Ultrasensitive Self-Powered Aptasensor Based on Enzyme Biofuel

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Ultrasensitive Self-Powered Aptasensor Based on Enzyme Bioful Cell and DNA Bioconjugate: A Facile and Powerful Tool for Antibiotic Residue Detection Panpan Gai, Chengcheng Gu, Ting Hou, and Feng Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b05109 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 17, 2017

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Analytical Chemistry

Ultrasensitive Self-Powered Aptasensor Based on Enzyme Biofuel Cell and DNA Bioconjugate: A Facile and Powerful Tool for Antibiotic Residue Detection

Panpan Gai, Chengcheng Gu, Ting Hou, and Feng Li*

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, P. R. China.

* Corresponding author. Tel/Fax: 86-532-86080855

E-mail: [email protected]

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ABSTRACT Herein, we reported a novel ultrasensitive one-compartment enzyme biofuel cells (EBFCs)-based self-powered aptasensing platform for antibiotic residue detection. By taking full advantage of the unique features of both EBFCs-based self-powered sensors and aptamers, the as-proposed aptasensing platform have the merits of simple instrumentation, anti-interference ability, high selectivity, and low cost. In this study, DNA bioconjugate, i.e. SiO2@gold nanoparticles-complementary strand of aptamer (SiO2@AuNPs-csDNA), was elaborately designed and played a key role in blocking the mass transport of glucose to the bioanode. While in the presence of the target antibiotic, SiO2@AuNPs-csDNA bioconjugate broke away from the bioanode due to the aptamer recognition of the target. Without the blocking of glucose by the DNA bioconjugate, a significantly elevated open circuit voltage of the EBFCs-based aptasensor was obtained, whose amplitude was dependent on the antibiotic concentration. In addition, this proposed aptasensor was the first reported self-powered aptasensing platform for antibiotic determination and featured with high sensitivity owing to the elaborate design of DNA bioconjugate modified bioanode of EBFC, which was superior to those previously reported in literature. Furthermore, due to the anti-interference ability and the excellent selectivity of the aptasensor, no special sample pretreatment was needed for the detection of antibiotics in milk samples. Therefore, the proposed EBFCs-based self-powered aptasensor has a great promise to be applied as a powerful tool for on-site assay in the field of food safety.

KEYWORDS Self-powered aptasensor; Enzyme biofuel cell; DNA bioconjugate; Antibiotic residue detection

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INTRODUCTION Enzyme biofuel cells (EBFCs) have attracted considerable attention because of their mild operation conditions and promising applications as implantable power sources.1,2 Generally, most of the development of EBFCs devoted to the fabrication of energy devices with improved power output, long-term stability and miniaturization.1 Recently, self-powered biosensors, a new application of EBFCs, have been developed as novel biosensing platforms, and the biosensors themselves provide the power for sensing. They have been successfully applied in determining biomolecules3-7 and toxic pollutants,8,9 catalyst screening,10 immunoassays,11-13 and cytosensing.14,15. Compared with their counterparts, self-powered biosensors based on EBFCs possessed the merits of no need for external power sources, excellent anti-interference performance, simple fabrication process, miniaturized size and low cost.16,17 Aptamers are specific oligonucleotide strands that are obtained via an in vitro process called SELEX (Systematic Evolution of Ligands by EXponential enrichment), and can bind to their targets with high affinity and selectivity. As compared to traditional recognition molecules, such as antibodies, aptamers have the unique features of simple synthesis, relatively small size, being easily functionalized, and excellent chemical stability during long-term storage, making them ideal molecular receptors and sensing elements for constructing sensing platforms.18 Inspired by the aforementioned developments, by coupling the merits of EBFCs and aptasensors, EBFCs-based self-powered aptasensing platforms with simple instrumentation (only two electrodes necessary), anti-interference ability, low cost, excellent selectivity, and good stability could be constructed, Thus, these unique characteristics of self-powered aptasensors make them promising candidates as powerful sensing tools.

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Antibiotic residue in animal products is an ever increasing concern in food safety in the last few decades.19 Long-term intake of food products containing antibiotic residues exceeding maximum residue limit (MRL) could cause possible complications, such as antibiotic resistance, allergic reaction, etc.20 Therefore, it is imperative to develop simple, quantitative and reliable methodologies for determining antibiotic residues in food of animal origin. To date, a great number of approaches for the determination of antibiotic residue in food products have been reported, including chromatography,21 surface enhanced Raman spectroscopy,22 colorimetry,23 immunoassay24 and electrochemical method.25 However, these methods suffered from such drawbacks as complicated sample treatment, expensive instrumentation and low sensitivity. Thereby, constructing real-time and portable sensing devices has become urgent demand of food safety monitoring systems. Moreover, to realize on-site assay, it is necessary to develop novel antibiotic residue biosensing platforms with simple construction, good specificity and high sensitivity. Herein, by taking full advantages of the unique features of both EBFCs-based self-powered sensors and aptamers, we proposed a novel ultrasensitive one-compartment EBFCs-based self-powered aptasensing platform for antibiotic residue detection. In this study, the DNA bioconjugate, SiO2@gold nanoparticles-complementary strand of aptamer (SiO2@AuNPs-csDNA), played a key role in blocking the mass transport of glucose to the bioanode. The SiO2@AuNPs-csDNA bioconjugate was elaborately designed by anchoring csDNA to the surface of SiO2@AuNPs via Au-S bond (Scheme 1A), which was subsequently immobilized onto the aptamer/GOx modified electrode through base pairing with the amino-DNA aptamer (NH2-DNA) pre-attached to the bioanode. In addition, to accelerate the electron transfer and to promote the electrical contacting between the enzyme and the electrode, AuNPs-modified carbon paper (CP) was employed as the robust substrate electrode, which possessed the advantages of 4


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high specific surface area, excellent conductivity and good biocompatibility. For the biocathode, polydopamine (PDA) film was first deposited on the AuNPs modified CP electrode, and then laccase was attached to the modified biocathode, in which PDA acted as a mediator for laccase/PDA/AuNPs biocathode-based bioelectrocatalytic reduction of oxygen.26 The principle of the self-powered aptasensing is shown in Scheme 1, in which ampicillin (AMP) was used as a representative antibiotic model. The membrane-less EBFCs-based self-powered aptasensor, composed of SiO2@AuNPs-csDNA bioconjugate/aptamer/GOx/AuNPs bioanode and laccase/PDA/AuNPs biocathode, was fabricated for detecting the antibiotic AMP. In the absence of the target (Scheme 1B), due to the steric hindrance effects, SiO2@AuNPs-csDNA bioconjugate prevented the mass transport of the fuel glucose from approaching the active sites of GOx, resulting in a low open circuit voltage (EOCV) of EBFCs. Whereas, in the presence of the target AMP (Scheme 1C), which was recognized by the aptamer, the SiO2@AuNPs-csDNA bioconjugate broke away from the bioanode due to the dehybridization of NH2-DNA and csDNA induced by the aptamer conformation change. In this case, without the blocking of the DNA bioconjugate, glucose could reach the active sites of GOx and the aptamer/GOx/AuNPs bioanode biocatalytic oxidation of glucose could be efficiently realized. In consequence, a significantly elevated EOCV of the aptasensor was obtained, whose amplitude was dependent on the AMP concentration. Therefore, sensitive determination of AMP could be realized. This effective and simple aptasensing strategy needed no external power sources, and exhibited high sensitivity and selectivity, thus holding a great promise to be applied as a powerful on-site assay tool for antibiotic residue detection.

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Scheme 1. Schematic illustration of the fabrication of DNA bioconjugate SiO2@AuNPs-csDNA (A) and EBFCs-based self-powered aptasensor for determination of AMP (B & C).

EXPERIMENTAL SECTION Materials and Reagents. DNA oligonucleotides were synthesized and HPLC-purified by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The sequence of the amino-DNA aptamer (NH2-DNA) was 5′-NH2-(CH2)6-TTT TGC GGG CGG TTG TAT AGC GG-3′. The sequence of cs-DNA, which was partially complementary to the aptamer, was 5′-SH-(CH2)6-TTT TTT TTT CCG CTA TAC AAC CGC C-3′. Glucose oxidase (GOx) from Aspergillus niger (EC 1.1.3.4, 158.9 units mg-1), 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 6-mercapto-1-hexanol (MCH), tetraethoxysilane (TEOS), 3-Aminopropyltriethoxysilane (APTES) and DL-Dithiothreitol (DTT) were purchased from Sigma-Aldrich (St. Louis, USA). β-D-Glucose was obtained from Tokyo Chemical Industry Co. Ltd. (Japan). Ampicillin (AMP), tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), amoxicillin, lincomycin, and tiamulin were obtained from Aladdin (Shanghai, China). Chloroauric acid (HAuCl4·4H2O), citric acid

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and phenylalanine were obtained from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). 0.1 M pH 7.4 phosphate buffer (PB) consisting of Na2HPO4 and NaH2PO4 were employed as the supporting electrolyte. AuNPs were prepared according to the literature by adding a sodium citrate solution to a boiling HAuCl4 solution.27 All reagents were of analytical grade and used without further purification. Milk samples were purchased from the local market. Ultrapure water (resistivity > 18.2 MΩ cm at 25 °C) obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA, USA) was used for all the experiments. Tris-HCl buffer (pH 7.4, 10 mM) containing 0.1 M NaCl was adopted for the preparation and hybridization of DNA stock solutions.

Apparatus and Instrumentation. Field emission scanning electron microscopy (FESEM) images were measured on a HITACHI S4800 SEM (Hitachi, Japan). Transmission electron microscopy (TEM) images were recorded on a HT7700 microscope (Hitachi, Japan) operated at 100 kV. Dynamic light scattering (DLS) was measured by 90Plus Zeta (Brookhaven, USA). Electrochemical impedance spectroscopy (EIS) was carried on an Autolab PGSTAT 302N electrochemical analyzer (Metrohm Autolab, The Netherlands) within a frequency range of 0.1 Hz to 100 kHz and with 2.5 mM K3Fe(CN)6/K4Fe(CN)6 used as the probe. Cyclic voltammetric measurement was performed on a CHI 660E electrochemical workstation (Shanghai CH Instrument Co., China) using a three-electrode system: the fabricated bioanode or biocathode as the working electrode, a Pt wire as the counter electrode, and an Ag/AgCl as the reference electrode. The open circuit voltage (EOCV) of EBFC was measured by connecting the bioanode and the biocathode placed in the electrolytic cell.

Synthesis of DNA Bioconjugate SiO2@AuNPs-csDNA. Firstly, amino-functionalized SiO2 spheres were synthesized according to our previous publication28 with slight modifications. Briefly, 4.5 7



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mL TEOS was added to the mixture of 9 mL NH3·H2O (28%), 16.25 mL ethanol and 24.75 mL water, and the mixture was stirred at room temperature for 3 h to obtain SiO2 spheres. Excessive TEOS and ammonia were removed by centrifugation (10000 rpm, 20 min), and the obtained precipitate was washed with ultrapure water and ethanol, and dried at 70 °C. Subsequently, 0.4 mL APTES was added dropwise to the obtained SiO2 sphere ethanol solution and stirred at room temperature for 6 h. To remove excess reactants, the solution was then centrifuged and washed at least four times with ethanol. Subsequently, 500 µL of 10 nM carboxy group modified AuNPs and 10 mg mL-1 EDC/NHS were incubated with 100 µL of 1 mg mL-1 amino-functionalized SiO2 sphere suspension overnight at room temperature. Surplus AuNPs were separated by centrifugation at a speed of 6500 rpm for 12.0 min. The supernatant was carefully removed and the precipitate was SiO2@AuNPs. And then, 1 µM csDNA activated with 1 mM DTT was incubated with the SiO2@AuNPs at room temperature for 2 h to bond to SiO2@AuNPs through Au-S bond. Following that, 10 µL of 1 mM MCH was injected into the suspension, and incubated for 30 min to block any possible residual sites on AuNPs, and then washed with PB for at least three times. After centrifugation, the precipitate was dispersed in PB (pH 7.4) to obtain the DNA bioconjugate SiO2@AuNPs-csDNA.

Preparation of SiO2@AuNPs–csDNA/aptamer/GOx/AuNPs Modified Bioanode. 50 µL of the as-prepared AuNPs (100 nM) was coated on the surface of the carbon paper electrode (0.5 cm × 0.5 cm) and dried at 37 °C for 2 h. Then the substrate electrode was immersed in a solution containing 10 mg mL-1 EDC/NHS for 30 min to activate the carboxyl group of AuNPs. After rinsing with ultrapure water to eliminate excess EDC and NHS, the activated electrode was coated with 50 µL of mixture containing 20 mg mL-1 GOx solution and 1 µM aptamer (NH2-DNA) at 4 °C for 12 h to obtain 8


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aptamer/GOx/AuNPs modified electrode through a condensation reaction between carboxyl groups on the AuNPs and terminal amino groups on the lysine residues of GOx and NH2-DNA. Subsequently, 50 µL of DNA bioconjugate

SiO2@AuNPs-csDNA was

dropped

onto

the

aforementioned

aptamer/GOx/AuNPs modified carbon paper electrode surface and incubated at 37 °C for 2 h to obtain the bioanode of EBFC. The electrode was stored at 4 °C when not in use.

Preparation of Laccase/PDA/AuNPs Modified Biocathode. 50 µL of the prepared AuNPs (100 nM) was coated on the surface of the carbon paper electrode. Then, the substrate electrode was dried at 37 °C. Subsequently, the substrate electrode was immersed into 6 mM dopamine solution (0.1 M PB, pH 8.5) for 3 h under stirring. After being rinsed with ultrapure water to eliminate excess dopamine, the activated electrode was incubated with 50 µL of laccase solution (30 mg mL-1) dissolved in PB (pH 7.4) at 4 °C for 12 h to obtain the laccase/PDA/AuNPs modified biocathode.

Self-Powered Aptasensing of AMP. A membrane-less glucose/O2 EBFC-based self-powered aptasensor was constructed by using the as-prepared bioanode and biocathode at room temperature. The supporting electrolyte was 0.1 M PB (pH 7.4) containing 5 mM glucose. The EOCV of the EBFC composed of the as-prepared SiO2@AuNPs-csDNA/aptamer/GOx/AuNPs modified bioanode and laccase/PDA/AuNPs modified biocathode was first measured in the supporting electrolyte, and subsequently the SiO2@AuNPs-csDNA/aptamer/GOx/AuNPs modified bioanode was soaked in 1 mL of the target AMP solution at a certain concentration and incubated for 30 min to capture the target. Then the electrode was taken out, and put back into the electrolytic cell, and the EOCV of EBFC was measured again. For real sample detection, no special sample pretreatment was needed; 100 µL of milk samples

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were diluted to 5 mL using PB (pH 7.4), and the EOCV was measured according to the aforementioned procedures.

RESULTS AND DISCUSSION Characterization of AuNPs Substrate Electrode. The performance of the EBFCs is crucial to the properties of the EBFCs-based self-powered biosensor. Notably, the efficient electrical communication between the enzyme and the substrate electrode played an important role in improving the performance of EBFCs. AuNPs with the merits of high specific surface area, excellent conductivity and good biocompatibility have been widely used to assist electron transfer. Thus, AuNPs modified CP was selected as the effective substrate electrode to facilitate the enzyme wiring. FESEM and TEM were used to characterize the morphology and the modification of CP by AuNPs. As compared to the bare CP (Figure 1A), the AuNPs modified CP was densely decorated by AuNPs (Figure 1B), which exhibited uniform structures (Figure 1C) with an average diameter of about 12 nm, evidently meeting the requirements for proper substrate electrode construction. Meanwhile, AuNPs also acted as the scaffolds for the enzyme and aptamer immobilization through a condensation reaction between the carboxyl groups on the AuNPs and the terminal amino groups on the lysine residues of GOx and NH2-DNA. In addition, EIS was performed to examine the conductivity of the AuNPs modified CP electrode. The charge-transfer resistance (Rct) of the [Fe(CN)6]3−/4− probe at AuNPs modified CP electrode decreased to only about 40 Ω (curve b in Figure 1D) compared to Rct of 2300 Ω for the bare CP electrode (Figure 1D, curve a) because of the excellent electron transfer ability of AuNPs, which would remarkably enhance the electron transport of the enzyme biocatalysis.29,30

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Figure 1. FESEM of (A) the bare CP and (B) the AuNPs modified CP. (C) TEM of AuNPs. (D) EIS of the bare CP electrode (a), AuNPs/CP electrode (b), aptamer/GOx/AuNPs modified CP electrode (c), DNA bioconjugate/aptamer/GOx/AuNPs/CP-functionalized bioanode before (d) and after (e) incubation with 1 nM AMP. Insets are the enlarged views of curve b and c (top), and the Randles equivalent circuit used to fit the EIS data (below).

Characterization of DNA Bioconjugate. The elaborate construction of the DNA bioconjugate, i.e. SiO2@AuNPs-csDNA, is the core components of the self-powered aptasensor. With their large surface for DNA immobilization and as a non-conductive material, SiO2@AuNPs was adopted as the obstacle to prevent glucose from approaching the active sites of GOx. The steric hindrance of the obtained bioconjugate would greatly reduce the biocatalytic oxidation of glucose and magnified the response signal for ultrasensitive aptasensing. Firstly, the morphology of SiO2 and SiO2@AuNPs were characterized by TEM. SiO2 exhibited a well-defined and highly uniform structure (Figure 2A), and multiple homogeneous AuNPs were decorated on the surface of SiO2 (Figure 2B), denoted as 11



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SiO2@AuNPs, laying the foundation of the csDNA assembly through Au-S bond. Meanwhile, the assmebly of csDNA to the surface of SiO2@AuNPs was characterized by the average hydrodynamic sizes. As shown in Figure 2C, the hydrodynamic size of SiO2@AuNPs from DLS analysis was 516±8 nm, while it increased to 638±10 nm for SiO2@AuNPs-csDNA, confirming the successful assembly of DNA on SiO2@AuNPs to form the DNA bioconjugate.

Figure 2. TEM of SiO2 (A), and SiO2@AuNPs (B); (C) Average hydrodynamic size characterization of SiO2@AuNPs and SiO2@AuNPs-csDNA by DLS

Characterization of Bioanode and Biocathode. The assembly process of the DNA bioconjugate-functionalized aptamer/GOx/AuNPs bioanode was firstly monitored by EIS. Compared to the AuNPs modified CP electrode (Figure 1B, curve b), the Rct significantly increased after GOx and NH2-DNA modification (Figure 1B, curve c), probably owing to the inert blocking layer formed by the biomolecules that hindered the electron transfer. After assembling SiO2@AuNPs-csDNA bioconjugate to the aptamer/GOx/AuNPs electrode, its steric hindrance effects further obstructed the approaching of

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the [Fe(CN)6]3−/4− probe to the bioanode surface, thus the Rct significantly increased to 1500 Ω (curve d in Figure 1B). The subsequent recognition of AMP by the aptamer caused DNA bioconjugate to liberate from the bioanode, and thus the accessibility of the redox probe to the conductive electrode interface resulted in a reduced Rct (curve e). In addition, the aforementioned modification steps were also characterized by FESEM. Compared to the AuNPs modified CP electrode (Figure 1B), a uniform biofilm formed on aptamer/GOx/AuNPs/CP electrode (Figure 3A) owing to the functionalization of NH2-DNA and GOx. What’s more, the uniform and dense coverage of SiO2@AuNPs-csDNA biocaonjugates on the aptamer/GOx/AuNPs/CP electrode (Figure 3B) not only indicated the efficient combination between DNA bioconjugates and the aptamer, but also demonstrated the feasibility of preventing mass transport of the glucose molecules to the electrode. More significantly, once the bioanode was incubated with 100 nM AMP, most of DNA bioconjugates were dissociated from the bioanode (Figure 3C) because of the effective recognition of the target AMP. Therefore, both the EIS results and the FESEM images clearly revealed the successful assembly of the self-powered bioanode.

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. Figure 3. FESEM of aptamer/GOx/AuNPs modified CP electrode (A), DNA bioconjugate/aptamer/ GOx/AuNPs/CP-functionalized bioanode before (B) and after (C) incubation with 100 nM AMP. Insert in (B) was the enlarged view of the bioanode. (D) FESEM of laccase/PDA/AuNPs biocathode. Cyclic voltammetry (CV) was also performed to examine the bioelectrocatalysis behaviour of the bioanode and the capture of the target antibiotics (Figure 4A). Compared to the control electrode, i.e. the DNA bioconjugate/aptamer/AuNPs/CP electrode, on which no redox peaks were observed whether glucose was present or not (Figure S1 in Supporting Information), the SiO2@AuNPs-csDNA/aptamer/ GOx/AuNPs/CP bioanode gave a pair of redox peaks at about −0.5 V in the absence of glucose (Figure 4A, curve a), attributed to the characteristic peak of GOx. While in the presence of glucose, there was almost no change for the anodic current (Figure 4A, curve b) because of the blocking of the mass transport of glucose by the DNA bioconjugate SiO2@AuNPs-csDNA. Once the target was recognized and captured, the anodic current remarkably increased as expected (Figure 4A, curve c), suggesting that the dissociation of the DNA bioconjugate from the bioanode enhanced the mass transport of glucose, and thus was beneficial to the biocatalysis oxidation of glucose by GOx. Therefore, both Rct and CV signal 14


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changes proved that it was the binding of AMP that released the DNA bioconjugate from the electrode surface. This target-induced signal response laid a solid foundation for AMP selective detection and quantification,

and

confirmed

the

feasibility

for

aptasensing

based

on

the

DNA

bioconjugate-functionalized bioanode. Meanwhile, as the electron acceptor in EBFCs, the biocathode was also a highly determinative factor for the performance of the aptasensor. Laccase, one of the most commonly used cathodic enzyme, was selected for catalyzing oxygen reduction. In this study, PDA was employed as an electron mediator of laccase for bioelectrocatalytic reduction of oxygen.26 The fabrication process was also characterized by FESEM. After the modification by laccase, the surface of the laccase/PDA/AuNPs/CP biocathode (Figure 3D) became rougher than that of the PDA/AuNPs/CP electrode (Figure S2 in Supporting Information), demonstrating the successful modification of the biocathode by laccase. Moreover, as shown in Figure 4B, a pair of redox peaks emerged at about 0.4 V (curve a), owing to the excellent electrochemical activity of PDA. Compared with that in deoxygenated electrolyte solution, in the air-saturated solution, the cathodic current remarkably increased with the assistance of the electron mediator PDA (curve b), demonstrating that the laccase/PDA/AuNPs biocathode possessed highly efficient catalytic activity for oxygen reduction and provided the prerequisite for fabricating high-performance EBFCs and ultrasensitive self-powered sensors. Furthermore, no crossover reactions occurred between the bioanode and the biocathode, making them suitable for the construction of the membrane-less EBFCs.

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Figure 4. (A) CVs of the DNA bioconjugate SiO2@AuNPs-csDNA/aptamer/GOx/AuNPs bioanode in PB (pH 7.4) without glucose (a) and with 5 mM glucose (b), and DNA bioconjugate SiO2@AuNPs-csDNA/aptamer/GOx/AuNPs bioanode after incubation with 1nM AMP in PB (pH 7.4) with 5 mM glucose (c). (B) CVs of laccase/PDA/AuNPs biocathode saturated with N2 (a) and air (b) in PB (pH 7.4).

Self-Powered Aptasensing of Antibiotic AMP. By coupling the DNA bioconjugate SiO2@AuNPs-csDNA-functionalized bioanode and the laccase/PDA/AuNPs biocathode, a self-powered aptasensor based on the one-compartment glucose/O2 EBFCs was constructed. As shown in Figure 5, in the absence of AMP, the EOCV of the assembled EBFCs-based aptasensor was about 0.20 V (curve a, Figure 5A). With the increase of the target AMP concentration, the EOCV of the aptasensor gradually increased (curve b-f, Figure 5A), due to the gradual dissociation of DNA bioconjugate SiO2@AuNPs-csDNA from the bioanode. The calibration curve was fitted between EOCV and the logarithm of the AMP concentration over a linear range of 10 pM to 100 nM. The linear equation was determined to be EOCV = 0.227 + 0.025logcAMP (correlation coefficient of R2 = 0.9959), with the limit of detection of 3 pM (based on S/N=3), which is superior to the MRL of official standard issued by Food and Drug Administration of the United States of America (10 µg/L or about 28 nM ) 31 and China (50 µg/kg or about 140 nM).32 It is also worth noting that, as shown in Table S1, the as-proposed 16


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self-powered aptasensing method showed outstanding analytical performance, with a limit of detection and a linear range superior or comparable to those of the methods for common antibiotics detection reported in literature.

Figure 5. (A) The Eocv of the aptasensor incubated with AMP with different concentrations: 0, 10 pM, 100 pM, 1 nM, 10 nM and 100 nM (curve a-f). (B) The plot of Eocv vs. the logarithm of the AMP concentration. Error bars represent the standard deviation of an average value from independent measurements of three aptasensors.

Specificity and Stability of AMP Assay. The specificity and stability for the aptasensor are also key features that determines its performance for analysing target analyte in real samples. In this case, six other antibiotics, namely tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), amoxicillin, lincomycin, and tiamulin, and two food additives (citric acid and phenylalanine) at 10 times concentration of AMP were selected as the negative controls to evaluate the selectivity of the aptasensor. As depicted in Figure 6, only in the presence of the target AMP, the EOCV of the aptasensor showed an elevated value. While the EOCV of the aptasensor was comparable to that of Blank when AMP was substituted by the predominant amount of the interfering substances. It was apparent that the interference was neglectable for the detection of target AMP due to the high binding specificity of the aptamer,

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demonstrating that the proposed aptasensor possessed excellent selectivity for discriminating target from complex samples. To characterize the stability of the self-powered aptasensor, six aptasensors were independently fabricated, and the EOCV response of each was measured intermittently after storage at 4 °C for certain periods of time. It was found that the relative standard deviation (RSD) of the current response was only 4.87%. The EOCV signals barely changed after storage for 10 days, and upon storage for 20 days, the average EOCV signal still remained 91.3% of the initial value, indicating that the as-proposed aptasensor had a good stability for bioassay.

Figure 6. Comparison of EOCV of the aptasensor in the presence of 1nM AMP or 10 nM interfering substances, in which “Blank” indicates the condition in the absence of both the target and the interfering substances. The error bars represent the standard deviation obtained from the measurements performed on three aptasensors.

AMP Detection in Real Samples. In order to evaluate its applicability in real sample analysis, our method was employed to determine AMP residues in milk. The assays were performed by means of standard addition. Due to the anti-interference ability and the excellent selectivity of the aptasensor, the

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milk samples did not need any special pretreatment and were only diluted to 5% of the original concentration. As shown in Table 1, the bioassay results obtained by the as-proposed method were consistent with those obtained by the standard LC-MS-MS method stated in the National Measurement Standards of People's Republic of China (GB/T 22975-2008) with AMP concentration equal to or higher than 2.00 nM, and more importantly our method exhibited better sensitivity, being able to detect AMP lower than 2.00 nM, which, however, could not be achieved by the standard method. Besides, the RSDs of 3.54%–5.26% and the recoveries of 95.0%–106.0% were achieved. Thus, this method shows great potential for practical use in determination of AMP residues in real samples. Moreover, to extend the practical application of the as-proposed aptasensor, it is necessary to further improve its analytical performance, including higher output performance for better sensitivity, miniaturization of the device for portable and/or on-site assay, and better stability of the EBFC, and researches along these lines are conducted in our laboratory.

Table 1. Measurement of AMP added to the milk samples by our method and the standard LC-MS-MS method. AMP Concentration (nM) Sample No.

Added

Standard Method

Proposed Method (n = 6)

RSD (%)

Recovery (%)

1

0.020

Not detected

0.019 ± 0.001

5.26

95.0

2

0.20

Not detected

0.21 ± 0.01

4.76

105.0

3

2.00

2.08

2.12 ± 0.09

4.24

106.0

4

20.00

20.03

19.51 ± 0.69

3.54

97.6

CONCLUTIONS In summary, we have constructed a novel ultrasensitive self-powered aptasensing platform based on the one-compartment EBFCs for determining the antibiotic residue in milk. DNA bioconjugate was elaborately designed and used as an amplification means for altering the biocatalytical activity of the 19



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bioanode, and thus ensured the high sensitivity of the AMP aptasensor, which was superior to other approaches reported previously. In addition, this proposed aptasensor was the first reported self-powered aptasensing platform for antibiotic determination and featured with excellent selectivity owing to the high specificity of the aptamer. Furthermore, no need for external power sources endows the aptasensor with simple construction, rendering it the ability of real-time and on-site bioassay. Therefore, the proposed EBFCs-based self-powered aptasensor has a great promise to be applied as a powerful tool for on-site assay in the field of food safety.

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ASSOCIATED CONTENT Supporting Information Additional information as noted in the text. The control experiments of bioanode; FESEM image of PDA/AuNPs/CP electrode; Comparison of analytical performance for antibiotics assay by our method and those reported in literature.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (F. Li) Tel/Fax: (86) 532-86080855 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We gratefully appreciate the financial support from the National Natural Science Foundation of China (21605092, 21675095 and 21575074), A Project of Shandong Province Higher Educational Science and Technology Program (J16LC07), State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1509), and the Special Foundation for Taishan Scholar of Shandong Province (No. ts201511052).

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For TOC only

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Ultrasensitive Self-Powered Aptasensor Based on Enzyme Biofuel

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