Biosensors and Bioelectronics 68 (2015) 783–790
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Magnetic-fluorescent-targeting multifunctional aptasensorfor highly sensitive and one-step rapid detection of ochratoxin A Chengquan Wang a,c, Jing Qian b, Kan Wang b, Kun Wang b,n, Qian Liu b, Xiaoya Dong b, Chengke Wang a, Xingyi Huang a,n a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China c Changzhou College of Information Technology, Changzhou 213164, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 5 December 2014 Received in revised form 5 February 2015 Accepted 6 February 2015 Available online 7 February 2015
A multifunctional aptasensor for highly sensitive and one-step rapid detection of ochratoxin A (OTA), has been developed using aptamer-conjugated magnetic beads (MBs) as the recognition and concentration element and a heavy CdTe quantum dots (QDs) as the label. Initially, the thiolated aptamer was conjugated on the Fe3O4@Au MBs through Au–S covalent binding. Subsequently, multiple CdTe QDs were loaded both in and on a versatile SiO2 nanocarrier to produce a large amplification factor of hybrid fluorescent nanoparticles (HFNPs) labeled complementary DNA (cDNA). The magnetic-fluorescent-targeting multifunctional aptasensor was thus fabricated by immobilizing the HFNPs onto MBs’ surface through the hybrid reaction between the aptamer and cDNA. This aptasensor can be produced at large scale in a single run, and then can be conveniently used for rapid detection of OTA through a one-step incubation procedure. The presence of OTA would trigger aptamer-OTA binding, resulting in the partial release of the HFNPs into bulk solution. After a simple magnetic separation, the supernatant liquid of the above solution contained a great number of CdTe QDs produced an intense fluorescence emission. Under the optimal conditions, the fluorescence intensity of the released HFNPs was proportional to the concentration of OTA in a wide range of 15 pg mL 1 –100 ng mL 1 with a detection limit of 5.4 pg mL 1 (S/ N ¼3). This multifunctional aptasensor represents a promising path toward routine quality control of food safety, and also creates the opportunity to develop aptasensors for other targets using this strategy. & 2015 Elsevier B.V. All rights reserved.
Keywords: Aptasensor Multifunctional Signal amplification Ochratoxin A Rapid detection
1. Introduction About 25% of the world’s agricultural commodities are contaminated by mycotoxins to a certain degree during crop growth, harvest, storage, or processing (Al-Taher et al., 2013). Ochratoxin A (OTA), one of the most toxic mycotoxins, is known to cause nephrotoxic, hepatotoxic, neurotoxic, teratogenic, and immunotoxic effects to human beings (Hayat et al., 2013a; Wu et al., 2012). Since OTA cannot be destroyed even when cooked at quite high temperature, it can easily enter the food chain such as those reached during baking bread or breakfast cereal production (Turner et al., 2009). Considering its severe toxic effects, the European Commission has fixed the maximum tolerated levels of OTA at 5 μ g kg 1 for raw cereal grains and 2 μg kg 1 for products derived n
Corresponding authors. Fax: þ 86 51188797308. E-mail addresses: [email protected] (K. Wang), [email protected] (X. Huang). http://dx.doi.org/10.1016/j.bios.2015.02.008 0956-5663/& 2015 Elsevier B.V. All rights reserved.
from cereals (Hayat et al., 2013b; Yang et al., 2012). The intensifying legislative framework worldwide as well as the increasing awareness about OTA has aroused the need for efficient analytical methods to ensure food safety issues and quality control (Hayat et al., 2012; Zhao et al., 2014). Chromatographic-based methods such as high-performance liquid chromatography (HPLC) (Flajs et al., 2009; Ghali et al., 2008) and LC coupled with fluorescence (Cigic and Prosen, 2009) or mass (Aresta et al., 2006) spectrometry have been extensively used as the confirmatory method in recent years. In spite of the numerous advantages that such techniques offer, their practical applications are often limited due to the associated high cost, complicated pretreatment procedures of samples, and requirement of technical skills and expensive instruments (Ahmed et al., 2007; Prabhakar et al., 2011). As an alternative, enzyme-linked immunosorbent assay (ELISA) (Yu et al., 2011; Turner et al., 2009) and other antibody-based electrochemical (Bone et al., 2010), fluorescent (Prieto-Simon et al., 2007), and surface plasmon resonance (Shankaran et al.,
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2007) immunosensors are also constructed for OTA detection. However, the preparation of antibody is a very long, complicated, and labor-intensive process via animal immunization. Besides, the antibody is susceptible to external conditions, especially temperature and preservation conditions (Rhouati et al., 2013). Aptamers are single stranded DNA or RNA molecules with certain sequence selected by the classic selection of ligands by exponential enrichment technique (Ma et al., 2013). Compared to antibody, aptamer is characterized by high affinity and specificity, smaller size, ease of synthesis, and high stability (Tong et al., 2012). Besides, aptamer can easily be modified with a variety of functionalities such as thiol groups, fluorescent labels, biotin, and enzymes, enabling the design of flexible sensing platforms (Hansen et al., 2006). Since the development of an aptamer specific for OTA by Cruz-Aguado and Penner (2008), many aptasensors for detecting OTA have been described combined with fluorescent (Chen et al., 2012; Sheng et al., 2011), electrochemical (Bonel et al., 2011; Zhang et al., 2012), and colorimetric transducers (Yang et al., 2011). These works open a new way for the detection of OTA mycotoxin, but the highly sensitive detection of OTA is difficult to achieve by a basic aptasensor with the 1:1 labeling ratio of the signaling label and OTA molecule (Lu et al., 2010). Signal amplification is an efficient way to improve the sensitivity of an aptasensor. Some signal amplification strategies such as rolling circle amplification (Tong et al., 2012; Huang et al., 2013), loop-mediated isothermal amplification (Yuan et al., 2014; Xie et al., 2014), and target recycling amplification performed by enzymes (Tong et al., 2011) have been proposed. Though these methods offer advantages in terms of signal amplification, their practical use might be hampered by their high operation cost, complicated operating process or rigorous operating conditions (Deng et al., 2009). Besides, aptamer-functionalized nanomaterials have been reported to amplify the signal in development of aptasensors (Kuang et al., 2010). Our group have already developed two electrochemical aptasensors for ultrasensitive OTA detection by employing Au nanoparticles (NPs) (Yang et al., 2014) and Au NPs/graphene nanocomposites (Jiang et al., 2014). Using these nanomterials might overcome some of the problems due to their high stability, low cost, and labeling convenience. In these proposals, however, each sample measurment needs tens hours aptasensor fabrication, which does not satisfy the simple, rapid, and high through-put detection requriments for current assays. Herein, magnetically controllable aptasensor combined with a heavy CdTe quantum dots (QDs) label, has been developed for the rapid and highly sensitive detection of trace OTA. Fe3O4@Au magnetic beads (MBs) with Au shell were synthesized for the immobilization of the thiolated aptamers. Multiple CdTe QDs were encapsulated in and coated on a versatile SiO2 nanocarrier to produce a large amplification factor of hybrid fluorescent NPs (HFNPs) labeled complementary DNA (cDNA) via a condensation reaction. The aptamer conjugated with MBs was then hybridized with cDNA labeled with HFNPs to generate a magnetic-fluorescent-targeting multifunctional aptasensor. The introduction of OTA would trigger aptamer-OTA binding, resulting in the partial release of the cDNA-HFNPs into bulk solution. After a simple magnetic separation, the supernatant liquid of the above solution contained a great number of CdTe QDs produced an increasing fluorescence emission with an increasing OTA concentration
2. Experimental section 2.1. Reagents and materials 3-mercaptopropionic acid (MPA, 99%), tetraethylorthosilicate (TEOS), Tris(hydroxymethyl)aminomethane (Tris), Tris (2-
chloroethyl) phosphate (TCEP), and 6-mercapto-1-hexanol (MCH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), poly (diallyldimethyl-ammonium chloride) (PDDA), (3-aminopropyl) triethoxysilane (APTS), OTA, fumonisin B1 (FB1), and aflatoxins B1 (AFB1) were obtained from Sigma-Aldrich. cDNA: 5ʹ-CCT TTA CGC CAC CCA CAC CCG ATC-NH2-3ʹ and aptamer: 5ʹ-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA–SH-3ʹ were purchased from Sangon Biotech Co., Ltd. (China). DNA oligonucleotide stock solutions were prepared with 10 mM Tris–HCl buffer (pH 7.4, containing 10 mM NaCl and 5 mM MgCl2) and kept frozen in dark. The Fe3O4 microspheres, the core–shell Fe3O4@Au MBs, and the MPAcapped CdTe QDs were prepared according to the descriptions of Wan et al. (2012), Wu et al. (2007), and Zhang et al. (2010), respectively (Supporting information). Double-distilled water was used throughout the study. 2.2. Instrumentation The morphology of the samples was checked by transmission electron microscopy (TEM) technique (JEOL 2100, JEOL, Japan). X-ray diffraction (XRD) spectra were carried out with a Bruker D8 ADVANCE diffractometer (Germany) with Cu Kα (λ ¼ 1.5406 Å) radiation. X-ray photoelectron spectroscopy (XPS) was performed on ESCALAB 250 multitechnique surface analysis system (Thermo Electron Co., USA). UV–vis absorption spectra were measured by UV-2450 spectrophotometer (Shimadzu, Japan). Fluorescence spectra were recorded on a Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan). Zeta potential was obtained using a Malvern Zetasizer Nano ZS instrument (ZEN 2600, England). All the photographs were taken using a Canon digital camera (IXUS 230 HS, China) under the illumination of a 365 nm UV lamp. 2.3. Preparation of aptamer-coupled Fe3O4@Au MBs Aptamer coupled Fe3O4@Au MBs (MB-aptamer) bioconjugations were synthesized according to previous reports (Zhou et al., 2013). Intially, 500 μL of 100 μM aptamer was added to 500 μL of 10 mM Tris–HCl buffer containing 100 mM TCEP to activate the aptamer for 1 h at 37 °C in a reciprocating oscillator. After removing excess TCEP, the freshly activated aptamer was added to 35 mL of Fe3O4@Au MBs and sonicated for 30 s, and then incubated for 30 min with gentle shaking at 37 °C. After the salt aging according to the description of Zhou et al. (2013), the MBaptamer bioconjugations were separated readily by a magnet to remove excess unbound aptamers. Finally, the MB-aptamer bioconjugations were treated with 35 mL of 2 μM MCH and kept at 37 °C for 1 h, again under stirring. After magnetic separation and washing, the resultant MB-aptamer bioconjugations were then redispersed in 35 mL of Tris–HCl buffer and stored at 4 °C for later use. 2.4. Preparation of cDNA-conjugated QD@SiO2@QD HFNPs Synthesis of the core–satellite QD@SiO2@QD HFNPs has been described in detail in Supporting information. To conjugate cDNA on the QD@SiO2@QD HFNPs, 8 mL of 10 mM Tris–HCl buffer containing 1 mM EDC and 1 mM NHS was added to 15 mL of the resulting QD@SiO2@QD HFNPs dispersion. After shaking for 1 h, 500 μL of 100 μM cDNA was added. After shaking at 37 °C overnight, the cDNA-conjugated QD@SiO2@QD HFNPs (cDNA-HFNPs) was centrifuged at 6500 rpm for 5 min to remove excess cDNA, and then redispersed in 15 mL of Tris–HCl buffer and stored at 4 °C for further use.
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2.5. Preparation of the multifunctional aptasensor The mixture of MB-aptamer and cDNA-HFNPs was keeping shaken for 1 h at 37 °C with the help of reciprocating oscillator. After magnetic separation and washing, the MB-aptamer/cDNAHFNPs bioconjugations were redispersed in 40 mL Tris–HCl buffer and used as the magnetic-fluorescent-targeting multifunctional aptasensor in further study. 2.6. Analytical procedure 400 μL of the resulting MB-aptamer/cDNA-HFNPs bioconjugations was added to a series of parallel centrifuge tubes with numbers. Then, 100 μL of OTA containing solution with various concentrations was added to the tubes. After incubation at 37 °C for 1 h, measured supernatant liquid was then separated by a magnetic field and washing with Tris–HCl buffer. Then, the tansformed supernatant liquid containing different amount of HFNPs was dilute to a final volume of 2 mL and its fluorescence (FL) intensity was recorded accordingly.
3. Results and discussion 3.1. Aptasensor fabrication and detection principle The whole process for the multifunctional aptasensor construction is illustrated in Scheme 1A. Specifically, the QD@SiO2 @QD HFNPs were synthesized by a multistep procedure involving the synthesis of CdTe QDs doped silica cores (steps 1 and 2), modification of the silica surface by PDDA (step 3), and final
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planting of the CdTe QDs on the surface of QD@SiO2 to botain QD@SiO2@QD HFNPs (step 4). The zeta potential of QD@SiO2 was – 25.9 mV, which indicated that the surface of the QD@SiO2 terminated by hydroxyl groups was negatively charged at neutral pH. The modification of the positively charged PDDA on QD@SiO2 switched the zeta potential to a positive value of 36.8 mV. The negatively charged MPA-capped CdTe QDs with the zeta potential of –34.1 mV were subsequently tethered electrostatically to the QD@SiO2 surface to result in QD@SiO2@QD HFNPs. By using the EDC/NHS chemistry, preparation of cDNA-HFNPs was achieved through the covalent binding interactions between the amino groups of cDNA and the carboxylic groups on the surface of MPAcapped CdTe QDs (step 5). After the formation of the duplex structure between aptamer on Fe3O4@Au MBs and cDNA labeled with QD@SiO2@QD HFNPs, the multifunctional aptasensor (MBaptamer/cDNA-HFNPs) was thus successful produced through the hybridization event (step 6). The working principle of the aptasensor is illustrated in Scheme 1B. Without OTA, the resulting MB-aptamer/cDNA-HFNPs can be separated readily by a magnet placed under the centrifuge tube. Therefore, the supernatant liquid contains no QD@SiO2@QD HFNPs and thus no fluorescence emission can be detected. However, the incubation of OTA with MB-aptamer/cDNA-HFNPs would trigger aptamer-OTA binding, resulting in the formation of the aptamer-OTA complex. This caused some cDNA-HFNPs dissociating from the Fe3O4@Au MBs. The more OTA molecules in the detection system, the more cDNA-HFNPs were combined with the target and released into bulk solution. Therefore, the FL intensity generated from the QD@SiO2@QD HFNPs in the supernatant liquid as a function of OTA concentration was measured accordingly. Since MB-aptamer/cDNA-HFNPs can be prepared at large scale in a
Scheme 1. Schematic illustration of the fabrication of the multifunctional aptasensor with a heavy CdTe quantum dots label (A) and its working principle for OTA detection (B).
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Fig. 1. TEM images of the as-prepared Fe3O4 microspheres (A) and Fe3O4@Au MBs (B), (C) XRD patterns of the as-prepared Fe3O4 microspheres (a) and Fe3O4@Au MBs (b), and (D) the UV–vis spectra of Fe3O4 microspheres (a) and Fe3O4@Au MBs (b) dispersed in water.
single run, this aptasensing system can be conveniently used for rapid and high through-put OTA detection through a one-step incubation procedure followed by a simple magnetic separation. 3.2. Characterization of the Fe3O4@Au MBs One can see that Fe3O4 microspheres were identifiable with an average diameter of 450 nm (Fig. 1A). The functionality of Fe3O4 microspheres with APTS would produce the Fe3O4 microspheres with amine groups. The amine groups on the APTS-coated Fe3O4 surface preferentially bind noble metals such as Au NPs, instead of other species (Freeman et al., 1995). After reduction of the adsorbed AuCl4– ions by citrate, an additional shell-building Au layer was decorated on the Fe3O4 microspheres’ surface, confirming the successful formation of the (Fe3O4)core–Aushell MBs (Fig. 1B). The as-prepared core–shell Fe3O4@Au MBs with an adaitional Au layer can improve the stability of the pristine Fe3O4 microspheres in biological conditions and provides a friendly microenvironment for efficiently covalent binding with the thiolated aptamer via the well-understood Au–S chemistry (Wu et al., 2007). The coated Au shell was further confirmed by typical XRD patterns (Fig. 1C). The Fe3O4 microspheres (curve a) exhibited diffraction peaks centered at 2θ values of 30.1°, 35.5°, 43.1°, 53.5°, 57.0°, and 62.6°, which were indexed to (220), (311), (400), (422), (511), and (440) planes of cubic structure of Fe3O4 (JCPDS no. 790419), respectively (Wan et al., 2012). Fe3O4@Au MBs (curve b) exhibited all the diffraction peaks of pristine Fe3O4 microspheres but with four additional diffraction peaks at 38.1°, 44.3°, 64.5°, and 77.6°, which were indexed to the (111), (200), (220), and (311) planes for the face-centered-cubic gold (JCPDS no. 04-0784) planes
of gold cubic phase, respectively (Zhang et al., 2008). This information indicated that a thin layer of Au shell, which was not thick enough to overlay the Fe3O4 diffraction peaks, was successfully deposited onto the surface of the Fe3O4 microspheres by the seed growth method. There is no obvious absorbance peak was observed for the Fe3O4 microspheres suspension (curve a in Fig. 1D). When the layer of Au was deposited onto the surface of Fe3O4 microspheres, an extra surface plasmon peak was clearly observed at 554 nm for the Fe3O4@Au MBs (curve b in Fig. 1D). This value is different from that previously reported for Au NPs with a value of 522 nm (Yang et al., 2014). The red shift of absorption peak in the spectrum of Fe3O4@Au MBs suspension can be ascribed to the fact that AuNPs bound strongly on the surface of Fe3O4 microspheres (Cui et al., 2011). The resulting Fe3O4@Au MBs displayed obvious color change before (photograph a in Fig. 1D) and after (photograph b in Fig. 1D) the Au shell building. As a result of the magnetite content, the resulting Fe3O4@Au MBs can quickly respond to external magnetic field (photograph c in Fig. 1D) in 30 s and quickly be redispersed homogeneously by hand-shaking. The excellent magnetic property enables Fe3O4@Au MBs to be used for simple and efficient magnetic separation. 3.3. Characterization of the QD@SiO2@QD HFNPs Fig. 2A shows a TEM image of QDs@SiO2 NPs with an average diameter of 150 nm. As can be observed, these QDs@SiO2 NPs are well-dispersed with a chemically clean and homogenized structure. It can be observed from QD@SiO2@QD HFNPs that a large number of dark dots have been coated on the surface of each silica
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Fig. 2. TEM images of the as-prepared QD@SiO2 (A) and QD@SiO2@QD (B), (C) XPS spectrum of the QD@SiO2@QD HFNPs, the UV–vis (D) and fluorescence (E) spectra of QD@SiO2 (a) and QD@SiO2@QD (b), and the corresponding photographs under daylight (inset of D) and UV illumination (inset of E).
core with excellent dispersivity (Fig. 2B). Furthermore, no bare QDs@SiO2 or free CdTe QDs was observed, which suggested the complete formation of the QD@SiO2@QD HFNPs. The wide scan XPS spectra of QD@SiO2@QD HFNPs (Fig. 2C) clearly indicated that the sample was composed of Cd, Te, Si, O, C, N, and S elements, and no peaks of other elements were observed. An absorption peak at 610 nm and an emission peak at 670 nm were observed for both QDs@SiO2 NPs (curve a in Fig. 2D) and QD@SiO2@QD HFNPs (curve b in Fig. 2D). They displayed a similar color as CdTe QDs in water (inset a in Fig. S1A) under daylight (inset in Fig. 2D) and emitted an intense red fluorescence under UV illumination (inset in Fig. 2E), consistent with that of CdTe QDs itself (inset b in Fig. S1A). It is interesting to notice a slight red shift in the peak emission of the QDs@SiO2 and QD@SiO2@QD HFNPs when compared with the CdTe QDs aqueous solution (Fig. S1A), might due to the strong binding interactions between the CdTe QDs and the silica. Moreover, there was 1.8 fold enhancement in the FL intensity for QD@SiO2@QD HFNPs (curve b in Fig. 2E) compared to that of the QDs@SiO2 NPs (curve a in Fig. 2E) at the same concentration of the silica spheres. That is
largely attributed to the fact that each QD@SiO2@QD HFNP can carry a large number of CdTe QDs not only in the silica core but also on the silica surface. As a label, this kind of QD@SiO2@QD HFNPs would be hugely beneficial for signal amplification. 3.4. Fabrication of the multifunctional aptasensor Compared with the spectrum of Fe3O4@Au MBs suspension (curve b in Fig. 1D), an additional peak was observed at 262 nm for MB-aptamer bioconjugations (curve b in Fig. 3A), similar to that of the free aptamer solution (curve a in Fig. 3A), indicating the successful coupling of aptamer on the Fe3O4@Au MBs surface. High-resolution XPS spectrum of P 2p was ascertained to confirm whether the amino-modified cDNA was conjugated successfully to the surface of the QD@SiO2@QD HFNPs. There was no clear P 2p characterized signal was observed in the scanning region for QD@SiO2@QD without cDNA coupling (curve a in Fig. 3B). In contrast, the normalized XPS P 2p line has a maximum signal at 133.1 eV for the cDNA-HFNPs bioconjugates in the same region (curve b in Fig. 3B), coming from the phosphate backbone in the
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Fig. 3. (A) The UV–vis spectra of free aptamer (a) and MB-aptamer (b), (B) XPS spectra of P 2p for QD@SiO2@QD before (a) and after (b) cDNA conjugation, (C) the fluorescence spectrum of cDNA-HFNPs, (D) the fluorescence spectra and typical photographs (inset) of Fe3O4@Au MBs suspension (a) and the MB-aptamer/cDNA-HFNPs bioconjugations (b).
cDNA (Wang et al., 2006). The conjugation of cDNA did not affect the fluorescence emission of QD@SiO2@QD obviously (Fig. 3C). The cDNA-HFNPs bioconjugation was homogeneous and bright red under UV irradiation (inset in Fig. 3C). The MB-aptamer displayed no emission peak in the scanning region without cDNA-HFNPs coupling (curve a in Fig. 3D). Because of the magnetic Fe3O4 core, the MB-aptamer conjugation can be directed to specific locations when manipulated by an external magnetic field, indeed, there was no red fluorescence was observed from the aggregations on the cuvette wall under UV irradiation (inset a in Fig. 3D). In contrast, the resultant MB-aptamer/ cDNA-HFNPs bioconjugations (curve b in Fig. 3D) displayed a strong fluorescence emission peak at 670 nm, consistent with that of the cDNA-HFNPs (Fig. 3C). Once the magnet is placed beside the vial, the MB-aptamer/cDNA-HFNPs bioconjugations quickly move and accumulate near it within few minutes, one can observe the bright red fluorescence of the aggregations on the cuvette wall with the bulk solution clear and transparent (inset b in Fig. 3D). With removal of the external magnet and hand-shaking, the aggregations would be rapidly redispersed again. Based on above experimental results, a magnetic-fluorescent-targeting multifunctional aptasensor for rapid OTA detection has been constructed successfully. The combination of magnetic, fluorescent, and targeting objects in a single aptasensing system is of extraordinary interest lies in its multifunctional applications where it is necessary to monitor and manipulate at the same time. 3.5. Optimization of detection conditions To generate a rapid-response and sensitive aptasensor with a low detection limit for OTA, it is significant to optimize important parameters involved in the aptasensor fabrication and detection process. Following the routine hybridization time, from 10 to 80 min were investigated and displayed in Fig. 4A. The FL intensity
of MB-aptamer/cDNA-HFNPs increased dramatically with the growth of the time before 60 min, and then leveled off at times longer than 60 min, so 60 min was used as the optimized time required for hybridization of cDNA and aptamer. Since DNA hybridization is a temperature-dependent process, the solution temperature for the hybridization between cDNA and aptamer was also optimized. Fig. 4B indicated that the FL intensity of MB-aptamer/cDNA-HFNPs reached the maximal value when the temperature was 37 °C, which was then used as the optimized temperature for the DNA hybridization. Another impomtant factor that affects DNA hybridization is the pH value of the supporting electrolyte. Based on physiological conditions, the solution pH values ranging from 6.0 to 8.5 were tested and the results shown in Fig. 4C. It is noted that the best performance was obtained in the Tris–HCl buffer solution with a pH value of 7.4. Since the reaction time of OTA with its aptamer was closely related to the released QD@SiO2@QD HFNPs in the bulk solution, the effect of the binding time greatly influenced the final sensing of the target molecules. As shown in Fig. 4D, it was apparent that the FL intensity of the released QD@SiO2@QD HFNPs obviously increased with the increasing binding time from 10 to 60 min and then reached a plateau in 60 min. This suggested that 60 min was enough and thus chosen as the incubation time in the following detection. 3.6. Analytical performance of the aptasensor Under the optimized conditions, a simple, rapid, and sensitive aptasensor for the fluorescence detection of OTA was developed. As expected, the intensity of the emission at 670 nm was increased successively upon the incubation with an increasing amount of OTA (Fig. 5A). The FL intensity ceased to increase beyond 500 ng mL 1 of OTA, indicating the binding reaction between OTA and its aptamer was nearly saturated at this concentration. By analyzing the FL intensity (I) with the concentrations of OTA
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2015), but lower than that of the recently reported electrochemical methods using rolling circle amplification (Tong et al., 2012; Huang et al., 2013), loop-mediated isothermal amplification (Yuan et al., 2014; Xie et al., 2014), and target recycling amplification (Tong et al., 2011). However, this proposal provides a simple and rapid way for parallel OTA detection through a one-step incubation procedure. 3.7. Selectivity and reproducibility The selectivity of this aptasensor was evaluated by the comparison of the sensing results of FB1, AFB1, and OTA. As shown in Fig. S2, the response signals to FB1 and AFB1 were neglectable
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(Fig. 5B), a good linear relationship was obtained between the FL intensities and the logarithm of the OTA concentrations (inset in Fig. 5B). The linear equation is I ¼–130.7þ 193.7 log (c/ng mL 1) (R2 ¼ 0.9895) over a wide concentration range of 15 pg mL 1–100 ng mL 1. The limit of detection (LOD) was calculated to be 5.4 pg mL 1 based on S/N ¼3. The analytical performances of the present aptasensor along with other aptasensors developed for OTA in literatures were all summarized in Table S1. As can be seen, the proposed aptasensor exhibits a broader linear range and its sensitivity is comparable to or higher than the corresponding values observed in most of the works (Bonel et al., 2011; Chen et al., 2012; Guo et al., 2011; Yang et al., 2011; Prabhakar et al., 2011; Wei et al., 2015; Zhu et al., 2015; Wang et al.,
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Fig. 5. (A) Fluorescence spectra of the released HFNPs corresponding to various OTA concentrations (from a to n: 0, 0.015, 0.05, 0.1, 0.5, 1, 5, 10, 30, 50, 100, 200, 300, and 500 ng mL 1). (B) The FL intensity in (A) was plotted as a function of concentration of OTA. Inset: the calibration curve of the OTA detection.
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with the concentration of 200 ng mL 1, while an obvious increase in response was observed for OTA with a concentration of 50 ng mL 1. This result suggested that the aptasensor possessed high selectivity. The reproducibility of the aptasensor was also investigated at the OTA concentration of 50 ng mL 1. The relative standard deviation (RSD) for five measurements was 5.3%, indicating that the designed aptasensor for OTA detection was highly reproducible. 3.8. Analytical application in real samples To further investigate the potential practical applications of the proposed method, this aptasensor was applied to peanut (without shell) from the local supermarket. The preparation of the real testing samples (Supporting information) was according to the descriptions of Chen et al. (2012) and Luna et al. (2013). Table S2 summarized the analytical results of OTA determination in the real peanut samples by using the as-prepared aptasensor. As can be seen, the recoveries of the spiked samples ranged from 96.0 up to 99.7%, with RSD lower than 6.4%, demonstrating that this multifunctional aptasensor can be used for OTA detection in real samples with the satisfied results.
4. Conclusions In this study, we describe the very promising and fascinating development of a highly sensitive aptasensor that combines magnetic-fluorescent-targeting multifunctionality. Target OTA can be concentrated by the MB-aptamer and the fluorescence signal can be effectively amplified by using a heavy CdTe QDs label loaded both in and on SiO2 nanocarrier. The use of core–shell Fe3O4 @Au MBs provided not only a friendly microenvironment for efficiently covalent bind with thiolated aptamer but also an efficient method for the separation of the HFNPs labels with the help of a magnetic field. Under optimal conditions, the correlation between OTA concentration and the fluorescence signal from the released QD@SiO2@QD labels was found to be linear from 15 pg mL 1 to 100 ng mL 1 with a LOD of 5.4 pg mL 1. As the MB-aptamer/ cDNA-HFNPs bioconjugations can be produced at large scale in a single run, this aptasensing system can be conveniently used for rapid OTA detection through a one-step incubation procedure followed by a simple magnetic separation. The aptasensor was successfully employed for detecting OTA in peanut samples, validating their great application potential in food quality control.
Acknowledgements This research work was supported by the National Natural Science Foundation of China (Nos. 21405063, 21175061, 21375050, and 31071549), the Natural Science Foundation of Jiangsu province (No. BK20130481), Special Fund for Agro-scientific Research in the Public Interest (No. 201003008-04), Qinglan Project, Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 14KJA550001), the Program Sponsored for Scientific Innovation Research of College Graduate in Jiangsu Province (KYLX_1072), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.02.008.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Magnetic-fluorescent-targeting multifunctional aptasensorfor highly sensitive and one-step rapid detection of ochratoxin A Chengquan Wang a,c, Jing Qian b, Kan Wang b, Kun Wang b,n, Qian Liu b, Xiaoya Dong b, Chengke Wang a, Xingyi Huang a,n a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China c Changzhou College of Information Technology, Changzhou 213164, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 5 December 2014 Received in revised form 5 February 2015 Accepted 6 February 2015 Available online 7 February 2015
A multifunctional aptasensor for highly sensitive and one-step rapid detection of ochratoxin A (OTA), has been developed using aptamer-conjugated magnetic beads (MBs) as the recognition and concentration element and a heavy CdTe quantum dots (QDs) as the label. Initially, the thiolated aptamer was conjugated on the Fe3O4@Au MBs through Au–S covalent binding. Subsequently, multiple CdTe QDs were loaded both in and on a versatile SiO2 nanocarrier to produce a large amplification factor of hybrid fluorescent nanoparticles (HFNPs) labeled complementary DNA (cDNA). The magnetic-fluorescent-targeting multifunctional aptasensor was thus fabricated by immobilizing the HFNPs onto MBs’ surface through the hybrid reaction between the aptamer and cDNA. This aptasensor can be produced at large scale in a single run, and then can be conveniently used for rapid detection of OTA through a one-step incubation procedure. The presence of OTA would trigger aptamer-OTA binding, resulting in the partial release of the HFNPs into bulk solution. After a simple magnetic separation, the supernatant liquid of the above solution contained a great number of CdTe QDs produced an intense fluorescence emission. Under the optimal conditions, the fluorescence intensity of the released HFNPs was proportional to the concentration of OTA in a wide range of 15 pg mL 1 –100 ng mL 1 with a detection limit of 5.4 pg mL 1 (S/ N ¼3). This multifunctional aptasensor represents a promising path toward routine quality control of food safety, and also creates the opportunity to develop aptasensors for other targets using this strategy. & 2015 Elsevier B.V. All rights reserved.
Keywords: Aptasensor Multifunctional Signal amplification Ochratoxin A Rapid detection
1. Introduction About 25% of the world’s agricultural commodities are contaminated by mycotoxins to a certain degree during crop growth, harvest, storage, or processing (Al-Taher et al., 2013). Ochratoxin A (OTA), one of the most toxic mycotoxins, is known to cause nephrotoxic, hepatotoxic, neurotoxic, teratogenic, and immunotoxic effects to human beings (Hayat et al., 2013a; Wu et al., 2012). Since OTA cannot be destroyed even when cooked at quite high temperature, it can easily enter the food chain such as those reached during baking bread or breakfast cereal production (Turner et al., 2009). Considering its severe toxic effects, the European Commission has fixed the maximum tolerated levels of OTA at 5 μ g kg 1 for raw cereal grains and 2 μg kg 1 for products derived n
Corresponding authors. Fax: þ 86 51188797308. E-mail addresses: [email protected] (K. Wang), [email protected] (X. Huang). http://dx.doi.org/10.1016/j.bios.2015.02.008 0956-5663/& 2015 Elsevier B.V. All rights reserved.
from cereals (Hayat et al., 2013b; Yang et al., 2012). The intensifying legislative framework worldwide as well as the increasing awareness about OTA has aroused the need for efficient analytical methods to ensure food safety issues and quality control (Hayat et al., 2012; Zhao et al., 2014). Chromatographic-based methods such as high-performance liquid chromatography (HPLC) (Flajs et al., 2009; Ghali et al., 2008) and LC coupled with fluorescence (Cigic and Prosen, 2009) or mass (Aresta et al., 2006) spectrometry have been extensively used as the confirmatory method in recent years. In spite of the numerous advantages that such techniques offer, their practical applications are often limited due to the associated high cost, complicated pretreatment procedures of samples, and requirement of technical skills and expensive instruments (Ahmed et al., 2007; Prabhakar et al., 2011). As an alternative, enzyme-linked immunosorbent assay (ELISA) (Yu et al., 2011; Turner et al., 2009) and other antibody-based electrochemical (Bone et al., 2010), fluorescent (Prieto-Simon et al., 2007), and surface plasmon resonance (Shankaran et al.,
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2007) immunosensors are also constructed for OTA detection. However, the preparation of antibody is a very long, complicated, and labor-intensive process via animal immunization. Besides, the antibody is susceptible to external conditions, especially temperature and preservation conditions (Rhouati et al., 2013). Aptamers are single stranded DNA or RNA molecules with certain sequence selected by the classic selection of ligands by exponential enrichment technique (Ma et al., 2013). Compared to antibody, aptamer is characterized by high affinity and specificity, smaller size, ease of synthesis, and high stability (Tong et al., 2012). Besides, aptamer can easily be modified with a variety of functionalities such as thiol groups, fluorescent labels, biotin, and enzymes, enabling the design of flexible sensing platforms (Hansen et al., 2006). Since the development of an aptamer specific for OTA by Cruz-Aguado and Penner (2008), many aptasensors for detecting OTA have been described combined with fluorescent (Chen et al., 2012; Sheng et al., 2011), electrochemical (Bonel et al., 2011; Zhang et al., 2012), and colorimetric transducers (Yang et al., 2011). These works open a new way for the detection of OTA mycotoxin, but the highly sensitive detection of OTA is difficult to achieve by a basic aptasensor with the 1:1 labeling ratio of the signaling label and OTA molecule (Lu et al., 2010). Signal amplification is an efficient way to improve the sensitivity of an aptasensor. Some signal amplification strategies such as rolling circle amplification (Tong et al., 2012; Huang et al., 2013), loop-mediated isothermal amplification (Yuan et al., 2014; Xie et al., 2014), and target recycling amplification performed by enzymes (Tong et al., 2011) have been proposed. Though these methods offer advantages in terms of signal amplification, their practical use might be hampered by their high operation cost, complicated operating process or rigorous operating conditions (Deng et al., 2009). Besides, aptamer-functionalized nanomaterials have been reported to amplify the signal in development of aptasensors (Kuang et al., 2010). Our group have already developed two electrochemical aptasensors for ultrasensitive OTA detection by employing Au nanoparticles (NPs) (Yang et al., 2014) and Au NPs/graphene nanocomposites (Jiang et al., 2014). Using these nanomterials might overcome some of the problems due to their high stability, low cost, and labeling convenience. In these proposals, however, each sample measurment needs tens hours aptasensor fabrication, which does not satisfy the simple, rapid, and high through-put detection requriments for current assays. Herein, magnetically controllable aptasensor combined with a heavy CdTe quantum dots (QDs) label, has been developed for the rapid and highly sensitive detection of trace OTA. Fe3O4@Au magnetic beads (MBs) with Au shell were synthesized for the immobilization of the thiolated aptamers. Multiple CdTe QDs were encapsulated in and coated on a versatile SiO2 nanocarrier to produce a large amplification factor of hybrid fluorescent NPs (HFNPs) labeled complementary DNA (cDNA) via a condensation reaction. The aptamer conjugated with MBs was then hybridized with cDNA labeled with HFNPs to generate a magnetic-fluorescent-targeting multifunctional aptasensor. The introduction of OTA would trigger aptamer-OTA binding, resulting in the partial release of the cDNA-HFNPs into bulk solution. After a simple magnetic separation, the supernatant liquid of the above solution contained a great number of CdTe QDs produced an increasing fluorescence emission with an increasing OTA concentration
2. Experimental section 2.1. Reagents and materials 3-mercaptopropionic acid (MPA, 99%), tetraethylorthosilicate (TEOS), Tris(hydroxymethyl)aminomethane (Tris), Tris (2-
chloroethyl) phosphate (TCEP), and 6-mercapto-1-hexanol (MCH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), poly (diallyldimethyl-ammonium chloride) (PDDA), (3-aminopropyl) triethoxysilane (APTS), OTA, fumonisin B1 (FB1), and aflatoxins B1 (AFB1) were obtained from Sigma-Aldrich. cDNA: 5ʹ-CCT TTA CGC CAC CCA CAC CCG ATC-NH2-3ʹ and aptamer: 5ʹ-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA–SH-3ʹ were purchased from Sangon Biotech Co., Ltd. (China). DNA oligonucleotide stock solutions were prepared with 10 mM Tris–HCl buffer (pH 7.4, containing 10 mM NaCl and 5 mM MgCl2) and kept frozen in dark. The Fe3O4 microspheres, the core–shell Fe3O4@Au MBs, and the MPAcapped CdTe QDs were prepared according to the descriptions of Wan et al. (2012), Wu et al. (2007), and Zhang et al. (2010), respectively (Supporting information). Double-distilled water was used throughout the study. 2.2. Instrumentation The morphology of the samples was checked by transmission electron microscopy (TEM) technique (JEOL 2100, JEOL, Japan). X-ray diffraction (XRD) spectra were carried out with a Bruker D8 ADVANCE diffractometer (Germany) with Cu Kα (λ ¼ 1.5406 Å) radiation. X-ray photoelectron spectroscopy (XPS) was performed on ESCALAB 250 multitechnique surface analysis system (Thermo Electron Co., USA). UV–vis absorption spectra were measured by UV-2450 spectrophotometer (Shimadzu, Japan). Fluorescence spectra were recorded on a Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan). Zeta potential was obtained using a Malvern Zetasizer Nano ZS instrument (ZEN 2600, England). All the photographs were taken using a Canon digital camera (IXUS 230 HS, China) under the illumination of a 365 nm UV lamp. 2.3. Preparation of aptamer-coupled Fe3O4@Au MBs Aptamer coupled Fe3O4@Au MBs (MB-aptamer) bioconjugations were synthesized according to previous reports (Zhou et al., 2013). Intially, 500 μL of 100 μM aptamer was added to 500 μL of 10 mM Tris–HCl buffer containing 100 mM TCEP to activate the aptamer for 1 h at 37 °C in a reciprocating oscillator. After removing excess TCEP, the freshly activated aptamer was added to 35 mL of Fe3O4@Au MBs and sonicated for 30 s, and then incubated for 30 min with gentle shaking at 37 °C. After the salt aging according to the description of Zhou et al. (2013), the MBaptamer bioconjugations were separated readily by a magnet to remove excess unbound aptamers. Finally, the MB-aptamer bioconjugations were treated with 35 mL of 2 μM MCH and kept at 37 °C for 1 h, again under stirring. After magnetic separation and washing, the resultant MB-aptamer bioconjugations were then redispersed in 35 mL of Tris–HCl buffer and stored at 4 °C for later use. 2.4. Preparation of cDNA-conjugated QD@SiO2@QD HFNPs Synthesis of the core–satellite QD@SiO2@QD HFNPs has been described in detail in Supporting information. To conjugate cDNA on the QD@SiO2@QD HFNPs, 8 mL of 10 mM Tris–HCl buffer containing 1 mM EDC and 1 mM NHS was added to 15 mL of the resulting QD@SiO2@QD HFNPs dispersion. After shaking for 1 h, 500 μL of 100 μM cDNA was added. After shaking at 37 °C overnight, the cDNA-conjugated QD@SiO2@QD HFNPs (cDNA-HFNPs) was centrifuged at 6500 rpm for 5 min to remove excess cDNA, and then redispersed in 15 mL of Tris–HCl buffer and stored at 4 °C for further use.
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2.5. Preparation of the multifunctional aptasensor The mixture of MB-aptamer and cDNA-HFNPs was keeping shaken for 1 h at 37 °C with the help of reciprocating oscillator. After magnetic separation and washing, the MB-aptamer/cDNAHFNPs bioconjugations were redispersed in 40 mL Tris–HCl buffer and used as the magnetic-fluorescent-targeting multifunctional aptasensor in further study. 2.6. Analytical procedure 400 μL of the resulting MB-aptamer/cDNA-HFNPs bioconjugations was added to a series of parallel centrifuge tubes with numbers. Then, 100 μL of OTA containing solution with various concentrations was added to the tubes. After incubation at 37 °C for 1 h, measured supernatant liquid was then separated by a magnetic field and washing with Tris–HCl buffer. Then, the tansformed supernatant liquid containing different amount of HFNPs was dilute to a final volume of 2 mL and its fluorescence (FL) intensity was recorded accordingly.
3. Results and discussion 3.1. Aptasensor fabrication and detection principle The whole process for the multifunctional aptasensor construction is illustrated in Scheme 1A. Specifically, the QD@SiO2 @QD HFNPs were synthesized by a multistep procedure involving the synthesis of CdTe QDs doped silica cores (steps 1 and 2), modification of the silica surface by PDDA (step 3), and final
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planting of the CdTe QDs on the surface of QD@SiO2 to botain QD@SiO2@QD HFNPs (step 4). The zeta potential of QD@SiO2 was – 25.9 mV, which indicated that the surface of the QD@SiO2 terminated by hydroxyl groups was negatively charged at neutral pH. The modification of the positively charged PDDA on QD@SiO2 switched the zeta potential to a positive value of 36.8 mV. The negatively charged MPA-capped CdTe QDs with the zeta potential of –34.1 mV were subsequently tethered electrostatically to the QD@SiO2 surface to result in QD@SiO2@QD HFNPs. By using the EDC/NHS chemistry, preparation of cDNA-HFNPs was achieved through the covalent binding interactions between the amino groups of cDNA and the carboxylic groups on the surface of MPAcapped CdTe QDs (step 5). After the formation of the duplex structure between aptamer on Fe3O4@Au MBs and cDNA labeled with QD@SiO2@QD HFNPs, the multifunctional aptasensor (MBaptamer/cDNA-HFNPs) was thus successful produced through the hybridization event (step 6). The working principle of the aptasensor is illustrated in Scheme 1B. Without OTA, the resulting MB-aptamer/cDNA-HFNPs can be separated readily by a magnet placed under the centrifuge tube. Therefore, the supernatant liquid contains no QD@SiO2@QD HFNPs and thus no fluorescence emission can be detected. However, the incubation of OTA with MB-aptamer/cDNA-HFNPs would trigger aptamer-OTA binding, resulting in the formation of the aptamer-OTA complex. This caused some cDNA-HFNPs dissociating from the Fe3O4@Au MBs. The more OTA molecules in the detection system, the more cDNA-HFNPs were combined with the target and released into bulk solution. Therefore, the FL intensity generated from the QD@SiO2@QD HFNPs in the supernatant liquid as a function of OTA concentration was measured accordingly. Since MB-aptamer/cDNA-HFNPs can be prepared at large scale in a
Scheme 1. Schematic illustration of the fabrication of the multifunctional aptasensor with a heavy CdTe quantum dots label (A) and its working principle for OTA detection (B).
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Fig. 1. TEM images of the as-prepared Fe3O4 microspheres (A) and Fe3O4@Au MBs (B), (C) XRD patterns of the as-prepared Fe3O4 microspheres (a) and Fe3O4@Au MBs (b), and (D) the UV–vis spectra of Fe3O4 microspheres (a) and Fe3O4@Au MBs (b) dispersed in water.
single run, this aptasensing system can be conveniently used for rapid and high through-put OTA detection through a one-step incubation procedure followed by a simple magnetic separation. 3.2. Characterization of the Fe3O4@Au MBs One can see that Fe3O4 microspheres were identifiable with an average diameter of 450 nm (Fig. 1A). The functionality of Fe3O4 microspheres with APTS would produce the Fe3O4 microspheres with amine groups. The amine groups on the APTS-coated Fe3O4 surface preferentially bind noble metals such as Au NPs, instead of other species (Freeman et al., 1995). After reduction of the adsorbed AuCl4– ions by citrate, an additional shell-building Au layer was decorated on the Fe3O4 microspheres’ surface, confirming the successful formation of the (Fe3O4)core–Aushell MBs (Fig. 1B). The as-prepared core–shell Fe3O4@Au MBs with an adaitional Au layer can improve the stability of the pristine Fe3O4 microspheres in biological conditions and provides a friendly microenvironment for efficiently covalent binding with the thiolated aptamer via the well-understood Au–S chemistry (Wu et al., 2007). The coated Au shell was further confirmed by typical XRD patterns (Fig. 1C). The Fe3O4 microspheres (curve a) exhibited diffraction peaks centered at 2θ values of 30.1°, 35.5°, 43.1°, 53.5°, 57.0°, and 62.6°, which were indexed to (220), (311), (400), (422), (511), and (440) planes of cubic structure of Fe3O4 (JCPDS no. 790419), respectively (Wan et al., 2012). Fe3O4@Au MBs (curve b) exhibited all the diffraction peaks of pristine Fe3O4 microspheres but with four additional diffraction peaks at 38.1°, 44.3°, 64.5°, and 77.6°, which were indexed to the (111), (200), (220), and (311) planes for the face-centered-cubic gold (JCPDS no. 04-0784) planes
of gold cubic phase, respectively (Zhang et al., 2008). This information indicated that a thin layer of Au shell, which was not thick enough to overlay the Fe3O4 diffraction peaks, was successfully deposited onto the surface of the Fe3O4 microspheres by the seed growth method. There is no obvious absorbance peak was observed for the Fe3O4 microspheres suspension (curve a in Fig. 1D). When the layer of Au was deposited onto the surface of Fe3O4 microspheres, an extra surface plasmon peak was clearly observed at 554 nm for the Fe3O4@Au MBs (curve b in Fig. 1D). This value is different from that previously reported for Au NPs with a value of 522 nm (Yang et al., 2014). The red shift of absorption peak in the spectrum of Fe3O4@Au MBs suspension can be ascribed to the fact that AuNPs bound strongly on the surface of Fe3O4 microspheres (Cui et al., 2011). The resulting Fe3O4@Au MBs displayed obvious color change before (photograph a in Fig. 1D) and after (photograph b in Fig. 1D) the Au shell building. As a result of the magnetite content, the resulting Fe3O4@Au MBs can quickly respond to external magnetic field (photograph c in Fig. 1D) in 30 s and quickly be redispersed homogeneously by hand-shaking. The excellent magnetic property enables Fe3O4@Au MBs to be used for simple and efficient magnetic separation. 3.3. Characterization of the QD@SiO2@QD HFNPs Fig. 2A shows a TEM image of QDs@SiO2 NPs with an average diameter of 150 nm. As can be observed, these QDs@SiO2 NPs are well-dispersed with a chemically clean and homogenized structure. It can be observed from QD@SiO2@QD HFNPs that a large number of dark dots have been coated on the surface of each silica
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Fig. 2. TEM images of the as-prepared QD@SiO2 (A) and QD@SiO2@QD (B), (C) XPS spectrum of the QD@SiO2@QD HFNPs, the UV–vis (D) and fluorescence (E) spectra of QD@SiO2 (a) and QD@SiO2@QD (b), and the corresponding photographs under daylight (inset of D) and UV illumination (inset of E).
core with excellent dispersivity (Fig. 2B). Furthermore, no bare QDs@SiO2 or free CdTe QDs was observed, which suggested the complete formation of the QD@SiO2@QD HFNPs. The wide scan XPS spectra of QD@SiO2@QD HFNPs (Fig. 2C) clearly indicated that the sample was composed of Cd, Te, Si, O, C, N, and S elements, and no peaks of other elements were observed. An absorption peak at 610 nm and an emission peak at 670 nm were observed for both QDs@SiO2 NPs (curve a in Fig. 2D) and QD@SiO2@QD HFNPs (curve b in Fig. 2D). They displayed a similar color as CdTe QDs in water (inset a in Fig. S1A) under daylight (inset in Fig. 2D) and emitted an intense red fluorescence under UV illumination (inset in Fig. 2E), consistent with that of CdTe QDs itself (inset b in Fig. S1A). It is interesting to notice a slight red shift in the peak emission of the QDs@SiO2 and QD@SiO2@QD HFNPs when compared with the CdTe QDs aqueous solution (Fig. S1A), might due to the strong binding interactions between the CdTe QDs and the silica. Moreover, there was 1.8 fold enhancement in the FL intensity for QD@SiO2@QD HFNPs (curve b in Fig. 2E) compared to that of the QDs@SiO2 NPs (curve a in Fig. 2E) at the same concentration of the silica spheres. That is
largely attributed to the fact that each QD@SiO2@QD HFNP can carry a large number of CdTe QDs not only in the silica core but also on the silica surface. As a label, this kind of QD@SiO2@QD HFNPs would be hugely beneficial for signal amplification. 3.4. Fabrication of the multifunctional aptasensor Compared with the spectrum of Fe3O4@Au MBs suspension (curve b in Fig. 1D), an additional peak was observed at 262 nm for MB-aptamer bioconjugations (curve b in Fig. 3A), similar to that of the free aptamer solution (curve a in Fig. 3A), indicating the successful coupling of aptamer on the Fe3O4@Au MBs surface. High-resolution XPS spectrum of P 2p was ascertained to confirm whether the amino-modified cDNA was conjugated successfully to the surface of the QD@SiO2@QD HFNPs. There was no clear P 2p characterized signal was observed in the scanning region for QD@SiO2@QD without cDNA coupling (curve a in Fig. 3B). In contrast, the normalized XPS P 2p line has a maximum signal at 133.1 eV for the cDNA-HFNPs bioconjugates in the same region (curve b in Fig. 3B), coming from the phosphate backbone in the
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Fig. 3. (A) The UV–vis spectra of free aptamer (a) and MB-aptamer (b), (B) XPS spectra of P 2p for QD@SiO2@QD before (a) and after (b) cDNA conjugation, (C) the fluorescence spectrum of cDNA-HFNPs, (D) the fluorescence spectra and typical photographs (inset) of Fe3O4@Au MBs suspension (a) and the MB-aptamer/cDNA-HFNPs bioconjugations (b).
cDNA (Wang et al., 2006). The conjugation of cDNA did not affect the fluorescence emission of QD@SiO2@QD obviously (Fig. 3C). The cDNA-HFNPs bioconjugation was homogeneous and bright red under UV irradiation (inset in Fig. 3C). The MB-aptamer displayed no emission peak in the scanning region without cDNA-HFNPs coupling (curve a in Fig. 3D). Because of the magnetic Fe3O4 core, the MB-aptamer conjugation can be directed to specific locations when manipulated by an external magnetic field, indeed, there was no red fluorescence was observed from the aggregations on the cuvette wall under UV irradiation (inset a in Fig. 3D). In contrast, the resultant MB-aptamer/ cDNA-HFNPs bioconjugations (curve b in Fig. 3D) displayed a strong fluorescence emission peak at 670 nm, consistent with that of the cDNA-HFNPs (Fig. 3C). Once the magnet is placed beside the vial, the MB-aptamer/cDNA-HFNPs bioconjugations quickly move and accumulate near it within few minutes, one can observe the bright red fluorescence of the aggregations on the cuvette wall with the bulk solution clear and transparent (inset b in Fig. 3D). With removal of the external magnet and hand-shaking, the aggregations would be rapidly redispersed again. Based on above experimental results, a magnetic-fluorescent-targeting multifunctional aptasensor for rapid OTA detection has been constructed successfully. The combination of magnetic, fluorescent, and targeting objects in a single aptasensing system is of extraordinary interest lies in its multifunctional applications where it is necessary to monitor and manipulate at the same time. 3.5. Optimization of detection conditions To generate a rapid-response and sensitive aptasensor with a low detection limit for OTA, it is significant to optimize important parameters involved in the aptasensor fabrication and detection process. Following the routine hybridization time, from 10 to 80 min were investigated and displayed in Fig. 4A. The FL intensity
of MB-aptamer/cDNA-HFNPs increased dramatically with the growth of the time before 60 min, and then leveled off at times longer than 60 min, so 60 min was used as the optimized time required for hybridization of cDNA and aptamer. Since DNA hybridization is a temperature-dependent process, the solution temperature for the hybridization between cDNA and aptamer was also optimized. Fig. 4B indicated that the FL intensity of MB-aptamer/cDNA-HFNPs reached the maximal value when the temperature was 37 °C, which was then used as the optimized temperature for the DNA hybridization. Another impomtant factor that affects DNA hybridization is the pH value of the supporting electrolyte. Based on physiological conditions, the solution pH values ranging from 6.0 to 8.5 were tested and the results shown in Fig. 4C. It is noted that the best performance was obtained in the Tris–HCl buffer solution with a pH value of 7.4. Since the reaction time of OTA with its aptamer was closely related to the released QD@SiO2@QD HFNPs in the bulk solution, the effect of the binding time greatly influenced the final sensing of the target molecules. As shown in Fig. 4D, it was apparent that the FL intensity of the released QD@SiO2@QD HFNPs obviously increased with the increasing binding time from 10 to 60 min and then reached a plateau in 60 min. This suggested that 60 min was enough and thus chosen as the incubation time in the following detection. 3.6. Analytical performance of the aptasensor Under the optimized conditions, a simple, rapid, and sensitive aptasensor for the fluorescence detection of OTA was developed. As expected, the intensity of the emission at 670 nm was increased successively upon the incubation with an increasing amount of OTA (Fig. 5A). The FL intensity ceased to increase beyond 500 ng mL 1 of OTA, indicating the binding reaction between OTA and its aptamer was nearly saturated at this concentration. By analyzing the FL intensity (I) with the concentrations of OTA
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Fig. 4. Effect of the hybridization time (A), hybridization temperature (B), and hybridization solution pH value for the performance of MB-aptamer/cDNA-HFNPs. (D) The fluorescence responses of the aptasensor under different binding time with OTA at the concentration of 100 ng mL 1.
2015), but lower than that of the recently reported electrochemical methods using rolling circle amplification (Tong et al., 2012; Huang et al., 2013), loop-mediated isothermal amplification (Yuan et al., 2014; Xie et al., 2014), and target recycling amplification (Tong et al., 2011). However, this proposal provides a simple and rapid way for parallel OTA detection through a one-step incubation procedure. 3.7. Selectivity and reproducibility The selectivity of this aptasensor was evaluated by the comparison of the sensing results of FB1, AFB1, and OTA. As shown in Fig. S2, the response signals to FB1 and AFB1 were neglectable
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(Fig. 5B), a good linear relationship was obtained between the FL intensities and the logarithm of the OTA concentrations (inset in Fig. 5B). The linear equation is I ¼–130.7þ 193.7 log (c/ng mL 1) (R2 ¼ 0.9895) over a wide concentration range of 15 pg mL 1–100 ng mL 1. The limit of detection (LOD) was calculated to be 5.4 pg mL 1 based on S/N ¼3. The analytical performances of the present aptasensor along with other aptasensors developed for OTA in literatures were all summarized in Table S1. As can be seen, the proposed aptasensor exhibits a broader linear range and its sensitivity is comparable to or higher than the corresponding values observed in most of the works (Bonel et al., 2011; Chen et al., 2012; Guo et al., 2011; Yang et al., 2011; Prabhakar et al., 2011; Wei et al., 2015; Zhu et al., 2015; Wang et al.,
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Fig. 5. (A) Fluorescence spectra of the released HFNPs corresponding to various OTA concentrations (from a to n: 0, 0.015, 0.05, 0.1, 0.5, 1, 5, 10, 30, 50, 100, 200, 300, and 500 ng mL 1). (B) The FL intensity in (A) was plotted as a function of concentration of OTA. Inset: the calibration curve of the OTA detection.
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with the concentration of 200 ng mL 1, while an obvious increase in response was observed for OTA with a concentration of 50 ng mL 1. This result suggested that the aptasensor possessed high selectivity. The reproducibility of the aptasensor was also investigated at the OTA concentration of 50 ng mL 1. The relative standard deviation (RSD) for five measurements was 5.3%, indicating that the designed aptasensor for OTA detection was highly reproducible. 3.8. Analytical application in real samples To further investigate the potential practical applications of the proposed method, this aptasensor was applied to peanut (without shell) from the local supermarket. The preparation of the real testing samples (Supporting information) was according to the descriptions of Chen et al. (2012) and Luna et al. (2013). Table S2 summarized the analytical results of OTA determination in the real peanut samples by using the as-prepared aptasensor. As can be seen, the recoveries of the spiked samples ranged from 96.0 up to 99.7%, with RSD lower than 6.4%, demonstrating that this multifunctional aptasensor can be used for OTA detection in real samples with the satisfied results.
4. Conclusions In this study, we describe the very promising and fascinating development of a highly sensitive aptasensor that combines magnetic-fluorescent-targeting multifunctionality. Target OTA can be concentrated by the MB-aptamer and the fluorescence signal can be effectively amplified by using a heavy CdTe QDs label loaded both in and on SiO2 nanocarrier. The use of core–shell Fe3O4 @Au MBs provided not only a friendly microenvironment for efficiently covalent bind with thiolated aptamer but also an efficient method for the separation of the HFNPs labels with the help of a magnetic field. Under optimal conditions, the correlation between OTA concentration and the fluorescence signal from the released QD@SiO2@QD labels was found to be linear from 15 pg mL 1 to 100 ng mL 1 with a LOD of 5.4 pg mL 1. As the MB-aptamer/ cDNA-HFNPs bioconjugations can be produced at large scale in a single run, this aptasensing system can be conveniently used for rapid OTA detection through a one-step incubation procedure followed by a simple magnetic separation. The aptasensor was successfully employed for detecting OTA in peanut samples, validating their great application potential in food quality control.
Acknowledgements This research work was supported by the National Natural Science Foundation of China (Nos. 21405063, 21175061, 21375050, and 31071549), the Natural Science Foundation of Jiangsu province (No. BK20130481), Special Fund for Agro-scientific Research in the Public Interest (No. 201003008-04), Qinglan Project, Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 14KJA550001), the Program Sponsored for Scientific Innovation Research of College Graduate in Jiangsu Province (KYLX_1072), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.02.008.
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