Enzymatic Fuel Cell-Based Self-Powered Homogeneous

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Enzymatic Fuel Cell-Based Self-Powered Homogeneous Immunosensing Platform via Target-Induced Glucose Release: An Appealing Alternative Strategy for Turn-On Melamine Assay Chengcheng Gu, Panpan Gai, Ting Hou, Haiyin Li, Changhui Xue, and Feng Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07104 • Publication Date (Web): 26 Sep 2017 Downloaded from http://pubs.acs.org on September 26, 2017

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ACS Applied Materials & Interfaces

Enzymatic Fuel Cell-Based Self-Powered Homogeneous Immunosensing Platform via Target-Induced Glucose Release: An Appealing Alternative Strategy for Turn-On Melamine Assay

Chengcheng Gu, Panpan Gai*, Ting Hou, Haiyin Li, Changhui Xue and Feng Li*

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

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

E-mails: [email protected] (F. Li); [email protected] (P. P. Gai)

1

ACS Paragon Plus Environment


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ABSTRACT Enzymatic fuel cell (EFC)-based self-powered biosensors have attracted considerable attention because of their unique feature of no need for extra power sources during the entire detection process, which endows them the merits of simplicity, rapidness, low cost, anti-interference and ease of use. Herein, we proposed, for the first time, an EFC-based self-powered homogeneous immunosensing platform by integrating the target-induced biofuel release and bioconjugate immunoassay for ultrasensitive melamine (ME) detection. In this design, the biofuel, i.e. glucose molecules, was entrapped in the pores of positively charged mesoporous silica nanoparticles (PMSN) and capped by the bio-gate AuNPs-labeled anti-ME antibody (AuNPs-Ab). The presence of the target ME triggered the entrapped glucose release due to the removal of the bio-gate via immunoreaction, which resulted in the transfer of electrons produced by glucose oxidation at the bioanode to the biocathode, and thus, the open circuit voltage of EFC-based self-powered immunosensor dramatically increased, realizing the ultrasensitive turn-on assay for ME. The limit of detection (LOD) for ME assay was down to 2.1 pM (S/N = 3), superior to those previously reported in literature. Notably, real milk samples need no special sample pretreatment for the detection of ME because of the good anti-interference ability of EFC-based self-powered biosensors and the excellent selectivity of the homogeneous immunoassay. Therefore, this appealing self-powered homogenous immunosensing platform holds great promise as a successful prototype of portable and on-site bioassay in the field of food safety.

KEYWORDS Enzymatic fuel cell; Self-powered homogenous immunosensing platform; Melamine detection; Target-induced glucose release; Turn-on assay

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INTRODUCTION Enzymatic fuel cells (EFCs) have aroused considerable interest for green energy-conversion due to their ability of harvesting electricity from renewable biofuels under mild working conditions.1-11 Recently, EFC-based self-powered biosensors, whose performance output is dependent on the concentration of target analyte, are novel biosensing platform ascribing to the unique advantages of no need for external power sources,

simple

instrumentation,

remarkable

anti-interference

performance,

low

cost

and

miniaturization12-13. As the robust alternatives for on-site assay, they have been successfully applied in molecular diagnostics,14-19 catalyst screening,20-21 immunoassays,22-25 cytosensing26-27 and drug release.28 Homogeneous immunoassays, which take place in solution and require neither steps of separating antigen or antibody from the samples nor washing, could endow the bioassay with simplicity, rapid response, and ease of miniaturization.29-30 Homogeneous electrochemical immunoassays have demonstrated to be a promising biosensing strategy due to their additional merits of excellent sensitivity, cost-effectiveness, and ease for use 31-32 Thus, combining the advantages of homogeneous electrochemical immunoassays and EFC-based self-powered biosensors will well meet the urgent demands for ultrasensitive, rapid, accurate and on-site detection. As we all know, melamine (ME) is commonly applied for manufacturing melamine formaldehyde resin and in plastic engineering.33-34 Unfortunately, the abundant amount of nonprotein nitrogen in ME has led to its illegal use in infant formula and milk products to obtain a false increase in protein content. Moreover, chronic consumption of products polluted by ME could induce kidney failure and even death, especially in babies and children with poor resistance to diseases.35 Therefore, it is imperative to develop a reliable and accurate system for detecting ME in ME-polluted products. To date, a variety of analytical methods have 3



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been used for assaying ME, including enzyme-linked immunosorbent assay (ELISA),36-37 surface enhanced Raman spectroscopy (SERS),38-39 gas chromatography/mass spectrometry (GC/MS),40-41 electrochemical methods33, 42-47 and colorimetric sensing strategies.48-49 However, most of the above methods suffered from the drawbacks of expensive experimental instrumentation, time-consuming operation, low sensitivity and/or complicated sample treatment. Thereby, there is an urgent demand for the development of simple, rapid, low-cost, sensitive, and reliable methods suitable for on-site assay of ME. Herein, we proposed, for the first time, an EFC-based self-powered homogeneous immunosensing platform for ultrasensitive detection of ME by combining the target-induced biofuel release and bioconjugate immunoassay. To construct this assay protocol, the biofuel, i.e. glucose molecules, was entrapped in the pores of positively charged mesoporous silica nanoparticles (PMSN) and capped by the AuNPs-labeled anti-ME antibody (AuNPs-mAb, as the bio-gate) with negative charges through the electrostatic attraction. As shown in Scheme 1, the one-compartment EFC-based self-powered immunosensor, composed of the GOD/CNT/AuNPs bioanode and the laccase/PDA/CNT/AuNPs biocathode, was assembled for detecting ME. In the absence of target ME, glucose could not approach the surface of the bioanode because it was encapsulated in the pores of PMSN, and the EFC only produced a low open circuit voltage (EOCV). Once the target ME was captured, the competitive displacement reaction occurred. Due to the fact that the specific antigen-antibody interaction between the target ME and AuNPs-mAb was superior to the electrostatic attraction between PMSN and AuNPs-mAb, AuNPs-mAb was liberated from the PMSN to bind to ME, and thus the bio-gate AuNPs-mAb was opened, releasing glucose originally entrapped in PMSN. Subsequently, the electrons generated by glucose oxidation would transfer to the biocathode, and the EOCV of the EFC-based self-powered immunosensor dramatically 4


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increased. As a consequence, ultrasensitive detection of ME was successfully realized through integrating target-responsive controlled release of the biofuel and the high biocatalytic ability of the biocathode on oxygen reduction. This appealing immunosensing protocol not only possesses the advantages of simplicity, rapidness, reliability and ultra-high sensitivity for bioassays, but also provides a successful prototype for on-site detection in food safety field.

Scheme 1. Schematic illustration of the principle of the glucose controlled self-powered homogeneous immunosensing strategy for ME detection.

EXPERIMENTAL SECTION Materials and Reagents. Mouse anti-melamine (1B12) antibody was purchased from Beijing Bioss Biotechnology Co. Ltd. (Beijing, China). Glucose oxidase (GOD) from Aspergillus niger (EC 1.1.3.4, 158.9

units

mg-1),

laccase

from

Trametes

versicolor

(EC

1.10.3.2,

1.07

units

mg-1),

cetyltrimethylammonium bromide (CTAB), tetraethoxysilane (TEOS), 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) melamine, urea, L-cysteine (L-cys), 5



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ascorbic acid (AA), bovine serum albumin (BSA), uric acid, lysozyme, lactose, lactate and poly (diallyldimethylammonium chloride) (PDDA, 20%, w/w in water, MW of 200 000~350 000) were all purchased from Sigma-Aldrich (St. Louis, U.S.A.). Carbon nanotubes (CNT) were obtained from Nanjing Jicang Nano Tech Co., Ltd (Nanjing, China). Carbon paper (CP) was purchased from Shanghai Hesen Electric Co., Ltd (Shanghai, China). β-D-Glucose was obtained from Tokyo Chemical Industry Co. Ltd. (Japan). Chloroauric acid (HAuCl4·4H2O) and tris(hydroxymethyl)aminomethane (Tris) was obtained from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). All other reagents were of analytical grade and used without further purification. AuNPs were prepared according to the literature by adding a sodium citrate solution to a boiling HAuCl4 solution.50 Ultrapure water (resistivity > 18.2 MΩ cm at 25 °C) obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA, U.S.A.) was used for all the experiments. A Tris-HCl buffer (pH 9.0, 0.01 M) was adopted for the preparation of Mouse Anti-Melamine (1B12) stock solutions. Phosphate buffer (PB) solution (pH 7.4, 0.1 M) consisting of Na2HPO4 and NaH2PO4 was used as the supporting electrolyte.

Apparatus and Instrumentation. Scanning electron microscopy (SEM) images were measured on a JEOL 7500F 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, U.S.A.). Electrochemical impedance spectroscopy (EIS) was carried out 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 [Fe(CN)6]3−/4− used as the probe. The concentration of the free glucose was measured by portable glucose meter (ACCU-CHEK®Performa). Cyclic voltammetric measurement was performed on a CHI 660E electrochemical workstation (Shanghai CH Instrument Co., 6


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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 EFC was measured by connecting the bioanode and the biocathode placed in the electrolytic cell. All experiments were carried out at room temperature (25 ± 1 °C).

Synthesis of PDDA-Modified MSN. Firstly, MSN was prepared according to our previous publication with slight modifications.51 Typically, 250 mg of CTAB and 875 µL of sodium hydroxide (2.0 M) were successively added into 120 mL of ultrapure water under stirring and kept stirring for 20 min at 80 °C. Subsequently, 1.25 mL of TEOS was injected dropwise into the above solution followed by vigorous stirring for 2 h to obtain white precipitates. Then the obtained product was filtered, washed with ultrapure water and methanol, and dried in air. Next, the excessive CTAB was removed by refluxing the precipitates in a mixture consisting of HCl and methanol for 10 h. The obtained MSN was filtered, washed with ultrapure water and methanol, and then dried at 60 °C. Next, PDDA-modified MSN was prepared as follows: 16 mg of the as-synthesized MSN was dispersed into 8.0 mL of 1% PDDA salt solution (0.02 M NaCl), and then ultrasonicaed for 30 min to form a homogeneous suspension of positively charged PDDA-modified MSN. Afterwards, the mixture was centrifuged (15 000 rpm, 10 min), washed with ultrapure water, and dried to obtain the positively charged PDDA-modified MSN (PMSN).

Preparation of Antibody Bioconjugate PMSN@AuNPs-mAb. Firstly, 20 µL of 40 µg mL-1 anti-ME antibody solution was added into 1.0 mL of AuNPs (10 nM) and incubated at 4 °C for 12 h to obtain the AuNPs-labeled anti-ME antibody (AuNPs-mAb) through Au-N bond. Meanwhile, 10 mg of PMSN was suspended into 1.0 mL of glucose solution (1.0 M), and the suspension was gently shaken 7



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overnight at room temperature to make glucose molecules diffuse into the pores of the PMSN. Subsequently, the obtained AuNPs-mAb was mixed with the suspension and incubated at room temperature for 4 h under gentle stirring, and as a result, AuNPs-mAb was adsorbed onto the surface of PMSN and capped on the pores through electrostatic interaction. Following that, surplus AuNPs-mAb and unloaded glucose were separated by centrifugation at 3 000 rpm for 2.0 min. Finally, the collected PMSN@AuNPs-mAb was dispersed into 1.0 mL of 0.01 M Tris-HCl buffer (pH 9.0), and stored at 4 °C for further use (The mAb is a kind of protein, whose isoelectric point is about 8.0. In pH 9.0 tris-HCl, the as-prepared AuNP-mAb is negatively charged).

Preparation and Electrochemical Characterization of GOD/CNT/AuNPs Bioanode. 8.0 mg of CNT was dispersed in 4.0 mL of 1% PDDA salt solution (0.02 M NaCl) followed by sonication for 30 min, resulting in a homogeneous suspension of positively charged CNT/PDDA. Residual PDDA was extracted by centrifugation (15 000 rpm, 10 min), and the isolated precipitate was washed with ultrapure water at least three times. Subsequently, the purified CNT/PDDA was mixed with 3.0 mL of AuNPs (10 nM) under gentle shaking overnight at room temperature. Then excessive AuNPs were removed by centrifugation (8 000 rpm, 10 min), and the CNT/AuNPs hybrid precipitate was resuspended in ultrapure water to bring the concentration to 1 mg mL-1. Following that, 50 µL of the as-prepared CNT/AuNPs hybrid suspension was spread on the surface of the CP 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 1 mg mL-1 EDC and 1 mg mL-1 NHS for 30 min to activate the carboxyl group of AuNPs. Afterwards, the activated electrode was rinsed with ultrapure water to eliminate excess EDC and NHS, and then incubated with 50 µL GOD solution (20 mg mL-1) at 4 °C for 12 h to obtain the bioanode of the EFC. The electrode was stored at 4 °C when not in 8


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use. As for the blank control experiment, i.e. in the absence of the target ME, 10 µL of PMSN@AuNPs-mAb was added to 5 mL of PB (pH 7.4, 0.1 M), CV experiments with the potential window ranging from -0.8 to 0 V (vs. Ag/AgCl) were measured. Then, 40 µL of the target ME with different concentrations was incubated with 10 µL of PMSN@AuNPs-mAb overnight at 4 °C before the electrochemical measurements. Subsequently, the above mixture was added to 5 mL of PB (pH 7.4, 0.1 M) and the CV experiments were conducted again. Preparation of Laccase/PDA/CNT/AuNPs Biocathode. 50 µL of the as-prepared CNT/AuNPs hybrid suspension was spread on the surface of the CP electrode and dried at 37 °C for 2 h. Subsequently, the substrate electrode was immersed into 6 mM dopamine solution (0.1 M PB, pH 8.5) under stirring for 3 h. After that, the activated electrode was rinsed with ultrapure water to eliminate excess dopamine and then incubated with 50 µL of laccase solution (30 mg mL-1) in PB (pH 7.4) at 4 °C for 12 h to obtain the laccase/PDA/CNT/AuNPs modified biocathode.

Fabrication of EFC-Based Self-Powered Immunosensor and EOCV Measurement. A membrane-less EFC-based self-powered immunosensor was assembled using the as-prepared bioanode and biocathode at room temperature. For the detection of the target ME, 40 µL of the target ME with different concentrations were incubated with 10 µL of PMSN@AuNPs-mAb overnight at 4 °C, respectively. Then the EOCV of the EFC was measured by adding the resulting mixture to the supporting electrolyte (0.1 M PB, pH 7.4).

RESULTS AND DISCUSSION Assembly and Characterization of Bioanode and Biocathode. The efficient electrical communication between the enzyme and the substrate electrode played an important role in the 9



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performance of the as-constructed EFCs. CNT/AuNPs, featured with high electrical conductivity, good biocompatibility, ease of functionalization, and high surface-area-to-volume ratio, have been widely applied to transfer electrons. In this case, CNT/AuNPs served not only as electrical wires to facilitate the electron transfer with high efficiency, but also as an immobilization support for redox enzymes. As shown in Figure 1A, the enzyme molecules could be bonded to the substrate electrode through a condensation reaction between the amino groups in enzymes and the carboxyl groups on the Au NPs. Herein, EIS was employed to monitor the assembling process of the bioanode and the biocathode. In comparison with the charge-transfer resistance (Rct) value of the bare CP electrode (Figure S1A), the CNT/AuNPs modified CP electrode showed much smaller Rct value (45 Ω, curve a in Figure 1B), indicating that the CNT/AuNPs had excellent electron transfer ability and could form good electronic and ionic conduction pathways between the substrate electrode and the electrolyte. After the formation of PDA on the surface of the CNT/AuNPs, the Rct value barely changed (curve b in Figure 1B), suggesting that the PDA also possessed good electron transport ability and could act as the electron mediator for laccase.52 Upon the modification with GOD or laccase, respectively, the GOD/CNT/AuNPs bioanode and the laccase/PDA/CNT/AuNPs biocathode showed elevated Rct values (curve c and curve d in Figure 1B) due to the additional barrier effects of the proteins on the electron transfer of the redox probe, which demonstrated the successful modification of the bioanode with GOD and the biocathode with laccase. In addition, the morphology of the different electrodes was characterized by SEM and TEM. As shown in Figure 1C and S1B, CNT/AuNPs were distributed evenly on the CP surface, and the insets in Figure 1C also showed that the CNT/AuNPs exhibited a well-defined structure and were decorated with uniform gold nanoparticles. The uniform distribution of Au NPs on CNTs favored the bioenzyme immobilization at their optimal positions on the 10


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electrodes, which further promoted the electrical communication between the enzyme and the electrode.53 The appearance of PDA nanoparticles suggested the successful polymerization of DA on the surface of CNT/AuNPs (Figure 1D). After the modification with the enzymes, the GOD/CNT/AuNPs/CP bioanode (Figure 1E) and the laccase/PDA/CNT/AuNPs/CP biocathode (Figure 1F) became much rougher than that of the substrate electrodes, demonstrating the effective assembly of the enzymes on the substrate electrodes.

Figure 1. (A) Schematic illustration of GOD/CNT/AuNPs modified CP bioanode and laccase/CNT/AuNPs modified CP biocathode. (B) EIS of (a) CNT/AuNPs/CP electrode, (b) PDA/CNT/AuNPs/CP electrode, (c) GOD/CNT/AuNPs/CP bioanode, and (d) laccase/PDA/CNT/AuNPs/CP biocathode. (C) SEM of CNT/AuNPs/CP electrode; insets in (C) were the enlarged views of the CNT/AuNPs/CP electrode and TEM of the CNT/AuNPs. (D) SEM of the PDA/CNT/AuNPs/CP electrode; inset in (D) was the enlarged view of the PDA/CNT/AuNPs/CP electrode. (E) SEM of the GOD/CNT/AuNPs/CP bioanode, and (F) SEM of the laccase/PDA/CNT/AuNPs/CP biocathode.

Characterization of PMSN@AuNPs-mAb Bioconjugate. The elaborate design of the antibody bioconjugate, i.e. PMSN@AuNPs-mAb, is the core component of the EFC-based self-powered immunosensor. To construct the PMSN@AuNPs-mAb bioconjugate through the electrostatic attraction, the 11



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positively charged PDDA-modified MSN and the negatively charged AuNPs-mAb were fabricated, respectively, whose surface charges were characterized by zeta potential. As shown in Figure 2A, the attachment of the positively charged PDDA on the MSN changed the zeta potential from -5.36 V, (column a) to +42.48 V (column b). Furthermore, the as-prepared AuNPs-mAb had a more negative zeta potential (-54.72 mV, column d) than that of the carboxyl modified AuNPs (-40.78 mV, column c) because the isoelectric point of the ME antibody was lower than pH 9.0 to afford a negatively charged species. Furthermore, to verify the adsorption of the negatively charged AuNPs-mAb onto the PMSN via the electrostatic interaction, the morphologies of PMSN and PMSN@AuNPs-mAb bioconjugate were characterized by TEM. Figure 2B and 2C show that plenty of AuNPs-mAbs were adsorbed on the surface of PMSN, which suggested that the AuNPs-mAbs could serve as the effective bio-gate via electrostatic adsorption to PMSN. In this case, the biofuel, i.e. glucose molecules, could be successfully entrapped in the pores of PMSN and capped by the AuNPs-mAb. In addition, DLS was also used to investigate the size distribution of PMSN and PMSN@AuNPs-mAb bioconjugate, and further to confirm the construction of the bioconjugate. As shown in Figure 2D, the hydrodynamic size of PMSN@AuNPs-mAb bioconjugate (166 nm, a) was evidently bigger than that of PMSN (102 nm, b), proving that the successful adsorption of AuNPs-mAb to the surface of PMSN. What’s more, the N2 adsorption-desorption isotherm in Figure 2E indicated that MSNs were porous with an average pore diameter of 3.4 nm, whereas, the dimension of a glucose molecule was 0.432 nm,54 which suggested that glucose molecules could be easily entrapped in the pores of PMSN. The encapsulated glucose was estimated to be about 33 mM, which was calculated by detecting the unloaded glucose concentration using a portable glucose meter. Furthermore, the dimension of AuNPs-mAb was also investigated by the DLS. As shown in Figure S2, the hydrodynamic size of the 12


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bare AuNPs was 15.7 nm (Figure S2a), while it increased to 26.3 nm for AuNPs-mAb (Figure S2b), confirming the successful assembly of AuNPs-mAb. The aforementioned results suggested that the entrapped glucose molecules in the pores of PMSN would firmly be capped by the bio-gate AuNPs-mAb. Brunauer–Emmett–Teller

(BET)

analyses

of

MSN,

PMSN,

PMSN-loaded

glucose

and

PMSN@AuNPs-mAb were carried out to further demonstrate the assembly and the encapsulation process. As shown in Figure 2F, the specific surface areas of MSN, PMSN, PMSN-loaded glucose and PMSN@AuNPs-mAb were 685 m2 g-1, 631 m2 g-1, 259 m2 g-1 and 224 m2 g-1, respectively. These results manifested that the mesopores of MSNs were filled with plenty of glucose molecules and then blocked by AuNPs-mAb successfully. As a consequence, PMSN@AuNPs-mAb bioconjugate was successfully assembled, which lays a solid foundation for the construction of the EFC-based self-powered immunosensor.

13



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Figure 2. (A) Zeta potentials of (a) MSN, (b) PMSN, (c) AuNPs, and (d) AuNPs-mAb. TEM images of (B) PMSN and (C) PMSN@AuNPs-mAb. (D) Average hydrodynamic size characterization of PMSN (a) and PMSN@AuNPs-mAb (b) by DLS. (E) Pore size distribution of MSNs. (F) N2 adsorption-desorption isotherm of (a) MSN, (b) PMSNs, (c) glucose-loaded PMSN, (d) PMSN@AuNPs-mAb.

Optimization for the Assembly of PMSN@AuNPs-mAb Bioconjugate. To achieve an optimal response signal of the EFC-based self-powered homogeneous immunoassay, the influence of different assembly conditions of the PMSN@AuNPs-mAb bioconjugate, including the amount of glucose and AuNPs-mAb, and the time of immunoreaction and release process, were investigated. The optimal experimental conditions were evaluated using the extent of the shift upwards at -0.6 V in the voltammograms (caused by the increase of glucose concentration) as the judging criteria.55-58 Firstly, the concentration of glucose incubated with PMSN was studied since the response signal comes from the glucose oxidation. As shown in Figure S3A, the bioelectrocatalysis current increased with the increment glucose concentration and leveled off when it was higher than 1.0 M. Thus, 1.0 M was used as the optimal glucose concentration for PMSN@AuNPs-mAb bioconjugate. Next, we also investigated the effect of the amount of AuNPs-mAb adsorbed on the surface of PMSN, which would greatly affect the sensitivity of the bioassay. As shown in Figure S3B, when the amount of AuNPs-mAb increased from 0.16 µg to 0.8 µg in the reaction system, the bioelectrocatalysis current increased, which suggested that higher amount of capping units would sealed more glucose molecules. But the bioelectrocatalysis current barely changed in the range of 0.8 µg to 1.2 µg, which attributed that the adsorption quantity of capping units reached saturation. Hence, 0.8 µg of AuNPs-mAb was selected in the PMSN@AuNPs-mAb bioconjugate reaction system. In addition, the influence of the immunoreaction time was studied, which was also the glucose 14


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release time, because glucose would be released as soon as ME was captured by the AuNPs-mAb. Figure S3C displays that the bioelectrocatalysis current increased with the longer reaction time up to 2 h and barely changed after 2 h. Therefore, 2 h was used for the immunoreaction and release time.

Characterization of Bioanode and Biocathode. Cyclic voltammetry (CV) was performed to investigate the bioelectrocatalysis behaviour of the GOD/CNT/AuNPs bioanode, and further to confirm the results of target-induced glucose release. In the absence of glucose (curve a in Figure 3A), as compared to the CNT/AuNPs electrode (Figure S4), the GOD/CNT/AuNPs bioanode showed a pair of well-defined redox peaks of GOD at about -0.5 V, which attributed to the characteristic peak of the enzyme cofactor flavin adenine dinucleotide (FAD) dissociated from the GOD.55, 59 With the addition of the glucose, the GOD

consumption of O2 (glucose + O2 →gluconolactone + H2O2) resulted in the decrease of the reductive current (Figure S5), which was in accordance with the reported references.55-59 The results confirmed that a portion of active GOD still remained at the electrode surface and the O2-mediated glucose oxidation occurred.55, 59 In the absence of ME, with PMSN@AuNPs-mAb being added, there was almost no change for the anodic current (Figure 3A, curve b) because the glucose molecules were entrapped in the pores of PMSN by the AuNPs-mAb. To further demonstrate that the glucose was firmly entrapped into the pores of PMSN with an acceptable shelf-time, CVs were performed at the bioanode in the presence of PMSN@AuNPs-mAb but in the absence of ME during a period of 10 days. As expected, the anodic current barely changed (Figure S6), which suggested that the glucose was firmly blocked into the pores of MSN and the oxidation reaction could not occur without free glucose. Once the target ME was captured, the O2-mediated glucose oxidation by the GOD occurred (Figure 3A, curve c),55-58 indicating that the bio-gate AuNPs-mAb was liberated from the surface of PMSN and resulted in glucose release from the pores of PMSN. This target-induced signal response played a key role in selective ME detection and quantification. In addition, the biocathode as the electron acceptor also had a significant influence on the performance of the immunosensor. In this work, laccase was employed as the cathodic enzyme for biocatalyzing oxygen reduction and PDA as the electron mediator to achieve indirect electron transfer between laccase and the surface of electrode. As indicated in Figure 3B, the biocathode gave a pair of redox peaks at about 0.4 V in

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the absence of O2 (curve a), which was caused by the electrochemical activity of PDA.52 In contrast, the cathodic current increased in the air-saturated solution (curve b) and further enhanced in the O2-saturated solution (curve c), indicating the laccase/PDA/CNT/AuNPs biocathode could efficiently catalyze the O2 reduction. To make it more suitable for practical applications, air-saturated solution was selected in our work. Furthermore, no crossover reactions between the bioanode and the biocathode occurred. Thus, the aforementioned results provide the precondition for constructing a high-performance EFC and highly sensitive immunosensor.

Figure 3. (A) CVs of the GOD/CNT/AuNPs bioanode in PB (pH 7.4) in the absence of glucose (a), and where bioconjugate PMSN@AuNPs-mAb before (b) and after (c) incubation with 100 nM ME. (B) CVs of the laccase/PDA/CNT/AuNPs biocathode saturated with N2 (a), air (b), and O2 (c) in PB (pH 7.4).

EFC-Based Self-Powered Immunosensor for ME Detection. Under the optimal experimental conditions, a membrane-less glucose/O2 EFC-based self-powered homogeneous immunosensing platform was fabricated by coupling the GOD/CNT/AuNPs bioanode and the laccase/PDA/CNT/AuNPs biocathode. Initially, to manifest the feasibility of the as-proposed self-powered immunosensors based on the target-induced glucose release, the variation of the EOCV based on the different “free-glucose” concentration was investigated. As expected, the EOCV gradually increased with the elevated “free glucose” concentration ranging from 1 mM to 10 mM but barely changed when it was higher than 10 mM. Furthermore, it showed a good linear relationship between the EOCV and the logarithm of the glucose concentration over a linear 16


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range of 1 mM to 10 mM (Figure S7), which suggested that the as-proposed strategy of ME-induced glucose release worked effectively. Subsequently, the maximal concentration of target-induced glucose release was also measured using a series of different ME concentration incubation with the bioconjugate PMSN@AuNPs-mAb. With the increase of the ME concentration, the amount of the released glucose increased, which was measured by the portable glucose meter. But when the ME concentration was higher than 100 nM, the released glucose concentration was barely changed, suggesting the glucose release reached maximum. And at this point, the maximal concentration of glucose released was about 5.5 mM, which was just in the linear range of strand curve in Figure S7 and further demonstrated the feasibility of the as-proposed strategy. For the detection of ME, as shown in Figure 4A, in the absence of target ME, the EOCV of the as-proposed immunosensor was about 0.21 V (curve a), and in the presence of ME, the EOCV of the immunosensor elevated with positive correlation with ME concentration (curve b-f). Furthermore, the linear curve was fitted between EOCV and the logarithm of the ME concentration over a linear range of 10 pM to 100 nM. The linear equation for ME was EOCV = 0.337 + 0.038logcME (correlation coefficient R2 = 0.9988), and a detection limit of 2.1 pM (S/N=3) was obtained, which was obviously superior to the Maximum Residue Limits (MRLs) of the official standards issued by both European Union (2.5 ppm or about 19.8 µM) and US Food and Drug Administration (FDA) (0.25 ppm or about 1.98 µM) and even much lower than that of the most sensitive method reported in literature (24 pM) (Table S1).

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Figure 4. (A) The EOCV of the as-proposed immunosensor incubated with ME with different concentrations: 0 (a), 10 pM (b), 100 pM (c), 1 nM (d), 10 nM (e) and 100 nM (f). (B) Variation of the EOCV as a function of ME concentration; inset was the linear relationship of EOCV vs. the concentration of the ME. Error bars represent the standard deviation of independent measurements of three immunosensors.

Specificity and Reproducibility of the ME Assay. In order to evaluate the specificity of the EFC-based self-powered immunosensor, possible interferences, such as, urea, L-cys, AA, BSA, uric acid, lysozyme, lactose and lactate were selected as the negative controls. As shown in Figure 5, the ∆EOCV of the immunosensor for ME assay were much bigger than that of the interferences (with the concentrations 100 times of that of ME), implying that the proposed EFC-based self-powered immunosensor had good selectivity for discriminating the target ME from other interfering substances owing to the high specific recognition of the homogenous immunoassays. Moreover, the reproducibility of the immunosensor was also investigated by repeating the measurements under the same conditions. The results showed that the relative standard deviation (RSD) was less than 6.9% for the determination of 1.0 nM ME using the six freshly fabricated immunosensors, demonstrating stable performance of the as-proposed immunosensor for ME bioassay. In addition, to further clarify the reproducibility of EOCV based on different sets of PMSN@AuNPs-mAb, the EOCV data based on five different sets of PMSN@AuNPs-mAb before and after

18


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capturing the target were detected. As shown in Figure S8, there are almost no change in the value of EOCV based on 5 different sets of PMSN@AuNPs-mAb both before and after capturing the target, respectively, further demonstrating the excellent reproducibility of the proposed immunosensors.

Figure 5. Comparison of ∆EOCV for the EFC in the presence of 1 nM ME and 100 nM interferences, respectively; ∆EOCV = EOCV- E0OCV, in which E0OCV was the blank signal of EFC. Error bars represent the standard deviation of an average value from independent measurements of three immunosensors.

ME Detection in Real Milk Samples. To verify its feasibility in practical analysis, the as-proposed immunosensor was adopted to detect ME in milk using the method of standard addition. Owing to the good anti-inference capability and high selectivity for the as-proposed immunosensor, it exhibited excellent performance even though the milk samples were only diluted to 5% of the original concentration without any pretreatment or separation. The good RSDs of 2.35%–6.12% and the recoveries of 97.0%–102.0% were achieved (see Table 1), suggesting that this method shows great promise for ME determination in real samples.

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Table 1. Measurement of ME added to the milk samples by means of standard addition. Sample No.

Added (nM)

Found (nM) (n = 6)

RSD (%)

Recovery (%)

1

0.05

0.049 ± 0.003

6.12

98.0

2

1.00

1.02 ± 0.05

4.90

102.0

3

2.00

1.94 ± 0.08

4.12

97.0

4

5.00

5.09 ± 0.12

2.35

101.8

5

10.00

10.10 ± 0.36

3.56

101.0

CONCLUSIONS In summary, we constructed a novel EFC-based self-powered homogeneous immunosensing platform for determining ME in milk via the strategy of target-induced glucose release. The as-proposed immunosensing platform not only combined the advantages of EFC-based self-powered biosensors and the homogeneous electrochemical immunoassay, but also exhibited broader linear response range and higher sensitivity for ME detection. In addition, the as-proposed EFC-based self-powered immunosensors has also been successfully applied in the detection of ME in real milk samples without any special pretreatment, which ascribed to the excellent anti-inference capability and high sensitivity. Therefore, this appealing immunosensing platform not only provides an ingenious strategy to achieve simple, rapid, reliable, and ultrasensitive ME bioassays, but also gives a successful prototype of portable and on-site detection in the field of food safety.

ASSOCIATED CONTENT Supporting Information Additional information as noted in the main text. The EIS and SEM image of the carbon paper; DLS of the AuNPs and AuNPs-mAb; A series of control experiments; Optimization of experimental conditions for bioconjugate; Comparison of analytical performance for ME assay by our method and those reported in literature. 20


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AUTHOR INFORMATION Corresponding Authors *E-mails: [email protected] (F. Li); [email protected] (P. P. Gai) Tel/Fax: (86) 532-86080855

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully appreciate the financial support from the National Natural Science Foundation of China (21605092, 21675095 and 21375072), the Natural Science Foundation of Shandong Province, China (ZR2016BQ08), A Project of Shandong Province Higher Educational Science and Technology Program (J16LC07 and J15LC08), the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (663-1117002), and the Special Foundation for Distinguished Taishan Scholar of Shandong Province (ts201511052).

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For TOC only

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Enzymatic Fuel Cell-Based Self-Powered Homogeneous

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