Fluorescent aptasensor for chloramphenicol detection using DIL-encapsulated liposome as nanotracer

Biosensors and Bioelectronics 81 (2016) 454–459

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Fluorescent aptasensor for chloramphenicol detection using DIL-encapsulated liposome as nanotracer Yang-Bao Miao a, Hong-Xia Ren b,c, Ning Gan a,n, Yuting Cao a,n, Tianhua Li a, Yijin Chen d a State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211, PR China b Key Laboratory of Asymmetric Synthesis and Chirotechnology of Sichuan Province, Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, PR China c University of Chinese Academy of Sciences, Beijing 10049, PR China d Faculty of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210000, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 19 January 2016 Received in revised form 12 March 2016 Accepted 15 March 2016 Available online 16 March 2016

A novel fluorescence aptasensor was successfully developed to respond to chloramphenicol (CAP) in food based on magnetic aptamer-liposome vesicle probe. In order to fabricate it, aptamer labeled on functionalized magnetic beads (MB) was firstly employed as capture adsorbent (MB-Apt), then SSB (singlestranded DNA binding protein) and DIL (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanineperchlorate) coimmobilized liposomes (SSB/DIL-Lip) was employed as vesicle signal tracer. The composite vesicle probe is formed between SSB/DIL-Lip and MB-Apt based on SSB's specific recognition towards aptamer on vesicle signal tracer. Upon the vesicle probe solution reacted with CAP, the aptamer on the magnetic beads preferentially bounded with CAP, and then released SSB/DIL-Lip vesicle signal tracer in the supernatant after magnetic separation. The released tracer can emit fluorescence which was correspondence with the concentration of the analyte. At the optimum conditions, the aptasensor exhibited a good linear response for CAP detection in the range of 0.003–10 nM with a detection limit of 1 pM. Importantly, the methodology was further validated for analyzing CAP in fish samples with consistent results obtained by ELISA kit, thus providing a promising approach for quantitative monitoring of CAP and significant anti-interference ability in food safety. & 2016 Elsevier B.V. All rights reserved.

Keywords: Fluorescence aptasensor Magnetic liposome vesicle signal tracer Single-stranded DNA binding protein DIL Chloramphenicol

1. Introduction To fabricate fluorescence assay forreal-time monitoring of water soluble antibiotic contaminant in food samples, for instance chloramphenicol (CAP), kanamycin (Kana), has attracted more and more attention during these years (Yan et al., 2015; Liu et al., 2015; Xing et al., 2015; Guo et al., 2015). Because, upon the overdose of CAP intake from food or medicinal, it can result in many serious negative impacts, including bone marrow suppression, gray baby syndrome, kidney damage and allergic reactions (Wang et al., 2010; Yu et al., 2014). Therefore, it is essential to develop sufficiently sensitive methods to detect CAP residues for food and clinical diagnosis. In order to fulfill the purpose, it's crucial to synthesize fluorescent biological probes (bio-probe) towards anayltes with high specificity, sensitivity, and simplicity of implementation (Zhang et al., 2015; Kaur and Choi, 2015). Moreover, it must have high capacity of resisting interference from the n

Corresponding authors. E-mail addresses: [email protected] (N. Gan), [email protected] (Y. Cao).

http://dx.doi.org/10.1016/j.bios.2016.03.034 0956-5663/& 2016 Elsevier B.V. All rights reserved.

complex matrix in food (Hidayat et al., 2013). As a result, various fluorescence biosensors employing aptamer as recognition probes have been developed in recent year, such as label-free analyte fluorescent aptamer sensor, sandwich type fluorescent aptamer sensor (Bogomolova and Aldissi, 2015; Yang et al., 2015). Due to the aptamer possess unique excellence in rapidity in vitro synthesis, highly specificity, and non-immunogenic usage (Xiao et al., 2005). Especially, fluorescent aptamer sensor have attracted great attention on account of their facile operation, high sensitivity, and high specificity toward a variety of targets. This is because aptamer has even higher affinity towards targets than antibody (Xiao et al., 2005). Among various species fluorescent bio-probes, small organic molecules, for instance 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanineperchlorate (DIL) (Verbovetski et al., 2002), rhodamine (Johnson et al., 1980), fluorescein (Vermes et al., 1995), are popular because they are rich in variety and easy to use (Xu and Lu, 2009). Nevertheless, it contain large fused polynuclear rings, which are inclined to aggregate when dispersed in aqueous solution, inflicting upon the notorious aggregation-caused quenching (ACQ). Other defects of there organic probes are their poor aqueous solubility, which are not suitable for detection of

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Scheme 1. Scheme of depicting the proposed biosensing of chloramphenicol based on magnetic vesicle probes with DIL and single-stranded DNA binding protein coimmobilized liposomes as nanotracer.

analyte in aqueous phase (Niu et al., 2009). Accordingly, the development of new types of aqueous and specific fluorescent aptamer probes for antibiotic contaminant is highly desirable. For the purpose of detection ultra-trace level of target, the liposomes was further employed for enhance the fluorescent signal. The liposomes, which are spherical lipid vesicles in magnitude of nanometer with a double layer membrane structure consisting of amphiphilic lipid molecules, provide a wide variety of attractive features (Klibanov et al., 1990). Because they are soluble in water and have a particular hydrophobic inner membrane and vesicle cavity which can encapsulate many hydrophobic molecules in this nanometer cavity, for example drugs (Juliano and Stamp, 1975), to improve their permeability stability into cells (Leserman et al., 1980), pharmacokinetics (Garg et al., 2006) and bio-distribution (Camelo et al., 2007). And its inner hydrophobic membrane can also be immobilized with many hydrophobic fluorescence probes to amplify signal (Camelo et al., 2007). Moreover, by choosing different precursors for preparing liposome, it's easy to fabricate grafting ligands, such as-COOH,-NH2, on its outer surface (Zhou et al., 2013). Thus, it's easy to label biological macromolecules, for instance protein (Caracciolo et al., 2015), antibody (Lozano et al., 2015), using some conventional coupling reagents, such as N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Therefore, it is very useful to employ liposome to encapsulate a large numbers of fluorescence probes and labeling biological proteins to fabricate sensitive fluorescence vesicle tracers (Caracciolo et al., 2015). However, the liposomes being applied for detection of small molecule antibiotics in food is still rarely reported. Meanwhile, how to quantitatively convert the amount of analyte to fluorescence signal is another important issue. The fabrication of a composite detection probe between capture probe and liposome based fluorescence tracers can fulfill the purpose. When the capture probe react with target, the tracers can be replaced in correspondence into supernatant for fluorescence detection. Among many studies, the fluorescence aptamer based tracers was connect with capture probe through hybridization between single strand aptamer with its complementary strand DNA (cDNA) (Zhao et al., 2015; Wang et al., 2015; Song et al., 2016). Thus usage of cDNA can enhance the fabrication complexity and price for the vesicle probes. In this research, we used CAP as model analyte for testing our fluorescence assay. And DIL (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanineperchlorate, which is lipophilic fluorescent dye to membrane.) and single-stranded DNA binding protein (SSB) was coimmoblized on liposomes to prepare highly sensitive fluorescence vesicle signal tracer (SSB/DIL-Lip). As far as we’re concerned, this nanotracer have been application in detection of antibiotics in food is still barely report. Firstly, DIL is a hydrophobic fluorescent

dye which can be well soluble in lipids (Ochi Ardebili et al., 2015). In addition, fluorescence of DIL is very weak before entering the membrane, when entering into the membrane, they can be inspired to strong fluorescence (Lu and Gursky, 2013). The reason of why we choose single-stranded DNA binding protein (SSB) labeling on liposome. It is according to the team of Kowalczykowski's study (Kowalczykowski et al., 1981). They demonstrated that SSB can specifically bind a single-stranded DNA based on immunereaction. Thus SSB can react specifically with single-stranded aptamer easily without using other coupling reagent. Therefore, a novel detection mechanism for the florescence assay was fabricated, which is based on competition replacement reaction between target analyte (CAP) and SSB in vesicle signal tracer with aptamer on capture adsorbent. Because CAP has higher affinity to aptamer than SSB, the fluorescence vesicle signal tracer can be quantitatively replaced into supernatant after the probes binding CAP. In order to reduce the matrix interference while detecting CAP residues in the food, we also use magnetic beads labeled with aptamer of CAP to fabricate magnetic capture adsorbent. Thus, when the capture adsorbent binds the target, it can be easily magnetically separated from the background. It's easy for manipulation and can reduce the procedures in pretreatment. The preparation steps of the vesicle probes and detection mechanism of the fluorescence assay is shown in Scheme 1. The vesicle probe for CAP was prepared through SSB specifically recognizes CAP aptamer between the vesicle signal tracer based on SSB/DIL-Lip and capture adsorbent using the CAP aptamer was modified on aminofunctionalized magnetic beads (MB). Upon the vesicle probe solution was mixed with CAP, the aptamer on the capture adsorbent preferentially bounded with CAP, and then released SSB/DIL-Lip complex to the supernatant after magnetic separation. The SSB/ DIL-Lip complex produced fluorescence, which was analyzed by fluorescence spectrometer. Moreover, the assay approach exhibited excellent analytical performance during the determination of CAP in real food samples.

2. Experimental 2.1. Reagents and chemicals The oligonucleotides used in this paper are as the following sequences: Aptamer (Mehta et al., 2011) (thiolated Apt bound to CAP), 5′(CH2)6-ACT TCA GTG AGT TGT CCC ACG GTC GGC GAG TCG GTG GTAG; was purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). DIL (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanineperchlorate), Amino-functionalized magnetic beads (MB) were purchased from Sigma-Aldrich (St. Louis, MO). Hexane, methanol and

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were incubated 3 h under rotation. Unbound aptamer was removed by magnetic separation. The vesicle probes were synthesized by adding 100 μL Apt/MB to 200 μL SSB/DIL-Lip. The mixture was stirred gently for 1 h at 37 °C. Then after magnetic separation, the precipitated vesicle probes were re-dispersed in 200 μL 0.1 mol/L PBS buffer solutions (pH 7.4) and stored in 4 °C.

chloroform were bought from Shanghai Lingfeng Chemical Reagent Co., Ltd. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[carboxy(polyethylene glycol)–2000] (ammonium salt) (DSPEPEG-NH2) were purchased from Avanti Polar Lipids, Phosphate buffer saline (PBS, pH 7.4, 0.1 M KH2PO4-K2HPO4, 0.1 M KCl) was used as washing and binding buffer. Tris-HCl buffer (0.1 M) containing 0.1 M NaCl and 5 mM MgCl2 (pH 7.4) was employed for preparation of DNA stock solutions. A 0.1 M PBS (pH 7.4) buffer containing K2HPO4 and KH2PO4 was used to washing solution. CB buffer (50 mM NH4-OAc, 1 mM CaCl2, and 1 mM MnCl2. Tris (2carboxyethyl) phosphine hydrochloride (TCEP), N-(3-dimethylaminopropyl)-N’-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), ethanesulfonic acid (MES) buffer were purchased from Sigma-Aldrich. All other reagents were analytical grade and were used without further purification. Double-distilled water was used throughout the study.

The manufacturing process of the fluorescence assay for CAP is declared in Scheme 1. Primarily, in order to detect CAP, 50 μL CAP (0.03–10 nM) was incubated with for 30 min at room temperature based on immune-reaction. After incubation, the solution was shaken plentifully for fluorescence development and the absorbance was determined at 565 nm by F-4600 spectrophotometer for quantitative analysis. For briefness of measurement, the experiments were performed at room temperature.

2.2. Apparatus

2.5. The detection of CAP in real fish samples

The transmission electron microscopic (TEM) image was obtained from a H600 transmission electron microscope (Hitachi, Japan). Scanning electron micrographs (SEM) were obtained from a S3400N scanning electron microscope (Hitachi, Japan). The UV– vis spectra were recorded by a UV-1800 spectrophotometer (Shimazu Co., Japan). Fluorescence measurements were carried out on an F-4600 spectrophotometer (Hitachi, Japan). Dynamic Light Scattering was carried out by the Malvern zetasizer Nano ZS90, Malvern instruments Ltd., (Malvin, UK).

Several fish specimen in supermarket was employed for proving the application of the proposed method. In the first instance, 2.0 g fish proof sample was weight and afterwards was placed in 50 mL centrifuge tube. Where after, 4 mL 3% (w/v) tri-chloroacetic acid aqueous solution was blended, followed by a whirl until smooth and then addition of 2 mL methanol and a thoroughly shake. After centrifugation 10 min at 4000 r/min, 3 mL the supernatant was dried at 50–60 °C under nitrogen atmosphere and then resolved in water for the detection by the developed aptasensor. We choose the extraction liquid in blank fish to testify the matrix interference in real samples. ELISA method was performed in Microplate Reader in 96-cell Plate according the protocol of kits.

2.3. The synthesis of composite fluorescence vesicle probe The liposome was prepared according to the procedure as previously reported (Zhou et al., 2013) with some modification. 24 μL of 0.1 M DSPC, 24 μL of 1.0 mM DSPE-PEG-NH2, and 15 μL of 50 mM cholesterol were added into the flask with 3 mL of chloroform, and then mixed uniformly at 35–45 °C. Then chloroform was volatilized by rotary evaporation under reduced pressure, and the resultant thin film adhering to the inside wall of flask was dried in argon atmosphere. Afterwards, 2 mL of phosphate buffer solution (pH ¼7.4) was added into the flask, followed by shaking at 30 °C to dissolve the thin film in PBS solution. The final solution was extruded through a 0.1-μm polycarbonate membrane filter back and forth for three times, and the products of Liposome complexes were kept at room temperature for further use. The liposome was labeled with the fluorescent probe as follows (Ochi Ardebili et al., 2015): To the liposome, DIL (50 μM) water was added in double distilled, while the solution was gently vortex-mixed, and the sterile mixture incubated at 37 °C for 8 h. The single-stranded DNA binding protein/liposome-DIL complexes (SSB/DIL-Lip) were prepared according to the reported methods (Bartczak and Kanaras, 2011). The liposome functionalized SSB were prepared via the reaction between the –NH2 of liposome and the –COOH of SSB by using EDC (20 mM) and NHS (40 mM) as coupling agents. 1 mg SSB was suspended in 1 mL doubled distilled water and sonicated to obtain a homogeneous solution. Then, the above collected amino-functionalized liposome were added and stirred for about 10 h. The SSB/DIL-Lip complexes were obtained by centrifugation. The Apt/MB complexes were prepared according to the reported methods. The amino-functionalized MB was activated at room temperature under gentle rotation for 1.5 h by 200 μL of 5% glutaraldehyde solution in CB (pH ¼ 7.0). The amino-functionalized Magnetic beads were then retained by magnetic separation and the solution was removed, followed by four washes each in 200 μL of CB. Subsequently, adding into amino-functionalized aptamer

2.4. Analytical procedure

3. Results and discussion 3.1. Construction and characterization of DIL, liposome, DIL-liposome, single-stranded DNA binding protein/DIL-liposome, aminofunctionalized magnetic beads, MB-Apt The prepared liposome was evaluated by transmission electron microscope (TEM) and scanning electron micrographs (SEM), UV– vis absorption. TEM (Fig. 1A) and SEM (Fig. 1B) images show that the liposome is highly monodispersed and uniform in size. The average size is estimated to be 8770.25. The DIL-liposome complexes (DIL-Lip) shown uniform in size (the detail shown in Fig. S1). Size polydispersity index and surface charge of liposome and DIL-Lip complexes were measured by a Zetasizer Nano-ZS respectively. The DIL-Lip complexes are uniform with a polydispersity index of 0.213 70.018 for DIL-Lip complexes (the detail shown in Fig. S2 and Table S1). Analysis of size distributions reveals the average size of 9273.8 nm for the DIL-Lip complexes (the detail shown in Fig. S1) much larger than that of 5 70.25 nm for pristine liposome (the detail shown in Fig. S1), suggesting the successful encapsulation of DIL inside the liposomes. Zeta potential is measured to be 43.5 70.72 mV and 38.97 0.61 for liposome and DIL-Lip complexes indicating that the DIL-Lip complexes are highly dispersible in aqueous solution (the detail shown in Table S1). The ultraviolet visible absorption spectra of Liposome (curve a), SSB (curve b), DIL(curve c), DIL-Lip (curve d) and SSB/DIL-Lip (curve e), were also shown in Fig. 1. Caecostenetroides as shown in Fig. 1C-a, liposome showed no obviously characteristic peak. Fig. 1C-d demonstrated maximum absorption peak at 525 and 571 nm which can be ascribed as DIL well into the liposomes (Ikeda et al., 2010). The SSB/DIL-Lip manifested maximum

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Fig. 1. TEM images of DIL-liposome (A) with ruler of 100 nm, (B) SEM images of DIL-liposome, (C) UV–vis spectra of Liposome (curve a), SSB (curve b), DIL curve c), DILliposome (curve d) and SSB/DIL-Lip (curve e), (D) UV–vis spectra of Amino-functionalized magnetic beads (Figure D-a), Aptamer (Figure D-b), MB-Apt (Figure D-c).

absorption peak at 301, 522 and 562 which can be ascribed as the SSB labeled on DIL-Lip. The ultraviolet visible absorption spectra of Amino-functionalized magnetic beads (MB) (Fig. 1d-a). And the SEM images shown Amino-functionalized MB (Fig. S3), Aptamer (Fig. 1d-b), MB-Apt (Fig. 1d-c). Fig. 1d-c demonstrated maximum absorption peak at 261 nm which can be ascribed as the Aptamer labled on MB (Ikawa et al., 2011). 3.2. Comparison of fluorescence aptasensor responses using SSB-DIL and DIL-Lip labels In order to evaluate the liposome can amplified signal by the fluorescence response, we chose two signal tags with varied DIL loading amount. Hence, we performed two control experiments by adding SSB-DIL and DIL-Lip as signal tags. An obvious difference between SSB-DIL and DIL-Lip is that the former has a higher loading capacity of DIL at same concentration 1 mg mL  1. As shown in Fig. S4-b, upon adding DIL-Lip, a significantly increased fluorescence intensity appeared, which was about 11-fold higher than Fig. S4-a. It showed that DIL can be excellent integrated into liposomes, which was due to the principle of similar compatibility. It can be concluded that liposome has excellent signal amplification effect to implement high sensitivity for the fluorescence aptasensor. 3.3. Optimization of experimental condition It was well-known that the fluorescence response was closely related to pH of detection solution, the optimal concentration of DIL, the reaction time between the probe and CAP, incubation temperature. Fig. S5 showed the effects of pH of detection solution (curve A), the optimal concentration of DIL (curve B), the reaction time between the probe and CAP (curve C),incubation temperature (curve D). Fig. S5 (A) demonstrated the fluorescence intensity of diffidence pH of detection solution and the maximum intensity

value at pH 7.4. Furthermore, based on the experimental results in Fig. S5 (C), 30 min was selected as the optimal incubation time. Fig. S5 (D), the values of incubation temperature from 20 °C to 45 °C was chosen for experiments. Results revealed that the temperature of 37.4 °C was optimal. It could be seen from Fig. S5 (B) that the FL intensity signal first increased and then smooth with the increasing of concentration of DIL from 20 to 60 μM with 10 mg/mL liposomes and the maximum fluorescence value was obtained at 50 μM of DIL. Fig. S6 demonstrated pH values effect on fluorescence intensity with DIL and single-stranded DNA binding protein coimmobilized liposomes. And based on the experimental results in Fig. S6, the experiment has no effect on pH with 6-9. Fig. S7 that the decay of the fluorescence properties of the DIL (black curve), SSB/DIL-Lip (red curve) with time. We observed the decay of the fluorescence properties of the sensor with time. But this decay is not obvious at 24 h. 3.4. Selectivity and specificity of aptasensor for CAP In order to further investigate the selectivity and specificity of the proposed analysis, we choose five main antibiotics maybe in food, including kanamycin (Kana), streptomyces erythreus (SE), oxytetracycline (OTC), gentamicin sulfate (GS) and chlortetracycline (CTC). Separately, the five other antibiotics were added into the vesicle probe with identical concentration (10 nM). As demonstrated in Fig. 2, only CAP can induce the prominence fluorescence enhancement, there is intimation that the aptasensor shows high selectivity for the target CAP species. 3.5. The detection mechanism and increased sensitivity performance To further demonstrate that if the CAP can replace SSB/DIL-Lip complex to the supernatant due to the aptamer on the capture adsorbent preferentially bounded with CAP. We used UV–vis spectrum to investigate the supernatant after the reaction with

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the relative standard deviation (RSD) was about 2.05%. And the sensor system can be employed for five repeated measurement of 0.003 nM of CAP and the relative standard deviation (RSD) was about 4.97%. The results represented that the proposed strategy can be used for the detection of a wide concentration range of CAP. 3.7. Analytical application

Fig. 2. The specificity and selectivity of the developed fluorescence aptasensor.

different concentration CAP. This is because DIL-Lip can show well UV absorption peak (Fig. 3A). The L-DIL displayed a strong UV absorption peak at 580 nm and a weak fluorescent peak at 522 nm in the supernatant. The absorption intensity of DIL-Lip gradually increased with the increasing CAP. As shown in Fig. 3, the absorption intensity value reduced linearly with the concentration of CAP varied from 0.01 to 10 nM. The relationship can be described as y ¼0.032ln(x) þ0.207. The correlation coefficient is 0.990, demonstrating a favorable linear relationship. This result showed when the vesicle probe solution was mixed with CAP, the aptamer on the capture adsorbent can preferentially bound with CAP, and then release SSB/DIL-Lip complex to the supernatant to emit strong fluorescence light. 3.6. Analytical performance of the fluorescence aptasensor The developed aptasensor was evaluated by a series of CAP concentrations based on the peak of fluorescence emission at 565 nm. As shown in Fig. 4, the fluorescence intensity value increased linearly with the concentration of CAP varied from 0.003 to 10 nM. The relationship can be depicted as y¼ 222.8þln CCAP (nM), y represents the fluorescence intensity, x represents the molar concentration of CAP. The correlation coefficient is 0.994, indicating a favorable linear relationship. The detection limit was 0.001 nM (S/N¼ 3). Such low detection limit could be put down to Liposome-encapsulated a multitude of DIL. The sensor system can be employed for five repeated measurement of 10 nM of CAP and

We also enumerated many recent literatures to detect CAP with not the same detection methods in Table 1. In our study, the detection limit was as low as 0.003 nM and the linear concentration range achieved 4 orders of magnitude. So as to investigate the practical application of the aptasensor, several seafood specimens obtained from supermarket in China was employed to prove the application of the proposed method. The feasibility of the applied bioanalysis strategy was evaluated with real fish samples. And the obtained results were contrasted with those of the ELISA methods. The results were given in Table S2. The results were almost uniform in those by ELISA method without significant difference (Ttest). The accuracy of CAP detection in fish samples was also valuated by determining the recovery of CAP by a criterion addition method, into which a known quantity of CAP (0.1, 1 nM, respectively) was added. As shown in Table 1, the recoveries were all between 90.00% and 105.56%, indicating a favorable accuracy of the proposed fluorescence aptasensor for CAP detection. It was distinctly demonstrated that the aptasensor was applicable to the detection of CAP in real seafood samples.

4. Conclusion In conclusion, we developed of a novel fluorescence aptasensor for CAP based on magnetic vesicle aptamer probes. CAP with an ultrahigh sensitivity (0.003 nM) can be detected because of the high sensitivity of the vesicle probes which contains many DIL inside liposome. We also fabricate a novel detection mechanism for the florescence assay, which is based on competition replacement reaction between CAP and SSB with aptamer. Because CAP has higher affinity to aptamer than SSB, the fluorescence vesicle signal tracer can be quantitatively replaced into supernatant after the probes binding CAP. Furthermore, the proposed method shown a high specificity to CAP based on the aptamer selectivity, and can be easily achieve matrix separation in the solution containing other antibiotic relevant proteins as interferes. More inspiringly, the proposed sensing strategy for CAP detection could be applied to CAP residues detection in real fish samples, which results was consistent with that from ELISA. In the scheme, we just

Fig. 3. (A) UV–vis spectra of replacement policy in different concentrations of CAP, (B) calibration plot in different concentrations of CAP.

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Fig. 4. (A) Fluorescence emission spectra of the proposed assay in the presence of different concentrations of CAP, (B) calibration plot in different concentrations of CAP, all the experiment under optimal conditions with excitation wavelength of 520 nm. Table 1 The current detection methods for CAP, linear range and the limit of detection. Method

Target analyte: CAP (nM) Linear range

Flow immunoassay 0.09–61.89 Fluorescence 100–70000 Electrochemical biosensor 0.10–2500 GC–MS 310–3095 HPLC-MS/MS 1.55–309.47 This method 0.003–10

References

LOD 0.06 33 0.02 155 0.001

(Berlina et al., 2013) (Tana et al., 2015) (Yadav et al., 2014) (Liu et al., 2014) (Wu et al., 2012) This work

Note: The symbol ‘-’ suggest that the sample could not be detected by the corresponding method.

used chloramphenicol as model for testify our assay. Moreover, the designed assay can be applied in detect other antibiotics when changing the aptamer on the composite probes, which revealed its potential for applications in food and environment.

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31070866), the Natural Science Foundation of Zhejiang (Y15B050002, Y16B050004, Pd2013088, LY13C200017), the Natural Science Foundation of Ningbo (2014A610184) and the K.C. Wong Magna Fund in Ningbo University, Non-profit project funded by State Administration of Grain (201313010) and a project funded by 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.2016.03.034.

References Bartczak, D., Kanaras, A.G., 2011. Langmuir 27, 10119–10123. Berlina, A.N., Taranova, N.A., Zherdev, A.V., Vengerov, Y.Y., Dzantiev, B.B., 2013. Anal. Bioanal. Chem. 405, 4997–5000. Bogomolova, A., Aldissi, M., 2015. Biosens. Bioelectron. 66, 290–296.

Caracciolo, G., Pozzi, D., Capriotti, A.L., Cavaliere, C., Piovesana, S., Amenitsch, H., Laganà, A., 2015. RSC Adv. 5, 5967–5975. Camelo, S., Lajavardi, L., Bochot, A., Goldenberg, B., Naud, M.C., Fattal, E., BeharCohen, F., de Kozak, Y., 2007. Mol. Vis. 13, 2263. Garg, M., Mishra, D., Agashe, H., Jain, N.K., 2006. J. Pharm. Pharmacol. 58, 459–468. Guo, W., Sun, N., Qin, X., Pei, M., Wang, L., 2015. Biosens. Bioelectron. 74, 691–697. Hidayat, B.J., Thygesen, L.G., Johansen, K.S., 2013. Cellulose 20, 1041–1055. Ikawa, Y., Touden, S., Furuta, H., 2011. Org. Biomol. Chem. 9, 8068–8078. Ikeda, A., Kawai, Y., Kikuchi, J.I., Akiyama, M., 2010. Chem. Commun. 46, 2847–2849. Johnson, L.V., Walsh, M.L., Chen, L.B., 1980. Proc. Natl. Acad. Sci. 77, 990–994. Juliano, R.L., Stamp, D., 1975. Biochem. Biophys. Res. Commun. 63, 651–658. Kaur, M., Choi, D.H., 2015. Chem. Soc. Rev. 44, 58–77. Klibanov, A.L., Maruyama, K., Torchilin, V.P., Huang, L., 1990. FEBS Lett. 268, 235–237. Kowalczykowski, S.C., Bear, D.G., Von Hippel, P.H., 1981. Enzymes 14, 373–444. Leserman, L.D., Barbet, J., Kourilsky, F., Weinstein, J.N., 1980. Nature 288, 602–604. Liu, Y., Yan, K., Okoth, O.K., Zhang, J., 2015. Biosens. Bioelectron. 74, 1016–1021. Liu, T., Xie, J., Zhao, J., Song, G., Hu, Y., 2014. Food Anal. Methods 7, 814–819. Lozano, N., Al-Ahmady, Z.S., Beziere, N.S., Ntziachristos, V., Kostarelos, K., 2015. Int. J. Pharm. 482, 2–10. Lu, M., Gursky, O., 2013. Biomol. Concepts 4, 501–518. Mehta, J., Van Dorst, B., Rouah-Martin, E., Herrebout, W., Scippo, M.L., Blust, R., Robbens, J., 2011. J. Biotechnol. 155, 361–369. Niu, H.T., Jiang, X., He, J., Cheng, J.P., 2009. Tetrahedron Lett. 50, 6668–6671. Ochi Ardebili, M.M., Amoabediny, G., Rezayat, S.M., Akbarzadeh, A., Ebrahimi, B., 2015. SSU_J. 23, 2000–2012. Song, Q., Peng, M., Wang, L., He, D., Ouyang, J., 2016. Biosens. Bioelectron. 77, 237–241. Tana, Z., Xu, H., Li, G., Yang, X., Choi, M.M., 2015. Spectrochim. Acta Part A: Mol. Biomol. Spectrosc. 149, 615–620. Verbovetski, I., Bychkov, H., Trahtemberg, U., Shapira, I., Hareuveni, M., Ben-Tal, O., Kutikov, I., Gill, O., Mevorach, D., 2002. J. Exp. Med. 196, 1553–1561. Vermes, I., Haanen, C., Steffens-Nakken, H., Reutellingsperger, C., 1995. J. Immunol. Methods 184, 39–51. Wang, L., Zhang, Y., Gao, X., Duan, Z., Wang, S., 2010. J. Agric. Food Chem. 58, 3265–3270. Wang, K., Liao, J., Yang, X., Zhao, M., Chen, M., Yao, W., Tan, W., Lan, X., 2015. Biosens. Bioelectron. 63, 172–177. Wu, J., Chen, L., Mao, P., Lu, Y., Wang, H., 2012. J. Sep. Sci. 35, 3586–3592. Xiao, Y., Lubin, A.A., Heeger, A.J., Plaxco, K.W., 2005. Angew. Chem. 117, 5592–5595. Xing, Y.P., Liu, C., Zhou, X.H., Shi, H.C., 2015. Sci. Rep., 5. Xu, W., Lu, Y., 2009. Anal. Chem. 82, 574–578. Yadav, S.K., Agrawal, B., Chandra, P., Goyal, R.N., 2014. Biosens. Bioelectron. 55, 337–342. Yan, Z., Gan, N., Wang, D., Cao, Y., Chen, M., Li, T., Chen, Y., 2015. Biosens. Bioelectron. 74, 718–724. Yang, C., Spinelli, N., Perrier, S., Defrancq, E., Peyrin, E., 2015. Anal. Chem. 87, 3139–3143. Yu, X., He, Y., Jiang, J., Cui, H., 2014. Anal. Chim. Acta 812, 236–242. Zhao, M., Zhuo, Y., Chai, Y.Q., Yuan, R., 2015. Biomaterials 52, 476–483. Zhang, B., Ge, C., Yao, J., Liu, Y., Xie, H., Fang, J., 2015. J. Am. Chem. Soc. 137, 757–769. Zhou, J., Wang, Q.X., Zhang, C.Y., 2013. J. Am. Chem. Soc. 135, 2056–2059.