Aptasensor based on the synergistic contributions of chitosan-gold nanoparticles, graphene-gold nanoparticles and multi-walled carbon nanotubes-cobalt phthalocyanine nanocomposites for kanamycin detection.

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Analyst Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: X. Sun, F. Li, G. Shen, J. Huang and X. Wang, Analyst, 2013, DOI: 10.1039/C3AN01840G.

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Analyst View Article Online

DOI: 10.1039/C3AN01840G

Aptasensor based on the synergistic contributions of chitosan-gold nanoparticles, graphene-gold nanoparticles and multi-walled carbon nanotubes-cobalt phthalocyanine nanocomposites for kanamycin Published on 14 October 2013. Downloaded by University of Virginia on 26/10/2013 11:00:34.

detection Xia Sun b, Falan Li b,Guanghui Shen b, Jiadong Huang a,c*, Xiangyou Wang b*

b School of Agriculture and Food Engineering, Shandong University of Technology, Zibo255049, P.R. China. c Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, P.R. China

*

Corresponding author: Professor Jiadong Huang and Professor Xiangyou Wang, Tel.: +86 531 89736122; Fax: +86 531 82769122 E-mail: [email protected] (J. Huang); [email protected] (X. Wang)

1

Analyst Accepted Manuscript

a College of Medicine and Life Sciences, University of Jinan, Jinan250022, P.R. China.

Analyst

Page 2 of 32 View Article Online

DOI: 10.1039/C3AN01840G

Abstract An electrochemical aptasensor was developed for the detection of kanamycin based on the

(GR-AuNPs) and multi-walled carbon nanotubes-cobalt phthalocyanine (MWCNTs-CoPc) nanocomposites. The aptasensor was prepared by sequentially dripping CS-AuNPs, GR-AuNPs and MWCNTs-CoPc nanocomposites onto gold electrode (GE) surface. During the above process, these nanomaterials showed remarkable synergistic effect towards aptasensor. CS-AuNPs, GR-AuNPs and MWCNTs-CoPc as nanocomposites mediator improved electron relay during entire electron transfer process and the aptasensor response speed. The electrochemical properties of the modified processes were characterized by cyclic voltammetry (CV). The morphologies of the nanocomposites were characterized by scanning electron microscopy (SEM). The experimental conditions such as concentration of aptamer, the time, the temperature and the pH were optimized. Based on the synergistic contributions of CS-AuNPs, GR-AuNPs and MWCNTs-CoPc nanocomposites, the proposed aptasensor displayed high sensitivity, highly specific, low detection limit (5.8×10-9 M) (S/N=3) and excellent stability. It was successfully applied to the detection of kanamycin in real milk spiked samples. Keywords: Aptasensor, Chitosan-gold nanoparticles, Graphene-gold nanoparticles, Multi-walled carbon nanotubes-cobalt phthalocyanine nanocomposites, Kanamycin detection.

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Published on 14 October 2013. Downloaded by University of Virginia on 26/10/2013 11:00:34.

synergistic contributions of chitosan-gold nanoparticles (CS-AuNPs), graphene-gold nanoparticles

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1. Introduction Kanamycin, an aminoglycoside antibiotic, is used to treat a wide variety of infections. In

inhibit protein synthesis.1 When it is abused in humans through intake of food or medicinal overprescribing, it will cause serious side effects, including toxicity to the kidneys, loss of hearing and allergic reactions to the drug.2 Therefore, it is critical to develop sufficiently sensitive methods to detect kanamycin residue for food safety and clinical diagnosis. Recently, many analytical methods, such as capillary electrophoresis,3 surface plasmon resonance,4 square-wave cathodic adsorptive stripping voltammetry,5 immunoassay,6 HPLC,7 the microbiological multi-residue system8 and immunological methods9 have been reported for the detection of kanamycin. However, most of those above-mentioned methods are often tedious, time consuming and require a great amount of solvents, reagents and expensive apparatus. Thus, a low cost, less reagent consumption, highly sensitive and selective technique for kanamycin detection is very desirable. Aptamers are RNA or DNA molecules with specific 3-D structures. They can be selected through an in vitro selection process called systematic evolution of ligands by exponential enrichment (SELEX), and they are capable of recognizing and binding a variety of targets ranging from small molecules to organisms. Owing to the unique advantages in chemical component, accurate, easily modified, vitro synthesis, high purity and chemically stable, aptamers have been used as a strong competitor of antibodies in analysis application.10 Moreover, due to their relative ease of tailored binding affinity, modification, isolation and easier storage,11 aptamers exhibit several unprecedented advantages as compared with antibodies. Various aptamer-based strategies and technologies for detections have been developed, such as surface plasmon resonance (SPR),12

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susceptible bacteria, the role of kanamycin is to bind 30 S subunit of the bacterial ribosome and

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quartz crystal microbalance (QCM),13 electrochemical detection14, colorimetric,15 fluorescence,16 chemiluminescent,17 dry-reagent strip18 and so on.

based on the enzymatic catalytic reaction. The commonly used enzymes served as labels in bioassays include horseradish peroxidase (HRP),19 glucose dehydrogenase20 and alkaline phosphatase.21 Among these enzymes labels, HRP is the most commonly used label since it is the smallest and most stable enzyme.22 Using HRP as the label enzyme, an electrochemical aptasensor for thrombin detection based on enzymatic reaction has been developed.19 Recently, a RNA aptasensor labeled with methylene blue has been reported for detection of kanamycin and some other aminoglycoside antibiotics in blood serum.23 To enhance the sensitivity of the electrochemical sensors, the composites of nanomaterials and conducting polymers have also been used.24 As a counterpart of graphite with well-separated 2D aromatic sheets, graphene possesses high electron mobility under ambient conditions25 and large specific surface area with low manufacturing cost.26 Recently, graphene-based composites materials have received increasing attention due to the synergistic contribution of two or more functional components and their potential applications.27 In addition, as excellent acceptors, gold nanoparticles (AuNPs) can allow ultra-efficient adsorption of proteins as a result of their exceptional properties, such as large surface area, superior mechanical properties, excellent conductivity and large thermal conductivity.28 Many investigations have demonstrated that graphene-gold nanoparticles (GR-AuNP) nanocomposites have remarkable electrochemical properties. Our group has also reported that GR-AuNPs can improve the electrochemical response and the effective surface area of the electrode.29 Moreover, some biocompatible materials such as

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In addition, enzyme is used as an electroactive label to introduce further signal amplification

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chitosan (CS), gold nanoparticles (AuNPs) and ionic liquid are also introduced into the modified layers to improve the stability of enzyme.30 CS is a typical biological macromolecule, a

slightly acidic solution allows the co-processing of CS with other biomolecules because its primary amines can be chemically cross-linked with other reagents. The -NH2 ligand of CS has been used to stabilize the AuNPs in the preparation process. The application of the CS-AuNPs nanocomposites has been reported and they can provide a suitable micro-environment for enzyme immobilization.31 Au nanoparticle also assemble through electrostatic interactions (as polyion complexes) on chitosan with solution of citrate-stabilized gold nanoparticles (negatively charged),32 and the nanoparticles can act as tiny conduction centers to facilitate electron transfer.33 Among the various types of nanomaterials, the MWCNTs possess a relatively well-characterized behavior in terms of electron transport mechanism34,35 and surface binding phenomenon.36 Recently, immobilization of metal phthalocyanine (MPc) at the surface of MWCNTs has been achieved by means of noncovalent adsorption.37 Moreover, MWCNTs and cobalt phthalocyanine (CoPc) (MWCNTs-CoPc) composites have been widely used to modify electrode and the enhancement effect of current response is significant.38 In this work, considering benefits of the CS-AuNPs, GR-AuNPs and MWCNTs-CoPc, we integrated them in a aptasensor to exploit the synergy contributions on the improvement of aptasensor characteristics. Kanamycin as a small molecular and an aptamer modified with different

group

(Atp

1:

5'-amino-TGGGGGTTGAGGCTAAGCCGAC-3';

Apt2:

5'-biotin-TGGGGGTTG AGGCTAAGCCGAC-3')39 were used to recognize kanamycin to form a sandwich

structure.

The

aptamers

(Apt1:

5

5'-NH2-(CH2)6-GGTTGGTGTGGTTGG-3';



polysaccharide copolymers of glucosamine and N-acetylglucosamine. The solubility of CS in

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Apt2:5'-NH2-(CH2)6-GGTTGGTGTGGTTGG-3) were reported to recognise different sites of thrombin. 40,41 The sequences of Apt1 and Apt2 were the same, and they can combine different part

5'-biotin-AAAAAAAAAATACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAG TAT-3';

Apt2:

5'-amino-AAAAAAAAAATACTCAGGGCACTGCAAGCAATTGTGGTCCC

AATGGGCTGAGTAT-3') were reported to recognise different sites of platelet-derived growth factor B-chain homodimer (PDGF-BB)42 The sequences of Apt1 and Apt2 were also the same modified with different group. Mutational experiments on the kanamycin aptamer revealed that kanamycin binds to its loop region (GG sequence).39 The loop region (GG sequence) of Atp1(5'-amino-TGGGGGTTGAGGCTAAGCCGAC-3')

and

Apt2(

5'-biotin-TGGGGGTTG

AGGCTAAGCCGAC-3') have a high affinity for kanamycin. Kanamycin can binds to loop region (GG sequence) of aptamer. Therefore, we suspected that two same aptamer can combine different part of one kanamycin( Fig. 1 (b), (c)), and the experiments showed that our guess is correct. To the best of our knowledge, such an aptasensor had not been reported. The proposed aptasensor had the advantages of high sensitivity and low background current. This electrochemical aptasensor was simple, rapid, sensitive and highly specific, and it was developed for sensitive detection of kanamycin in real milk samples. (Fig. 1) 2. Experimental 2.1 Reagents and chemicals NaH2PO4·2H2O and Na2HPO4·12H2O were purchased from Beijing Chemical Technology Co., Ltd. (Beijing, China). Graphene (GR), kanamycin sulfate, neomycin sulfate, chlortetracycline

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of one thrombin to form a sandwich structure. An aptamers with sandwich structure (Apt1:

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hydrochloride and gentamicin sulfate were all purchased from Jingchun Co., Ltd. (Shanghai, China). Chitosan (CS), DNA oligonucleotides modified with biotin and amino group (Atp 1:

CCGAC-3') were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The aptamer sequences specific for kanamycin, which are identified by Song et al. via the SELEX process from a library (1 × 1015~1 × 1016 molecules, 100 mL) of single-stranded DNA randomized at 40 contiguous positions Chloroauric acid (HAuCl4) and ethanol were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cobalt phthalocyanine (CoPc) was obtained from Alfa Aesar (Ward Hill, MA). Multi-walled carbon nanotubes (MWCNTs) were purchased from Sigma-Aldrich (St. Louis, USA). All other chemicals were analytical reagent grade. All the solutions were prepared with ultrapure water which was purified with a Milli-Q purification system (Branstead, USA). 2.2 Apparatus Electrochemical measurements were carried out with a CHI660C workstation (China). A three-electrode configuration was employed, consisting of a gold electrode (GE, φ = 2 mm) serving as the working electrode, a saturated calomel electrode (SCE) and a platinum wire serving as the reference and counter electrodes, respectively. Solution pH value was measured by a FE 20 Mettler-Toledo pH meter (Switzerland). Ultrasonication was performed using a KQ-100E ultrasonic cleaner (Kunshan, China). Scanning electron micrographs (SEM) was studied by JSM-6360LV SEM (Japan). All electrochemical experiments were carried out at room temperature (RT).

2.3 Preparation of CS-AuNPs nanocomposites

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5'-amino-TGGGGGTTGAGGCTAAGCCGAC-3'; Apt 2: 5'-biotin-TGGGGGTTGAGGCTAAG

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The CS was prepared according to the reference.43 The CS-AuNPs nanocomposites were prepared as follows: all glasswares used in the preparation were thoroughly cleaned in aqua regia

1% CS (W/V) acetic acid solution was added into the stirring 200 µl of 1% HAuCl4 (W/V) aqueous solution. After stirring for about 60 min, 100 µl of KBH4 (0.4 mol/L) was added slowly. The solution quickly turned into a ruby-red solution, indicating the formation of the AuNPs. After continuing vigorously stirring the solution for 1 h, the as-prepared nanocomposites were obtained. The resulting solution of colloidal particles was stored in a brown bottle and kept at 4 °C. The resulting nanocomposites was used for all the characterizations and experiments. 2.4 Preparation of GR-AuNPs nanocomposites The synthesis of GR-AuNPs nanomaterials consisted of the following three steps according to previous method.44 Firstly, graphene oxide was prepared from natural flake graphite power by the modified Hummers method.45 1 g graphite powder was added to a big beaker containing 0.5 g sodium nitrate and 23 mL 98% H2SO4, then the mixture were stirred for 30 min in an ice-salt bath at 0 °C.. 3 g KMnO4 was slowly added into the solution at 20 °C.. After being stirred for 2 h, the solution temperature was raised gradually to 35 °C in a water bath and remained for more than 30 min. Then 46 mL ultrapure water was added to the solution and heated to 98 °C for 15 min. After adding 140 mL ultrapure water to dilute the solution, 30% H2O2 was added into the mixture until the color of the suspension changed to brilliant yellow. The reaction mixture was filtered and washed with HCl aqueous solution and ultrapure water. Finally, the obtained solid was dried at 50°C for 24 h and we got graphite oxide. Secondly, polyvinyl pyrrolidone-protected graphene (PVP-protected GR) was prepared. It was prepared as follows: The prepared graphite oxide (5 mg)

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(3 parts HCl, 1 part HNO3), rinsed in triply ultrapure water and oven-dried prior to use. 20 ml of

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was then dispersed into 20 mL of ultrapure water ultrasonication for 2 h. After the dispersed solution was sonicated until it became translucent with no visible particulate matter. Then 80 mg

stirred for 12 h. The above mixture was mixed with 7 µL of hydrazine solution (50 wt% in water) and 80 µL of ammonia solution (25 wt% in water), respectively. Then the mixture was stirred for 1 h at the temperature of 95 °C and we got the black dispersion. At last, it was centrifuged for 10 min and dissolved in 2.5 mL of ultrapure water. Thirdly, GR-AuNPs were synthesized by NaBH4 reduction method: 250 µL of HAuCl4 (1 wt%) solution was added into 2.5 mL of PVP-protected GR obtained above and the mixture was adequately stirred at RT for 0.5 h. Afterwards, 12.5 µL of NaBH4 solution (0.2 mol/L) was added into the mixture solution under stirring. After continuously stirred for 1 h at RT, the color of the solution was dark brown and we got GR-AuNPs. 2.5 Preparation of MWCNTs-CoPc nanocomposites The MWCNTs were purified by adopting the conditions described previously in the literature.46 MWCNTs were chemically shortened and functionalized with carboxylic acid groups on their tips. 50 mg of MWCNTs was suspended in a 40 mL mixtures of concentrated sulfuric acid and nitric acid (3:1, v/v). After ultrasonic agitation for 4 h, the mixture was washed with ultrapure water by centrifugation (10,000 rpm) until the resulting solution was neutral. MWCNTs-CoPc nanocomposites were formed by mixing CoPc (2 mg) in ultrapure water (1 mL) with a 2 mg/mL of MWCNTs solution, followed by ultrasonication for 30 min.47 Then, MWCNTs-CoPc nanocomposites were obtained. 2.6 The fabrication of the aptasensor

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of polyvinyl pyrrolidone (PVP) was added into the resulting dispersion and continuously was

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Prior to modification, the gold electrodes were polished with 0.3 µm alumina powder and sonicated in ultrapure water. Then, electrodes were cleaned in piranha solution (v/v, 7:3 H2SO4 /

ultrapure water. Next, the electrodes were electrochemically cleaned in 0.5 M H2SO4 in the potential scanning between -0.5 V and 1.5 V for 5 min, followed by washing them with ultrapure water and drying under nitrogen. After dried at RT, 6 µL of CS-AuNPs nanocomposites film was pipetted onto the surface of the GE. Then the modified electrode (CS-AuNPs/GE) was dried at RT, 6 µL of the GR-AuNPs nanocomposites film was cast onto the pre-treated substrate and dried in 2 h at RT. At last, 6 µL of the MWCNTs-CoPc was dropped onto the above modified electrode. Then the MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/GE was obtained. The aptamer (Apt1) was assembled on a modified GE surface, while the other aptamer (Apt2) was modified with biotin at 5'-end, which could be further labeled with streptavidin-horseradish peroxidase (SA-HRP). Following that,

6

µL of the aminated

kanamycin Apt1

was

dropped onto

the

MWCNTs-CoPc/GR-AuNPs/CS-AuNPs modified electrode. The electrode was incubated with 0.5% BSA at RT for 0.5 h in order to block possible remaining active sites and avoid the non-specific adsorption. Finally, 6 µL of the prepared Kana-SA-HRP-Apt2 mixture were dropped onto the modified electrode surface and incubated for 1 h. The resulted aptasensor was stored in the 0.1 M PBS (pH 7.5) at 4 °C when not in use. After every adsorption step, the modified electrode was thoroughly rinsed with ultrapure water and dried with nitrogen. The stepwise preparation of the aptasensor was shown in Scheme 1 of Supporting information.

2.7 Electrochemical measurements Electrochemical measurements were done in a conventional electrochemical cell at 37 °C.

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H2O2; warning: piranha solution reacts violently with organic solvents) for 20 min, washed with

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Cyclic voltammograms (CVs) were performed in 0.01 M PBS (pH 7.4) containing 0.1 M KCl from -0.2 to 0.6 V at a scan rate of 100 mV/s. The electrochemical differential pulse voltammetry

-0.05 V to 0.4 V with a pulse height of 50 mV, the step height and the frequency were kept as 4 mV and 15 Hz, respectively. DPV measurements of modified GE were carried out in PBS containing 1 mM hydroquinone (HQ) and 1 mM H2O2. Upon binding with kanamycin, the HRP-labeled aptamer/kanamycin complex on the electrode would increase the reduction current of HQ in the presence of H2O2. The sensitivity and the specificity of the proposed electrochemical aptasensor were investigated by DPV. 3. Results and discussion

3.1 SEM characterization of modified electrodes

The morphologies and microstructures of the as-prepared different films were studied by SEM observation (Fig. 2). When the first CS-AuNPs layer was assembled, gold nanoclusters were deposited on the multilayer film, many different diameter clusters were observed (Fig. 2 (a)). Fig. 2 (b) presented the SEM image of GR-AuNPs nanocomposites. The SEM view of GR-AuNPs clearly illustrated a number of bright dots positioned over the entire surface, particularly at the folded edges and wrinkle sites, suggesting that the AuNPs were indeed linked to the surface of the GR sheets. As shown in Fig. 2 (c), the SEM image revealed that MWCNTs were well distributed on the surface of GE with the forming of small bundles (tubes). Fig. 2.(d) presented the SEM image of GR-AuNPs nanocomposites immobilized on the CS-AuNPs/GE, which was familiar to the SEM view of GR-AuNPs. As shown in Fig. 2 (e), the SEM image revealed that view and the SEM view of MWCNTs-CoPc is similar, when MWCNTs-CoPc was immobilized on the 11



(DPV) measurements were carried out under the following conditions: The voltage scanned from

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GR-AuNPs/CS-AuNPs/GE. Therefore it could be concluded that these nanocomposites had been dispersed on the surface of GE to form nanocomposites film successfully. (Fig. 2)

In this paper, CVs were used in the study to investigate the processes of the modification of electrodes. The cyclic voltammograms (CVs) of differently modified electrodes were presented in Fig. 3 A. It could be seen that the bare GE had obvious redox peak (Fig. 3 A (a)). After the CS-AuNPs (Fig. 3 A (b)) was immobilized on the bare GE, a larger current response was exhibited. The reason might be that AuNPs can effectively increase surface area and the current response of electrode.

With

the

addition

of

6

µL

GR-AuNPs

on

the

CS-AuNPs/GE,

the

GR-AuNPs/CS-AuNPs/GE exhibited a much higher current (Fig. 3 A (c)) response due to the synergistic effects of the good electrical conductivity of GR and AuNPs. After MWCNTs-CoPc (Fig. 3 A (d)) was immobilized on the GR-AuNPs/CS-AuNPs/GE, a larger current response was exhibited. It was found that sensitivity of the aptasensor was more 6-fold better than those of CS-AuNPs (Fig. 3 B), GR-AuNPs and MWCNTs-CoPc used alone (After the CS-AuNPs was alone immobilized on the bare GE, the current only increased 12.5 µA. After the GR-AuNPs was alone immobilized on the bare GE, the current only increased 18.3 µA. The MWCNTs-CoPc was alone immobilized on the bare GE, the current only increased 8.1 µA. After MWCNTs-CoPc was immobilized on the GR-AuNPs/CS-AuNPs/GE, the current increased 231.3 µA. ). This could be ascribed to the improvement of the conductivity between graphene nanosheets in the GR-AuNPs due to the introduction of the CS-AuNPs nanocomposites. Fast charge transfer was obtained from MWCNTs-CoPc to graphene sheets and CS-AuNPs due to unique electrochemical properties of

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3.2 Fabrication mechanism of the sensor

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the MWCNTs-CoPc. When the Apt1 (Fig. 3 A (e)) and BSA (Fig. 3 A (f)) were modified on the MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/GE surface, the peak current decreased in sequence,

transfer, and blocked non-specific sites. The self-assembly of Kana-SA-HRP-Apt2 on BSA/Apt1/MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/GE the voltammetric characteristics of HQ at Kana-SA-HRP-Apt2/BSA/Apt1/MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/GE

changed

dramatically (Fig. 3 A (g)). This result demonstrated that SA-HRP was successfully labelled to the aptamer complex and maintained its eletrocatalytic activity to H2O2 since the increased reduction current at Kana-SA-HRP-Apt2/BSA/Apt1 was due to the catalytic effect of HRP. HQ serves as an effective mediator of shuttling electrons between the electrode surface and the redox center of HRP. As shown in the following mechanism, H2O2 in the solution is firstly reduced by the immobilized HRP (Red). Then the HRP can be regenerated with the aid of the mediator, while the mediator HQ can be oxidized to BQ in the enzymatic reaction, leading to the disappearance of the HQ oxidation peak. Finally, the BQ can be reduced back to HQ by receiving electrons from the electrode. H2O2 + HRP(Red) → HRP(Ox) + H2O HRP(Ox) + HQ → BQ + HRP(Red) BQ + 2H+ + 2e→ HQ Therefore, HQ is recycled in the system, leading to a big increase of the reduction current. The increase of the reduction current was related to the amount of SA-HPR on the electrode, further related to the concentration of kanamycin. (Fig. 3)

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which suggested the aptamer and BSA severely reduced effective area and active sites for electron

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3.3 Optimization of the analytical parameters The experimental parameters were optimized in terms of aptamer concentration, incubation time，temperature and pH. The optimum concentration of capturing Apt1 was determined by drop

activated surface of MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/GE. The aptamer modified probe was treated with 10 µM kanamycin in 0.1 M PBS (pH 7.4). Then, DPVs were recorded for different concentrations of aptamer-modified sensors. The effect of aptamer concentrations on the responses was shown in Fig. 4 (a). The current response increased with increasing aptamer concentrations. However, there was no significant increase in the response that was observed for a concentration of aptamer above 5.0 µM, due to the saturation of the active sites for immobilizing aptamer. Thus, an aptamer concentration of 5.0 µM was used for sensor fabrication. To investigate the effect of incubation time with respect to kanamycin uptake, the Apt/MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/GE was immersed in 0.1 M PBS (pH 7.4) containing 10 µM kanamycin for different time interval (0-120 min) and DPVs were recorded for the captured kanamycin. The relationship between the sensor response and the incubation time was shown in Fig. 4 (b). The current response increased with the increasing incubation time and reached a platform at 60 min. However, there was no significant increase in the peak current observed after 60 min due to the saturation of the active sites for kanamycin binding. Thus, 60 min was selected as the adequate incubation time for the Apt/MWCNTs-CoPc/GR-AuNPs /CS-AuNPs/GE to interact with kanamycin. Further, the effect of incubation temperature was investigated at the temperatures ranging from 10 °C to 60 °C. The signal responses of the assay to the different incubation temperatures are shown in Fig. 4 (c). The peak current increased significantly as the temperature increased from 10 14



coating with different concentrations (0.5, 1.0, 2.0, 5.0, 10.0, 15.0, 20.0 µM) of aptamer onto the

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°C to 30 °C and then suddenly decreased over 30 °C. The decrease of the sensor response over 30 °C might be due to the partial denaturation of the aptamer structure at the elevated temperatures.

temperature. In addition, the effect of pH on the sensor response was examined by recording DPVs for the kanamycin-captured electrode in 0.1 M PBS. The current increased as the medium pH increasing from 5.0 to 9.0 and then decreased when the pH values were higher than 7.4 (Fig. 4 (d)). The maximum current was observed at a pH of 7.4. Therefore, all subsequent experiments were performed in 0.1 M PBS of pH 7.4. Detection parameters, such as H2O2 concentration and HQ concentration could also affect the detection sensitivity of the assay. A series of optimizations for these parameters were performed to improve the sensitivity of aptasensor. As shown in Fig. 4 (e), the peak current of the proposed aptasensor increased until 10 mM HQ. The current responses to a saturation value over 10 mM HQ. As shown in Fig. 3 (f), the current response increased with the increase of H2O2 concentration and leveled off to a saturation value at 8 mM of H2O2 when the concentration of HQ was 10 mM. Therefore, pH 7.4, 10 mM HQ and 8 mM H2O2 were chosen as the optimization of the detection system. (Fig. 4) 3.4. Calibration curve The calibration plots for kanamycin detection with the prepared aptasensor under optimal experimental conditions were shown in Fig. 5. A gradual increase in current was observed with the increasing kanamycin concentration and the corresponding calibration curve exhibited a good

15



Taking the stability of the aptamer into consideration, 30 °C was selected as the optimal

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linearity. The changes of the oxidation peak current response (∆I) of the aptasensor were found to be proportional to the kanamycin concentration in linear ranges from 10.0 to 150.0 ng/mL, with a

was 0.9987, respectively. Compared to other reported colorimetric methods, the proposed aptasensor exhibited higher sensitivity with a lower detection limit (Table 1).39,48 The results of this comparison were shown in Table 1 of the Supplemental Information． (Fig. 5) (Table 1) 3.5. Selectivity, reproducibility, repeatability and stability of the aptasensor Selectivity is an important property of the aptasensor. It was evaluated by measuring the DPV response. We introduced four kanamycin analogues such as oxytetracycline (QTC), neomycin sulfate (NEO), chloromycetin (CHI), chlortetracycline (CTC) and gentamicin sulfate (GM) to investigate

the

selectivity

performance

of

the

sensor.

The

Apt/MWCNTs-CoPc

/GR-AuNPs/CS-AuNPs/GE was incubated in 0.1 M PBS (pH 7.4) containing 10.0 mM kanamycin and 100 mM interfering antibiotics for 60 min. After washing the electrode with PBS, further experiments were conducted in 0.1 M PBS (pH 7.4) using DPV. The current responses for kanamycin in the absence and presence of other antibiotics were obtained (Fig. 6 A (a) of the Supplemental Information.). It was observed that in the presence of these interferents, the change in current was negligible, indicating that the selectivity of the developed aptasensor for kanamycin was good. To further confirm the selectivity of this assay, the cross reactivity of other kanamycin analogues was tested. The experimental results were shown in Fig. 6 A (b). As evident from Fig. 6 A (b), kanamycin (10 mmol/L) showed a much stronger current response, while almost negligible

16



detection limit of 5.8 nM (S/N=3). The linear slope was 0.5428 and the correlation coefficients

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electrochemical changes were detected for any of kanamycin analogues (100 mmol/L) tested in its mixture. Because the current response was induced by the specific interaction of the aptamer and

could not combine with kanamycin analogues. All of the above results confirmed that there was insignificant cross reactivity for other kanamycin analogues and the developed aptasensor could be used to determinate kanamycin with high specificity. Therefore, the corre-sponding current responses of kanamycin analogues were almost negligible. The reproducibility of the aptasensor was conducted by determining 10 µmol/L kanamycin solution with six aptasensors which were fabricated with the same procedure. The relative standard deviation (RSD) of 3.8% was obtained, which indicated that the aptasensor had a good reproducibility. To investigate the repeatability of the aptasensor, five aptasensors fabricated independently under the same conditions were examined. After using the five aptasensors for 3 times continuously, the RSD of 4.2% was observed. It indicated that the aptasensor had good repeatability. Stability is a key parameter for the application and development of the sensor. Under optimal conditions, the aptasensor was measured by CV for a 60-cycle successive scan, and a 2.63% deviation of the initial response was observed (Fig. 6 B). This result indicated that the proposed aptasensor had a high stability. To investigate further the stability of the sensor, we prepared four aptasensors and stored them for 15 days at 4 °C. Then we used them to detect the 10 µmol/L kanamycin. The current response decreased by about 7%. It suggested that the developed aptasensor possessed good stability.

17



the target molecule, the results showed that these aptamers could only recognize kanamycin and

Analyst


DOI: 10.1039/C3AN01840G

(Fig. 6) 3.6. Determination of kanamycin in real samples Although the proposed sensor showed good selectivity towards kanamycin, it is worth

milk is of considerable interest, since milk is one of the most heavily regulated products in food industry because of the risk of having veterinary medicine residue. The milk samples used in this study were all purchased from a supermarket in China. The milk sample was firstly filtered through a sterile milipore membrane (0.20 µm)49 and diluted five times with PBS. Further, kanamycin standard solution was spiked into the diluted milk, making the final concentrations of 0.05, 0.1, 0.5, 1.0, 2.0 µM kanamycin and then experiments were carried out according to the aforementioned optimized conditions for kanamycin detection with the developed aptamer sensor. The kanamycin concentration recoveries were between 97.18%-103.1% (Table 2), which clearly indicated that the aptasensor was available for the detection of kanamycin in real milk sample. (Table 2) 4. Conclusions In this paper, we have developed an ultrasensitive and highly specific electrochemical aptasensor for kanamycin detection based on aptamer/target/aptamer configuration. The detection limit down to 5.8×10-9 M for kanamycin has been achieved by the reaction of H2O2 and HQ catalyzed by HRP. This could be ascribed to improvement of the conductivity between graphene nanosheets in the GR-AuNPs due to introduction of the AuNPs, fast charge transfer from MWCNTs-CoPc to the graphene sheets and AuNPs due to unique electrochemical properties of the MWCNTs-CoPc, and good biocompatibility of the AuNPs for horseradish peroxidase. The

18



exploring the analytical utility of sensor for a practical application. Detection of kanamycin in

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aptasensor possessed high sensitivity, good reproducibility and cost-effective. In addition, the proposed sensor was successfully applied for kanamycin detection in milk sample. It could be a

Acknowledgements This work was supported by the National Natural Science Foundation of the People’s Republic of China (No. 31171700 and 31101296), the National High Technology Research and Development Program of China (National 863 Program of China) (No. 2012AA101604), the Natural Science Foundation of Shandong Province (No.ZR2010DQ025) and the Shandong Province Higher Educational Science and Technology Program (No. J10LB14). References 1 J. Wirmer and E. Westhof, Methods Enzymol., 2006, 415, 180-202. 2 R. Oertel, V. Neumeister and W. Kirch, J. Chromatogr. A, 2004, 1058, 197-201. 3 E. Kaale, A. V. Schepdael, E. Roets and J. Hoogmartens, Electrophoresis, 2003, 24, 1119-1125. 4 M. Frasconi, R. Tel-Vered, M. Riskin and I. Willner, Anal. Chem., 2010, 82, 2512-2519. 5 J. L. Yan and Russ. J. Electrochem., 2008, 44, 1334-1338. 6 E. M. G. Loomans, J. Wiltenburg, M. Koets and A. Amerongen, J. Agric. Food Chem., 2003, 51, 587-593. 7 Y. X. Zhou, W. J. Yang, L. Y. Zhang and Z. Y. Wang, J. Liq. Chromatogr. Related Technol., 2007, 30, 1603-1615. 8 Althaus, M. I. Berrugab, A. Monteroc, M. Rocac and M. P. Molinac, Anal. Chim. Acta, 2009, 632, 156-162.

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Figure captions Scheme 1. The stepwise preparation of the aptasensor.

kanamycin; (c) Sandwich structure model. Fig. 2. SEM images of (a) CS-AuNPs/GE; (b) GR-AuNPs/GE; (c) MWCNTs-CoPc/GE; (d) GR-AuNPs/CS-AuNPs/GE; (e) MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/GE. Fig. 3. (A): (a) CV of the bare GE; (b) CS-AuNPs/GE; (c) GR-AuNPs/CS-AuNPs/GE; (d) MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/GE; (e) Apt1/MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/ GE;

(f)

BSA/Apt1/MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/GE;

(g)

Kana-SA-HRP-Apt2

/BSA/Apt1/MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/GE. (B): (a) CV of the bare GE; (b) CS-AuNPs/GE; (c) GR-AuNPs/GE; (d) MWCNTs- CoPc/GE; (e) MWCNTs-CoPc/GR-AuNPs/CS-AuNPs/GE. Fig. 4. The optimization of experimental parameter: the influence of (a) aptamer concentration, (b) the incubation time, (c) the temperature of working, (d) pH of working, (e) HQ concentration, (f) H2O2 concentration. Fig. 5. (a.b) The calibration curve of aptasensor. Fig. 6. (A) The specificity of the aptasensor; (B) The stability analysis of aptasensor

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Fig. 1 (a) Predicted secondary structure of the kanamycin aptamer; (b) The molecular structure of


Scheme 1



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Fig. 1


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d

Fig. 2



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e

A




View Article Online

B

Fig. 3


a

c

e f

Fig. 4



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b

d

a




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b

Fig. 5

A a




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B

b

Fig. 6

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Method of detection

Limit of detection (M)

Fluorescence detection ELISA ELISA Colorimetric detection Aptasensor detection Aptasensor detection

8.95× 10-9-2.56× 10-8 4.15 × 10-9 2.1× 10-8

Linear range (M)

References

2.7× 10-8-6.7× 10-5

Yu et al., 2009

-

Watanabe et al., 1999 Loomans et al., 2003

2.5 × 10-8

-

Song et al., 2011

9.4 ± 0.4 × 10-9

5 × 10-8-9× 10-6

Zhu et al., 2012

5.8 × 10-9

1 × 10-8-1.5 × 10-7

this work



Table 1 Comparison with other electrochemistry methods in the determination of kanamycin.

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DOI: 10.1039/C3AN01840G

Milk Found (µM)

Added (µM)

Total found (µM)

Recovery (%)

Not detected Not detected Not detected Not detected Not detected

0.05 0.1 0.5 1 2

0.0512 0.0987 0.4972 1.1031 1.9435

102.4 98.7 99.44 103.1 97.18



Table 2 The detection of real samples of kanamycin by Apt/MWCNTs-CoPc/GRAuNPs/CS-AuNPs/GE.

Phthalocyanine Doped Metal Oxide Nanoparticles on Multiwalled Carbon Nanotubes Platform for the detection of Dopamine.

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An aptamer-based biosensor for sensitive thrombin detection with phthalocyanine@SiO2 mesoporous nanoparticles.

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Fabrication of copper nanoparticles decorated multiwalled carbon nanotubes as a high performance electrochemical sensor for the detection of neotame.

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Non-enzymatic detection of urea using unmodified gold nanoparticles based aptasensor.

Fluorescent carbon nanoparticles for the fluorescent detection of metal ions.

Synergistic bifunctional catalyst design based on perovskite oxide nanoparticles and intertwined carbon nanotubes for rechargeable zinc-air battery applications.

Impedimetric aptasensor for ochratoxin A determination based on Au nanoparticles stabilized with hyper-branched polymer.

Three-Dimensional Conductive Nanocomposites Based on Multiwalled Carbon Nanotube Networks and PEDOT:PSS as a Flexible Transparent Electrode for Optoelectronics.

Gold Nanoparticles dotted Reduction Graphene Oxide Nanocomposite Based Electrochemical Aptasensor for Selective, Rapid, Sensitive and Congener-Specific PCB77 Detection.

Disaggregation of heteroaggregates composed of multiwalled carbon nanotubes and hematite nanoparticles.

An ultrasensitive homogeneous aptasensor for kanamycin based on upconversion fluorescence resonance energy transfer.

multi-walled carbon nanotubes nanohybrids.

Gum ghatti and Fe₃O₄ magnetic nanoparticles based nanocomposites for the effective adsorption of rhodamine B.