Biosensors and Bioelectronics 66 (2015) 554–558
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Building heterogeneous core–satellite chiral assemblies for ultrasensitive toxin detection Xueli Zhao, Xiaoling Wu, Liguang Xu n, Wei Ma, Hua Kuang, Libing Wang, Chuanlai Xu State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 October 2014 Received in revised form 4 December 2014 Accepted 8 December 2014 Available online 9 December 2014
A chiral-aptasensor for ochratoxin A (OTA) detection based on Au core–Ag nanoparticle satellite assemblies was fabricated for the first time. High yields of Au core–Ag NP satellite assemblies were prepared in the aqueous phase and the optical properties of the Au core–Ag NP satellite assemblies were investigated in detail. Because of the different concentrations of the OTA target, the assembly degree of the architecture varied which led to the corresponding chiral signals. The developed method for OTA detection with excellent linear range from 1 to 50 pg/mL showed high selectivity for OTA and the limit of detection as low as 0.16 pg/mL. The feasibility of this method was demonstrated by performing recovery experiment using negative red wine samples, excellent recovery ranged from 90% to 105% was achieved, and indicated promising applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: Core–satellite Circular dichroism Toxin Aptasensor Detection
1. Introduction Ochratoxin A (OTA), one of the most poisonous mycotoxins, is widely-found residue in food raw materials and may cause cancer, immunosuppression and immunotoxicity (Chen et al., 2014; Haighton et al., 2012). As OTA poses a high risk to human health, OTA detection have been attracting more attentions (Duarte et al., 2011). Conventional methods for OTA quantification are mostly based on instruments, such as liquid chromatography–tandem mass spectrometry (LC–MS/MS) and high-performance liquid chromatography connected to tandem mass spectrometry (HPLC– MS/MS) (Bazin et al., 2013). However, skilled operators, expensive instruments and complicated sample preparation have restricted their wide application. Enzyme-linked immunosorbent assay (ELISA) (Li et al., 2013) and electrochemical methods (Kuang et al., 2010; Olcer et al., 2014; Vidal et al., 2013) have also been developed for OTA biosensing. However, these methods either involve tedious processes, stringent sample pretreatments, or showed low sensitivity (Liu et al., 2008). To improve the sensitivity of OTA detection, surface-enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR) have been used and showed a limit of detection (LOD) of 1 nM (Park et al., 2014). Other studies on OTA detection have been based on signal amplification technology (such as rolling chain amplification and loop-mediated n
Corresponding author. E-mail address: [email protected] (L. Xu).
http://dx.doi.org/10.1016/j.bios.2014.12.021 0956-5663/& 2014 Elsevier B.V. All rights reserved.
isothermal amplification), however, the complexity of the operating procedure has limited their practical application and on-site OTA analysis (Chen et al., 2014; Huang et al., 2013). Nanoparticle (NP) core–satellite assemblies, a unique architecture assembled by nanomaterials, has attracted the attention of researchers (Chou et al., 2014; Mucic et al., 1998; Pal et al., 2009; Yoon et al., 2012). Lazarides and co-workers reported a reconfigurable Au core (50 nm)–Au satellite (13 nm) using DNA linkers. Based on the trypsin-mediated disassembly of the Au core (50 nm)–Au satellite (50 nm or 30 nm), Lee’s group established a colorimetric sensor for protease detection on a two-dimensional glass substrate (Waldeisen et al., 2011). Bach’s group showed a sensitive near-IR SERS sensor with a LOD below 1 nM, based on a high density of hot-spots of bottom-up core–satellite nanostructures (Zheng et al., 2013). Our previous works evaluated Au nanorod (NR) core–Au nanoparticle (NP)-satellite assemblies, investigated their chirality and bioproperties, and demonstrated their potential in biosensing (Xu et al., 2013, 2012). In recent years, chirality has increased in popularity in nanotechnology. A large number of chiral geometries such as dimers and pyramids have been reported (Wu et al., 2013; Yan et al., 2012). The generating mechanism of circular dichroism (CD) was investigated and chiral sensors for detection in the biological, food and medical fields were established (Govorov et al., 2010; Ma et al., 2013a; Wu et al., 2013). In the present study, chiral Au core– Ag NP satellite assemblies were fabricated and the chiral signals showed that this architecture had intense chiral optical response with increased the number of satellite NP, and based on this
X. Zhao et al. / Biosensors and Bioelectronics 66 (2015) 554–558
principle, the chiral aptasensor with high sensitivity and selectivity for OTA detection was developed.
2. Experimental section 2.1. Materials The thiolated DNA oligonucleotides (OTA aptamer and its complementary sequence) were manufactured by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., purified by HPLC (the purity of DNA used in this study were all greater than 98%). These oligonucleotides were dissolved in TE buffer (Shanghai Sangon) to give a final concentration of 50 μM. OTA, deoxynivalenol (DON), aflatoxin B1 (AFB1), aflatoxin M1 (AFM1), fumonisin (FB), and ochratoxin B (OTB) were purchased from SigmaAldrich. Deionized (DI) water obtained using a Milli-Q device (18.2 MΩ, Millipore, Molsheim, France) was used throughout this work. The detailed sequences of the oligonucleotides are as follows: OTA-aptamer: 5′-SH-TTTTTTTTTTGATCGGGTGTGGGTGGCGTAAAGGGAG CATCGG-3′ OTA-complementary: 5′-SH- TTTTTTTTTTCCGATGCTCCCT-3′
555
2.5. Preparation of single-stranded DNA modified-Au NPs and Ag NPs Au NPs were modified with OTA aptamer at a coupling ratio of 200 to 1. Briefly, Au NPs (35 72 nm) were concentrated ten times and then resuspended in 0.01 M phosphate buffer at a final concentration of 15 nM. Thiolated OTA aptamer (50 μM, dissolved in TE buffer) was added to the Au NPs solution (final aptamer concentration was 3 μM), 1 μL of NaCl (5 M) was added to 100 μL of the solution to increase the concentration of NaCl to 50 mM after 2 h. And then, this process was repeated at one more increment of 0.05 M NaCl thereafter until a concentration of 0.3 M NaCl was reached (Demers et al., 2002; Rosi et al., 2006). The mixed solution was left to stand for 12 h to complete the single-stranded functionalization process. The particles were centrifuged (5000 rpm, 10 min) and resuspended in 0.01 M Tris–HCl buffer, washed for three times. Ag NPs were modified with OTA complementary at a coupling ratio of 5 to 1. Ag NPs were concentrated ten times and then resuspended in 0.01 M Tris–HCl buffer at a final concentration of 100 nM. Thiolated OTA complementary (50 μM, dissolved in TE buffer) was added to the Ag NPs solution (final aptamer concentration was 0.5 μM) and left to stand for 12 h to complete the functionalization process. The particles were centrifuged (14,000 rpm, 15 min) and resuspended in 0.01 M Tris–HCl buffer, washed for three times.
2.2. Instrumentation 2.6. Fabrication of the Au core–Ag NP satellite sensor The ultraviolet–visible (UV–vis) spectrum was obtained using a UNICO 2100 PC UV–vis spectrophotometer. Transmission electron microscopy (TEM) images were acquired using a JEOL JEM-2100 operating at an acceleration voltage of 200 kV. The CD spectrum was scanned by MOS-450/AF-Circular Dichroism at a scanning speed of 1 nm/s and the path length of quartz glass cuvette was 1 cm.
Firstly, 30 μL of Au NP-OTA aptamer and 150 μL of Ag NP-OTA complementary were mixed, followed by 3 min of gentle shaking. Six OTA standards (0, 10, 20, 100, 200, and 500 pg/mL) were reconstituted with deionized water. 20 μL of the six standard solutions were respectively added to the mixed solution and mixed well. The solutions were then incubated at room temperature (25 °C) for 8 h.
2.3. Synthesis of gold nanoparticles Gold nanoparticles (Au NPs, 3572 nm) were synthesized by a seed-mediated growth method. Seeds (Au NPs of 13 71.5 nm) were synthesized by citrate reduction. Briefly, 2 mL of trisodium citrate (38.8 mM) was quickly added to a boiling solution of HAuCl4 (40 mL, 0.5 mM) with vigorous stirring and refluxed until there were no more color changes in the solution. 7.5 mL of ascorbic acid (5.3 mM) was injected into the mixed solution (2 mL– 10 mM HAuCl4, 0.1 mM–10 mM AgNO3, 42.5 mL H2O, 3.75 mL seeds) with a constant-flow pump at a speed of 0.6 mL/min under vigorous stirring. To make sure the Au NPs could be well-dispersed at high ionic strength solution, bis(p-sulfonatophenyl) phenylphosphine dihydrate, dipotassium salt (BPS) was used to modify the particles. 3 mg BPS was added to 100 mL of Au NP solution and stirred at room temperature for 8 h (Loweth et al., 1999).
3. Results and discussion 3.1. Establishment of heterogeneous core-satellites chiral-aptasensor To achieve the Au core–Ag NP satellite assemblies, Au NPs (35 72 nm) and Ag NPs (8 71 nm) were functionalized with the aptamer for OTA and partially complementary sequences (Scheme 1 and Fig. S1), respectively, according to the previous method (Xu et al., 2006). The coupling molar ratio of Au NP to OTA
2.4. Synthesis of silver nanoparticles Silver nanoparticles (Ag NPs, 8 71 nm) were prepared by a routine method with modifications (Liu et al., 2009; Sun and Xia 2003; Yan et al., 2012). Typically, freshly prepared NaBH4 (0.45 mL, 0.1 M) and poly (N-vinyl-2-pyrrolidone) (PVP) (5 mL, 1% by weight) were added to 20 mL of distilled water (in a water-ice bath), followed by the simultaneous injection of AgNO3 (5 mL, 10 mM) and PVP (5 mL, 1% by weight) into the mixture using a constant-flow pump at a rate of 30 mL/h under high-speed stirring. The reaction solution was kept at 80 °C for 2 h to remove the unreacted NaBH4 and then stored at 4 °C.
Scheme 1. Scheme of chiral-aptasensor for the detection of OTA based on Au core– Ag nanoparticle satellite assemblies.
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Fig. 1. TEM images of Au core–Ag nanoparticle satellite assemblies with different concentrations of OTA. (a) 0 pg/mL, (b) 1 pg/mL, (c) 2 pg/mL, (d) 10 pg/mL, (e) 20 pg/mL, and (f) 50 pg/mL. Scale bar was 50 nm.
aptamer was 1:200, and the molar ratio of Ag NP to OTA complementary was 1:5. Then, the Au NP-OTA aptamer and Ag NP-OTA complementary were mixed together for hybridization at room temperature. As shown in Fig. S2, the number of Ag satellites per Au core gradually increased as prolonging the hybridization time. The assembly reaction mainly occurred in the first 4 h, and was completely finished 4 h later. The obtained Au core–Ag NP satellite assemblies showed a high yield of 85% according to statistics obtained by TEM, with a few individual Ag NPs remaining in the colloids (Fig. 1a and Fig. S2f). The optical properties of the Au core–Ag NP satellite assemblies were then measured. As shown in Fig. 2, two peaks of the Au core– Ag NP satellite assemblies were observed in the visible region. Both of these peaks showed a slight red shift (2 nm of red-shift), compared with individual Au NPs and Ag NPs. The slight red-shift was attributed to the surface plasmon coupling of the Au core and Ag NP satellite assemblies in a large gap (53 bp), which was consistent with a previous study (Yoon et al., 2012). In comparison, there was no red-shift for the mixture of Au NP-OTA aptamer and Ag-OTA complementary or the mixture of Au NPs and Ag NPs, which further indicated the successful fabrication of the Au core and Ag NP satellite assemblies. Besides the ultraviolet–visible properties, the Au core–Ag NP satellite assemblies were observed to display a strong CD signal in the visible range (Fig. 2 and 3a). There were two plasmonic chiroptical peaks in their CD spectra, one was at the plasmonic band of Ag (400 nm) with an intensity of 21 70.2 m deg, and the other was at the plasmonic band of Au (527 nm) with an intensity of 11.570.1 m deg. From the results shown in Fig. 2, the controls before the fabrication of Au core–Ag NP satellite assemblies showed no or weak CD signals, indicating that their plasmonic CD properties originated from the Au core–Ag
Fig. 2. Chiroptical activities of Au core–Ag nanoparticle satellite assemblies. CD (a) and UV–vis (b) spectra of Au core–Ag nanoparticle satellite assemblies, NPs modified with DNA (AuNP-OTA aptamer and Ag-OTA complementary), mixture of NPs modified with DNA (AuNP-OTA aptamer and Ag-OTA complementary), NPs (Au NPs and Ag NPs) and mixture of NPs (Au NPs and Ag NPs).
NP satellite assemblies. According to the previous published papers, the intense plasmonic CD signal was probably attributed to the asymmetric assembly of the nanostructure and the plasmon-
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Fig. 3. OTA detection based on CD chiroplasmonic technique with Au core–Ag nanoparticle satellite assemblies. (a) CD spectra for different concentrations of OTA and (b) the corresponding CD calibration curve for OTA detection. The calibration curve was obtained from ΣCD¼ (CD400 nm þ CD527 nm) as a logarithmic function of OTA concentrations.
enhancement of chiral DNAs located in the hot spot region which was confirmed by the increased intensity of the CD signals with the assembly of Au core–Ag satellites (Fig. 1 and 3a) (Xu et al., 2013; Zhou et al., 2011; Zhu et al., 2013). It may be concluded that the chiroptical activity of Au core–Ag NP satellite assemblies was related to the plasmon-enhancement of chiral DNA and the dissymmetry of the collective system. Considering the different CD signals with various degrees of assembly, a chiral-aptasensor was established for OTA detection. As shown in Scheme 1, in the absence of OTA target, OTA aptamer functionalized Au NPs were assembled with OTA complementarymodified Ag NPs, leading to the formation of Au core–Ag NP satellite assembly with the intense CD signal. However, in the presence of OTA targets, they competed with the complementary sequence modified on the surface of Ag NPs, resulting in incomplete assemblies of core–satellites with low CD signals. As shown in Fig. 1, the degree of assembly decreased with increased OTA concentration. In the absence of OTA (Fig. 1a), the structure of the Au core–Ag satellite was complete and almost no individual Ag NPs were observed. In contrast, there were no core– satellites at a high concentration of OTA (50 pg/mL, Fig. 1f). The CD and UV–vis spectra at different concentrations of the target are shown in Fig. 3a and Fig. S3, respectively. The UV–vis spectra were not different to each other, while the CD spectra were different. With increased concentration of OTA, the CD intensity (at the band
of 527 nm and 400 nm) was significantly decreased. Calibration curves were plotted with ΣCD (CD400 nm þCD527 nm) as a logarithmic function of OTA concentration, and exhibited a good linear range from 1 to 50 pg/mL with a linear relationship of R2 ¼ 0.997 (Fig. 3b). Furthermore, the LOD (LOD ¼ 3.3SD/S, where SD is the standard deviation of the response and S is the slope of the calibration curve, each data point was repeated for five times to calculate the mean value as the final point for calibration curve) was calculated to be 0.16 pg/mL, which was almost sixty times lower than that obtained by ELISA (0.01 ng/mL, Table S1). Compared with other detection methods, the LOD of the as-fabricated sensor (0.16 pg/mL) was ideal sensor for OTA detection without complex amplification procedures (Table S1). 3.2. Selectivity of the as-fabricated sensor for OTA detection To evaluate the selectivity of this assay for OTA detection, five other mycotoxins, such as deoxynivalenol (DON), aflatoxin B1 (AFB1), aflatoxin M1 (AFM1), fumonisin (FB), and ochratoxin B (OTB), were chosen for the specificity analysis. The concentration of these five mycotoxins was 1 μg/mL, which was one thousand times the concentration of OTA. All other assay procedures were the same as those for OTA detection. The ΣCD signals of OTA were lower than those of other targets (Fig. 4). Another control experiment without target was also performed, and showed no changes in CD signals, which further confirmed the high selectivity of this developed method for OTA detection. 3.3. Analysis of OTA in real samples We also demonstrated the feasibility of this method by performing recovery experiments using OTA-spiked red wine samples. The red wine samples were negative, which was confirmed by HPLC. Following pretreatment of the red wine samples (Ma et al., 2013b), OTA standard solutions were added at a final concentration of 2, 5, 10, and 20 pg/mL. As shown in Table 1, the recovery ranged from 90% to 105%, indicating the developed method possessed excellent recovery. Therefore, the developed chiral aptasensor can be used for the detection of OTA in red wine samples.
4. Conclusions Fig. 4. Selectivity of the chiral-aptasensor toward OTA against other mycotoxins. The ΣCD values of the proposed sensor after the hybridization with different targets: OTA (1 ng/mL), DON (1 μg/mL), AFB 1 (1 μg/mL), AFM 1 (1 μg/mL), FB (1 μg/mL), and OTB (1 μg/mL). Control was red wine without any targets spiked in.
In summary, we developed a novel chiral-aptasensor for the detection of OTA, based on the fabrication of chiral Au core–Ag NP satellite assemblies. The LOD was as low as 0.16 pg/mL without a
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Table 1 Recovery of OTA spiked in red wine samples (n ¼5). Sample
Spiked concentration (pg/mL)
Detected concentration Meana 7 SDb (pg/mL)
Recovery (%)
Red wine
2 5 10 20
2.17 0.2 4.5 7 0.2 9.4 7 0.6 20.4 7 0.8
105 90 94 102
a b
The mean of five experiments. SD ¼standard deviation.
complicated signal amplification procedure. The developed sensor also had high specificity and good practicability, and is promising for the determination of OTA in real samples.
Acknowledgements This work is financially supported by the Key Programs from MOST (2012BAK08B01 and 2012BAD29B04).
Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.12.021.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Building heterogeneous core–satellite chiral assemblies for ultrasensitive toxin detection Xueli Zhao, Xiaoling Wu, Liguang Xu n, Wei Ma, Hua Kuang, Libing Wang, Chuanlai Xu State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 October 2014 Received in revised form 4 December 2014 Accepted 8 December 2014 Available online 9 December 2014
A chiral-aptasensor for ochratoxin A (OTA) detection based on Au core–Ag nanoparticle satellite assemblies was fabricated for the first time. High yields of Au core–Ag NP satellite assemblies were prepared in the aqueous phase and the optical properties of the Au core–Ag NP satellite assemblies were investigated in detail. Because of the different concentrations of the OTA target, the assembly degree of the architecture varied which led to the corresponding chiral signals. The developed method for OTA detection with excellent linear range from 1 to 50 pg/mL showed high selectivity for OTA and the limit of detection as low as 0.16 pg/mL. The feasibility of this method was demonstrated by performing recovery experiment using negative red wine samples, excellent recovery ranged from 90% to 105% was achieved, and indicated promising applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: Core–satellite Circular dichroism Toxin Aptasensor Detection
1. Introduction Ochratoxin A (OTA), one of the most poisonous mycotoxins, is widely-found residue in food raw materials and may cause cancer, immunosuppression and immunotoxicity (Chen et al., 2014; Haighton et al., 2012). As OTA poses a high risk to human health, OTA detection have been attracting more attentions (Duarte et al., 2011). Conventional methods for OTA quantification are mostly based on instruments, such as liquid chromatography–tandem mass spectrometry (LC–MS/MS) and high-performance liquid chromatography connected to tandem mass spectrometry (HPLC– MS/MS) (Bazin et al., 2013). However, skilled operators, expensive instruments and complicated sample preparation have restricted their wide application. Enzyme-linked immunosorbent assay (ELISA) (Li et al., 2013) and electrochemical methods (Kuang et al., 2010; Olcer et al., 2014; Vidal et al., 2013) have also been developed for OTA biosensing. However, these methods either involve tedious processes, stringent sample pretreatments, or showed low sensitivity (Liu et al., 2008). To improve the sensitivity of OTA detection, surface-enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR) have been used and showed a limit of detection (LOD) of 1 nM (Park et al., 2014). Other studies on OTA detection have been based on signal amplification technology (such as rolling chain amplification and loop-mediated n
Corresponding author. E-mail address: [email protected] (L. Xu).
http://dx.doi.org/10.1016/j.bios.2014.12.021 0956-5663/& 2014 Elsevier B.V. All rights reserved.
isothermal amplification), however, the complexity of the operating procedure has limited their practical application and on-site OTA analysis (Chen et al., 2014; Huang et al., 2013). Nanoparticle (NP) core–satellite assemblies, a unique architecture assembled by nanomaterials, has attracted the attention of researchers (Chou et al., 2014; Mucic et al., 1998; Pal et al., 2009; Yoon et al., 2012). Lazarides and co-workers reported a reconfigurable Au core (50 nm)–Au satellite (13 nm) using DNA linkers. Based on the trypsin-mediated disassembly of the Au core (50 nm)–Au satellite (50 nm or 30 nm), Lee’s group established a colorimetric sensor for protease detection on a two-dimensional glass substrate (Waldeisen et al., 2011). Bach’s group showed a sensitive near-IR SERS sensor with a LOD below 1 nM, based on a high density of hot-spots of bottom-up core–satellite nanostructures (Zheng et al., 2013). Our previous works evaluated Au nanorod (NR) core–Au nanoparticle (NP)-satellite assemblies, investigated their chirality and bioproperties, and demonstrated their potential in biosensing (Xu et al., 2013, 2012). In recent years, chirality has increased in popularity in nanotechnology. A large number of chiral geometries such as dimers and pyramids have been reported (Wu et al., 2013; Yan et al., 2012). The generating mechanism of circular dichroism (CD) was investigated and chiral sensors for detection in the biological, food and medical fields were established (Govorov et al., 2010; Ma et al., 2013a; Wu et al., 2013). In the present study, chiral Au core– Ag NP satellite assemblies were fabricated and the chiral signals showed that this architecture had intense chiral optical response with increased the number of satellite NP, and based on this
X. Zhao et al. / Biosensors and Bioelectronics 66 (2015) 554–558
principle, the chiral aptasensor with high sensitivity and selectivity for OTA detection was developed.
2. Experimental section 2.1. Materials The thiolated DNA oligonucleotides (OTA aptamer and its complementary sequence) were manufactured by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., purified by HPLC (the purity of DNA used in this study were all greater than 98%). These oligonucleotides were dissolved in TE buffer (Shanghai Sangon) to give a final concentration of 50 μM. OTA, deoxynivalenol (DON), aflatoxin B1 (AFB1), aflatoxin M1 (AFM1), fumonisin (FB), and ochratoxin B (OTB) were purchased from SigmaAldrich. Deionized (DI) water obtained using a Milli-Q device (18.2 MΩ, Millipore, Molsheim, France) was used throughout this work. The detailed sequences of the oligonucleotides are as follows: OTA-aptamer: 5′-SH-TTTTTTTTTTGATCGGGTGTGGGTGGCGTAAAGGGAG CATCGG-3′ OTA-complementary: 5′-SH- TTTTTTTTTTCCGATGCTCCCT-3′
555
2.5. Preparation of single-stranded DNA modified-Au NPs and Ag NPs Au NPs were modified with OTA aptamer at a coupling ratio of 200 to 1. Briefly, Au NPs (35 72 nm) were concentrated ten times and then resuspended in 0.01 M phosphate buffer at a final concentration of 15 nM. Thiolated OTA aptamer (50 μM, dissolved in TE buffer) was added to the Au NPs solution (final aptamer concentration was 3 μM), 1 μL of NaCl (5 M) was added to 100 μL of the solution to increase the concentration of NaCl to 50 mM after 2 h. And then, this process was repeated at one more increment of 0.05 M NaCl thereafter until a concentration of 0.3 M NaCl was reached (Demers et al., 2002; Rosi et al., 2006). The mixed solution was left to stand for 12 h to complete the single-stranded functionalization process. The particles were centrifuged (5000 rpm, 10 min) and resuspended in 0.01 M Tris–HCl buffer, washed for three times. Ag NPs were modified with OTA complementary at a coupling ratio of 5 to 1. Ag NPs were concentrated ten times and then resuspended in 0.01 M Tris–HCl buffer at a final concentration of 100 nM. Thiolated OTA complementary (50 μM, dissolved in TE buffer) was added to the Ag NPs solution (final aptamer concentration was 0.5 μM) and left to stand for 12 h to complete the functionalization process. The particles were centrifuged (14,000 rpm, 15 min) and resuspended in 0.01 M Tris–HCl buffer, washed for three times.
2.2. Instrumentation 2.6. Fabrication of the Au core–Ag NP satellite sensor The ultraviolet–visible (UV–vis) spectrum was obtained using a UNICO 2100 PC UV–vis spectrophotometer. Transmission electron microscopy (TEM) images were acquired using a JEOL JEM-2100 operating at an acceleration voltage of 200 kV. The CD spectrum was scanned by MOS-450/AF-Circular Dichroism at a scanning speed of 1 nm/s and the path length of quartz glass cuvette was 1 cm.
Firstly, 30 μL of Au NP-OTA aptamer and 150 μL of Ag NP-OTA complementary were mixed, followed by 3 min of gentle shaking. Six OTA standards (0, 10, 20, 100, 200, and 500 pg/mL) were reconstituted with deionized water. 20 μL of the six standard solutions were respectively added to the mixed solution and mixed well. The solutions were then incubated at room temperature (25 °C) for 8 h.
2.3. Synthesis of gold nanoparticles Gold nanoparticles (Au NPs, 3572 nm) were synthesized by a seed-mediated growth method. Seeds (Au NPs of 13 71.5 nm) were synthesized by citrate reduction. Briefly, 2 mL of trisodium citrate (38.8 mM) was quickly added to a boiling solution of HAuCl4 (40 mL, 0.5 mM) with vigorous stirring and refluxed until there were no more color changes in the solution. 7.5 mL of ascorbic acid (5.3 mM) was injected into the mixed solution (2 mL– 10 mM HAuCl4, 0.1 mM–10 mM AgNO3, 42.5 mL H2O, 3.75 mL seeds) with a constant-flow pump at a speed of 0.6 mL/min under vigorous stirring. To make sure the Au NPs could be well-dispersed at high ionic strength solution, bis(p-sulfonatophenyl) phenylphosphine dihydrate, dipotassium salt (BPS) was used to modify the particles. 3 mg BPS was added to 100 mL of Au NP solution and stirred at room temperature for 8 h (Loweth et al., 1999).
3. Results and discussion 3.1. Establishment of heterogeneous core-satellites chiral-aptasensor To achieve the Au core–Ag NP satellite assemblies, Au NPs (35 72 nm) and Ag NPs (8 71 nm) were functionalized with the aptamer for OTA and partially complementary sequences (Scheme 1 and Fig. S1), respectively, according to the previous method (Xu et al., 2006). The coupling molar ratio of Au NP to OTA
2.4. Synthesis of silver nanoparticles Silver nanoparticles (Ag NPs, 8 71 nm) were prepared by a routine method with modifications (Liu et al., 2009; Sun and Xia 2003; Yan et al., 2012). Typically, freshly prepared NaBH4 (0.45 mL, 0.1 M) and poly (N-vinyl-2-pyrrolidone) (PVP) (5 mL, 1% by weight) were added to 20 mL of distilled water (in a water-ice bath), followed by the simultaneous injection of AgNO3 (5 mL, 10 mM) and PVP (5 mL, 1% by weight) into the mixture using a constant-flow pump at a rate of 30 mL/h under high-speed stirring. The reaction solution was kept at 80 °C for 2 h to remove the unreacted NaBH4 and then stored at 4 °C.
Scheme 1. Scheme of chiral-aptasensor for the detection of OTA based on Au core– Ag nanoparticle satellite assemblies.
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Fig. 1. TEM images of Au core–Ag nanoparticle satellite assemblies with different concentrations of OTA. (a) 0 pg/mL, (b) 1 pg/mL, (c) 2 pg/mL, (d) 10 pg/mL, (e) 20 pg/mL, and (f) 50 pg/mL. Scale bar was 50 nm.
aptamer was 1:200, and the molar ratio of Ag NP to OTA complementary was 1:5. Then, the Au NP-OTA aptamer and Ag NP-OTA complementary were mixed together for hybridization at room temperature. As shown in Fig. S2, the number of Ag satellites per Au core gradually increased as prolonging the hybridization time. The assembly reaction mainly occurred in the first 4 h, and was completely finished 4 h later. The obtained Au core–Ag NP satellite assemblies showed a high yield of 85% according to statistics obtained by TEM, with a few individual Ag NPs remaining in the colloids (Fig. 1a and Fig. S2f). The optical properties of the Au core–Ag NP satellite assemblies were then measured. As shown in Fig. 2, two peaks of the Au core– Ag NP satellite assemblies were observed in the visible region. Both of these peaks showed a slight red shift (2 nm of red-shift), compared with individual Au NPs and Ag NPs. The slight red-shift was attributed to the surface plasmon coupling of the Au core and Ag NP satellite assemblies in a large gap (53 bp), which was consistent with a previous study (Yoon et al., 2012). In comparison, there was no red-shift for the mixture of Au NP-OTA aptamer and Ag-OTA complementary or the mixture of Au NPs and Ag NPs, which further indicated the successful fabrication of the Au core and Ag NP satellite assemblies. Besides the ultraviolet–visible properties, the Au core–Ag NP satellite assemblies were observed to display a strong CD signal in the visible range (Fig. 2 and 3a). There were two plasmonic chiroptical peaks in their CD spectra, one was at the plasmonic band of Ag (400 nm) with an intensity of 21 70.2 m deg, and the other was at the plasmonic band of Au (527 nm) with an intensity of 11.570.1 m deg. From the results shown in Fig. 2, the controls before the fabrication of Au core–Ag NP satellite assemblies showed no or weak CD signals, indicating that their plasmonic CD properties originated from the Au core–Ag
Fig. 2. Chiroptical activities of Au core–Ag nanoparticle satellite assemblies. CD (a) and UV–vis (b) spectra of Au core–Ag nanoparticle satellite assemblies, NPs modified with DNA (AuNP-OTA aptamer and Ag-OTA complementary), mixture of NPs modified with DNA (AuNP-OTA aptamer and Ag-OTA complementary), NPs (Au NPs and Ag NPs) and mixture of NPs (Au NPs and Ag NPs).
NP satellite assemblies. According to the previous published papers, the intense plasmonic CD signal was probably attributed to the asymmetric assembly of the nanostructure and the plasmon-
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Fig. 3. OTA detection based on CD chiroplasmonic technique with Au core–Ag nanoparticle satellite assemblies. (a) CD spectra for different concentrations of OTA and (b) the corresponding CD calibration curve for OTA detection. The calibration curve was obtained from ΣCD¼ (CD400 nm þ CD527 nm) as a logarithmic function of OTA concentrations.
enhancement of chiral DNAs located in the hot spot region which was confirmed by the increased intensity of the CD signals with the assembly of Au core–Ag satellites (Fig. 1 and 3a) (Xu et al., 2013; Zhou et al., 2011; Zhu et al., 2013). It may be concluded that the chiroptical activity of Au core–Ag NP satellite assemblies was related to the plasmon-enhancement of chiral DNA and the dissymmetry of the collective system. Considering the different CD signals with various degrees of assembly, a chiral-aptasensor was established for OTA detection. As shown in Scheme 1, in the absence of OTA target, OTA aptamer functionalized Au NPs were assembled with OTA complementarymodified Ag NPs, leading to the formation of Au core–Ag NP satellite assembly with the intense CD signal. However, in the presence of OTA targets, they competed with the complementary sequence modified on the surface of Ag NPs, resulting in incomplete assemblies of core–satellites with low CD signals. As shown in Fig. 1, the degree of assembly decreased with increased OTA concentration. In the absence of OTA (Fig. 1a), the structure of the Au core–Ag satellite was complete and almost no individual Ag NPs were observed. In contrast, there were no core– satellites at a high concentration of OTA (50 pg/mL, Fig. 1f). The CD and UV–vis spectra at different concentrations of the target are shown in Fig. 3a and Fig. S3, respectively. The UV–vis spectra were not different to each other, while the CD spectra were different. With increased concentration of OTA, the CD intensity (at the band
of 527 nm and 400 nm) was significantly decreased. Calibration curves were plotted with ΣCD (CD400 nm þCD527 nm) as a logarithmic function of OTA concentration, and exhibited a good linear range from 1 to 50 pg/mL with a linear relationship of R2 ¼ 0.997 (Fig. 3b). Furthermore, the LOD (LOD ¼ 3.3SD/S, where SD is the standard deviation of the response and S is the slope of the calibration curve, each data point was repeated for five times to calculate the mean value as the final point for calibration curve) was calculated to be 0.16 pg/mL, which was almost sixty times lower than that obtained by ELISA (0.01 ng/mL, Table S1). Compared with other detection methods, the LOD of the as-fabricated sensor (0.16 pg/mL) was ideal sensor for OTA detection without complex amplification procedures (Table S1). 3.2. Selectivity of the as-fabricated sensor for OTA detection To evaluate the selectivity of this assay for OTA detection, five other mycotoxins, such as deoxynivalenol (DON), aflatoxin B1 (AFB1), aflatoxin M1 (AFM1), fumonisin (FB), and ochratoxin B (OTB), were chosen for the specificity analysis. The concentration of these five mycotoxins was 1 μg/mL, which was one thousand times the concentration of OTA. All other assay procedures were the same as those for OTA detection. The ΣCD signals of OTA were lower than those of other targets (Fig. 4). Another control experiment without target was also performed, and showed no changes in CD signals, which further confirmed the high selectivity of this developed method for OTA detection. 3.3. Analysis of OTA in real samples We also demonstrated the feasibility of this method by performing recovery experiments using OTA-spiked red wine samples. The red wine samples were negative, which was confirmed by HPLC. Following pretreatment of the red wine samples (Ma et al., 2013b), OTA standard solutions were added at a final concentration of 2, 5, 10, and 20 pg/mL. As shown in Table 1, the recovery ranged from 90% to 105%, indicating the developed method possessed excellent recovery. Therefore, the developed chiral aptasensor can be used for the detection of OTA in red wine samples.
4. Conclusions Fig. 4. Selectivity of the chiral-aptasensor toward OTA against other mycotoxins. The ΣCD values of the proposed sensor after the hybridization with different targets: OTA (1 ng/mL), DON (1 μg/mL), AFB 1 (1 μg/mL), AFM 1 (1 μg/mL), FB (1 μg/mL), and OTB (1 μg/mL). Control was red wine without any targets spiked in.
In summary, we developed a novel chiral-aptasensor for the detection of OTA, based on the fabrication of chiral Au core–Ag NP satellite assemblies. The LOD was as low as 0.16 pg/mL without a
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Table 1 Recovery of OTA spiked in red wine samples (n ¼5). Sample
Spiked concentration (pg/mL)
Detected concentration Meana 7 SDb (pg/mL)
Recovery (%)
Red wine
2 5 10 20
2.17 0.2 4.5 7 0.2 9.4 7 0.6 20.4 7 0.8
105 90 94 102
a b
The mean of five experiments. SD ¼standard deviation.
complicated signal amplification procedure. The developed sensor also had high specificity and good practicability, and is promising for the determination of OTA in real samples.
Acknowledgements This work is financially supported by the Key Programs from MOST (2012BAK08B01 and 2012BAD29B04).
Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.12.021.
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