A novel electrochemical aptasensor for sensitive detection of streptomycin based on gold nanoparticle-functionalized magnetic multi-walled carbon nanotubes and nanoporous PtTi alloy

Abstract

A facile electrochemical aptasensor based on a novel signal amplification strategy was employed for sensitive and selective detection of streptomycin for the first time. The aptasensor was prepared by sequentially dripping a gold nanoparticle-functionalized magnetic multi-walled carbon nanotube (Au@MWCNTs–Fe₃O₄) composite and nanoporous PtTi (NP-PtTi) alloy onto a glassy carbon electrode (GCE) surface. The NP-PtTi alloy with a uniform nanoporous structure was successfully fabricated by selectively de-alloying Al from a PtTiAl alloy in HCl solution. The results demonstrated that the Au@MWCNTs–Fe₃O₄/NP-PtTi composite not only showed a remarkable synergistic effect towards the aptasensor, but also provided a beneficial immobilization platform for the streptomycin aptamer carrier. Under optimal conditions, a wide linear range from 0.05 to 100 ng mL⁻¹ and a detection limit of 7.8 pg mL⁻¹ were obtained. The designed aptasensor showed an excellent analytical performance with good reproducibility, high selectivity and stability. Finally, the proposed aptasensor was successfully used for the detection of streptomycin in a real sample.

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1. Introduction

Streptomycin is an aminoglycoside antimicrobial agent which is produced by Streptomyces griseus.^1–3 It has been broadly used in veterinary and human treatment of Gram-negative infectious diseases.^2–4 The abuse of streptomycin could result in the presence of this antibiotic in animal-derived foods and serious side effects such as nephrotoxicity and ototoxicity.^1–3 To protect the security of customers, the European Union (EU) has established maximum residue limits (MRLs) for streptomycin in food, such as 125 ng mL⁻¹ for milk.^2,3

Recently, a series of methods have been developed for the quantitative detection of streptomycin, such as high performance liquid chromatography (HPLC),^5,6 gas chromatography mass spectrometry (GC-MS),^3,4 liquid chromatography mass spectrometry (LC-MS)⁷ and enzyme-linked immunosorbent assay (ELISA).⁸ Although these reported methods are both sensitive and accurate, they require long and tedious pretreatment of samples, expensive laboratory instruments and professional operators. Aptamers are short single-stranded DNA or RNA oligonucleotides selected from random-sequence nucleic acid libraries by an in vitro evolution process.^9,10 Nowadays, electrochemical aptamer-based sensors (aptasensors) based on a specific interaction between the aptamer and target molecules have attracted considerable interest for their highly sensitive and specific detection of antibiotics.^11,12 Meanwhile, the modification technology of the electrode is the crucial step, which affects the signal intensity of the electrochemical detection and the immobilization amounts of biomolecules. Thus, it is necessary to develop a facile immobilization method to form efficient matrices to improve the performance of aptasensors.

Efficiently immobilizing specific biomolecules on a substrate plays a critical role in improving the sensitivity of the electrochemical aptasensor. Nanoporous metal materials with well-defined pore sizes and ultrahigh surface area-to-volume ratios including Au, Ag, Cu, Pd, and Pt-based metals^13–18 have been employed in electrochemical biosensors for binding to amine-containing molecules. It has been proven that nanoporous metals are ideal materials which can form immobilization matrices easily and amplify signals.^10,11 It is noted that the fabrication of nanoporous materials with a large specific surface area by a simple de-alloying process in HCl solution has been rarely studied.^14,15 In this paper, the NP-PtTi alloy with a uniform nanoporous structure was successfully fabricated, which increased the larger specific surface area and provided a beneficial immobilization platform for the streptomycin aptamer carrier.

To improve the sensitivity of the electrochemical analysis, a variety of nanomaterials were used in the fabrication of the aptasensors to achieve signal amplification. Magnetic nanomaterials have drawn considerable attention because of their great potential applications in the field of biosensors.¹⁹ Ferriferrous oxide (Fe₃O₄) is a type of magnetic nanoparticle that is environmentally friendly, and exhibits good electrical properties due to the electron transfer between Fe²⁺ and Fe³⁺.²⁰ Moreover, ferriferrous oxide nanoparticles (Fe₃O₄ NPs) have been reported as nanocarriers to immobilize antibodies or deoxyribonucleic acid in electrochemical immunosensors.^21,22 A series of nanomaterials based on Fe₃O₄ NPs have been designed for a signal amplification strategy in the previously reported literature, such as ferrocene-functionalized iron oxide nanoparticles,²³ dumbbell-like Pt–Fe₃O₄ nanoparticles²⁴ and dumbbell-like Au–Fe₃O₄ nanoparticles.²⁵ Multi-walled carbon nanotubes (MWCNTs) have gained increasing interests in a great variety of research fields due to their high electrical conductivity, chemical stability and remarkable mechanical strength.²⁶ The surface functionalization of MWCNTs could provide extra capability to intensify the chemical modification.^27,28 Magnetic multi-walled carbon nanotube nanocomposites (MWCNTs–Fe₃O₄) have a large surface area, higher conductivity properties and exceptional adsorption capability to capture lead ions (Pb²⁺).¹⁹ Moreover, gold nanoparticles (Au NPs), as excellent carriers,^29,30 can allow ultra efficient adsorption of proteins as a result of their exceptional properties, such as a large surface area, superior mechanical properties, excellent conductivity and good biocompatibility. Au NPs not only increase the quantity of the immobilized streptomycin aptamer, but also preserve the specific activity of the biomolecules.³¹ Inspired by those works, a Au@MWCNTs–Fe₃O₄ composite with a larger surface area and uniform structure was successfully fabricated as an effective load matrix for the immobilization of NP-PtTi alloy and the electrochemical signal was significantly enhanced. Chitosan (CS) has been widely used as an electrode modification material because of its excellent film-forming ability, adsorption capability and biocompatibility.^10,11 In this paper, CS as a good solvent might improve the dispersion and stability of the Au@MWCNTs–Fe₃O₄/NP-PtTi composite, which was beneficial for the detection of the streptomycin antibiotic.

In this work, we have developed a Au@MWCNTs–Fe₃O₄/NP-PtTi composite-modified GCE as a novel system for the preparation of an electrochemical sensing platform. Based on the good conductivity of MWCNTs, Au NPs and Fe₃O₄ NPs, we prepared a Au@MWCNTs–Fe₃O₄ composite to modify the electrode. The Au@MWCNTs–Fe₃O₄/NP-PtTi composite constructed an effective biomolecule immobilization matrix with unblocked conductive pathways for electron transfer. More importantly, this was the first report on the use of a Au@MWCNTs–Fe₃O₄/NP-PtTi nanocomposite leading to significant signal improvement in electrochemical sensors. We investigated various experimental conditions that may influence the performance of the aptasensor. Under optimal conditions, the aptasensor displayed excellent sensitivity, selectivity, reproducibility and stability. Moreover, this developed aptasensor was applied successfully in the determination of streptomycin in real samples.

2. Materials and methods

2.1. Materials

MWCNTs with a purity of 95% were obtained from Beijing Dekedaojin Technology Co., Ltd (China). Ferric chloride (FeCl₃·6H₂O), NaH₂PO₄, Na₂HPO₄·3H₂O, K₃Fe(CN)₆, K₄Fe(CN)₆·3H₂O, NaCl, trisodium citrate, ethylene glycol, sodium acetate (NaAc) and CS were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Gold chloride (HAuCl₄·4H₂O), bovine serum albumin (BSA), dihydrostreptomycin (DHSRT), streptomycin, terramycin, kanamycin sulfate and neomycin sulfate were purchased from Aladdin Chemical Reagent Co., Ltd (Beijing, China). A streptomycin aptamer modified with an amino group at the 5′ position, namely 5′-NH₂-TAG GGA ATT CGT CGA CGG ATC CGG GGT CTG GTG TTC TGC TTT GTT CTG TCG GGT CGT CTG CAG GTC GAC GCA TGC GCC G-3′, was synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China). The streptomycin aptamer was selected by SELEX that could specifically bind with streptomycin.^3,32,33 All other chemicals were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). All the solutions were prepared with ultrapure water which was purified with a Millipore water purification system (≥18 MΩ cm, Milli-Q, Millipore).

2.2. Apparatus

Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were carried out with a CHI 760E electrochemical workstation (Chenhua Instruments Co., Shanghai, China). Electrochemical impedance measurements (EIS) were performed on a Zennium electrochemical workstation (Zahner, Germany). A conventional three-electrode system consisted of a glassy carbon working electrode (GCE, Φ = 3 mm), a platinum wire counter electrode and a KCl saturated Ag/AgCl reference electrode. The morphologies and energy dispersive X-ray spectroscopy (EDS) of the samples were characterized by QUANTA PEG 250 field emission scanning electron microscopy (SEM).

2.3. Synthesis of the Au@MWCNTs–Fe₃O₄ composite

MWCNTs–Fe₃O₄ was fabricated according to the literature.^34,35 0.5 g FeCl₃·6H₂O and 0.2 g MWCNTs were dissolved in 20 mL of ethylene glycol to form a homogeneous solution, followed by the addition of 1.5 g NaAc. The mixture was stirred vigorously for 30 min and then sealed in a Teflon-lined stainless steel autoclave. The autoclave was heated to and maintained at 180 °C for 24 h, and allowed to cool to room temperature (RT). The resulting black powder was obtained after being washed several times and dried at 35 °C under high vacuum overnight.

A mixture of MWCNTs–Fe₃O₄ (20 mg) and the prepared Au NP solution (40 mL) was shaken for 12 h.³⁴ The Au NPs were synthesized according to the literature.^36,37 The final product was obtained by being washed several times and dried at 35 °C under high vacuum overnight. 5 mg Au@MWCNTs–Fe₃O₄ composite was dissolved in 1 mL of 1 wt% CS under sonication to obtain a homogeneous suspension.

2.4. Fabrication of the aptasensor and electrochemical measurements

The bare GCE was firstly polished with 0.3 and 0.05 μm alumina slurry successively, and then thoroughly washed with ethanol and ultrapure water. 6 μL Au@MWCNTs–Fe₃O₄ suspension (5 mg mL⁻¹) was dropped onto the surface of the GCE and dried at RT. Next, 6 μL NP-PtTi suspension (2 mg mL⁻¹) was deposited on the surface of Au@MWCNTs–Fe₃O₄/GCE. The NP-PtTi alloy was successfully fabricated by simple de-alloying of a PtTiAl source alloy in HCl solution.^15,38 2 mg NP-PtTi alloy was dispersed in 1 mL of 1 wt% CS under sonication to obtain a homogeneous suspension. After thoroughly washing with 0.1 M PBS (pH 7.4), 10 μL of 5 μmol L⁻¹ aptamer was assembled on the surface of NP-PtTi/Au@MWCNTs–Fe₃O₄/GCE and kept standing overnight. The aptamer-modified electrode was thoroughly washed with PBS to remove the loosely adsorbed aptamer. Subsequently, 10 μL of 1% BSA was incubated at 4 °C for 2 h to block the possible remaining active sites of the NP-PtTi alloy and to avoid non-specific adsorption. After washing again with PBS, the obtained electrode was incubated with different concentrations of streptomycin solution for 2 h. Finally, the as-prepared electrodes were stored in a refrigerator when not used. A schematic illustration of the stepwise modification procedure of the aptasensor is displayed in Scheme 1.

Scheme 1 Schematic diagram of the streptomycin aptasensor.

All electrochemical measurements including CV, EIS and DPV were recorded in 0.1 M PBS containing 5.0 mM [Fe(CN)₆]^3−/4− and 0.2 M KCl. DPV was conducted from −0.2 to 0.6 V with a modulation amplitude of 0.05 V, a pulse width of 0.05 s and sample width of 0.0167 s.

3. Results and discussion

3.1. Characterization of the prepared NP-PtTi alloy

The morphologies of the prepared NP-PtTi alloy were examined using SEM and TEM. It is observed that the NP-PtTi alloy has an interconnected spongy morphology with a ligament size less than 10 nm (Fig. 1A), which was useful for increasing the special surface areas.^10,15 Fig. 1B shows a HRTEM image of the NP-PtTi sample. The clear contrast of the bright pores and the dark ligaments further confirm the formation of a three-dimensional (3D) interconnected network structure, which was beneficial for the mass and electron transport during electrochemical sensing. It was consistent with the SEM observation. These results indicated that NP-PtTi alloy with no porous structure has been fabricated successfully by a one-step de-alloying procedure.^15,38

Fig. 1 (A) SEM and (B) HRTEM of NP-PtTi alloy.

3.2. Characterization of the different nanomaterials

SEM images of MWCNTs–Fe₃O₄, Au@MWCNTs–Fe₃O₄ and Au@MWCNTs–Fe₃O₄/NP-PtTi were helpful to identify successful synthesis by observing the morphologies. MWCNTs were entangled with one another, which could be attributed to π–π stacking interactions between the MWCNTs.^11,37 It is observed that numbers of Fe₃O₄ NPs were distributed on the surface of the MWCNTs (Fig. 2A), suggesting the successful fabrication of Fe₃O₄ NPs on the surface of the MWCNTs.^34,36 The SEM image of Au@MWCNTs–Fe₃O₄ (Fig. 2B) shows that lots of Au NPs with a uniform diameter of about 25 nm were loaded on the surface of MWCNTs–Fe₃O₄, which could provide the larger surface area. When the NP-PtTi alloy was coated on the surface of Au@MWCNTs–Fe₃O₄, irregular bright dots were obviously observed (Fig. 2C) because of the difference in particle size between Au NPs and NP-PtTi. The grain size distribution of the NP-PtTi alloy is shown in Fig. S1.† The average size of the grains was 64.7 nm.^15,38 The surface morphology of NP-PtTi/Au@MWCNTs–Fe₃O₄ was changed, suggesting that the NP-PtTi alloy was successfully deposited onto the surface of the Au@MWCNTs–Fe₃O₄ composite.

Fig. 2 SEM of (A) MWCNTs–Fe₃O₄, (B) Au@MWCNTs–Fe₃O₄ and (C) Au@MWCNTs–Fe₃O₄/NP-PtTi; EDS of (D) MWCNTs–Fe₃O₄, (E) Au@MWCNTs–Fe₃O₄ and (F) Au@MWCNTs–Fe₃O₄/NP-PtTi.

The EDS spectra of MWCNTs–Fe₃O₄, Au@MWCNTs–Fe₃O₄ and Au@MWCNTs–Fe₃O₄/NP-PtTi further explained what kinds of elements were contained in these nanomaterials. The spectrum of MWCNTs–Fe₃O₄ (Fig. 2D) confirms the presence of Fe₃O₄ in the composition due to obvious Fe and O elements existing. It is obvious that Fe, Au and O elements can be observed (Fig. 2E), which clearly confirms the successful synthesis of Au@MWCNTs–Fe₃O₄. When the NP-PtTi alloy was coated on the surface of Au@MWCNTs–Fe₃O₄, Fe, Au, O, Pt and a small amount of Ti were obviously observed (Fig. 2F). All the results indicated that the Au@MWCNTs–Fe₃O₄/NP-PtTi composite has been fabricated successfully.

3.3. Electrochemical characterization of the aptasensor

CV is used to investigate the electron transmission process of the modified electrode. The electrochemical behaviors of various modified electrodes were studied by taking Fe(CN)₆^3−/4− as the electrochemical probe. In order to investigate the synergistic effect of the Au@MWCNTs–Fe₃O₄ composite, CV results of Au NPs/GCE, Fe₃O₄ NPs/GCE, MWCNTs/GCE, MWCNTs–Au NPs/GCE, MWCNTs–Fe₃O₄/GCE and Au@MWCNTs–Fe₃O₄/GCE are shown in Fig. 3A. The bare GCE had an obvious redox peak (curve a), indicating a reversible electrochemical process. It can be clearly seen that the peak current was larger than that of the bare GCE after coating with Au NPs (curve b), Fe₃O₄ NPs (curve c) or MWCNTs (curve d), suggesting that the introduction of Au NPs, Fe₃O₄ NPs or MWCNTs played an important role in enhancing conductivity.^10,11 The peak current of MWCNTs–Au NPs/GCE (curve e) or MWCNTs–Fe₃O₄/GCE (curve f) was larger than that of MWCNTs/GCE, suggesting that the introduction of Au NPs or Fe₃O₄ NPs played an important role in the enhancement of its conductivity and active electrode area.^10,11 It is clearly seen that the current of MWCNTs–Fe₃O₄/GCE was superior to that of MWCNTs–Au NPs/GCE. It might be attributed to MWCNTs–Au NPs having a relatively poorer electron transfer ability than that of MWCNTs–Fe₃O₄.^34,36 Moreover, Au@MWCNTs–Fe₃O₄/GCE exhibited a much greater current response (curve g) due to the synergistic amplification effect of the good electrical conductivity of MWCNTs, Au NPs and Fe₃O₄ NPs.^10,25,37 All those results confirmed that Au@MWCNTs–Fe₃O₄ not only offered a biocompatible surface for biomolecule loading, but also provided a sensitive electric interface for further sensing.^10,11

Fig. 3 (A) CV (scan rate: 100 mV s⁻¹) of 5.0 mM [Fe(CN)₆]^3−/4− in 0.1 M PBS with 0.2 M KCl at (a) bare GCE, (b) Au NPs/GCE, (c) Fe₃O₄ NPs/GCE, (d) MWCNTs/GCE, (e) MWCNTs–Au NPs/GCE, (f) MWCNTs–Fe₃O₄/GCE and (g) Au@MWCNTs–Fe₃O₄/GCE; (B) CV (scan rate: 100 mV s⁻¹) and (C) EIS of (a) bare GCE, (b) Au@MWCNTs–Fe₃O₄/GCE, (c) NP-PtTi/MWCNTs–Au NPs/GCE, (d) aptamer/NP-PtTi/Au@MWCNTs–Fe₃O₄/GCE, (e) BSA/aptamer/NP-PtTi/Au@MWCNTs–Fe₃O₄/GCE and (f) streptomycin/BSA/aptamer/NP-PtTi/Au@MWCNTs–Fe₃O₄/GCE. Measurements were performed in 0.1 M PBS containing 5.0 mM [Fe(CN)₆]^3−/4− and 0.2 M KCl.

The fabrication of the proposed aptasensor was characterized by CV and EIS. As shown in Fig. 3B, it can be seen that the bare GCE had an obvious redox peak (curve a). After treatment with Au@MWCNTs–Fe₃O₄ (curve b), the current increased about 486 μA, suggesting that the nanocomposite made the electron transfer easier. When the NP-PtTi alloy was immobilized on Au@MWCNTs–Fe₃O₄/GCE, a larger current response was exhibited (curve c). It is found that the intensity of the Au@MWCNTs–Fe₃O₄/NP-PtTi modified electrode was 1.2-fold larger than that of the Au@MWCNTs–Fe₃O₄ modified GCE, indicating that a Au@MWCNTs–Fe₃O₄/NP-PtTi film on the GCE could effectively increase the surface area and the current response of the electrode due to the synergistic effects of the good electrical conductivity of Au@MWCNTs–Fe₃O₄ and NP-PtTi.^10,34,36,37 After the capture of 5 μmol L⁻¹ aptamer, the peak current of [Fe(CN)₆]^3−/4− at aptamer/NP-PtTi/Au@MWCNTs–Fe₃O₄/GCE (curve d) became much smaller than that at NP-PtTi/Au@MWCNTs–Fe₃O₄/GCE, suggesting that the aptamer could generate an insulating layer and hinder electron transfer. BSA was employed to block extra active sites and avoid non-specific adsorption^11,29 and a successive decrease in the current was obtained (curve e). Finally, when 10 ng mL⁻¹ streptomycin solution was captured, the peak current decreased again (curve f), indicating that the formation of the streptomycin–aptamer complex^10,11,29,37 hindered the interfacial electron transfer to the modified electrode surface.

EIS is another suitable method for characterizing the features of surface-modified electrodes. Fig. 3C shows the Nyquist plots of different modified electrodes. In EIS, the semicircle diameter equals the charge transfer resistance (R_ct).^10,25,34 It is easy to see that the bare GCE shows a relatively large resistance (curve a). After the deposition of Au@MWCNTs–Fe₃O₄, R_ct of the electrode decreased (curve b), indicating that the Au@MWCNTs–Fe₃O₄ composite could accelerate the electron transfer. It is easy to find that R_ct of NP-PtTi/Au@MWCNTs–Fe₃O₄/GCE (curve c) was smaller than that of Au@MWCNTs–Fe₃O₄/GCE, suggesting that the synergic amplification effect of NP-PtTi and MWCNTs could improve the electron transfer process on the surface of the aptasensor.^10,11,37 When the aptamer was assembled on NP-PtTi/Au@MWCNTs–Fe₃O₄/GCE, R_ct was increased significantly (curve d), suggesting that the aptamer was immobilized on the electrode. Moreover, the capture of BSA and 10 ng mL⁻¹ streptomycin resulted in a further increase of R_ct (curves e and f), implying that electron transfer became more difficult. This result was consistent with the result of CV. All these observations confirmed that the Au@MWCNTs–Fe₃O₄/NP-PtTi composite could provide a sensitive modified electrode substrate and greatly increase the effective area of the electrode to load more biomolecules.

3.4. Optimization of experimental conditions

In order to increase the sensitivity and selectivity of the aptasensor, experimental parameters such as the atomic percent ratio of Fe and Au, incubation time and pH value were optimized systematically. The effect of the atomic percent ratio of Fe and Au in the Au@MWCNTs–Fe₃O₄ composite on the response of the aptasensor toward streptomycin was tested. As shown in Fig. S2A,† the highest value of electrochemical response was achieved at 1 : 0.5 among the different ratios ranging from 1 : 0.3 to 1 : 0.7. It might be attributed to MWCNTs–Au NPs having a relatively poorer electron transfer ability than that of MWCNTs–Fe₃O₄, so as to counteract the advantage of MWCNTs–Fe₃O₄.^34,36 Thus, the ratio of 1 : 0.5 was selected as the optimal condition to prepare the aptasensor.

The effect of the pH value of the measuring solution was investigated. Fig. S2B† shows the curves corresponding to the DPV detection of streptomycin in 0.1 M PBS based on the changes of current intensity (ΔI) from before and after streptomycin incubation. As shown in Fig. S2B,† ΔI increased with the increasing pH value from 6.2 to 7.4, and then decreased over pH 7.4. The optimal current response was obtained at pH 7.4, indicating that the aptamer could not combine well with streptomycin in strong acidic or alkaline solutions. Thus, pH 7.4 was employed throughout the detection process.

The incubation time is also an important parameter for the aptasensor to achieve maximized current signal. A series of BSA/aptamer/NP-PtTi/Au@MWCNTs–Fe₃O₄/GCEs were incubated with 10 ng mL⁻¹ streptomycin solution for 20, 30, 40, 60, 90, 120, 150 and 180 min, respectively. As shown in Fig. S2C,† ΔI increased significantly with the increase of incubation time and was inclined to level off after 120 min, which suggested that the building of a streptomycin–aptamer complex on the electrode surface reached saturation. Therefore, the optimal incubation time was 120 min.

3.5. Calibration curve of the aptasensor

Under the optimum conditions, the aptasensors were incubated in different concentrations of streptomycin solutions and the DPV responses of the proposed aptasensors were recorded. DPV was conducted from −0.2 to 0.6 V with a modulation amplitude of 0.05 V, a pulse width of 0.05 s and sample width of 0.0167 s. As shown in the inset of Fig. 4, the DPV current decreased gradually with the increase of the streptomycin concentration. The calibration curve shows a good linear relationship between ΔI and the concentrations of streptomycin solutions in the range of 0.05 to 100 ng mL⁻¹ with a correlation coefficient of 0.9992. The linear equation could be fitted as ΔI (μA) = 3.14526c + 15.432219 (ng mL⁻¹). The detection limit of streptomycin was 7.8 pg mL⁻¹ (S/N = 3). The proposed method for the determination of streptomycin is compared with the previously reported methods^1,3,39–42 in Table S1.† The proposed aptasensor offered the advantages of a wide linear range, shortened analysis time, and simplified operation with no need for expensive instrumentation and consumption of large amounts of reagent.

Fig. 4 Calibration curve of DPV peak currents for different streptomycin concentrations from 0.05 to 100 ng mL⁻¹. The inset shows typical DPV responses of the aptasensor to different concentrations of streptomycin ((a–h) 0, 0.05, 5, 10, 25, 50, 75, 100 ng mL⁻¹).

Under optimum conditions, the proposed approach displayed a wider linear range and lower limit of detection, which was attributed to four factors: (1) the Au@MWCNTs–Fe₃O₄ composite was the first prepared one to modify the electrodes, and was successfully used as an ideal material for the deposition of NP-PtTi alloy; (2) Au@MWCNTs–Fe₃O₄ provided a smoothly conductive pathway for electron transfer due to the synergistic amplification effect of the three kinds of nanomaterials, namely MWCNTs, Au NPs and Fe₃O₄ NPs; (3) the Au@MWCNTs–Fe₃O₄/NP-PtTi composite with excellent conductivity and a larger surface area was used as an effective load matrix for the deposition of biomolecules, which could play a key role in improving the capability of electron transfer; (4) the stability of the Au@MWCNTs–Fe₃O₄/NP-PtTi film was good and the streptomycin aptamer could firmly attach to the modified electrode surface via the interaction between the –NH₂ group of the aptamer and Pt.

3.6. The stability, selectivity and reproducibility of the aptasensor

The stability of the aptasensor is a key factor in practical application. To test the stability of the aptasensor, five electrodes prepared under the same conditions were utilized to measure 10 ng mL⁻¹ of streptomycin and stored at 4 °C before used. The results showed that the current responses of the aptasensor retained 93.7% of the initial response after 3 weeks, demonstrating that the proposed aptasensor exhibited sufficient stability for the detection of streptomycin.

Selectivity is another important criterion for electrochemical aptasensors. The current responses of the as-prepared aptasensors to 10 ng mL⁻¹ solutions of streptomycin, DHSRT, terramycin, kanamycin sulfate, neomycin sulfate, and the mixture of 10 ng mL⁻¹ streptomycin solution and 100 ng mL⁻¹ interfering substance solutions were studied. As shown in Fig. 5, streptomycin and DHSRT showed a much stronger current response (Fig. 5a and b) compared to that of the interferents (Fig. 5c–e). The primary structure of DHSRT is similar to that of streptomycin, but DHSRT contains a hydroxyl-methyl group instead of the aldehyde group of streptomycin.^43,44 Moreover, the mixtures of streptomycin and interferents also showed a much stronger current response (Fig. 5f–i). It is observed that the presence of these interferents almost did not change the current, confirming that the aptamer could only recognize streptomycin and DHSRT^43,44 and could not combine with the interferents. To further investigate the selectivity of the aptasensor, a study of interferences was performed using an oxytetracycline aptamer,⁴⁵ kanamycin aptamer^10,11 and neomycin aptamer⁴⁶ to measure 10 ng mL⁻¹ streptomycin solution. The as-prepared electrodes were incubated in different aptamer solutions and DPV responses of the proposed aptasensors were recorded. As shown in Fig. S3,† the streptomycin aptamer showed a much stronger current response (Fig. S3a†). In addition, mixtures of the streptomycin aptamer and other aptamer also showed a much stronger current response (Fig. S3e–g†). It is noticeable that a weak current response was obtained in the presence of those aptamers (Fig. S3b–d†), indicating that streptomycin could only recognize the streptomycin aptamer and could not combine with other aptamers. All those results confirmed that the developed aptasensor had excellent selectivity.

Fig. 5 DPV current responses of the aptasensor for the detection of (a) 10 ng mL⁻¹ streptomycin, (b) 10 ng mL⁻¹ DHSRT, (c) 10 ng mL⁻¹ terramycin, (d) 10 ng mL⁻¹ kanamycin sulfate, (e) 10 ng mL⁻¹ neomycin sulfate, (f) 100 ng mL⁻¹ DHSRT + 10 ng mL⁻¹ streptomycin, (g) 100 ng mL⁻¹ terramycin + 10 ng mL⁻¹ streptomycin, (h) 100 ng mL⁻¹ kanamycin sulfate + 10 ng mL⁻¹ streptomycin, (i) 100 ng mL⁻¹ neomycin sulfate + 10 ng mL⁻¹ streptomycin. Error bars are standard deviations across three repeat experiments.

The reproducibility of the developed aptasensor for streptomycin was investigated with intra-assay and inter-assay precision. The intra-assay precision of the aptasensor was evaluated by analyzing three streptomycin levels for six replicate measurements. The relative standard deviations (RSDs) were 4.1%, 3.5% and 3.9% for 5.0 ng mL⁻¹, 10.0 ng mL⁻¹ and 25.0 ng mL⁻¹ streptomycin, respectively. Similarly, the inter-assay precision was evaluated by analyzing the same streptomycin level with six aptasensors made using the same conditions independently. The RSDs were 3.2%, 4.4% and 4.0% for 5.0 ng mL⁻¹, 10.0 ng mL⁻¹ and 25.0 ng mL⁻¹ streptomycin, respectively. Thus, the reproducibility of the proposed aptasensor was acceptable.

3.7. Determination of streptomycin in real samples

To evaluate the analytical reliability and application potentiality of the proposed aptasensor, it was used for determining the recoveries of different concentrations of streptomycin in milk samples by an external standard method. The milk solution was firstly centrifuged at 12 000 rpm for 10 min to remove fat. Then, the obtained supernatant was diluted ten times with PBS. Furthermore, streptomycin standard solution was spiked into the diluted supernatant to prepare the final concentrations of 0.05, 0.5, 1.0, 5.0 and 8.0 ng mL⁻¹. The streptomycin standard solution was 0.5, 5.0, 10.0, 50.0 and 80.0 ng mL⁻¹. Finally, experiments were carried out according to the aforementioned optimized conditions. The recovery was in the range from 97.3% to 105.2% and the RSD was in the range from 2.79% to 4.01% (Table 1). Thus, the proposed aptasensor can be effectively applied in the quantitative determination of streptomycin in milk.

Table 1 Streptomycin determination in milk by the proposed aptasensor

Milk found (ng mL^−1)	Streptomycin added in milk (ng mL^−1)	Streptomycin determined by the electrochemical method (ng mL^−1 ± RSD%)	Recovery (%)
Not detected	0.05	0.052 ± 2.79	104.0
Not detected	0.5	0.526 ± 3.03	105.2
Not detected	1.0	0.973 ± 3.25	97.3
Not detected	5.0	5.121 ± 4.01	102.4
Not detected	8.0	7.965 ± 2.93	99.6

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4. Conclusions

In this work, a novel label-free electrochemical aptasensor was developed for sensitive detection of the streptomycin antibiotic using Au@MWCNTs–Fe₃O₄/NP-PtTi as the matrix. The prepared aptasensor offered several advantages: (1) the Au@MWCNTs–Fe₃O₄/NP-PtTi composite not only improved the amperometric signal significantly, but also provided abundant binding sites for the immobilization of biomolecules; (2) the constructed aptasensor with higher sensitivity exhibited a wide linear range for streptomycin from 0.05 to 100 ng mL⁻¹ with a low detection limit of 7.8 pg mL⁻¹; (3) the constructed aptasensor offered the advantages of improved stability, high selectivity and reproducibility. In addition, the resulting aptasensor was successfully applied for streptomycin detection in real milk samples. Thus, the presented aptasensor has potential applications for streptomycin detection in the field of food analysis.

Acknowledgements

This work was supported financially by Shandong Provincial Natural Science Foundation, China (Grant No. ZR2012BL11), and Shandong Provincial Science and Technology Development Plan Project, China (Grant No. 2013GGX10705).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra02029a

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