells, proteins, low-molecularweight inorganic or organic substrates, usually contains unmodified DNA and RNA, which is isolated through a selection process in vitro or systematic evolution of ligands by exponential enrichment (SELEX) (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Simultaneously, aptamer are indeed able to bind their complementary DNA sequence, and form a duplex structure (Jhaveri et al., 2000; Lin and Patel, 1997). Compared with other natural receptors such as antibodies and enzymes, aptamer showed various unapproachable advantages mainly including easy operation of synthesis and readily acquired from commercial sources (Wilson and Szostak, 1999), facilitating their satisfactory chemical stability. Additionally, aptamer generally exhibited high specificity and affinity towards their targets, and their structural flexibility allows for adaptation of undergoing significant conformational changes once aptamer and targets specifically bind with each other (Nutiu and Li, 2005; Sefah et al., 2009). Hence, these attractive superiorities result in potentiating them as ideal biosensing molecules (Jayasena, 1999; Tang and Breaker, 1997), and aptamer have broadened ways as sensors in fields of assaying metal ions, small
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molecules, proteins and cancer cell, etc. (Liu et al., 2009; Sun et al., 2011; Zhou et al., 2010; Zhou et al., 2012). Fluorescent nanomaterials, especially gold nanoclusters (AuNCs), silver nanoclusters (AgNCs) and copper nanoclusters (CuNCs), have appeared as powerful and sensitive probes (Gerion et al., 2003; Guo et al., 2010; Huang et al., 2008) and attracted numerous attentions. Among these nanoclusters, oligonucleotidestabilized AgNCs (DNA-AgNCs) are usually synthesized by NaBH4 mediating reductions of Ag ions in the presence of oligonucleotides, and biocompatible and low toxic (Yu et al., 2009). Besides, most of DNA-AgNCs were obtained by cytosine-rich DNA and exhibited notable spectral and photophysical properties due to the binding interactions between silver and cytosine (Ono et al., 2008; Robichaud et al., 2005), and introducing various DNA sequences provided diverse fluorescence colors of DNA-AgNCs (Gwinn et al., 2008; Richards et al., 2008). Again, DNA not only serves as templates, but also plays roles for molecular recognition. All these advantages of DNA-AgNCs suggested their promising value for analytical applications (Li et al., 2012). Combinations of fluorescent nanomaterials and biochemical molecules have exhibited great prospects for biochemical applications, since both the unique structural and photophysical performance and excellent properties have been effectively integrated (Zhang et al., 2012; Zhang et al.,
2011a; Zhang et al., 2011b). Again, employing nanomaterials may improve the sensitivity and accuracy of biosensors. Moreover, nano-sized devices generally response faster due to mass transport occurring over shorter distances. Surface-enhanced fluorescence (SEF) on metal nanostructures (Lakowicz, 2005), usually happens while a fluorophore locating near the surface of metallic nanoparticles (Antunes et al., 2001; Kulakovich et al., 2002; Parfenov et al., 2003). SEF is believed to occur through the coupling of the fluorophores with radiating plasmons from the metallic particles (Kawasaki and Mine, 2005; Wells et al., 2012; Zhang et al., 2005). Nevertheless, fluorescence is also quenched once the fluorophore is excessively close to the metal core, and maximum enhancement occurs at about 10 nm away from the metal surface (Kulakovich et al., 2002). Additionally, the size of the metal core has also been considered as a critical factor affecting enhancement of the fuorophore on nanoparticles (Gryczynski et al., 2002; Lakowicz, 2001; Malicka et al., 2002). Fortunately, gold nanoparticles functions as one major member of metallic particles for fluorescence enhancement as well as silver nanoclusters playing the role as the fluorophore. In this contribution, we successfully combined AgNCs and AuNPs to build up a SEF-based strategy for detecting CEA, and the assaying mechanism was illustrated in detail in Fig. 1A.
Fig. 1. (A) Schematic illustration of SEF occurring: AuNPs enhanced the fluorescence intensity of AgNCs on the basis of complementary DNA hybridization by introducing CEA–aptamer linking AuNPs with AgNCs together; (B) schematic illustration of the creative strategy for assaying CEA: more competitive interactions emerged once CEA and CEA–aptamer coexisted, leading to SEF failed and their fluorescence decreased.
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Specifically, AuNPs were modified with a half-complementary DNA for CEA–aptamer. Meanwhile, the AgNCs were synthesized by another DNA containing half-complementary sequence towards the aptamer. Once the CEA–aptamer was introduced, AuNPs will be linked with AgNCs together through the hybridization of DNA, thus a SEF-system formed. Crucially, the distance from the core of AuNPs to AgNCs was 25 bases estimated as about 10 nm (Yang et al., 2013; Zhang et al., 2005), providing the possibility for enhanced-fluorescence appearing (Lakowicz, 2001). Conversely, AuNPs and AgNCs showed apart from each other in the absence of CEA–aptamer, leading to scarce variation of the fluorescence signal (Fig. S1). Interestingly, CEA preferred to bind with its corresponding aptamer to form complexes rather than the interactions of between aptamer and DNA, while CEA and its aptamer were simultaneously added into the solution of AuNPs and AgNCs (Fig. 1B). Ultimately, we have established a novel detection approach for CEA based on this competitive strategy. Accordingly, this proposed method was employed by assaying CEA with a linear relationship of 0.01 ng mL 1 to 1 ng mL 1 and a detection limit of 3 pg mL 1, suggesting its satisfactory sensitivity. Importantly, the practicality of the current assay was further validated by supplementing CEA in human blood samples, indicating its application future.
2. Experimental 2.1. Apparatus All fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) with excitation slit set at 10 nm band pass and emission at 10 nm band pass in 1 cm 1 cm quartz cell. Meanwhile, UV/vis absorption spectra were recorded by a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). The high-resolution transmission electron microscopy (HR-TEM) images were obtained by using a TECNAI G2 F20 microscope (Hillsbroro, US) at 200 kV. Cell images were acquired by using an Olympus fluorescence microscope 1-71 (Tokyo, Japan). A Fangzhong pHS-3C digital pH meter (Chengdu, China) was employed to measure pH values of all the aqueous solutions and a vortex mixer QL-901 (Haimen, China) for blending solutions. Zeta potential of AuNPs solutions was performed at 25 °C using dynamic laser light scattering (Malvern, UK). The thermostatic water bath (DF-101s) was purchased from Gongyi Instrument Co., Ltd. (Hennan, China).
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0502-P ultrapure water system (Chongqing, China) was applied during the following experiments. 2.3. Preparation of DNA-templated silver nanoclusters Synthesis of DNA-AgNCs was according to the reported together with slight modification (Lan et al., 2011). In brief, AgNO3 solution (1 mM) was initially added to DNA solution (50 μM,160 μL) prepared in phosphate buffer (20 mM, pH 7.0) to provide an Ag þ -to-DNA molar ratio of 6:1, and this mixture was incubated in the dark ice-bath for 15 min. Subsequently, NaBH4 (2 mM, 15 μL) was added into the mixture under vigorous shaking, and the reactions were kept at 25 °C for 3 h. Following the previous procedures, fluorescent DNA-AgNCs were finally produced with fluorescence emission at 540 nm (excitation at 473 nm). 2.4. Synthesis of AuNPs AuNPs were prepared according to a previously described method as well as the corresponding concentration of 13 nM (Zhao et al., 2006). Specifically, trisodium citrate (2.5 mL, 38.8 mM) was added to a boiling solution of HAuCl4 (25 mL, 1 mM) accompanied by its color varying from pale yellow to deep red within a short while. Then, the mixture was heated to reflux for another 30 min, ensuring completed reductions. After slowly cooling to room temperature, the nanoparticles obtained here were filtered through a 0.22 μm filter membrane. 2.5. Modification of AuNPs To be specific, AuNPs modified with thiolated DNA proceeded on the basis of a published report (Zhao et al., 2007). Particularly, it started by mixing the AuNPs solution (600 μL, 13 nM) with thiolmodified oligonucleotide (280 μL, 6.6 μM), and the mixture was incubated at room temperature for 45 h. Then, Tris-HCl buffer (10 mL, 1 M, pH 7.4) and aqueous NaCl (90 μL, 1 M) were added into the mixture above, and further incubated for another 28 h. Again, additions of Tris-HCl buffer (5 μL, 1 M, pH 7.4) and aqueous NaCl (50 μL, 5 M) were performed for one more time, and the mixture was incubated for 18 h under room temperature. Next, this solution was separated by centrifuging at 20,000 rpm for 15 min, and the precipitated AuNPs were collected followed by centrifuging with wash buffer (2 1 mL; 20 mm Tris-HCl, pH 7.4, 300 mM NaCl) to remove other impurity. Eventually, the AuNPs obtained here were redispersed in wash buffer (600 mL) for following applications.
2.2. Chemicals and materials 2.6. Detection of CEA CEA, AFP, CA125, CA15-3 were purchased from Boaosen Biotech Co., Ltd. (Beijing, China). Thrombin, tyrosinase, dopamine, glucose oxidase, heparin sodium, bovine serum albumin (BSA) glucose, folic acid, norepinephrine (NE), bile acid and GSH were obtained from Sangon Biotechnonlog Co., Ltd. (Shanghai, China). All the DNA were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China), and their corresponding sequences were as follows: 5′-ATACCAGCTTATTCAATT-3′ (CEA–aptamer); 5′-AAGCTGGTATCCCCCCC-SH-3′ (modified on the surfaces of AuNPs); 5′CCCCCCCCCCCCAATTGAAT-3′ (for synthesizing AgNCs). Metal ions (Fe3 þ , Ca2 þ , K þ , Na þ ), disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4), trihydroxymethyl aminomethane (Tris), HCl, sodium citrate (CA), sodium chloride (NaCl), sodium borohydride (NaBH4), hydrogen tetrachloroaurate trihydrate (HAuCl4), silver nitrate (AgNO3) were purchased from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). Ultrapure water, 18.25 MΩ cm, produced with an Aquapro AWL-
At the very beginning, 50 μL AgNCs, 50 μL AuNPs (13 mM) and CEA–aptamer (70 μL, 50 μM) were successively pipetted into a 1.5mL vial. Next, an appropriate volume of CEA working solution or sample solution was introduced, and then diluted to 250 μL with Milli-Q purified water along with thoroughly vortexing. Finally, this mixture remained reacting at 37 °C for 30 min and subjected to fluorescence measurements. 2.7. Preparation of blood samples Human serum samples from three healthy volunteers were originally collected from Southwest University Hospital (Chongqing, China). Towards the recovery experiments, impurities of the blood samples were removed by centrifugation (10,000 rpm, 10 min). Then, the supernatant was collected into a 1.5-mL vial and supplemented with standard CEA solutions (0.3 ng, 0.6 ng,
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1.0 ng). Finally, the serum samples were respectively subjected to this proposed enhanced-fluorescence strategy.
data. Taken together, these results suggested DNA were successfully immobilized around the surface of AuNPs. 3.2. Establishing the assaying strategy
3. Results and discussion 3.1. Characterization of AgNCs and AuNPs To characterize this synthesized AgNCs, their corresponding maximum excitation and emission spectra were explored as 473 nm and 540 nm, respectively (Fig. 2A). Meanwhile, as indicated in Fig. 2A, this AgNCs prepared emitted obvious red fluorescence (photograph||) under UV light (365 nm) while appearing as light brown transparent under daylight (photograph I), suggesting satisfactory AgNCs obtained. Simultaneously, HEp-2 cells were incubated in the absence and presence of AgNCs for 30 min (Fig. 2B and C), and the obviously fluorescent signal was observed compared with that without AgNCs, proving their applicability of this nanoclusters for cell imaging. Accordingly, to evaluate multiple DNA assembly onto the AuNPs surface and their related stability, further characterization were performed. In particular, spectrophotometric analysis revealed only a slight change in AuNPs' peak absorbance, confirming successful retention of AuNPs stability modified by thiolated DNA (Luo et al., 2011) (Fig. S2). Furthermore, HR-TEM was employed to directly observe the morphology of the AuNPs with or without modifications, as clearly shown in Fig. 2D and F, both types of AuNPs obtained here appeared as sphere and well-dispersed. For the size distribution analysis, the diameter of unmodified-AuNPs existed in the range of 13.1 70.5 nm determined by dynamic light scattering (DLS), whereas DNA modified-AuNPs growing to the size of 15.4 70.7 nm (Fig. 2E and G), claiming their efficient modification. Besides, DNA-modified AuNPs showed more negative ζ-potentials ( 37.3 71.5) than that of AuNPs without modification ( 5.39 71.9) obtained from ζ-potential measurement (Table S1), indicating general agreement with both HR-TEM images and DLS
Towards building up a more sensitive method by improving the fluorescence of AgNCs, we were trying to construct a SEF system based on DNA modified-AuNPs and AgNCs linked by CEA–aptamer. As shown in Fig. 3A, the fluorescence intensity of AgNCs together with AuNPs has been obviously enhanced about 3-fold once CEA– aptamer (50 μm L 1) introduced. In striking contrast, their signal varied scarcely while no additions of CEA–aptamer (Fig. 3A), demonstrating that CEA–aptamer effectively linked AgNCs with AuNPs and caused SEF happened. Followed by introducing different concentrations of CEA–aptamer into the solution of AgNCs without AuNPs, there is no obvious fluorescence change observed (Fig. 3B), indicating that the enhanced-fluorescence was induced by efficient SEF rather than CEA–aptamer only. Again, the absorbance signal of DNA modified-AuNPs showed slight change in the presence of CEA–aptamer only, whereas the absorbance intensity of AgNCs was enhanced in the presence of both DNA modifiedAuNPs and CEA–aptamer (Fig. 3C). Taken all above together, these data proved the fluorescence intensity of the hypothetical method was dramatically enhanced by CEA–aptamer linking AgNCs with DNA modified-AuNPs together, and SEF indeed occurring. To achieve the ultimate purpose of detecting CEA, further experiments were designed. As shown in Fig. 3D, the fluorescence intensity of this SEF system decreased to about 50% accompanied by 0.5 ng mL 1 CEA added, suggesting it can be applied for assaying CEA. 3.3. Optimization of detection conditions Considering incubation time of CEA interacting with CEA– aptamer playing a key role, further explorations were provided. As shown in Fig. S3, the enhanced fluorescence of the analytical
Fig. 2. (A) The maximum excitation and emission spectra of DNA-AgNCs (inset: photographs of under visible light (�) and UV light (�) respectively); Hep-2 cells were incubated in the absence (B) and presence (C) of AgNCs for 30 min; HR-TEM images of AuNPs (D) and DNA modified-AuNPs (F); DLS data of AuNPs (E) and DNA modifiedAuNPs (G).
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Fig. 3. (A) FL spectra of AgNCs, AgNCs with CEA–aptamer, AgNCs and AuNPs in the presence and absence of CEA–aptamer; (B) FL spectra of AgNCs by adding various concentrations of CEA–aptamer; (C) UV–vis absorption spectra of AuNPs, AuNPs with CEA–aptamer, AgNCs, the mixture of AgNCs, AuNPs and CEA–aptamer; (D) FL spectra of the mixture including AgNCs, AuNPs and CEA–aptamer in the presence or absence of 0.5 ng mL 1 CEA.
system returned to the original-level signal of AgNCs together with AuNPs, while the incubation time reached 30 min after CEA (0.1 ng mL 1) engaging in the mixture of AgNCs, AuNPs and CEA– aptamer, suggesting 30 min can serving as the optimal incubation time. Additionally, 37 °C were determined as the reaction temperature owing to their biochemical nature and occurring in human bodies. Consequently, 30 min and 37 °C were finally selected as optimized analytical conditions.
3.4. Selectivity and interference from coexisting substance Next, the selectivity of this proposed strategy was evaluated by investigating responses to other eight related proteins including AFP, CA125, CA15-3, thrombin, BSA, tyrosinase, dopamine, Glucose oxidase (5 ng mL 1 for each) upon optimal conditions for the case of 0.8 ng mL 1 CEA. By comparing (F0 F)/F of CEA with that of other proteins (F0 and F correspond to the fluorescence intensity of
Fig. 4. (A) Selectivity of the proposed method by comparing CEA (0.8 ng mL 1) with other related proteins such as AFP, CA125, CA15-3, thrombin, BSA, tyrosinase, dopamine, Glucose oxidase (5 ng mL 1 for each); (B) influence of coexisting compounds and ions on the fluorescence of the system including AgNCs, AuNPs, CEA–aptamer and CEA by introducing glucose, folic acid, heparin sodium, norepinephrine, bile acid, Na þ , K þ , Ca2 þ , Fe3 þ (1 ng mL 1 for each).
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Fig.5. (A) Fluorescence emission spectra of AgNCs and AuNPs with different concentrations of CEA–aptamer; (B) plot of F/F0 versus the logarithm concentrations of CEA– aptamer; (C) fluorescence spectra of AgNCs, AuNPs and CEA–aptamer with various concentrations of CEA; (D) plot of F/F0 versus concentrations of CEA from 0.01 ng mL 1 to 1 ng mL 1.
AgNCs, AuNPs, CEA–aptamer before or after adding CEA, respectively), CEA was clearly distinguished from others (Fig. 4A), indicating that the proposed method implied satisfactory selectivity. To explore the influence originated from compounds and ions in physiological circumstances towards detecting CEA (0.5 ng mL 1), various substance such as bilirubin, bile acid, folic acid, heparin sodium, glucose, norepinephrine, Na þ K þ , Ca2 þ , and Fe3 þ (1 ng mL 1 for each) were separately introduced into current detection procedures. As Fig. 4B indicated, these biological compounds and ions showed scarce effect on the fluorescence intensity of the analytical system, describing its acceptable endurance for interference. 3.5. Detection of CEA–aptamer and CEA Fundamentally, the fluorescence intensity of AgNCs combing with AuNPs increased in accord with the amounts of CEA–aptamer (Fig. 5A), and the relevant signals regularly increased versus concentrations of 12.25 nM–50 μM (R2 ¼0.967) with the detection Table 1 Recoveries of CEA in human serum samples detected by the proposed method (n¼6). Sample
Supplemented (ng) Measured (ng) Recovery (%) RSD (%)
Human serum blood
0.3 0.6 1
0.294 0.615 0.953
98.16 102.5 95.31
0.843 0.685 0.751
limit of 4.8 nM at a signal-to-noise ratio of 3 (Fig. 5B). Accordingly, the final concentration of CEA–aptamer was fixed to 50 μM to obtain the maximal enhanced-fluorescence and improve the sensitivity during the following detections. Based on the established strategy, the standard curve for this method was established by a series concentrations of CEA (0.01, 0.05, 0.1, 0.3, 0.5, 0.7, 0.9, 1.0 ng mL 1). As shown in Fig. 5C and D, the fluorescence intensity decreased proportionally with the amount of CEA introduced. The fluorescence intensity decrease (F/F0) versus CEA concentrations displayed a linear range from 0.01 ng mL 1 to 0.1 ng mL 1 (R2 ¼0.984) together with the detection limit of 3 pg mL 1 at a signal-to-noise ratio of 3, suggesting the sensitivity and promising of this analytical strategy. To our knowledge, the linear range and the detection limit here are obviously lower than that of the previous methods (Table S2), demonstrating that our method can match the requirement of assaying CEA towards various purpose.
3.6. Analysis of blood samples For testing the practicality of this developed approach, standard recovery experiments were performed in human blood samples (Table 1). As listed, the recoveries of all samples were obtained as 98.16%, 102.5%, 95.31% respectively, proving little inference from substrates of the blood. Thus, the proposed method may broaden ways for practical detections of CEA in real samples.
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4. Conclusions In summary, we here successfully built up a creative SEF system by employing AuNPs to enhance the fluorescence intensity of AgNCs on the basis of complementary DNA hybridization, and further applied this system for assaying CEA due to its more competitive interactions with CEA–aptamer by comparison to hybridization between complementary DNA sequences. In particular, the fluorescence signals of AgNCs were first effectively enhanced by AuNPs, and introductions of CEA subsequently competed with the complementary DNA for interacting with CEA–aptamer, leading to SEF destroyed and the enhanced-fluorescence decreased. Importantly, the decrease of fluorescence intensity permitted detecting CEA in a linear range of 0.01 ng mL 1 to 0.1 ng mL 1 together with a detection limit of 3 pg mL 1 at a signal-to-noise ratio of 3, indicating its sensitivity of this proposed strategy. Eventually, a sensitive and selective approach towards CEA has been constructed. Additionally, this innovative strategy was subjected to detect CEA in human blood samples by recovery experiments, demonstrating its potentiality for diverse applications. Overall, the unique combination of AgNCs and AuNPs by virtue of aptamer has provided a valuable model for sensitively assaying other trace targets.
Acknowledgements We gratefully acknowledge financial support by National Natural Science Foundation of China (31100981), Research Fund for the Doctoral Program of Higher Education of China (20110182120014), Natural Science Foundation Project of CQ CSTC (cstc2013jcyjA10117), Fundamental Research Funds for the Central Universities (XDJK2013B038), and Program for Innovative Research Team in University of Chongqing (2013).
Appendix A. Suplementary materials Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.09.029.
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