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Magnetic Graphene Nanosheet-Based Microfluidic Device for Homogeneous Real-Time Electronic Monitoring of Pyrophosphatase Activity Using Enzymatic Hydrolysate-Induced Release of Copper Ion Youxiu Lin, Qian Zhou, Juan Li, Jian Shu, Zhenli Qiu, Yuping Lin, and Dianping Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04005 • Publication Date (Web): 26 Nov 2015 Downloaded from http://pubs.acs.org on November 26, 2015

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Analytical Chemistry

Magnetic

Graphene

Nanosheet-Based

Microfluidic

Device

for

Homogeneous Real-Time Electronic Monitoring of Pyrophosphatase Activity Using Enzymatic Hydrolysate-Induced Release of Copper Ion

Youxiu Lin, Qian Zhou, Juan Li,* Jian Shu, Zhenli Qiu, Yuping Lin, and Dianping Tang*

Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, People's Republic of China

CORRESPONDING AUTHOR INFORMATION Phone: +86-591-2286 6125; fax: +86-591-2286 6135; e-mail: [email protected] (D. Tang)

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ABSTRACT: A novel flow-through microfluidic device based on magneto-controlled graphene sensing platform was designed for homogeneous electronic monitoring of pyrophosphatase (PPase) activity, coupling with enzymatic hydrolysate-induced release of inorganic copper ion (Cu2+) from Cu2+-coordinated pyrophosphate ions (Cu2+-PPi) complex. Magnetic graphene nanosheets (MGNS) functionalized with negatively charged Nafion were synthesized by using the wet-chemistry method. The Cu2+-PPi complexes were prepared on the basis of the coordination reaction between copper ion and inorganic pyrophosphate ions. Upon target PPase introduction into the detection system, the analyte initially hydrolyzed pyrophosphate ions into phosphate ions, and released the electroactive copper ions from Cu2+-PPi complexes. The released copper ions could be readily captured through the negatively charged Nafion on the magnetic graphene nanosheets, which could be quantitatively monitored by using the stripping voltammetry on the flow-through detection cell with an external magnet. Under optimal conditions, the obtained electrochemical signal exhibited a high dependence on PPase activity within a dynamic range from 0.1 to 20 mU mL-1, and allowed the detection at a concentration as low as 0.05 mU mL-1. Coefficients of variation for reproducibility of the intra-assay and inter-assay were below 7.6 and 9.8%, respectively. The inhibition efficiency of sodium fluoride (NaF) also received good results in pyrophosphatase inhibitor screening research. In addition, the methodology afforded good specificity and selectivity, simplification, and low cost without the need of sample separations and multiple washing steps, thus representing a user-friendly protocol for practical utilization in quantitative PPase activity.

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Enzymes are remarkable molecular devices that determine the pattern of chemical transformations in biological systems. Enzymatic reactions play very important roles in almost all living organisms. Even in a signal cell, there exist thousands of enzymatic reactions to provide resources and energy for normal metabolism and proliferation.1,2 Inorganic pyrophosphatase (PPase, a kind of ubiquitous hydrolytic enzyme) can specifically catalyze the conversion of one-molecular pyrophosphate (P2O74−, PPi) to two orthophosphate ions (PO43-, Pi).3 Typically, the PPase-catalyzed PPi hydrolysis is a high-energy discharge reaction, and thus can be employed to some energetically unfavorable biochemical reactions to drive/or complement these reactions.4,5 Additionally, PPase has been reported on the relationship with some biological reactions (e.g., lipid synthesis and degradation, phosphorus metabolism, carbohydrate metabolism, DNA synthesis, calcium absorption and bone formation)6,7 and clinical diseases (e.g., lung adenocarcinomas, hyperthyroidism and colorectal cancer).8-10 To keep pace with expectations in future point-of-care testing, there is the request for more flexible, yet highly sensitive, quantitative, and easy-to-use methods for the rapid screening of PPase activity. Recently, substantial efforts have been made worldwide in the assay field to simplify the assay process with the purpose of manufacturing portable and affordable devices, while preserving the essential benefits in sensitivity, robustness, broad applicability and suitability to automation.11-13 Deng et al. designed a real-time colorimetric assay of PPase activity based on reversibly competitive coordination of Cu2+ between nanogold-labeled cysteine and pyrophosphate ion.14 The Yang's group reported a highly sensitive real-time assay of inorganic PPase activity based on the fluorescent gold nanoclusters.15 Unfavorably, the above-mentioned colorimetric/fluorescent assays for monitoring PPase activity often involved the nanostructure-based labeling strategies. Recently, Xu et al. developed a label-free fluorometric method for inorganic PPase activity on the basis of Cu+-catalyzed alkyne-azide cycloaddition click reaction.16 Undoubtedly, the additional components including sodium ascorbate, 3-azidocoumarins and propargyl alkyne were inevitably introduced during the measurement. To tackle this concern, an alternative sensing strategy that is based on an electrochemical detection principle and does not require an additional label or multiple reactions would be advantageous because of simple instrumentation and easy signal quantification. In this regard, the rapidly emerging research field of homogeneous assay format 3



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provides excitingly new possibilities for advance development of new analytical tools and instrumentation.17,18 Generally speaking, the type of homogeneous assay depends on the designed detection principle that is modulated and turned on/off as a result of the binding reaction.19.20 To successfully develop a unique homogeneous assay mode for monitoring the PPase activity, there are two basal issues to realize the application of electrochemical technique. The first key aspect is to design a highly efficient signal-generation tag. Routine approaches usually consist of ligand-conjugated enzymes or electroactive materials.21-23 Inspiringly, metal ions (e.g., cadmium, copper, zinc, and lead) could exhibit specific voltammetric characteristics at different applied potentials.24,25 Bai et al. employed Cu2+ ion as the electroactive indicator to successfully construct an electrochemical Cu2+ sensor.26 Gao et al. utilized the encapsulated Cu2+ ions into the dendrimer as the signal tags for sensitive electrochemical stripping detection of DNA hybridization.27 Significantly, Cu2+ ion could be coordinated in the presence of pyrophosphate (PPi) on the basis of the strong interaction between PPi and Cu2+. In contrast, the coordination compound could not be formed between orthophosphate ion (Pi) and Cu2+. To this end, the chemical transformation between PPi and Pi could be controlled by the added PPase. Our motivation in this work is to design a new class of homogeneous assay protocol for the detection of PPase activity based on enzymatic hydrolysate-induced Cu2+ release from the Cu2+-PPi complex. Another important issue is to design a high-throughout and reusable electrochemical sensing interface for development of homogeneous assay format. Magneto-controlled molecular electronics usually examine the effect of an external magnetic field on the electrochemical signal of the sensing interface.28-30 Recently, our group fabricated a series of magnetic bead-based sensing platforms for the detection of biomarkers or small molecules.31-37 In these cases, magnetic beads were not only used as the immobilization support for the labeling of biomolecules, but also employed for rapid separation and detection of the biomolecules. Herein we synthesize the honeycomb-like magnetic graphene nanosheets, functionalized with negatively charged Nafion, for the capture of positively charged Cu2+ ions after interaction with target PPase and the Cu2+-PPi complex (Scheme 1). The system consists of a magneto-controlled microfluidic device, Cu2+-PPi complexes and Nafion- modified magnetic graphene nanosheets. PPase activity is quantitatively 4


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determined on the basis of the stripping voltammetry by target-triggered release of Cu2+ ion from the Cu2+-PPi complex. The detectable signal derives from the released Cu2+. Attraction of functionalized magnetic graphene nanosheets to the probe surface with an external magnet activates to the electrical contact between the captured Cu2+ and the electrode, thus causing the sensor circuit to switch on. Positioning the magnet above the detection cell retracts the magnetic nanosheets from the substrate surface, and the detection circuit is switched off, thereby resulting in the reutilization of the sensing interface. Such a low-cost and flexible homogeneous sensing system with a flow-through micro-device provides a simple, rapid and readily reusable protocol for quantitative monitoring of inorganic PPase activity.

Scheme 1. Schematic illustration of magnetic graphene nanosheet-based magneto-controlled microfluidic device for homogeneous electrochemical determination of pyrophosphatase activity using enzymatic hydrolysate-induced release of copper ion (Nafion-MGO: Nafion-modified magnetic graphene nanosheets; PPase: pyrophosphatase).

■EXPERIMENTAL SECTION Materials and Reagents. Inorganic pyrophosphatase (PPase, EC 3.6.1.1, MW 71 kDa, pI: 4.75) from baker's yeast (S. cerevisiae) (≥ 90%, HPLC, lyophilized powder, ≥ 1000 units mg-1 protein) (Note: One-unit PPase can liberate 1.0-μmol inorganic orthophosphate per min at pH 7.2 at 25 ºC referring to Sigma's unit definition), and Nafion® 117 solution (~5% in a mixture of lower aliphatic alcohols and water) were purchased from Sigma-Aldrich (Shanghai, China). Sodium pyrophosphate and copper sulfate pentahydrate (CuSO4∙5H2O) were achieved from Beijing Chem. Inc. (Beijing, 5



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China). Graphene oxide nanosheets (product no.: 763713, powder or flakes, carbon: 42.0 – 52.0%, sulfur: ≤ 3.0%, formula weight: 4239.48 g mol-1, 250 mg in glass bottle) were achieved from Sigma-Aldrich. HEPES buffer [10 mM 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid containing 50 mM KNO3, Shanghai Chem. Re. Inc.] was used as the supporting electrolyte. All other chemicals were at least of analytical grade and used without further purification. Ultrapure water was prepared on a Millipore purification system (18.2 MΩ cm-1, Milli-Q). All experiments and measurements were carried out at room temperature (RT, 25 ± 1.0 ºC) unless otherwise statemented. Preparation of Nafion-Modified Magnetic Graphene Nanosheets. Before modification with Nafion, magnetic graphene nanosheets (MGO, i.e., graphene nanosheets coated with Fe3O4 nanoparticles) were prepared through the wet chemistry method, as described in our previous work.32 Briefly, 15-mg graphene oxide nanosheets and 60-mg FeCl3 were first added to 10 mL of 10 mg mL-1 NaOH in diethylene glycol (99 wt %, Alfa Aesar). After stirring for 60 min at RT, the resulting mixture was transfer to an oil bath and heated to 220 ºC for 30 min with stirring under the protection of nitrogen. Following that, 5-mL diethylene glycol at 70 ºC was quickly injected into the oil bath and further heated to 220 ºC for another 60 min. Finally, the as-synthesized magnetic graphene nanosheets were acquired by centrifugation (8,000g) and washing with ethanol. Next, magnetic graphene nanosheets were employed as the substrates for modification of Nafion to form negatively charged nanostructures. Prior to experiment, magnetic graphene nanosheets were initially dried in a vacuum at 80 ºC for 60 min. Following that, magnetic graphene nanosheets (10 mg) were thrown into 5.0 mL of 1.0 wt % Nafion in the ethanol and vigorously stirred for 6 h at RT to make negatively charged Nafion coat on the MGO through the physical adsorption. Afterwards, Nafion-modified MGO nanostructures were separated and purified with the aid of an external magnet. The resultant nanostructures were washed three times with ultrapure water to remove the excess Nafion. Finally, the obtained Nafion-coated MGO nanostructures (denoted as Nafion-MGO) were dispersed into 1.0 mL of 10 mM pH 7.2 HEPES buffer, and preserved at RT for further use with a final concentration of ~10 mg mL-1.

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Flow-Through Homogeneous Electronic Monitoring of PPase Activity. Scheme 1 gives the schematic illustration of magnetic graphene nanosheets-based microfluidic device for homogeneous real-time electronic monitoring of PPase activity by using enzymatic hydrolysate-induced release of copper ion. All electrochemical measurements were carried out on an AutoLab electrochemical workstation (μAUTIII, Eco Chemie B.V., The Netherlands) with a conventional three-electrode system comprising an indium tin oxide (ITO, 5 wt % In2O3 + SnO2) working electrode, a platinum wire auxiliary electrode and an Ag/AgCl reference electrode. The microfluidic device consisted of a six-way valve equipped with a 1.0-mL syringe pump and connected through a Teflon tube to the detection cell. The ITO electrode was installed at the bottom of the cell, and an external permanent BaFe12O19 magnet with pot shape (10 mm in diameter and 5 mm in depth, 410 – 430 mT) was placed under the ITO electrode. The carrier solution (HEPES buffer, 10 mM, pH 7.2), Nafion-MGO (10 mg mL-1), Cu2+-coordinated pyrophosphate ion (P2O74-, PPi) complex (Cu2+-PPi) and target PPase were introduced at a flow rate of 100 μL min-1 via a control valve-based injection loop, respectively. Target PPase was directly injected into the detection cell by using microsyringe, followed by Nafion-MGO and Cu2+-PPi. Before electrochemical measurement, Cu2+-coordinated pyrophosphate ion (P2O74-, PPi) complex (Cu2+-PPi) was prepared by means of mixing 2-mM Cu2+ ion and 2-mM PPi at a volume ratio of 4 : 9 in 500-μL HEPES buffer (10 mM, pH 7.2). The assay mainly consisted of the following steps: (i) 100-μL Nafion-MGO (10 mg mL-1) was flowed into the detection cell and collected on the ITO electrode with an external magnet, (ii) after washing with pH 7.2 HEPES buffer, 100 μL of above-prepared Cu2+-PPi mixture was injected into the cell, (iii) 50 μL of target PPase with different concentrations was added into the detection cell and incubated for 35 min at RT (Note: During this process, pyrophosphate ions were hydrolyzed into phosphate ions and released the electroactive copper ions from Cu2+-PPi complexes), and (iv) the square-wave anodic-stripping voltammogram (SWASV) was registered in an applied potential range from -200 to 400 mV with a potential step of 4 mV, a frequency of 25 Hz and an amplitude of 25 mV. The peak current at +68 mV was collected as the sensor signal. All incubations and measurements were performed by using stopped-flow technique. Analyses were always made in triplicate.

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■ RESULTS AND DISCUSSION Construction and Characteristics of Flow-Through Homogeneous Sensing Strategy. To develop a high-throughout detection scheme for quantitative monitoring of biomolecules, design of a simple and sensitive sensing platform without the need of sample separation and multiple washing steps would be advantageous. In this work, target PPase activity is determined in a flow-through homogeneous assay mode by coupling with a magneto-controlled microfluidic device. Incubation of target analyte with Cu2+-PPi complex and electrochemical measurement are implemented in the same detection cell. More importantly, our designed sensing interface can be repeatedly utilized via controlling a removable external magnet toward magnetic graphene nanosheets (Please see the detailed information on the reutilization at the last paragraph in the Introduction section). Fe3O4 nanoparticles were attached on the surface of graphene oxide nanosheets by in-situ precipitation method. Nafion-modified magnetic graphene nanosheets with negative charge mainly derived from the –SO3- groups of Nafion molecule, while the Cu2+-PPi complexes [i.e., Cu(P2O7)26−complex] were formed by coordination reaction between Cu2+ ions and PPi molecules. The electrochemical signal originated from free Cu2+ ion in the detection solution. In the absence of target PPase, Cu2+ ions were coordinated into the Cu2+-PPi complexes, which hindered capture of negatively charged magnetic graphene nanosheets, thus resulting in a weak electrochemical signal. Upon target PPase introduction, the bioactive enzyme initially hydrolyzed PPi into Pi, which resulted in the release of Cu2+ ion from the complexes and freely dispersed in the detection solution. With the assistance of negatively charged Nafion, the released Cu2+ ion could be captured onto the magnetic graphene nanosheets on the basis of opposite-charged adsorption technique. The capture Cu2+ ions could be detected by using stripping voltammetry, thereby causing a relatively strong electrochemical signal in comparison with background current. The signal depended on the concentration of target PPase in the sample. After each run, the ITO interface was easily regenerated via detaching the external magnet from the detection device. To realize our design, the successful synthesis of Nafion-modified magnetic graphene nanosheets should be very pivotal. In our previous work,32 we employed transmission electron 8


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microscope (TEM, Model H-7650, Hitachi Instruments, Japan) and X-ray photoelectron spectroscope (XPS, Thermo Fisher Scientific, Model Escalab 250 Spectrometer, Al Kα, 1486.6 eV) to characterize magnetic graphene nanosheets. To further investigate whether Nafion was modified onto magnetic graphene nanosheets, magnetic graphene nanosheets before and after modification with Nafion molecules were characterized by using scanning electron microscope (SEM, Hitachi S4800, Japan) (Figure 1A-B). Figure 1A gives typical SEM image of our synthesized MGO, and a large number of nanoparticles could be obviously observed on the MGO. When Nafion was modified on the MGO, however, the surface of magnetic graphene nanosheets became rougher than that of unmodified MGO (Figure 1B). Moreover, Nafion-modified magnetic graphene nanosheets could be homogeneously dispersed in pH 7.2 HEPES buffer (Figure 1C, left). As seen from right photograph in Figure 1C, significantly, Nafion-modified MGO was rapidly separated with the help of an external magnet. These results could provide a feasible convenience for the development of homogeneously magneto-controlled microfluidic detection mode.

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Figure 1. (A,B) SEM images of (A) magnetic graphene nanosheets and (B) Nafion-modified magnetic graphene nanosheets, respectively; (C) real sample for Nafion-MGO without (left) and with (right) the help of an external magnet; and (D) FTIR spectra of (a) MGO, (b) Nafion and (c) Nafion-MGO, respectively.

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Further, we also utilized Fourier transform infrared (FTIR, Vector 22, Bruker, USA) spectra of Nafion-modified MGO (Figure 1D). Curve 'a' represents FTIR spectra of the as-synthesized MGO. Typically, the distinct IR peaks of the side chains of Nafion polymer can be observed by the stretching vibrations of –COC- linkages around 970 cm-1 (curve 'b').38-40 Its hydrophilic ionic clusters (-SO3H) and hydropobic backbones (-CF2-) exhibit their symmetric stretching vibrations at 1075 and 1156 cm-1, respectively. And their asymmetric stretching vibrations correspond to the superimposed absorption peak occurring around 1232 cm-1. Compared with those of Nafion and MGO alone, these IR peaks of Nafion-MGO displayed a broadening and/or shifting phenomenon (curve 'c'), indicating the existence of strong interaction between MGO and Nafion. These spectral changes revealed that the successful modification of magnetic graphene nanosheets with Nafion. Control Tests. Certainly, another important precondition for design of homogeneous sensing strategy lies on formation of Cu2+-PPi complex (That is to say, whether the presence of PPi could obscure free Cu2+ ion for the generation of electrochemical signal). To monitor this issue, the same-concentration Cu2+ or PPi was detected in pH 7.2 HEPES buffer on our designed microfluidic device by using square-wave anodic-stripping voltammetry under the same conditions (Figure 2A). A strong electrochemical signal was obtained in the presence of only Cu2+ ion (curve 'a'), while no voltammetric peak current was observed in the PPi detection solution (curve 'c'), indicating that Cu2+ ion was a good electroactive signal-generation tag. After the mixture of Cu2+ ion with PPi, favorably, the voltammetric peak current largely decreased in comparison with that of Cu2+ alone (curve 'b'). According to previous reports,14,16 the decrease in the peak current should be ascribed to the formation of Cu2+-PPi complexes. The results indirectly indicated that Cu2+-coordinated PPi complex could not be captured by negatively charged MGO, which provided the proof-of-concept of enzymatic hydrolysate-induced Cu2+ release for the development of electrochemical sensing protocol. Generally speaking, PPase can catalyze the conversion of one-molecular PPi into two phosphate ions. Undoubtedly, HPO42−and H2PO4− ions are simultaneously present in pH 7.2 HEPES buffer. 10


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Thus, the effects of PO43-, HPO42−and H2PO4− ions on the electrochemical signal of our designed sensing strategy should be studied. As shown in Figure 2B, the voltammetric peak currents of Cu2+ + PO43- (curve 'b'), Cu2+ + HPO42− (curve 'c') and Cu2+ + H2PO4− (curve 'd') were almost the same as that of Cu2+ ion alone (curve 'a'). Only if Cu2+ and PPi were simultaneously existed, the peak current largely decreased (curve 'e'). The results indicated that Cu2+-based coordination complex derived from the reaction between Cu2+ ion and PPi. In this work, the electrochemical signal mainly originated from the captured Cu2+ by negatively charged Nafion. As control test, we also investigated the effect of magnetic graphene nanosheets with and without Nafion on the electrochemical signal of our strategy in pH 7.2 HEPES containing 1.0-mM Cu2+. As indicated from Figure 2C, a strong electrochemical signal was acquired on the Nafion-MGO (curve 'a'). In contrast, the absence of Nafion on the MGO could only exhibit a low voltammetric peak current (curve 'b'). Therefore, the as-prepared Nafion-MGO could be used as the capture substrate for free Cu2+ ion in the solution.

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Figure 2. (A) Voltammetric responses of magneto-controlled microfluidic device toward (a) Cu2+, (b) Cu2+ + PPi and (c) PPi; (B) Voltammetric responses of magneto-controlled microfluidic device toward (a) Cu2+, (b) Cu2+ + PO43-, (c) Cu2+ + HPO42−, (d) Cu2+ + H2PO4− and (e) Cu2+ + PPi; (C) Voltammetric responses of magneto-controlled microfluidic device toward 1.0 mM Cu2+ by using (a) Nafion-MGO and MGO as the capture substrate, respectively; and (D) Voltammetric responses of (a) Cu2+-PPi, (b) Cu2+ + PPase, (c) PPi + PPase and (d) Cu2+-PPi + 10 mU mL-1 PPase. The supporting electrolyte was pH 7.2 HEPES buffer containing 50 mM KNO3.

Logically, one important concern arises to whether our strategy could be used for the detection of target PPase. To demonstrate this issue, 10 mU mL-1 PPase was measured in the microfluidic device based on Cu2+-PPi and Nafion-MGO. As seen from curve 'a' in Figure 2D, the mixture containing Cu2+-PPi complexes only gave a weak electrochemical signal, indicating that most Cu2+ ions were chelated into the complex. Upon addition of target PPase into the detection cell, the peak current increased (Figure 2D, curve 'd'), which was attributed to enzymatic 12


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hydrolysate-induced Cu2+ release from the Cu2+-PPi complex. Meanwhile, we also observed that the added PPase could not coordinate Cu2+ ion (Figure 2D, curve 'b') in comparison with curve 'a'. No voltammetric peak current was appeared toward the mixture containing PPi and PPase (Figure 2D, curve 'c'). Based on these results, we could make a conclusion that target PPase could hydrolyze PPi into Pi, and released the coordinated Cu2+ ion from the complex. The released Cu2+ ions could be captured by negatively charged Nafion-MGO, thereby resulting in the increase of the electrochemical signal. Optimization of Experimental Conditions. As described above, the electrochemical signal derived from the captured Cu2+ ions by negatively charged Nafion on the MGO. Hence, the coated amount of Nafion on the MGO would directly affect the signal of detectable electronic signal. A low-amount Nafion would not facilitate the capture of free Cu2+ ions. Vice versus, a high-amount Nafion would hinder the electron transfer between the solution and ITO electrode. Importantly, the high-concentration Nafion increased the viscosity of Nafion-MGO suspension, which was unfavorable for the development of homogeneous microfluidic device. As shown in Figure 3A, an optimal voltammetric peak current was achieved at 5.0 mL of 1.0 wt % Nafion (10 mU mL-1 PPase as an example in this case). Thus, mixture of 10-mg MGO with 5.0 mL of 1.0 wt % Nafion was used for the preparation of Nafion-MGO in this work. As seen from Scheme 1, another factor influencing the homogeneous detection mode depended on the preparation of Cu2+-PPi complex. Although excess Cu2+ ions were conducive for formation of Cu2+-PPi complexes, it was easy to produce a high background signal. On the contrary, a low-concentration Cu2+ ion could reduce the background signal. However, it was unfavorable for the detection of low-concentration PPase because excess PPi substrates would consume some PPase molecules. Figure 3B shows the effects of different-volume ration between 2-mM Cu2+ and 2-mM PPi on the electrochemical signal of our designed sensing strategy (10 mU mL-1 PPase in this case). Experimental results indicated that a maximum change in the current relative to background signal was achieved at a volume ration of 4 : 9 for Cu2+ and PPi. Therefore, 2-mM Cu2+ ion and 2-mM PPi at a volume ratio of 4 : 9 was utilized to synthesize the Cu2+-PPi complex in 500-μL HEPES buffer containing 50 mM KNO3 (10 mM, pH 7.2).

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As a kind of proteins, PPase has an isoelectronic point of ~4.75. In order to avoid the adsorption of PPase onto negatively charged Nafion-MGO, PPase with negative charge would be preferable. That is to say, pH of HEPES buffer should be higher than 4.75. Certainly, it is very important to maintain the bioactivity of PPase. As indicated from Figure 3C, a strong electrochemical signal could be acquired at pH 7.2 HEPES buffer. A high or low pH would decrease the electrochemical signal. So, pH 7.2 HEPES buffer was used as the supporting electrolyte for the determination of PPase activity throughout this work.

Figure 3. Effects of (A) Nafion volume (1.0 wt %) for preparation of Nafion-MGO with 10-mg MGO, (B) volume ratio of 2-mM Cu2+ and 2-mM PPi for synthesis of Cu2+-PPi complexes in 500-μL HEPES buffer, (C) pH of HEPES buffer and (D) hydrolytic time for PPase toward Cu2+-PPi (i.e., capture time of Nafion-MGO toward free Cu2+ ions) on the electrochemical signal of magneto-controlled microfluidic device (10 mU mL-1 PPase used in this case).

Usually, it takes some time for PPase to hydrolyze the PPi into Pi. Figure 3D gives the effects of different hydrolytic times on the voltammetric peak current of homogeneous sensing strategy. The 14


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currents initially increased with the increasing hydrolytic time, and then tended to level off after 35 min. To save the assay time, 35 min was utilized for enzymatic hydrolysate-induced Cu2+ release from the Nafion-MGO complexes. Quantitative Monitoring of PPase Activity. Under optimal conditions, PPase bioactivity was evaluated by coupling with Nafion-MGO/Cu2+-PPi-based microfluidic device. The incubation and measurement were carried out in the same detection cell at the stopped-flow state. Figure 4A shows voltammetric responses of magneto-controlled microfluidic device toward different-concentration PPase concentrations in pH 7.2 HEPES buffer containing 50 mM KNO3. The voltammetric peak currents increased with the increasing PPase concentrations in the sample. A linear correlation between peak current and PPase activity was observed within the dynamic working range from 0.1 to 20 mU mL-1 with a detection limit (LOD) of 0.05 mU mL-1 at signal-to-noise ratios of 3 (Figure 4B). The regression equation could be fitted to y (µA) = 4.1681 × ln C[PPase] + 4.8687 (mU mL-1, R2 = 0.9946, n = 8). Obviously, the LOD of our designed strategy was even lower than those of previous reports, e.g., click chemistry-based fluorometric method (LOD: 0.2 mU mL-1),16 gold nanocluster-based fluorescent assay (1.0 mU mL-1),15 label-free colorimetric assay (27 mU mL-1),41 and nanogold-based colorimetric assay (10 mU mL-1).14 Significantly, the microfluidic device was capable of continuously carrying out all steps within less than 40 min per sample, including incubation, enzymatic reaction and electrochemical measurement.

Figure 4. (A) Voltammetric responses of Nafion-MGO-based magneto-controlled microfluidic device for homogeneous electronic monitoring of different PPase activities in pH 7.2 HEPES containing 50 mM KNO3, and (B) the corresponding calibration plots. Each data point represents the average value of three 15



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measurements, and the error bar represents the 95% confidence interval of the mean for electrochemical signal; error bars are standard error of the mean (1s) values.

Reproducibility, Precision and Specificity. The reproducibility and precision of our system were evaluated via monitoring 0.1, 1.0 and 10 mU mL-1 PPase (i.e., high, middle and low) by using identical batches of Cu2+-PPi and Nafion-MGO, respectively. Experimental results indicated that the relative standard deviations (RSDs) of using the same-batch Cu2+-PPi and Nafion-MGO were 5.3, 7.6, and 6.2% (n = 3) for 0.1, 1.0 and 10 mU mL-1 PPase, respectively. The batch-to-batch reproducibility and precision toward the above-mentioned analytes were also investigated by using the same assay mode, and the RSDs were 9.8, 9.3 and 7.4% (n = 3), respectively. These results suggested a good reproducibility and precision of magneto-controlled homogeneous assay.

Figure 5. (A) Specificity of Nafion-MGO-based magneto-controlled microfluidic device against target PPase (0.5 mU mL-1), GOx (50 mU mL-1), HAS (50 ng mL-1), DNase I (50 mU mL-1) and PSA (50 ng mL-1) (Note: The mixture contained all the above-mentioned analytes); (B-D) Voltammetric response curves of Nafion-MGO-based magneto-controlled microfluidic device in pH 7.2 HEPES buffer towards different 16


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components: (B) background signal (curve 'a0') and 0.5 mU mL-1 PPase (curve 'a'), (C) 50 mM Na+ (curve 'b0') and 50 mM Na+ + 0.5 mU mL-1 PPase (curve 'b'), and (D) 50 mM Pb2+ (curve 'c0') and 50 mM Pb2+ + 0.5 mU mL-1 PPase (curve 'b').

To investigate the specificity of the developed sensing strategy, several proteins including glucose oxidase (GOx), human serum albumin (HSA), DNase I and prostate-specific antigen (PSA), were evaluated by using our system under the same conditions. As shown in Figure 5A, the currents obtained from GOx (50 mU mL-1), HAS (50 ng mL-1), DNase I (50 mU mL-1) and PSA (50 ng mL-1) alone were almost the same as the background signal. A strong electrochemical signal was only obtained toward target PPase (0.5 mU mL-1). More happily, their mixture with target PPase did not cause a significant increase in the current relative to target PPase alone. Therefore, the specificity of this system was relatively satisfactory toward other possible proteins. Actually, metal ions (e.g., K+, Na+, Ca2+, Mg2+ and Fe2+) are also present in the real samples (e.g, human serum sample) beside proteins. During this measurement, positively charged metal ions might be adsorbed on the Nafion-MGO. Considering the future application, we further investigated the effects of other metal ions on the electrochemical signal of this system. In this case, we chose the non-electroactive metal ion (e.g., Na+) and electroactive metal ion (e.g., Pb2+) as an example for this evaluation. As seen from curves 'b0' and 'b' in Figure 5C, the presence of Na+ ion did not change the peak current in comparison with curves 'a0' and 'a' in Figure 5B, regardless of coexistence or not. Although Pb2+ ion could cause the increase in the peak current (Figure 5D, curves 'c0' and 'c'), the peak potential was obviously different from Cu2+ ion. Importantly, the peak current of Cu2+ ion in the mixture (Figure 5D, curve 'c') at +68 mV were almost the same as those of Cu2+ ion alone (Figure 5B, curve 'a'). Hence, we could monitor PPase activity in the real samples by evaluating the current of Cu2+ characteristic peak at +68 mV. These results revealed that our strategy had a high selectivity and specificity toward target PPase. Inhibition Assay. As is well-known, sodium fluoride (NaF) is an effective inhibitor for PPase owing to the formation of the [F- - PPase] intermediate, thus resulting in an instant decrease in the enzymatic activity.42,43 Using NaF as a model inhibitor toward PPase activity, the validity of this system in screening the inhibition of PPase was evaluated through adding different-concentration NaF into the incubation solution during the measurement (Figure 6). As control test, pure NaF was 17



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directly injected into the detection solution containing Cu2+-PPi in the absence of PPase. As seen from column 'b', the electrochemical signal in the presence of NaF was almost the same as the background signal (column 'a'), indicating that NaF could not cause the dissociation of Cu2+-PPi complex. This result also suggested that NaF could be used for evaluating the inhibitor efficiency of PPase. Initially, 25 µL of 20 mU mL-1 PPase was incubated with 25-µL NaF in pH 7.2 HEPES buffer with different concentrations for 60 min at 37 ºC (Note: The concentration of PPase after dilution became 10 mU mL-1). Thereafter, the resulting mixture was detected by above-mentioned method. As shown from Figure 6, the voltammetric peak currents gradually decreased with the increasing NaF concentrations, indicating the potential application of the developed assay system for studies of PPase inhibition. Furthermore, we also monitored the effects of sodium phosphate on the voltammetric signal of our strategy because sodium phosphate is typically added to foods to keep them moist during storage. As shown in Figure 6, introduction of sodium phosphate did not significantly cause the change in the voltammetric signal. Hence, sodium phosphate did not affect the assay sensitivity.

Figure 6. Inhibition of PPase activity (10 mU mL-1 PPase used in this case) toward different-concentration NaF standards (NaF: 2.0 μM; NaF1: 0.4 μM; NaF2: 0.8 μM; NaF3: 1.2 μM; NaF4: 1.6 μM; NaF5: 2.0 μM) and 2.0 μM Na3PO4 by using Nafion-MGO-based magneto-controlled microfluidic device.

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■ CONCLUSIONS In the present paper, we successfully demonstrated the development of advanced homogeneous electrochemical assay mode for sensitive and rapid screening of PPase activity by coupling with a magneto-controlled microfluidic device. Apart from high sensitivity and low detection limit, the microfluidic device exhibited some unique analytical characteristics as follows: (i) the assay could be carried out at one step without the requirement of sample separation and multiple washing steps, (ii) the sensing interface could be repeatedly utilized by detaching the permanent magnet from the detection device, (iii) enzymatic hydrolysate-induced Cu2+ release from Cu2+-PPi complex provided a signal-on detection mode, and (iv) the system offered promise for label-free, simple, cost-effective analysis of biological samples. Importantly, the magneto-controlled microfluidic device could be suitable for use in the mass production of miniature and automation for the detection of other biomolecules. Nevertheless, only one disadvantage of our reported technology is the interfering effect of other cations with redox activity or close to the stripping potential of copper ion during the measurement. To fulfill the potential application, future work should focus on improving scheme of voltammetric detection, e.g., by preparing a modified electrode for specifically capturing reaction with copper ion.

■ACKNOWLEDGEMENT This work was financially supported by the National Natural Science Foundation of China (Grant nos.: 41176079 and 21475025), the National Science Foundation of Fujian Province (Grant no.: 2014J07001), and Key Science Project (type A) of the Fujian Provincial Department of Education, China (Grant no.: JA12021).

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■ FOR ONLY TOC

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Magnetic Graphene Nanosheet-Based Microfluidic Device for

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