Talanta 139 (2015) 226–232
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An aptamer-based signal-on bio-assay for sensitive and selective detection of Kanamycin A by using gold nanoparticles Jing Chen a, Zhaohui Li a,n, Jia Ge a, Ran Yang a, Lin Zhang a, Ling-bo Qu a,b,n, Hong-qi Wang c, Ling Zhang c a
College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China School of Chemistry & Chemical Engineering, Henan University of Technology, Zhengzhou 450001, PR China c Institute of Quality Standard and Testing Technology for Agroproducts, Henan Academy of Agricultural Science, Zhengzhou 450002, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 December 2014 Received in revised form 10 February 2015 Accepted 19 February 2015 Available online 26 February 2015
In this study, a simple and sensitive aptamer-based fluorescence method for the detection of Kanamycin A by using gold nanoparticles (AuNPs) has been developed. In this assay, AuNPs were utilized as DNA nanocarrier as well as efficient fluorescence quencher. In the absence of Kanamycin A, dye-labeled aptamer could be adsorbed onto the surface of AuNPs and the fluorescence signal was quenched. In the presence of Kanamycin A, the specific binding between dye-labeled aptamer and its target induced the formation of rigid structure, which led to dye-labeled aptamer releasing from the surface of AuNPs and the fluorescence intensity was recovered consequently. Under optimum conditions, calibration modeling showed that the analytical linear range covered from 0.8 nM to 350 nM and the detection limit of 0.3 nM was realized successfully. This proposed bio-assay also showed high selectivity over other antibiotics. Meanwhile, this strategy was further used to determine the concentrations of Kanamycin A in milk sample with satisfying results. & 2015 Elsevier B.V. All rights reserved.
Keywords: Aptamer Fluorescence Gold nanoparticles Kanamycin A
1. Introduction Kanamycin, an aminoglycoside antibiotic produced by the fermentation of Streptomyces kanamyceticus, is widely used to treat a wide variety of infections. Moreover, Kanamycin disturbs bacteria protein synthesis by binding to the 30S subunit of ribosome, which results in codon misreading [1] and translocation inhibiting [2]. Kanamycin contains Kanamycin A as main active component and only small amounts of structurally related components such as Kanamycin B and C [3]. Like the other aminoglycosides, Kanamycin exhibits comparatively narrow safety margin and may cause many side effects, such as loss of hearing, toxicity to kidneys, and allergic reactions to drug [4,5]. In addition, the utilization of Kanamycin might produce residues in food and subsequently bring great threat to the health of human beings. Therefore, the development of a facile and selective method for sensitive detection of Kanamycin is very important. During the past decades, a variety of methods have been developed for the determination of Kanamycin including fluorescence detection [6], high performance liquid chromatography (HPLC) [7–9], capillary electrophoresis [10–12], enzyme-linked immunosorbent assay (ELISA) [13–16], surface plasmon resonance n
Corresponding authors. Tel.: þ 86 37167783126. E-mail addresses: [email protected] (Z. Li), [email protected] (L.-b. Qu).
http://dx.doi.org/10.1016/j.talanta.2015.02.036 0039-9140/& 2015 Elsevier B.V. All rights reserved.
(SPR) [17–19], etc. These techniques have shown several advantages such as low detection limit and high sensitivity. However, there are still some hindrances including time consuming, low selectivity, and the requirement of expensive apparatus. Therefore, it is desirable to develop facile, cost-effective and rapid assays for the detection of Kanamycin with favorable sensitivity and selectivity. Aptamers, short single-stranded oligonucleotides, are generated from an in vitro method known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) [20,21]. Aptamers possess high recognition ability to bind to their targets such as small molecules, proteins as well as cells with high affinity and specificity [22–28]. Compared to antibodies, aptamers offer many advantages such as low cost, inherent selectivity, and high stability. Recently, Ban and coworkers [29] have selected an aptamer for Kanamycin A (Ky2, 5′-TGG GGG TTG AGG CTA AGC CGA-3′), which showed stronger binding affinity in contrast with another reported aptamer [30,31]. Subsequently, electrochemical methods [32–34], fluorescence assays [35,36], cantilever assay [37], and spectrophotometry [38,39] were developed for the detection of Kanamycin A by using this Ky2 aptamer. These procedures have shown many advantages including good selectivity. However, the utilization of aptamer for Kanamycin A detection is still remaining at a very early stage and has great possibility to be used in food analysis. As is well known, gold nanoparticles (AuNPs) have been widely used in immunoassay, bio-chip and bio-assay read out techniques
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due to their long-term stability, friendly bio-compatibility, and the unique optical properties. Fluorescence quenching is a commonly observed consequence when fluorophores are appended onto AuNPs, leading to fluorescence resonance energy transfer (FRET) from the organic donor to the AuNPs acceptor [40–43]. Taking full advantages of AuNPs as well as aptamer probes, herein we developed a simple FRET system for homogeneous determination of Kanamycin A in milk sample by using AuNPs and dye-labeled Ky2 aptamer. In the absence of Kanamycin A, the FAM-labeled Ky2 could be adsorbed onto the surface of AuNPs and the fluorescence signal was quenched thoroughly. Comparatively, in the presence of Kanamycin A, the specific binding between the dye-labeled Ky2 with its target induces the formation of rigid hairpin structure, which led to FAM-labeled oligonucleotide releasing from the surface of the AuNPs and the fluorescence intensity was recovered consequently. This aptamer-based bio-assay exhibited an obviously improved selectivity over the common antibiotics and high sensitive detection ability in a wide linear rage with a low detection limit of 0.3 nM, which is far beyond the maximum residue limits for Kanamycin in milk (288 nM) established by European Union [44]. Meanwhile, this strategy was further used to determinate the concentrations of Kanamycin A in milk sample with satisfying results, which indicates that this proposed bio-assay holds a great potential to be used in food inspection and also biomedical studies.
2. Experimental section 2.1. Chemicals and apparatus Kanamycin sulfate (Kanamycin A) was obtained from Dr. Ehrenstorfer GmbH (Germany). HAuCl4 4H2O was purchased from SigmaAldrich (Shanghai, China). Streptomycin sulfate, Gentamycin sulfate, Tetracycline hydrochloride, Sulfadimethoxine, and Lincomycin were bought from National Institutes for Food and Drug Control (Beijing, China). All the other chemicals were of analytical grade and used without any further purification. FAM-labeled Ky2 (5′-FAM-TGG GGG TTG AGG CTA AGC CGA-3′) [29] were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Oligonucleotide stock solutions (100 mM) were prepared with a TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) and kept at 4 °C. Kanamycin A, Streptomycin sulfate, Gentamycin sulfate, Tetracycline hydrochloride, Sulfadimethoxine, and Lincomycin stock solutions (0.01 M) were prepared with ultrapure water and kept at 4 °C. 0.1 M of PB buffer (0.2 M KCl, pH 8.2) was prepared by sodium hydrogen phosphate, sodium dihydrogen phosphate and potassium chloride. All solutions were prepared using ultrapure water, which was obtained through a Millipore Milli-Q water purification system (Billerica, MA, USA) with an electric resistance 418.2 MΩ. The UV–vis spectra and fluorescence spectra were recorded on T6 UV–vis Spectrophotometer (Purkinje General, Beijing, China) and F-4600 fluorescence spectrophotometer (Hitachi, Japan), respectively. The binding affinity study was investigated on the microscale thermophoresis instrument (Nano Temper Technologies GmbH, Munich, Germany). All of the pH values were measured by a PHS-3C precision pH meter (Leici Devices Factory of Shanghai, China). 2.2. Kd confirmation by microscale thermophoresis 100 nM of FAM-labeled Ky2 aptamer solution was prepared in PB (0.01 M, pH 8.0). A serial dilution of Kanamycin A (50 μM to 8 nM) in PB buffer was prepared and mixed with the above labeled aptamer solution with the volume ratio of 1:1. Ky2 aptamer and Kanamycin A were incubated for 20 min at room temperature followed by being introduced into the microscale thermophoresis (MST). MST measurements were performed in standard glass
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capillaries (Nano Temper Technologies) fixed with 80% LED power and 40% MST power. Data analysis and curve fitting were carried out using Nano Temper Analysis software accompanied with MST. 2.3. Synthesis of gold nanoparticles All glassware for the synthesis were thoroughly cleaned in saturated chromic acid solution, rinsed with ultrapure water and dried before use. Gold nanoparticles (AuNPs) were synthesized by trisodium citrate reduction of HAuCl4 4H2O according to the literature reported previously [45]. Briefly, 0.75 mL of 1.0% trisodium citrate was rapidly added to a boiled 50 mL of 0.01% HAuCl4 solution under magnetic stirring. The solution color turned from light yellow to black blue and finally to wine red. Boiling and stirring were continued for 10 min followed by the removing of heating mantle and keep magnetic stirring for another 15 min. After being cooled down to room temperature, the solution were filtered through a 0.22 μm membrane and stored at 4 °C before use. The prepared AuNPs was characterized by UV vis-spectroscopy with an absorption maximum at 523 nm. The concentration of AuNPs was approximately 3 nM estimated by the absorbance at 523 nm using an extinction coefficient of 4.2 108 M 1 cm 1. 2.4. Procedures for Kanamycin A detection In a 100 mL reaction volume, 10 mL Ky2 aptamer (500 nM), 50 mL AuNPs solution (3 nM), 10 mL of 0.1 M PB (0.2 M KCl, pH 8.2) buffer, and 20 mL ultrapure water were mixed uniformly by vortex and incubated for 10 min at room temperature. Then 10 mL Kanamycin A solution with different concentrations (6 10 6 M, 4.5 10 6 M, 3.5 10 6 M, 3 10 6 M, 2 10 6 M, 1.5 10 6 M, 1 10 6 M, 8 10 7 M, 6 10 7 M, 4 10 7 M, 2 10 7 M, 1 10 7 M, 8 10 8 M, 6 10 8 M, 4 10 8 M, 2 10 8 M, 1 10 8 M, 8 10 9 M, 6 10 9 M, 4 10 9 M, 2 10 9 M) or other antibiotics (3.5 10 5 M) were added into the above solution and incubated for 2 h at 4 °C followed by the fluorescence measurement with excitation at 480 nm. 2.5. Preparation and analysis of milk samples To investigate the capability of this method for the determination of Kanamycin A in real samples, different concentrations of Kanamycin A in milk were further analyzed. Skim milk was bought from a local store and a brief pretreatment was required. According to government standard GB/T 22969-2008 of China, 0.8 g of the milk was blended with 3 mL of phosphoric acid and 0.3 mL of trichloroacetic acid. The solution was then intensive mixed and centrifuged for 10 min at 4000 rpm. The supernatant was poured into a benzene sulfonic acid type solid phase extraction column, which was pretreated with 5 mL of methanol and 10 mL of ultrapure water. After the sample passed through the column, 0.5 mL of methanol and 1.0 mL of ultrapure water was added to remove any effluent, followed by the addition of 2 mL phosphate buffer (0.2 M, pH 8.5) to obtain the treated milk sample. The sample was then diluted 10 folds in PB and spiked with different amounts of Kanamycin A to reach the final concentrations of 10 nM, 80 nM, and 250 nM, respectively. The detection procedure was the same as those described in the abovementioned experiment for Kanamycin A detection in PB buffer.
3. Results and discussion 3.1. Sensing mechanism This proposed strategy for the detection of Kanamycin A is demonstrated in Scheme 1, which is based on the conformational
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[AB]:
[AB] ⎛ = 1/2* ⎜⎜ ( [A 0] + [B0 ] + Kd ) ⎝
Scheme 1. Schematic illustration of Kanamycin A detection by using FAM-labeled Ky2 aptamer and gold nanoparticles.
change of Ky2 aptamer as well as fluorescence quenching by gold nanoparticles. In the absence of Kanamycin A, the FAM-labeled Ky2 aptamer existed in a random-coiled conformation as described by Ban and coworkers [29] and stuck onto the surface of AuNPs easily, which is leading to efficient FRET between FAM and AuNPs and the fluorescence of aptamer probes was quenched. Comparatively in the presence of Kanamycin A, the specific binding between FAM-labeled aptamer and its target induces the formation of a duplex hairpin (stem–loop) structure, which possessed much lower affinity to AuNPs and so released from their surface consequently. Therefore, the fluorescence recovered and could likely be attributed to the increasing of Kanamycin A concentrations.
Aptamer sequence of Kanamycin A was chosen in this work according to the literature reported [29]. In order to further prove the binding affinity of this aptamer to Kanamycin A, MST measurement was conducted as well. MST is a highly sensitive probe for many kinds of binding-induced interactions such as molecular size, charge, hydration shell or conformation [46]. Recently, MST has been applied for ions, small molecules, nucleic acids, peptides, proteins and crude cell lysate studies [47–51]. MST detection was based on the directed movement of molecules along a temperature gradient, an effect named “thermophoresis”. When the aqueous solution is heated locally, the molecules begin to move along the temperature gradient from the locally heated regions to the cold regions. As the movement reaches an equilibrium state, the spatial concentration change for a given thermal gradient (ΔT) is expressed by the equation.
(1)
where Chot is the molecular concentration in hot regions; Ccold is the molecular concentration in cold regions; ST is the Soret coefficient. The normalized fluorescence Fnorm mainly depends on this concentration ratio, and a temperature gradient of fluorescently labeled aptamer. Fnorm ¼ Fhot/Fcold ¼1 (ST dF/dT)ΔT¼ Chot/Ccold (dF/dT)ΔT. Within the titration experiment, Fnorm changes according to the following equation.
Fnorm = (1−x) F (A)norm + xF (AB)norm
2
0
0
d
0
0
⎟⎟ ⎠
(3)
[A0] is kept constant during the experiments and [B0] is varied by titration. The signal obtained in the measurement directly corresponds to the fluorescent molecules that formed the complex x¼ [AB]/[A0], which could be easily fitted with the derived formula to obtain Kd. The binding curve of a labeled DNA-aptamer to Kanamycin A was shown in Fig. 1. Plotting Fnorm against different concentrations of Kanamycin A resulted in a binding curve from which the dissociation constant Kd ¼11572.76 nM could be derived. This result is close to the literature value of 78.8 nM from fluorescence measurement [29]. The determined Hill coefficients of n¼1.2 (EC50¼138 nM) was obtained from MST directly, which indicated that more than one Kanamycin A molecule could bind to one Ky2 aptamer. 3.3. Optimization of the experimental conditions
3.2. Kd determination of Ky2 aptamer by using MST
Chot/Ccold = exp(−S T ΔT ) ≈ 1−S T ΔT
1/2 ⎞
(([A ] + [B ] + K ) −4 *[A ]* [B ] )
−
In order to optimize the experimental conditions, a series of measurements were conducted in this work. Firstly, pH dependence of fluorescence response of this bio-assay was investigated. As shown in Fig. 2a, the fluorescence signal increased gradually with the increasing of pH from 7.0 to 8.2. When the pH was higher than 8.2, the fluorescence signal decreased again. Therefore, pH 8.2 was employed as the optimal pH to obtain a high sensitivity throughout the experiments. A further study was performed to investigate the PB concentration dependence of fluorescence response of this bioassay. As shown in Fig. 2b, the highest fluorescence recovery efficiency was obtained at 10 mM. Therefore, 10 mM was chosen as the optimized concentration of PB buffer for all the experiments. Ionic strength plays an important role in aptamer conformation change from ssDNA to a hairpin region. As depicted in Fig. 2c, the degree of fluorescence intensity recovery increased when the KCl concentration was increased from 5 to 20 mM. When the concentration of KCl was higher than 20 mM, the fluorescence signal decreased again. Thus, 20 mM KCl was used as the optimized concentration. It was found that the amount of AuNPs has a large influence on the
(2)
where F(A)norm is normalized fluorescence of the unbound fluorescent molecule A (fluorescently labeled aptamer), F(AB)norm is normalized fluorescence of the bound fluorescent complex and x is the fraction of fluorescent molecules that formed the complex. The dissociation constant Kd is obtained by fitting the binding curve with the quadratic solution for the fraction of fluorescent molecule that formed the complex, calculated from the law of mass action. The dissociation constant is Kd ¼[A]n[B]/[AB], where [A] is the concentration of free fluorescent molecule, [B] is the concentration of free titrant (Kanamycin A), and [AB] is the concentration of the bound fluorescent complex. The free concentration of A and B are [A] ¼[A0] [AB] and [B] ¼[B0] [AB], for which [A0] is the analytical concentration of A and [B0] is the analytical concentration of B. This leads to a quadratic fitting function for
Fig. 1. Binding curves of Ky2 aptamer with Kanamycin A obtained from MST fixed with 80% LED power and 40% MST power. The dye labeled aptamer concentration kept constant at 50 nM. Concentration of Kanamycin A was ranging from 50 mM to 0.8 nM.
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Fig. 3. (a) Influence of AuNPs concentrations on the fluorescence of FAM-Ky2 aptamer in the absence and presence of Kanamycin A; (b) relative fluorescence intensity of the bio-assay, where F and F0 were the fluorescence intensity in the absence and presence of Kanamycin A, respectively.
Fig. 2. Effects of (a) pH, (b) buffer concentration, and (c) ionic strength on relative fluorescence intensity ((F F0)/F0), where F and F0 were the fluorescence intensity in the absence and presence of Kanamycin A, respectively. All experiments were performed in the presence of 50 nM Ky2 aptamer and 1.2 nM AuNPs. 250 nM Kanamycin A was selected under excitation at 480 nm.
fluorescence quenching. Fig. 3a shows the change of the fluorescence intensity when AuNPs concentration ranges from 0.3 nM to 2.1 nM with 50 nM probes either in the absence or the presence of Kanamycin A. When the AuNPs concentration ranges from 0.3 nM to 1.5 nM, increased relative fluorescence intensity ((F0 F)/F0) were observed (Fig. 3b), where F0 and F were the fluorescence intensity with and without Kanamycin A, respectively. As more AuNPs were added, no more fluorescence recovery could be further obtained and so 1.5 nM AuNPs was chosen as the optimized amount for the following experiments. The kinetic behavior of this designed aptamerAuNPs bio-assay was also investigated by monitoring the fluorescence intensity as a function of time. As shown in Fig. 4a, the fluorescence intensity deceased rapidly with the reaction time up to 10 min and then reached an equilibration. Therefore, 10 min was chosen as the optimum quenching time. The incubation time after the introduction of Kanamycin A into the solution to recover the fluorescence of system was also investigated and the results were depicted in Fig. 4b. The fluorescence recovery reached an equilibration over 2 h due to the saturation of the active sites for Kanamycin A binding.
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3.4. Sensitivity determination of this bio-assay Under optimum assay conditions, different concentrations of Kanamycin A from 0.8 to 600 nM were introduced to the buffer to evaluate the sensitivity of this bio-assay. Fig. 5a shows the fluorescence spectra of FAM-Ky2/AuNPs in the presence of different Kanamycin A concentrations in PB buffer solution. Fig. 5b is the fluorescence intensity plotted against the Kanamycin A concentration. However, when Kanamycin A concentration was higher than 350 nM, the fluorescence intensity was not further enhanced and a plateau is reached, because the interaction between Ky2 and Kanamycin A reached a balance and saturation. Furthermore, two linear relationships between fluorescence intensity and Kanamycin A concentration were obtained in the ranges of Kanamycin A
Fig. 4. (a) Kinetic study of fluorescence quenching of FAM-Ky2 aptamer in the presence of 1.5 nM AuNPs; (b) time-dependent fluorescence recovery of FAM-Ky2 aptamer in AuNPs solution with the addition of 250 nM Kanamycin A.
Fig. 5. (a) Fluorescence emission spectra of FAM-aptamer/AuNPs with different concentration of Kanamycin A (a–s): 0, 0.8 nM, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 40 nM, 60 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 450 nM, and 600 nM in PB buffer (10 mM, 20 mM KCl, pH 8.2); (b) the fluorescence intensity plotted against the Kanamycin A concentration; (c) the fluorescence recovery with respect to the concentration of Kanamycin A ranging from 0.8 nM to 20 nM; (d) the fluorescence recovery with respect to the concentration of Kanamycin A ranging from 40 nM to 350 nM. All experiments were performed in the presence of 50 nM Ky2 aptamer and 1.5 nM AuNPs, and the excitation wavelength was 480 nm.
concentration as 0.8–20 nM (Fig. 5c) and 40–350 nM (Fig. 5d). These two linear regression equations were y¼0.02198þ0.01279x (R2 ¼0.9977) and y¼0.2772þ0.00204x (R2 ¼ 0.9978), respectively,
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Table 1 Comparison with currently available methods for the detection of Kanamycin.
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Table 2 Determination of Kanamycin A in milk samples.
Method of detection
Limit of detection (M)
References
Sample
Added (M)
Found (M)
Recovery (%)
RSD (%)
Fluorescence detection ELISA ELISA SPR SPR Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection
8.95 10 9–2.56 10 8 4.15 10 9 2.1 10 8 2.0 10 9 1.0 7 0.10 10 12 2.5 10 8 9.4 7 0.4 10 9 5.8 10 9 8.6 10 9 1.0 10 5 1.43 10 7 1.8 10 11 1.0 10 9 1.49 10 9 3.0 10 10
Yu et al. [16] Watanabe et al. [13] Loomans et al. [14] Wang et al. [17] Frasconi et al. [19] Song et al. [29] Zhu et al. [32] Sun et al. [33] Li et al. [34] Bai et al. [37] Leung et al. [35] Li et al. [36] Zhou et al. [38] Sharma et al. [39] This work
1 2 3
1 10 8 8 10 8 2.5 10 7
1.05 10 8 7.77 10 8 2.55 10 7
105.1 99.2 102.1
3.47 1.34 4.23
system and there was little fluorescence recovery with the addition of Kanamycin A (Fig. S1). These results indicated that the proposed bioanalytical system had good selectivity toward Kanamycin A, which was contributed by the nature of high selectivity of aptamer probes.
3.6. Assay of Kanamycin A in milk samples To determine the feasibility of this bioanalytical system for possible applications, this proposed assay was further used to detect different concentrations of Kanamycin A in real milk samples. The recoveries and relative standard deviation (RSD) at various spiking levels for Kanamycin A in milk are listed in Table 2. As can be seen, Kanamycin A recovery was in the range of 99.2– 105.1% with RSDs below 4.5% for milk sample. The results showed that this proposed method had offered good possibilities for sensitive Kanamycin A detection in food samples.
4. Conclusions
Fig. 6. Selectivity of the assay system for Kanamycin A. The concentrations of Kanamycin A and other antibiotics are 350 nM and 3.5 mM in PB buffer (10 mM, 20 mM KCl, pH 8.2). All experiments were performed in the presence of 50 nM Ky2 aptamer and 1.5 nM AuNPs.
where y represented the relative intensity ((F F0)/F0) and x represented the concentration of Kanamycin A. In addition, the detection limit of 0.3 nM was realized according to the 3s rule. Compared with the reported methods for Kanamycin detection, this proposed bioassay exhibited relative high sensitivity, which was shown in Table 1. For consumer protection, European Union has established that the maximum residue limit for Kanamycin in milk should be 288 nM (http://www.emea.europa.eu). As the detection limit was much lower than the maximum residue limit, this proposed method has a great potential to be used for the analysis of the small volume Kanamycin A residues in milk as well as other food samples. 3.5. Selectivity inspection of this bio-assay To investigate the selectivity of this bioanalytical system towards Kanamycin A analysis, a list of 10-fold other antibiotics including Gentamycin sulfate (Aminoglycoside antibiotics), Streptomycin sulfate (Aminoglycoside antibiotics), Tetracycline hydrochloride (Tetracycline), Sulfadimethoxine (Sulfonamides), and Lincomycin (Lincosamide antibiotics) were inspected. As can be seen from Fig. 6, a fluorescence enhancement of (F F0)/F0 ¼ 1.05 was obtained in the presence of Kanamycin A. In contrast, the fluorescence signals had little changes in the presence of 10-fold other antibiotics. Meanwhile, FAM-labeled Ampicillin aptamer (FAM-AMP4) was utilized as a control probe in this
In summary, a simple fluorescence bioanalytical system for sensitive and selective detection of Kanamycin A was developed by using gold nanoparticles and FAM-labeled Ky2 aptamer probes. In the presence of Kanamycin A, the aptamer would release from AuNPs surface and fluorescence signal was recovered consequently. This bio-assay showed good sensing performances for Kanamycin A in linear ranges from 40–350 nM to 0.8–20 nM with a limit detection of 0.3 nM. Due to the highly specific recognition between aptamer and Kanamycin A, this sensing strategy showed strong ability of resistance to the interference of other antibiotics. More importantly, this proposed assay was successfully used for the determination of Kanamycin A in milk samples with satisfying results, which indicated that this bio-assay could be utilized as a sensitive, specific, low-cost, and easy-to-use system for the detection of Kanamycin A regarding food safety, public health security as well as medical diagnosis.
Acknowledgment This work was supported by the National Natural Science Foundation of China (21205108), the Scientific Research Foundation for the Returned Overseas Chinese Scholars (State Education Ministry of China), the Technology Foundation for Selected Overseas Chinese Scholars (Ministry of Personnel of China), and Special Fund for Agroscientific Research in the Public Interest (201203094).
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.talanta.2015.02.036.
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An aptamer-based signal-on bio-assay for sensitive and selective detection of Kanamycin A by using gold nanoparticles Jing Chen a, Zhaohui Li a,n, Jia Ge a, Ran Yang a, Lin Zhang a, Ling-bo Qu a,b,n, Hong-qi Wang c, Ling Zhang c a
College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China School of Chemistry & Chemical Engineering, Henan University of Technology, Zhengzhou 450001, PR China c Institute of Quality Standard and Testing Technology for Agroproducts, Henan Academy of Agricultural Science, Zhengzhou 450002, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 December 2014 Received in revised form 10 February 2015 Accepted 19 February 2015 Available online 26 February 2015
In this study, a simple and sensitive aptamer-based fluorescence method for the detection of Kanamycin A by using gold nanoparticles (AuNPs) has been developed. In this assay, AuNPs were utilized as DNA nanocarrier as well as efficient fluorescence quencher. In the absence of Kanamycin A, dye-labeled aptamer could be adsorbed onto the surface of AuNPs and the fluorescence signal was quenched. In the presence of Kanamycin A, the specific binding between dye-labeled aptamer and its target induced the formation of rigid structure, which led to dye-labeled aptamer releasing from the surface of AuNPs and the fluorescence intensity was recovered consequently. Under optimum conditions, calibration modeling showed that the analytical linear range covered from 0.8 nM to 350 nM and the detection limit of 0.3 nM was realized successfully. This proposed bio-assay also showed high selectivity over other antibiotics. Meanwhile, this strategy was further used to determine the concentrations of Kanamycin A in milk sample with satisfying results. & 2015 Elsevier B.V. All rights reserved.
Keywords: Aptamer Fluorescence Gold nanoparticles Kanamycin A
1. Introduction Kanamycin, an aminoglycoside antibiotic produced by the fermentation of Streptomyces kanamyceticus, is widely used to treat a wide variety of infections. Moreover, Kanamycin disturbs bacteria protein synthesis by binding to the 30S subunit of ribosome, which results in codon misreading [1] and translocation inhibiting [2]. Kanamycin contains Kanamycin A as main active component and only small amounts of structurally related components such as Kanamycin B and C [3]. Like the other aminoglycosides, Kanamycin exhibits comparatively narrow safety margin and may cause many side effects, such as loss of hearing, toxicity to kidneys, and allergic reactions to drug [4,5]. In addition, the utilization of Kanamycin might produce residues in food and subsequently bring great threat to the health of human beings. Therefore, the development of a facile and selective method for sensitive detection of Kanamycin is very important. During the past decades, a variety of methods have been developed for the determination of Kanamycin including fluorescence detection [6], high performance liquid chromatography (HPLC) [7–9], capillary electrophoresis [10–12], enzyme-linked immunosorbent assay (ELISA) [13–16], surface plasmon resonance n
Corresponding authors. Tel.: þ 86 37167783126. E-mail addresses: [email protected] (Z. Li), [email protected] (L.-b. Qu).
http://dx.doi.org/10.1016/j.talanta.2015.02.036 0039-9140/& 2015 Elsevier B.V. All rights reserved.
(SPR) [17–19], etc. These techniques have shown several advantages such as low detection limit and high sensitivity. However, there are still some hindrances including time consuming, low selectivity, and the requirement of expensive apparatus. Therefore, it is desirable to develop facile, cost-effective and rapid assays for the detection of Kanamycin with favorable sensitivity and selectivity. Aptamers, short single-stranded oligonucleotides, are generated from an in vitro method known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) [20,21]. Aptamers possess high recognition ability to bind to their targets such as small molecules, proteins as well as cells with high affinity and specificity [22–28]. Compared to antibodies, aptamers offer many advantages such as low cost, inherent selectivity, and high stability. Recently, Ban and coworkers [29] have selected an aptamer for Kanamycin A (Ky2, 5′-TGG GGG TTG AGG CTA AGC CGA-3′), which showed stronger binding affinity in contrast with another reported aptamer [30,31]. Subsequently, electrochemical methods [32–34], fluorescence assays [35,36], cantilever assay [37], and spectrophotometry [38,39] were developed for the detection of Kanamycin A by using this Ky2 aptamer. These procedures have shown many advantages including good selectivity. However, the utilization of aptamer for Kanamycin A detection is still remaining at a very early stage and has great possibility to be used in food analysis. As is well known, gold nanoparticles (AuNPs) have been widely used in immunoassay, bio-chip and bio-assay read out techniques
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due to their long-term stability, friendly bio-compatibility, and the unique optical properties. Fluorescence quenching is a commonly observed consequence when fluorophores are appended onto AuNPs, leading to fluorescence resonance energy transfer (FRET) from the organic donor to the AuNPs acceptor [40–43]. Taking full advantages of AuNPs as well as aptamer probes, herein we developed a simple FRET system for homogeneous determination of Kanamycin A in milk sample by using AuNPs and dye-labeled Ky2 aptamer. In the absence of Kanamycin A, the FAM-labeled Ky2 could be adsorbed onto the surface of AuNPs and the fluorescence signal was quenched thoroughly. Comparatively, in the presence of Kanamycin A, the specific binding between the dye-labeled Ky2 with its target induces the formation of rigid hairpin structure, which led to FAM-labeled oligonucleotide releasing from the surface of the AuNPs and the fluorescence intensity was recovered consequently. This aptamer-based bio-assay exhibited an obviously improved selectivity over the common antibiotics and high sensitive detection ability in a wide linear rage with a low detection limit of 0.3 nM, which is far beyond the maximum residue limits for Kanamycin in milk (288 nM) established by European Union [44]. Meanwhile, this strategy was further used to determinate the concentrations of Kanamycin A in milk sample with satisfying results, which indicates that this proposed bio-assay holds a great potential to be used in food inspection and also biomedical studies.
2. Experimental section 2.1. Chemicals and apparatus Kanamycin sulfate (Kanamycin A) was obtained from Dr. Ehrenstorfer GmbH (Germany). HAuCl4 4H2O was purchased from SigmaAldrich (Shanghai, China). Streptomycin sulfate, Gentamycin sulfate, Tetracycline hydrochloride, Sulfadimethoxine, and Lincomycin were bought from National Institutes for Food and Drug Control (Beijing, China). All the other chemicals were of analytical grade and used without any further purification. FAM-labeled Ky2 (5′-FAM-TGG GGG TTG AGG CTA AGC CGA-3′) [29] were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Oligonucleotide stock solutions (100 mM) were prepared with a TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) and kept at 4 °C. Kanamycin A, Streptomycin sulfate, Gentamycin sulfate, Tetracycline hydrochloride, Sulfadimethoxine, and Lincomycin stock solutions (0.01 M) were prepared with ultrapure water and kept at 4 °C. 0.1 M of PB buffer (0.2 M KCl, pH 8.2) was prepared by sodium hydrogen phosphate, sodium dihydrogen phosphate and potassium chloride. All solutions were prepared using ultrapure water, which was obtained through a Millipore Milli-Q water purification system (Billerica, MA, USA) with an electric resistance 418.2 MΩ. The UV–vis spectra and fluorescence spectra were recorded on T6 UV–vis Spectrophotometer (Purkinje General, Beijing, China) and F-4600 fluorescence spectrophotometer (Hitachi, Japan), respectively. The binding affinity study was investigated on the microscale thermophoresis instrument (Nano Temper Technologies GmbH, Munich, Germany). All of the pH values were measured by a PHS-3C precision pH meter (Leici Devices Factory of Shanghai, China). 2.2. Kd confirmation by microscale thermophoresis 100 nM of FAM-labeled Ky2 aptamer solution was prepared in PB (0.01 M, pH 8.0). A serial dilution of Kanamycin A (50 μM to 8 nM) in PB buffer was prepared and mixed with the above labeled aptamer solution with the volume ratio of 1:1. Ky2 aptamer and Kanamycin A were incubated for 20 min at room temperature followed by being introduced into the microscale thermophoresis (MST). MST measurements were performed in standard glass
227
capillaries (Nano Temper Technologies) fixed with 80% LED power and 40% MST power. Data analysis and curve fitting were carried out using Nano Temper Analysis software accompanied with MST. 2.3. Synthesis of gold nanoparticles All glassware for the synthesis were thoroughly cleaned in saturated chromic acid solution, rinsed with ultrapure water and dried before use. Gold nanoparticles (AuNPs) were synthesized by trisodium citrate reduction of HAuCl4 4H2O according to the literature reported previously [45]. Briefly, 0.75 mL of 1.0% trisodium citrate was rapidly added to a boiled 50 mL of 0.01% HAuCl4 solution under magnetic stirring. The solution color turned from light yellow to black blue and finally to wine red. Boiling and stirring were continued for 10 min followed by the removing of heating mantle and keep magnetic stirring for another 15 min. After being cooled down to room temperature, the solution were filtered through a 0.22 μm membrane and stored at 4 °C before use. The prepared AuNPs was characterized by UV vis-spectroscopy with an absorption maximum at 523 nm. The concentration of AuNPs was approximately 3 nM estimated by the absorbance at 523 nm using an extinction coefficient of 4.2 108 M 1 cm 1. 2.4. Procedures for Kanamycin A detection In a 100 mL reaction volume, 10 mL Ky2 aptamer (500 nM), 50 mL AuNPs solution (3 nM), 10 mL of 0.1 M PB (0.2 M KCl, pH 8.2) buffer, and 20 mL ultrapure water were mixed uniformly by vortex and incubated for 10 min at room temperature. Then 10 mL Kanamycin A solution with different concentrations (6 10 6 M, 4.5 10 6 M, 3.5 10 6 M, 3 10 6 M, 2 10 6 M, 1.5 10 6 M, 1 10 6 M, 8 10 7 M, 6 10 7 M, 4 10 7 M, 2 10 7 M, 1 10 7 M, 8 10 8 M, 6 10 8 M, 4 10 8 M, 2 10 8 M, 1 10 8 M, 8 10 9 M, 6 10 9 M, 4 10 9 M, 2 10 9 M) or other antibiotics (3.5 10 5 M) were added into the above solution and incubated for 2 h at 4 °C followed by the fluorescence measurement with excitation at 480 nm. 2.5. Preparation and analysis of milk samples To investigate the capability of this method for the determination of Kanamycin A in real samples, different concentrations of Kanamycin A in milk were further analyzed. Skim milk was bought from a local store and a brief pretreatment was required. According to government standard GB/T 22969-2008 of China, 0.8 g of the milk was blended with 3 mL of phosphoric acid and 0.3 mL of trichloroacetic acid. The solution was then intensive mixed and centrifuged for 10 min at 4000 rpm. The supernatant was poured into a benzene sulfonic acid type solid phase extraction column, which was pretreated with 5 mL of methanol and 10 mL of ultrapure water. After the sample passed through the column, 0.5 mL of methanol and 1.0 mL of ultrapure water was added to remove any effluent, followed by the addition of 2 mL phosphate buffer (0.2 M, pH 8.5) to obtain the treated milk sample. The sample was then diluted 10 folds in PB and spiked with different amounts of Kanamycin A to reach the final concentrations of 10 nM, 80 nM, and 250 nM, respectively. The detection procedure was the same as those described in the abovementioned experiment for Kanamycin A detection in PB buffer.
3. Results and discussion 3.1. Sensing mechanism This proposed strategy for the detection of Kanamycin A is demonstrated in Scheme 1, which is based on the conformational
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[AB]:
[AB] ⎛ = 1/2* ⎜⎜ ( [A 0] + [B0 ] + Kd ) ⎝
Scheme 1. Schematic illustration of Kanamycin A detection by using FAM-labeled Ky2 aptamer and gold nanoparticles.
change of Ky2 aptamer as well as fluorescence quenching by gold nanoparticles. In the absence of Kanamycin A, the FAM-labeled Ky2 aptamer existed in a random-coiled conformation as described by Ban and coworkers [29] and stuck onto the surface of AuNPs easily, which is leading to efficient FRET between FAM and AuNPs and the fluorescence of aptamer probes was quenched. Comparatively in the presence of Kanamycin A, the specific binding between FAM-labeled aptamer and its target induces the formation of a duplex hairpin (stem–loop) structure, which possessed much lower affinity to AuNPs and so released from their surface consequently. Therefore, the fluorescence recovered and could likely be attributed to the increasing of Kanamycin A concentrations.
Aptamer sequence of Kanamycin A was chosen in this work according to the literature reported [29]. In order to further prove the binding affinity of this aptamer to Kanamycin A, MST measurement was conducted as well. MST is a highly sensitive probe for many kinds of binding-induced interactions such as molecular size, charge, hydration shell or conformation [46]. Recently, MST has been applied for ions, small molecules, nucleic acids, peptides, proteins and crude cell lysate studies [47–51]. MST detection was based on the directed movement of molecules along a temperature gradient, an effect named “thermophoresis”. When the aqueous solution is heated locally, the molecules begin to move along the temperature gradient from the locally heated regions to the cold regions. As the movement reaches an equilibrium state, the spatial concentration change for a given thermal gradient (ΔT) is expressed by the equation.
(1)
where Chot is the molecular concentration in hot regions; Ccold is the molecular concentration in cold regions; ST is the Soret coefficient. The normalized fluorescence Fnorm mainly depends on this concentration ratio, and a temperature gradient of fluorescently labeled aptamer. Fnorm ¼ Fhot/Fcold ¼1 (ST dF/dT)ΔT¼ Chot/Ccold (dF/dT)ΔT. Within the titration experiment, Fnorm changes according to the following equation.
Fnorm = (1−x) F (A)norm + xF (AB)norm
2
0
0
d
0
0
⎟⎟ ⎠
(3)
[A0] is kept constant during the experiments and [B0] is varied by titration. The signal obtained in the measurement directly corresponds to the fluorescent molecules that formed the complex x¼ [AB]/[A0], which could be easily fitted with the derived formula to obtain Kd. The binding curve of a labeled DNA-aptamer to Kanamycin A was shown in Fig. 1. Plotting Fnorm against different concentrations of Kanamycin A resulted in a binding curve from which the dissociation constant Kd ¼11572.76 nM could be derived. This result is close to the literature value of 78.8 nM from fluorescence measurement [29]. The determined Hill coefficients of n¼1.2 (EC50¼138 nM) was obtained from MST directly, which indicated that more than one Kanamycin A molecule could bind to one Ky2 aptamer. 3.3. Optimization of the experimental conditions
3.2. Kd determination of Ky2 aptamer by using MST
Chot/Ccold = exp(−S T ΔT ) ≈ 1−S T ΔT
1/2 ⎞
(([A ] + [B ] + K ) −4 *[A ]* [B ] )
−
In order to optimize the experimental conditions, a series of measurements were conducted in this work. Firstly, pH dependence of fluorescence response of this bio-assay was investigated. As shown in Fig. 2a, the fluorescence signal increased gradually with the increasing of pH from 7.0 to 8.2. When the pH was higher than 8.2, the fluorescence signal decreased again. Therefore, pH 8.2 was employed as the optimal pH to obtain a high sensitivity throughout the experiments. A further study was performed to investigate the PB concentration dependence of fluorescence response of this bioassay. As shown in Fig. 2b, the highest fluorescence recovery efficiency was obtained at 10 mM. Therefore, 10 mM was chosen as the optimized concentration of PB buffer for all the experiments. Ionic strength plays an important role in aptamer conformation change from ssDNA to a hairpin region. As depicted in Fig. 2c, the degree of fluorescence intensity recovery increased when the KCl concentration was increased from 5 to 20 mM. When the concentration of KCl was higher than 20 mM, the fluorescence signal decreased again. Thus, 20 mM KCl was used as the optimized concentration. It was found that the amount of AuNPs has a large influence on the
(2)
where F(A)norm is normalized fluorescence of the unbound fluorescent molecule A (fluorescently labeled aptamer), F(AB)norm is normalized fluorescence of the bound fluorescent complex and x is the fraction of fluorescent molecules that formed the complex. The dissociation constant Kd is obtained by fitting the binding curve with the quadratic solution for the fraction of fluorescent molecule that formed the complex, calculated from the law of mass action. The dissociation constant is Kd ¼[A]n[B]/[AB], where [A] is the concentration of free fluorescent molecule, [B] is the concentration of free titrant (Kanamycin A), and [AB] is the concentration of the bound fluorescent complex. The free concentration of A and B are [A] ¼[A0] [AB] and [B] ¼[B0] [AB], for which [A0] is the analytical concentration of A and [B0] is the analytical concentration of B. This leads to a quadratic fitting function for
Fig. 1. Binding curves of Ky2 aptamer with Kanamycin A obtained from MST fixed with 80% LED power and 40% MST power. The dye labeled aptamer concentration kept constant at 50 nM. Concentration of Kanamycin A was ranging from 50 mM to 0.8 nM.
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Fig. 3. (a) Influence of AuNPs concentrations on the fluorescence of FAM-Ky2 aptamer in the absence and presence of Kanamycin A; (b) relative fluorescence intensity of the bio-assay, where F and F0 were the fluorescence intensity in the absence and presence of Kanamycin A, respectively.
Fig. 2. Effects of (a) pH, (b) buffer concentration, and (c) ionic strength on relative fluorescence intensity ((F F0)/F0), where F and F0 were the fluorescence intensity in the absence and presence of Kanamycin A, respectively. All experiments were performed in the presence of 50 nM Ky2 aptamer and 1.2 nM AuNPs. 250 nM Kanamycin A was selected under excitation at 480 nm.
fluorescence quenching. Fig. 3a shows the change of the fluorescence intensity when AuNPs concentration ranges from 0.3 nM to 2.1 nM with 50 nM probes either in the absence or the presence of Kanamycin A. When the AuNPs concentration ranges from 0.3 nM to 1.5 nM, increased relative fluorescence intensity ((F0 F)/F0) were observed (Fig. 3b), where F0 and F were the fluorescence intensity with and without Kanamycin A, respectively. As more AuNPs were added, no more fluorescence recovery could be further obtained and so 1.5 nM AuNPs was chosen as the optimized amount for the following experiments. The kinetic behavior of this designed aptamerAuNPs bio-assay was also investigated by monitoring the fluorescence intensity as a function of time. As shown in Fig. 4a, the fluorescence intensity deceased rapidly with the reaction time up to 10 min and then reached an equilibration. Therefore, 10 min was chosen as the optimum quenching time. The incubation time after the introduction of Kanamycin A into the solution to recover the fluorescence of system was also investigated and the results were depicted in Fig. 4b. The fluorescence recovery reached an equilibration over 2 h due to the saturation of the active sites for Kanamycin A binding.
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3.4. Sensitivity determination of this bio-assay Under optimum assay conditions, different concentrations of Kanamycin A from 0.8 to 600 nM were introduced to the buffer to evaluate the sensitivity of this bio-assay. Fig. 5a shows the fluorescence spectra of FAM-Ky2/AuNPs in the presence of different Kanamycin A concentrations in PB buffer solution. Fig. 5b is the fluorescence intensity plotted against the Kanamycin A concentration. However, when Kanamycin A concentration was higher than 350 nM, the fluorescence intensity was not further enhanced and a plateau is reached, because the interaction between Ky2 and Kanamycin A reached a balance and saturation. Furthermore, two linear relationships between fluorescence intensity and Kanamycin A concentration were obtained in the ranges of Kanamycin A
Fig. 4. (a) Kinetic study of fluorescence quenching of FAM-Ky2 aptamer in the presence of 1.5 nM AuNPs; (b) time-dependent fluorescence recovery of FAM-Ky2 aptamer in AuNPs solution with the addition of 250 nM Kanamycin A.
Fig. 5. (a) Fluorescence emission spectra of FAM-aptamer/AuNPs with different concentration of Kanamycin A (a–s): 0, 0.8 nM, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 40 nM, 60 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 450 nM, and 600 nM in PB buffer (10 mM, 20 mM KCl, pH 8.2); (b) the fluorescence intensity plotted against the Kanamycin A concentration; (c) the fluorescence recovery with respect to the concentration of Kanamycin A ranging from 0.8 nM to 20 nM; (d) the fluorescence recovery with respect to the concentration of Kanamycin A ranging from 40 nM to 350 nM. All experiments were performed in the presence of 50 nM Ky2 aptamer and 1.5 nM AuNPs, and the excitation wavelength was 480 nm.
concentration as 0.8–20 nM (Fig. 5c) and 40–350 nM (Fig. 5d). These two linear regression equations were y¼0.02198þ0.01279x (R2 ¼0.9977) and y¼0.2772þ0.00204x (R2 ¼ 0.9978), respectively,
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Table 1 Comparison with currently available methods for the detection of Kanamycin.
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Table 2 Determination of Kanamycin A in milk samples.
Method of detection
Limit of detection (M)
References
Sample
Added (M)
Found (M)
Recovery (%)
RSD (%)
Fluorescence detection ELISA ELISA SPR SPR Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection Aptasensor detection
8.95 10 9–2.56 10 8 4.15 10 9 2.1 10 8 2.0 10 9 1.0 7 0.10 10 12 2.5 10 8 9.4 7 0.4 10 9 5.8 10 9 8.6 10 9 1.0 10 5 1.43 10 7 1.8 10 11 1.0 10 9 1.49 10 9 3.0 10 10
Yu et al. [16] Watanabe et al. [13] Loomans et al. [14] Wang et al. [17] Frasconi et al. [19] Song et al. [29] Zhu et al. [32] Sun et al. [33] Li et al. [34] Bai et al. [37] Leung et al. [35] Li et al. [36] Zhou et al. [38] Sharma et al. [39] This work
1 2 3
1 10 8 8 10 8 2.5 10 7
1.05 10 8 7.77 10 8 2.55 10 7
105.1 99.2 102.1
3.47 1.34 4.23
system and there was little fluorescence recovery with the addition of Kanamycin A (Fig. S1). These results indicated that the proposed bioanalytical system had good selectivity toward Kanamycin A, which was contributed by the nature of high selectivity of aptamer probes.
3.6. Assay of Kanamycin A in milk samples To determine the feasibility of this bioanalytical system for possible applications, this proposed assay was further used to detect different concentrations of Kanamycin A in real milk samples. The recoveries and relative standard deviation (RSD) at various spiking levels for Kanamycin A in milk are listed in Table 2. As can be seen, Kanamycin A recovery was in the range of 99.2– 105.1% with RSDs below 4.5% for milk sample. The results showed that this proposed method had offered good possibilities for sensitive Kanamycin A detection in food samples.
4. Conclusions
Fig. 6. Selectivity of the assay system for Kanamycin A. The concentrations of Kanamycin A and other antibiotics are 350 nM and 3.5 mM in PB buffer (10 mM, 20 mM KCl, pH 8.2). All experiments were performed in the presence of 50 nM Ky2 aptamer and 1.5 nM AuNPs.
where y represented the relative intensity ((F F0)/F0) and x represented the concentration of Kanamycin A. In addition, the detection limit of 0.3 nM was realized according to the 3s rule. Compared with the reported methods for Kanamycin detection, this proposed bioassay exhibited relative high sensitivity, which was shown in Table 1. For consumer protection, European Union has established that the maximum residue limit for Kanamycin in milk should be 288 nM (http://www.emea.europa.eu). As the detection limit was much lower than the maximum residue limit, this proposed method has a great potential to be used for the analysis of the small volume Kanamycin A residues in milk as well as other food samples. 3.5. Selectivity inspection of this bio-assay To investigate the selectivity of this bioanalytical system towards Kanamycin A analysis, a list of 10-fold other antibiotics including Gentamycin sulfate (Aminoglycoside antibiotics), Streptomycin sulfate (Aminoglycoside antibiotics), Tetracycline hydrochloride (Tetracycline), Sulfadimethoxine (Sulfonamides), and Lincomycin (Lincosamide antibiotics) were inspected. As can be seen from Fig. 6, a fluorescence enhancement of (F F0)/F0 ¼ 1.05 was obtained in the presence of Kanamycin A. In contrast, the fluorescence signals had little changes in the presence of 10-fold other antibiotics. Meanwhile, FAM-labeled Ampicillin aptamer (FAM-AMP4) was utilized as a control probe in this
In summary, a simple fluorescence bioanalytical system for sensitive and selective detection of Kanamycin A was developed by using gold nanoparticles and FAM-labeled Ky2 aptamer probes. In the presence of Kanamycin A, the aptamer would release from AuNPs surface and fluorescence signal was recovered consequently. This bio-assay showed good sensing performances for Kanamycin A in linear ranges from 40–350 nM to 0.8–20 nM with a limit detection of 0.3 nM. Due to the highly specific recognition between aptamer and Kanamycin A, this sensing strategy showed strong ability of resistance to the interference of other antibiotics. More importantly, this proposed assay was successfully used for the determination of Kanamycin A in milk samples with satisfying results, which indicated that this bio-assay could be utilized as a sensitive, specific, low-cost, and easy-to-use system for the detection of Kanamycin A regarding food safety, public health security as well as medical diagnosis.
Acknowledgment This work was supported by the National Natural Science Foundation of China (21205108), the Scientific Research Foundation for the Returned Overseas Chinese Scholars (State Education Ministry of China), the Technology Foundation for Selected Overseas Chinese Scholars (Ministry of Personnel of China), and Special Fund for Agroscientific Research in the Public Interest (201203094).
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.talanta.2015.02.036.
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