Research Article www.acsami.org
Paper-Based Analytical Devices Relying on Visible-Light-Enhanced Glucose/Air Biofuel Cells Kaiqing Wu,† Yan Zhang,† Yanhu Wang,† Shenguang Ge,‡ Mei Yan,† Jinghua Yu,*,† and Xianrang Song§ †
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, P. R. China Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, University of Jinan, Jinan 250022, P.R. China § Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Jinan 250117, P.R. China ‡
S Supporting Information *
ABSTRACT: A strategy that combines visible-light-enhanced biofuel cells (BFCs) and electrochemical immunosensor into paper-based analytical devices was proposed for sensitive detection of the carbohydrate antigen 15-3 (CA15-3). The gold nanoparticle modified paper electrode with large surface area and good conductibility was applied as an effective matrix for primary antibodies. The glucose dehydrogenase (GDH) modified gold−silver bimetallic nanoparticles were used as bioanodic biocatalyst and signal magnification label. Poly(terthiophene) (pTTh), a photoresponsive conducting polymer, served as catalyst in cathode for the reduction of oxygen upon illumination by visible light. In the bioanode, electrons were generated through the oxidation of glucose catalyzed by GDH. The amount of electrons is determined by the amount of GDH, which finally depended on the amount of CA15-3. In the cathode, electrons from the bioanode could combine with the generated holes in the HOMO energy level of cathode catalysts pTTh. Meanwhile, the high energy level photoexcited electrons were generated in the LUMO energy level and involved in the oxygen reduction reaction, finally resulting in an increasing current and a decreasing overpotential. According to the current signal, simple and efficient detection of CA15-3 was achieved. KEYWORDS: paper, biofuel cells, visible light, conducting polymer, signal magnification
1. INTRODUCTION Paper, one of the easily prepared materials, is widely distributed everywhere and has turned into a potential substrate for its advantages, including being renewable, inexpensive, and biocompatible.1−4 To date, paper-based devices (μ-PADs) have opened up broad prospects on application v including the determination of glucose, lactate, uric acid in biological samples,5,6 medical and environmental diagnosis.7−10 Typically, naked-eye observations on color change,11,12 fluorescence,13,14 and photoelectrochemical15 or electrochemical methods16,17 are integrated in μ-PADs to detect analyte. There is no doubt that the above assays are significant and effective in actual detection of analyte. However, the drawbacks of complicated operation and usage of derivatization agents limit their wide applications. Considering the kinds of questions that go on in here, more effort would be made toward establishing economical, mass-producible, and portable μ-PADs. Biofuel cells (BFCs), powered by fuel, applied enzymes, metal, or microbes as biocatalysts, operating in mild media at ambient temperature, are new and potential power sources in the current study. Considerable attention has been paid to the potential for using these as in vivo power sources for micropumps, pacemakers, and so forth.18−21 Generally, BFCs © 2015 American Chemical Society
are composed of two bioelectrocatalytic electrodes, named as anode and cathode, used for oxidation of the fuel and reduction of the oxidizer, respectively. As BFCs, the electrical power is produced by the simultaneous oxidation and reduction processes. Recently, researchers noted that a hybrid fuel cell, combining a solar cell and a BFC in a strategy, can offer more power than each of the cells working individually.22 Inspired by this thought, a novel and powerful BFC induced by fuel and enhanced by visible-light irradiation could be constructed. In the fabrication process of BFCs, redox enzymes or noble metals or alloys23−27 are commonly adopted to participate in cathodic reaction. Although they were widely used in biofuel cell cathodes, they also suffered from the poor stability and high cost of redox enzymes and metal-based nanomaterials. In recent years, metal materials involving metal oxide nanomaterials or their composites have been used for oxidation−reduction.28−31 Although the metal materials show good catalytic properties, they are only active in acidic or alkaline solution. Considering that a neutral environment or physiological fluid is needed for Received: June 24, 2015 Accepted: October 19, 2015 Published: October 19, 2015 24330
DOI: 10.1021/acsami.5b07698 ACS Appl. Mater. Interfaces 2015, 7, 24330−24337
Research Article
ACS Applied Materials & Interfaces
2.3. Fabrication of the μ-PADs. The preparation of the μ-PADs (details can be found in Supporting Information) was modified on the basis of our previous work.36 The paper-based device was composed of a single layer of bare paper (the black one, to avoid the interference from external natural light) and three layers of patterned rectangular papers with the same size (15.0 mm × 15.0 mm), named as anodic tab (the blue one), reservoir tab (the red one), and cathodic tab (the green one) (shown in Scheme 1A). The diameters of circular
detection of cancers, the top priority is to search for alternative and friendly catalysts. Recently, conducting polymers have obtained considerable attention and are widely studied due to their good environmental stability, electrical conductivity, and electronic properties.32,33 In more recent research works, by combining the electrocatalytic activity and photophysical properties, conducting polymers have become efficient lightenhanced catalysts for oxygen reduction reaction.34 Among various conducting polymers, polyterthiophene (pTTh) shows superior outstanding optical properties and more prominent environmental stability than that from either polymer.35 The exceptional properties of pTTh made a potential alternative catalyst to trigger a cathodic reaction. Zhang and co-workers have established light-enhanced BFCs using expensive and hardly trimmed indium tin oxide (ITO) glass as support for the growth of pTTh in the cathode.34 In contrast, we used robust, inexpensive, and reliable paper as substrate for the growth of Au and pTTh. Herein, for the first time, we constructed a novel μ-PAD, which was powered by a visible-light-enhanced BFC that applied a gold nanocomposite (Au) modified paper electrode (Au-PE) as support for both bioanode and cathode. The Au-PE improved the conductivity of the paper sample zone, promoted electron transfer, and provided abundant sites for capture antibodies (Ab1) loading. Gold (Au)−silver (Ag) bimetallic nanoparticles (Au@Ag BMPs) were employed for immobilizing the GDH and secondary antibody (Ab2). When the prepared bioanode was exposed to a solution containing glucose and NADH, the GDH catalyzed the conversion of glucose into gluconolactone, protons, and electrons. Under the visible-light illumination, the pTTh worked efficiently for oxygen reduction at the cathode. According to the current signal, which was indirectly dependent on the concentration of sample, simple CA15-3 detection can be achieved successfully.
Scheme 1. (A) Shape and Size of the μ-PADs without the Screen-Printed Electrode, (B) One Side of the μ-PADs with Disk-like Carbon Electrode, and (C) the Reverse of the B Side Arcuate Carbon Electrode
2. EXPERIMENTAL SECTION
hydrophilic zones on anodic tab, reservoir tab, and cathodic tab are 6, 6, and 8 mm, respectively. The zone of the folding line, without printing, was used to provide an area for folding and ensure that the liquid flow smoothly between each hydrophilic zone after folding. A disk-like carbon electrode (5.0 mm) was screen-printed onto the circle hydrophilic zones of the anodic tab (Scheme 1B, Figure S1) and an arcuate carbon electrode was screen-printed onto the circle hydrophilic zones of cathodic tab (Scheme 1C, Figure S2). 2.4. Construction of Bioanode and Cathode. The gold nanocomposite modified paper bioanode electrode (Au-PBAE) and gold nanocomposite modified paper cathode electrode (Au-PCE) were prepared by Au grown on each hydrophilic zone (details can be found in Supporting Information), which was located in the middle of the PBAE/PCE. The bioanode (Scheme 2, right) was fabricated by means of attaching antibodies (Ab1) on Au layer via the interaction between amino on Ab1 and Au on Au-PBAE. Briefly, one drop of 20 μL of Ab1 was added on the porous Au-PBAE and held for 40 min at room tempreture. After a rinse with PBS, to remove physically absorbed Ab1, a drop of 10 μL of blocking solution was then applied to Au-PBAE and incubated for 40 min to block the remaining active sites. While the cathode (Scheme 2, left) was fabricated by in situ electropolymerized TTh on the Au-PCE in acetonitrile solution containing 0.1 M LiClO4 and 50 mM TTh at potentiostatic 1.1 V, the amount of pTTh was controlled by a fixed electrodeposition charge of 0.65 C cm−2 (detail information could be found in Supporting Information). Finally, bioanode and cathode were stored at 4 °C for further use. 2.5. Assay Procedure of the Visible-Light-Enhanced BFCsBased Immunosensor. To detect the CA15-3, the bioanode was drop casted by 10 μL of CA15-3 with different concentrations, followed by incubation for 25 min at 37 °C. Then, 10 μL of GDH-
2.1. Chemicals. NAD+-dependent GDH from Thermoplasma acidophilum (E.C.1.1.1.47, 183 U mg−1) and NAD+/NADH were received from Sigma-Aldrich Chemical Co. Glucose was supplied by Beijing Chemical Reagent Co. (Beijing). CA15-3, Ab1, and Ab2 were procured from Shanghai Linc-Bio Science Co. Ltd. (Shanghai, China). Lithium perchlorate (LiClO4) and terthiophene (TTh) were donated by Sinopharm Chemical Regent Co., Ltd. Whatman chromatography paper #2 (pure cellulose paper) was obtained from GE Healthcare Worldwide (Pudong Shanghai) and cut to proper size for further use. The blocking buffer was adjusted to 0.05% Tween and 0.5% BSA in phosphate buffer solution (PBS, pH 7.0) prior to use. Ultrapure water (≥18 MΩ.cm) is used for the preparation of aqueous solutions unless indicated. 2.2. Preparation of GDH-Au@Ag BMPs-Ab2 Bioconjugate. A 5 mg portion of Au@Ag BMPs (details in Supporting Information) was dissolved thoroughly in 10 mL of PBS (10.0 mM, pH 7.0) by sonication in a sonicator bath at atmospheric environment. The bioconjugates were freshly prepared by addition of Ab2 (500 μg mL−1, 50 μL) and GDH (100 μg mL−1, 50 μL) into Au@Ag BMPs solution (1 mL). The solution was incubated for 2 h with gentle mixing at atmospheric environment, and then the obtained GDH-Au@Ag BMPs-Ab2 bioconjugate was redispersed in 5 mL of blocking solution by continuous sonication. After 2 h, the as-synthesized solution was centrifuged at 15 000 rpm for 15 min to remove the supernatant. The bioconjugates were obtained and washed with PBS four times for further purification. Finally, the obtained bioconjugates from above were stored at 4 °C in atmospheric environment for later use. GDHAb2 bioconjugates were prepared using the same procedures as described in section 2.2 without adding Au@Ag BMPs for comparison. 24331
DOI: 10.1021/acsami.5b07698 ACS Appl. Mater. Interfaces 2015, 7, 24330−24337
Research Article
ACS Applied Materials & Interfaces Scheme 2. Fabrication Process of the VDBFCI on the Origami Device
Figure 1. (A) SEM image of the Au@Ag BMPs, (B) EDS of the Au@Ag BMPs, SEM images of (C) bare PBAE/PCE, (D) Au-PBAE/PCE, and (E) pTTh/Au-PCE. (F) UV−vis absorption spectra of ITO glass (black line) and pTTh on ITO glass (red line). Au@Ag BMPs-Ab2 was added to get a visible-light-enhanced BFCsbased immunosensor (VDBFCI). All modification processes were carried out in an origami device. Finally, the electrolyte solution including PBS (10.0 mM, pH 7.0) containing 50 mM glucose and 10 mM NAD+/NADH was added into the origami device to generate the power output. The light intensity detected at the electrode surface is 15 mW cm−2.
surface, and the producers with an average diameter of 150 nm. Figure 1B showed the corresponding energy dispersive spectrometer (EDS) of the Au@Ag BMPs, which consisted of 12.4% of Au and 87.6% of Ag elements, testifying that the material was successfully synthesized. For the sake of making the paper conductive, the Au layer was used in the immunoassay by stepwise growth on the paper. Prior to the growth of Au, rough and porous cellulose fibers with high surface area were found in Figure 1C. Subsequently, a dense and continuous conducting Au layer was fabricated successfully on the cellulose fiber surfaces after 10 min of growth based on the self-catalytic reduction mechanism (Figure 1D). The Au layer should grow through the paper to the back to make the
3. RESULTS AND DISCUSSION 3.1. Characterization of the Au@Ag BMPs, Bioanode, and Cathode. The morphological features of Au@Ag BMPs were directly depicted by scanning electron microscope (SEM) image in Figure 1A. It is clearly seen that the Au@Ag BMPs were distributed well and owned an unsmooth and uneven 24332
DOI: 10.1021/acsami.5b07698 ACS Appl. Mater. Interfaces 2015, 7, 24330−24337
Research Article
ACS Applied Materials & Interfaces
energy level of the cathode catalysts and involved in the oxygen reduction reaction, finally resulting in an increasing current and a decreasing overpotential.39 GDH enzyme was directly immobilized on the surface of Au@Ag BMPs and applied as oxidant for the oxidation of glucose in the bioanode; thus, the current response of the VDBFCI was directly related to the amount of the immobilized Ab2-Au@Ag BMPs-GDH. After incubation with 5 U mL−1 of CA15-3 and then GDH-Au@Ag BMPs-Ab2, the VDBFCI showed a large current intensity, when electrolyte solution was dropped on the origami device (Figure S4, curve c). In contrast, in the absence of Au@Ag BMPs during the incubation, the VDBFCI showed a low current intensity (curve b). The result indicated that the Au@Ag BMPs could provide large surface area for loading more GDH and enhancing the catalytic performance of GDH toward NAD+/NADH. The GDH-Au@ Ag BMPs-Ab2 bioconjugates were not directly immobilized on a gold layer or the screen-printed carbon electrode. They were indirectly immobilized on the bioanode through linking with CA15-3. Thus, the amount of the immobilized Ab2-Au@Ag BMPs-GDH was dependent on the concentration of CA15-3. With different concentrations of CA15-3 (0, 5, and 20 U mL−1), a greater amount of CA15-3 immobilized on the AuPBAE can indirectly immobilize more Ab2-Au@Ag BMPsGDH on the bioanode, and a higher current response (compare curves a, c, and d) was observed as a result. The possible mechanism of the VDBFCI was proposed to be as follows:
hydrophilic zones current-conducting. The positive and negative side of the Au-PBAE/PCE was measured by an Agilent U1251B hangheld digital multimeter to ensure the conductibility is acceptable. The pTTh was in situ electropolymerized on paper through electrochemical potentiostatic deposition and acted as cathodic catalyst. The as-prepared pTTh with continuous and rough film was attached on the cellulose fibers in Figure 1E. The wrinkled film could effectively increase the electrode area, while improving the efficiency of light utilization. Figure 1F showed the UV−vis absorption spectra of the pTTh (on the ITO glass). As can be observed, the spectrum of the pTTh film was in the range 420−600 nm, mainly in visible-light regions. 3.2. Electrochemical Characterization of Bioanode and Cathode. Electrochemical impedance spectroscopy (EIS) is a widespread and hypersensitized method to study the interface properties of the electrode surface, providing vast quantities of information about the surface.37 EIS of bioanode/ cathode was performed in a background solution of 5.0 mM [Fe(CN)6]3−/4− solution containing 0.5 M KCl. As indicated in Figure S3A, electron transfer resistance of the electrode decreases after its modification. It was clear that a relatively small length of this diameter (curve a) was observed at bare PBAE. Interestingly, when the paper was covered with a Au layer, the corresponding semicircle of the plot (curve b) was reduced, which was attributed to the Au layer with high conductivity and could facilitate the electron transfer. When Ab1 capture antibodies were assembled onto the Au-PBAE, a higher resistance was observed in curve c. The phenomenon was due to the protein layer slowing down the transmission rate of ferricyanide toward the Au-PBAE surface, indicating that the protein layer was an inert electron blocking layer between the electrode surface and electrolytic solution. Therefore, the immobilization of protein would increase the resistance as the experiment proceeded. Just as expected, with the joining of BSA and CA15-3, the resistance gradually increased (curves d and e), revealing the successful assembly of the proteins on the modified Au-PBAE. At last, when the GDH-Au@Ag BMPs-Ab2 bioconjugate was added into the modified Au-PBAE, the rate of electron transfer would experience a deep slow down, and a higher resistance appeared as a result (curve f). For the PCE, when the paper (curve a) was covered with a Au layer, the corresponding semicircle of the plot (curve b) was reduced. After the immobilization of pTTh on the paper, slight increase in the Ret value was observed (curve c), indicating the successful immobilization of pTTh on the paper (Figure S3B). 3.3. Possible Mechanism for the VDBFCI. The VDBFCI was activated by the addition of electrolyte solution. At the beginning, GDH-Au@Ag BMPs-Ab2 was bound with CA15-3 and immobilized on the bioanode. The electrons were generated through the oxidation of glucose at the GDHbased bioanode, and traveled to the cathode through the external circuit. The electrons from the bioanode induced the reaction of oxygen reduction in the pTTh-cathode. Without illumination, however, the reaction of cathodic reduction was slow and commenced at a negative potential because the electrons from bioanode had a relatively low energy level. Unlike other cathode catalysts, photosensitive conductive polymer PTTh could generate a large amount of electrons and holes upon light illumination. When the BFCs worked, the generated holes in the HOMO energy level could combine with the electrons from the bioanode. Meanwhile, the high energy level photoexcited electrons were generated in the LUMO
GDH
glucose ⎯⎯⎯⎯⎯→ gluconolactone + NADH + NAD
NADH → NAD+ + H+ + 2e− visible
pTTh ⎯⎯⎯⎯⎯→ pTTh(e−) + pTTh(h+) light
pTTh(h+) + e−(from bioanode) → pTTh
4pTTh(e−) + O2 + 4H+ → 2H 2O
3.4. Optimization of the Conditions. The goal of the research was to establish high performance of VDBFCI for the detection of the target molecule, so that a wide spectrum of parameters must be optimized. The value of pH plays a marked role in the performance of the immunoassay. The effect of pH on the current intensity of this VDBFCI was shown in Figure S5A. It could be noted that the current intensity increased with the increasing pH value and reached a maximum value at pH 7.0, indicating that the bioactivity of the immobilized protein could not stay stable in both highly acidic and alkaline surroundings.38 The effect of incubation time on the immunoassay was investigated. In an atmospheric environment, the dependence of incubation time on current intensity for 15 U mL−1 of CA15-3 was shown in Figure S5B. Current intensity rose quickly and reached a constant state after 25 min, indicating that the target analyte to the capture antibody was saturated, binding on the immunosensor surface. Thus, 25 min was applied as an appropriate incubation time in the further study. The concentration of glucose was another influencing factor, which could directly influence the current intensity. As can be seen in Figure S5C, the current increased with a series of glucose concentrations up to 50 mM, and changed little over this concentration. Finally, 50 mM glucose was applied as optimized concentration for anodic oxidation reaction in this work. 24333
DOI: 10.1021/acsami.5b07698 ACS Appl. Mater. Interfaces 2015, 7, 24330−24337
Research Article
ACS Applied Materials & Interfaces
Figure 2. (A) Polarization curves of the bioanode in air-saturated PBS electrolyte (10.0 mM, pH 7.0) containing 10 mM NAD+/NADH and (a) 0, (b) 30, and (c) 50 mM glucose in the presence of 15 U mL−1 CA15-3. (B) Polarization curves of the cathode in air-saturated electrolyte solution (b) without and (c) with illumination. (a) N2-saturated and (d) O2-saturated electrolyte solution with illumination.
Figure 3. (A) Relationship between current intensity of biofuel cell and concentration of CA15-3 in the as-prepared VDBFCI. (B) The polarization curves (black line) and dependence of the power density (red line) on the cell voltage in electrolyte solution with illumination.
3.5. Performance of Bioanode and Cathode. With assembly of the as-prepared paper-based bioanode and pTTh cathode to form a photoenhanced glucose/air BFCs on paper, the performance of BFCs was investigated. To evaluate the performance of the bioanode, polarization curves were measured by employing linear sweep voltammograms as testing method. Figure 2A showed the relationship between GDHbased bioanode and electrolyte solution with different glucose concentrations. As can be seen, the catalytic potential commences at −0.16 ± 0.01 V, and a peak current value of 117 μA cm−2 was observed at 0.06 V in PBS (pH 7.0) containing 50 mM glucose in the presence of 15 U mL−1 of CA15-3 (RSD = 2.2%, n = 6). The cathode is a vital part of the fuel cell and plays a significant role in the performance of the VDBFCI. In the asprepared VDBFCI, illumination takes effect in the oxidation reduction reaction in the cathode. To prove the effect of light illumination, the catalytic activity of the pTTh cathode in dark (curve b) and light (curve c) conditions was researched in Figure 2B. The image revealed that the catalytic current was enhanced and reached 47 μA cm−2 (RSD = 1.1%, n = 6), and the onset potential was 0.47 ± 0.01 V with light illumination. Meanwhile, we exposed pTTh in different oxygen environments to investigate the responses toward oxygen contents. As can be seen from Figure 2B, at N2-saturated conditions (curve a), no catalytic current was obviously exhibited. In the presence of O2, the cathode exhibited high electrocatalytic activity: in airsaturated electrolyte (curve c) or O2-saturated electrolyte (curve d). These results indicated that the enzyme, immobilized on the Au@Ag BMPs and then introduced to the bioanode, possessed excellent oxidizing ability toward glucose, and could be used as anodic catalyst in this VDBFCI. Furthermore, O2-saturated electrolyte exhibited much higher cathodic catalytic current
than the others, suggesting that pTTh had powerful capability to catalyze oxygen reduction upon visible-light illumination. The goal of this work was establishing a simple, practical VDBFCI, so that air-saturated electrolyte was chosen as the oxidant rather than a sample with an oxygen bubbling device. 3.6. Analytical Performance. To investigate the performance of the as-prepared immunoassay, the modified electrode was exposed to 5.0 μL of CA15-3 solutions. As shown in Figure 3A, under optimal experimental conditions, the current intensity of the immunoassay gradually increased with the concentration of CA15-3, indicating that more antigen-CA15-3 could immobilize more antibody molecules and enzymes. As a result, a good linear relationship can be observed between the current intensity and the CA15-3 concentrations. The equation of the calibration curve was ΔI = 3.076 + 1.585CCA‑153 (U mL−1, CA15-3 concentrations range from 0.01 to 50 U mL−1) with a correlation coefficient of 0.996. A comparison between the as-proposed VDBFCI and other reported CA15-3 assays was performed and shown in Table 1: the as-proposed VDBFCI with a detection limit of 0.004 U mL−1 at a signalto-noise ratio of 3 had a better acceptability than others. Table 1. Comparison of major characteristics of some methods used in the determination of CEA
24334
detection method
linear range (U mL−1)
detection limit (U mL−1)
electrochemical electrochemical quartz crystal microbalance surface plasmon resonance electrochemiluminescence electrochemical
0.02−60 0.1−20 0.5−26 1−40 0.1−120 0.01−50
0.008 0.012 none 0.025 0.033 0.004
ref
time
39 40 41 42 43 this work
2014 2013 2014 2010 2015
DOI: 10.1021/acsami.5b07698 ACS Appl. Mater. Interfaces 2015, 7, 24330−24337
Research Article
ACS Applied Materials & Interfaces
stability of the proposed VDBFCI was assayed while switching from dark to light. Figure S7 showed the power output of VDBFCI in electrolyte solution in the presence of 50 U mL−1 of CA15-3. As can be seen from the figure, there was almost no power output loss after 12 cycles.
Furthermore, we investigated the dependence of the power density and current density on cell voltage of this VDBFCI in the presence of 50 U mL−1 of CA15-3 in air-saturated electrolyte solution. As shown in Figure 3B, the as-proposed VDBFCI possesses a maximum power density of 70.2 μW cm−2 and an open circuit voltage of 0.61 V in the supporting electrolyte irradiated by visible light of 15 mW cm−2. 3.7. Real Serum Sample Analysis. To effectively monitor disease recurrence and improve the surviving rate of patients, tumor markers must be detected in an accurate, fast, and sensitive procedure. The feasibility of the designed VDBFCI was evaluated according to the comparison between the assay results of CA15-3 in human serum samples, using the proposed method and the reference values obtained by the commercially available electrochemistry analyzer, respectively. Given that the levels of tumor markers should not be beyond the calibration ranges, high levels of serum samples could be diluted with PBS (10.0 mM, pH 7.0) to an appropriate conclusion before use. As shown in Table 2, no significant difference was observed
4. CONCLUSIONS On the whole, this paper described a visible-light-enhanced glucose/air BFCs sensing system using Au-paper as the bioanode and cathode substrate. PTTh was in situ synthesized in the cathode and acted as cathodic catalyst, enhanced by visible light in the BFCs sensing system, with noble metals and usntable enzymes being avoided. Au@Ag BMPs with large surface were used for loading amount of GDH to achieve the bioanodic biocatalyst and signal amplification in the bioanode. In the presence of glucose as fuel supplemented by NAD+ and visible-light irradiation, current would generate and could be tested in the external circuit. The visible-light-enhanced BFCbased immunosensor exhibited highly sensitive amperometric response to CA15-3, good reproducibility, and acceptable precision. Given the advantages of the visible-light-enhanced BFC-based immunosensor, we anticipate that the user-friendly, portable, environmentally stable, and novel and powerful immunosensor provides an opportunity for CA15-3 detection and a promising way for accurate detection of other biological molecules.
Table 2. Assay Results of Clinical Serum Samples (1−4) Using the Proposed and Reference Methods a
−1
proposed method (U mL ) reference methoda (U mL−1) relative error (%) a
1
2
3
4
0.54 0.57 −5.26
4.32 4.27 1.17
9.66 9.51 1.58
23.45 23.24 0.90
■
Average of 11 measurements.
ASSOCIATED CONTENT
S Supporting Information *
between the two methods, indicating that the as-prepared VDBFCI could be applied to the clinical determination of CA15-3 levels in real samples with available results. 3.8. Reproducibility, Specificity, and Stability of the VDBFCI. Variation coefficients (CVs) of interassay were also the outstanding values for investigating the reproducibility of the immunoassay. With the same operation conditions, the CVs of interassay were investigated on the basis of five VDBFCIs, which were prepared in different batches. Results showed that the CVs were 8.3%, 7.5%, and 6.8%, which were acceptable in the work. Thus, the VDBFCI was verified to have an acceptable precision and reproducibility. To confirm that the observed electrochemistry response was due to the specific antibody−antigen interaction, the selectivity of the immunoassay was studied. In view of different tumor markers that may influence the selectivity of the immunoassay, possible structurally related interference in human serum, such as carcinoembryonic antigen (CEA), prostate specific antigen (PSA), and human chorionic gonadotrophin (HCG), was taken into account. As can be seen in Figure S6, the three pure interference antigens offered very similar current intensity with the blank solution. Additionally, the immunosensor was also incubated with CA15-3 containing the three interference solutions, and the current intensity was almost the same as that obtained from the CA15-3 only. The obtained results indicating that a slight influence was made by the three tumor markers and the interference degree of variability were acceptable. Long-term storage stability of the proposed VDBFCI was an important index that should be considered. Results show that no obvious change was observed after 4 days at 4 °C. When the storage time was prolonged to 4 weeks, measured at intervals of 4 days, the response changed slightly, indicating that the proposed VDBFCI could effectively work after a long storage. Meanwhile, the long-term operation
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b07698. Experimental details and additional characterization results (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +86-531-82767161. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (21475052, 21175058), Natural Science Foundation of Shandong Province, China (ZR2015JL006), and Technology Development Plan of Shandong Province, China (Grant No. 2014GGX103012).
■
REFERENCES
(1) Amin, G.; Zaman, S.; Zainelabdin, A.; Nur, O.; Willander, M. ZnO Nanorods-Polymer Hybrid White Light Emitting Diode Grown on a Disposable Paper Substrate. Phys. Status Solidi RRL 2011, 5, 71− 73. (2) Zhang, Y.; Ge, L.; Li, M.; Yan, M.; Ge, S. G.; Yu, J. H.; Song, X. R.; Cao, B. Q. Flexible Paper-Based ZnO Nanorod Light-Emitting Diodes Induced Multiplexed Photoelectrochemical Immunoassay. Chem. Commun. 2014, 50, 1417−1419. (3) Kumar, S.; Kaushik, S.; Pratap, R.; Raghavan, S. Graphene on Paper: A Simple, Low-Cost Chemical Sensing Platform. ACS Appl. Mater. Interfaces 2015, 7, 2189−2194. (4) Mahadeva, S. K.; Walus, K.; Stoeber, B. Paper as a Platform for Sensing Applications and Other Devices: A Review. ACS Appl. Mater. Interfaces 2015, 7, 8345−8362. 24335
DOI: 10.1021/acsami.5b07698 ACS Appl. Mater. Interfaces 2015, 7, 24330−24337
Research Article
ACS Applied Materials & Interfaces
Compressed Carbon Nanotube-Enzyme Electrodes. Nat. Commun. 2011, 2, 370. (25) Ramanavicius, A.; Kausaite-Minkstimiene, A.; MorkvenaiteVilkonciene, I.; Genys, P.; Mikhailova, R.; Semashko, T.; Voronovic, J.; Ramanaviciene, A. Biofuel Cell Based on Glucose Oxidase from Penicillium Funiculosum 46.1 and Horseradish Peroxidase. Chem. Eng. J. 2015, 264, 165−173. (26) Wang, Y. H.; Ge, L.; Ma, C.; Kong, Q. K.; Yan, M.; Ge, S. G.; Yu, J. H. Self-Powered and Sensitive DNA Detection in a ThreeDimensional Origami-Based Biofuel Cell Based on a Porous Pt-Paper Cathode. Chem. - Eur. J. 2014, 20, 12453−12462. (27) Zhao, Y.; Fan, L. Z.; Gao, D. M.; Ren, J. L.; Hong, B. HighPower Non-Enzymatic Glucose Biofuel Cells Based on ThreeDimensional Platinum Nanoclusters Immobilized on Multiwalled Carbon Nanotubes. Electrochim. Acta 2014, 145, 159−169. (28) Guo, S. J.; Zhang, S.; Sun, S. H. Tuning Nanoparticle Catalysis for the Oxygen Reduction Reaction. Angew. Chem. 2013, 125, 8686− 8705. (29) Liang, Y. G.; Li, Y. G.; Wang, H. L.; Zhou, J. G.; Wang, J.; Regier, T.; Dai, H. J. Co3O4 Nanocrystals on Graphene as a Synergistic Catalyst for Oxygen Reduction Reaction. Nat. Mater. 2011, 10, 780− 786. (30) Wei, W.; Liang, H. W.; Parvez, K.; Zhuang, X. D.; Feng, X. L.; Müllen, K. Nitrogen-Doped Carbon Nanosheets with Size-Defined Mesopores as Highly Efficient Metal-Free Catalyst for the Oxygen Reduction Reaction. Angew. Chem., Int. Ed. 2014, 53, 1570−1574. (31) Zhao, Y.; Watanabe, K.; Hashimoto, K. Self-Supporting Oxygen Reduction Electrocatalysts Made from a Nitrogen-Rich Network Polymer. J. Am. Chem. Soc. 2012, 134, 19528−19531. (32) Novak, P.; Muller, K.; Santhanam, K. S. V.; Haas, O. Electrochemically Active Polymers for Rechargeable Batteries. Chem. Rev. 1997, 97, 207−281. (33) Abdelwahab, A. A.; Koh, W.C. A.; Noh, H.-B.; Shim, Y.-B. A Selective Nitric Oxide Nanocomposite Biosensor Based on Direct Electron Transfer of Microperoxidase: Removal of Interferences by Co-immobilized Enzymes. Biosens. Bioelectron. 2010, 26, 1080−1086. (34) Zhang, L. L.; Xu, Z. K.; Lou, B. H.; Han, L.; Zhang, X. W.; Dong, S. J. Visible-Light-Enhanced Electrocatalysis and Bioelectrocatalysis Coupled in a Miniature Glucose/Air Biofuel Cell. ChemSusChem 2014, 7, 2427−2431. (35) Too, C. O.; Wallace, G. G.; Burrell, A. K.; Collis, G. E.; Officer, D. L.; Boge, E. W.; Brodie, S. G.; Evans, E. J. Photovoltaic Devices Based on Polythiophenes and Substituted Polythiophenes. Synth. Met. 2001, 123, 53−60. (36) Wang, Y. H.; Ge, L.; Wang, P. P.; Yan, M.; Yu, J. H.; Ge, S. G. A Three-Dimensional Origami-Based Immuno-Biofuel Cell for Selfpowered, Low-Cost, and Sensitive Point-of-Care Testing. Chem. Commun. 2014, 50, 1947−1949. (37) Lin, X. Y.; Ni, Y. N.; Kokot, S. A Novel Electrochemical Sensor for the Analysis of β-agonists: the Poly (acid chrome blue K)/ Graphene Oxide-Nafion/Glassy Carbon Electrode. J. Hazard. Mater. 2013, 260, 508−517. (38) Tang, D. P.; Ren, J. J. In Situ Amplified Electrochemical Immunoassay for Carcinoembryonic Antigen Using Horseradish Peroxidase-Encapsulated Nanogold Hollow Microspheres as Labels. Anal. Chem. 2008, 80, 8064−8070. (39) Zhao, L. F.; Wei, Q.; Wu, H.; Dou, J. K.; Li, H. Ionic Liquid Functionalized Graphene Based Immuno sensor for Sensitive Detection of Carbohydrate Antigen 15−3 Integrated with Cd2+Functionalized Nanoporous TiO2 as Labels. Biosens. Bioelectron. 2014, 59, 75−80. (40) Li, H.; He, J.; Li, S. J.; Turner, A. P. F. Electrochemical Immunosensor with N-Doped Graphene-Modified Electrode for Label-Free Detection of the Breast Cancer Biomarker CA 15−3. Biosens. Bioelectron. 2013, 43, 25−29. (41) Wang, X. H.; Yu, H. Y.; Lu, D. D.; Zhang, J.; Deng, W. W. Label Free Detection of the Breast Cancer Biomarker CA15.3 Using ZnO Nanorods Coated Quartz Crystal Microbalance. Sens. Actuators, B 2014, 195, 630−634.
(5) Dungchai, W.; Chailapakul, O.; Henry, C. S. Electrochemical Detection for Paper-Based Microfluidics. Anal. Chem. 2009, 81, 5821− 5826. (6) Chen, X.; Chen, J.; Wang, F. B.; Xiang, X.; Luo, M.; Ji, X. H.; He, Z. K. Determination of Glucose and Uric Acid with Bienzyme Colorimetry on Microfluidic Paper-Based Analysis Devices. Biosens. Bioelectron. 2012, 35, 363−368. (7) Lu, Y.; Shi, W. W.; Qin, J. H.; Lin, B. C. Fabrication and Characterization of Paper-Based Microfluidics Prepared in Nitrocellulose Membrane by Wax Printing. Anal. Chem. 2010, 82, 329−335. (8) Hou, S. Y.; Chen, H. K.; Cheng, H. C.; Huang, C. Y. Development of Zeptomole and Attomolar Detection Sensitivity of Biotin-Peptide Using a Dot-Blot Goldnanoparticle Immunoassay. Anal. Chem. 2007, 79, 980−985. (9) Chin, C. D.; Linder, V.; Sia, S. K. Lab-on-a-Chip Devices for Global Health: Past Studies and Future Opportunities. Lab Chip 2007, 7, 41−57. (10) Martinez, A. W.; Phillips, S. T.; Wiley, B. J.; Gupta, M.; Whitesides, G. M. FLASH: A Rapid Method for Prototyping PaperBased Microfluidic Devices. Lab Chip 2008, 8, 2146−2150. (11) Cate, D. M.; Dungchai, W.; Cunningham, J. C.; Volckens, J.; Henry, C. S. Simple, Distance-Based Measurement for Paper Analytical Devices. Lab Chip 2013, 13, 2397−2404. (12) Jokerst, J. C.; Adkins, J. A.; Bisha, B.; Mentele, M. M.; Goodridge, L. D.; Henry, C. S. Development of a Paper-Based Analytical Device for Colorimetric Detection of Select Foodborne Pathogens. Anal. Chem. 2012, 84, 2900−2907. (13) Liu, H.; Crooks, R. M. Three-Dimensional Paper Microfluidic Devices Assembled Using the Principles of Origami. J. Am. Chem. Soc. 2011, 133, 17564−17566. (14) Thom, N. K.; Lewis, G. G.; Yeung, K.; Phillips, S. T. Quantitative Fluorescence Sssays Using a Self-Powered Paper-Based Microfluidic Device and a Camera-Equipped Cellular Phone. RSC Adv. 2014, 4, 1334−1340. (15) Ge, L.; Wang, P. P.; Ge, S. G.; Li, N. Q.; Yu, J. H.; Yan, M.; Huang, J. D. Photoelectrochemical Lab-on-Paper Device Based on an Integrated Paper Supercapacitor and Internal Light Source. Anal. Chem. 2013, 85, 3961−3970. (16) Cunningham, J. C.; Brenes, N. J.; Crooks, R. M. Paper Electrochemical Device for Detection of DNA and Thrombin by Target-Induced Conformational Switching. Anal. Chem. 2014, 86, 6166−6170. (17) Lankelma, J.; Nie, Z.; Carrilho, E.; Whitesides, G. M. Paperbased Analytical Device for Electrochemical Flow-Injection Analysis of Glucose in Urine. Anal. Chem. 2012, 84, 4147−4152. (18) Barton, S. C.; Gallaway, J.; Atanassov, P. Enzymatic Biofuel Cells for Implantable and Microscale Devices. Chem. Rev. 2004, 104, 4867− 4886. (19) Katz, E.; MacVittie, K. Implanted Biofuel Cells Operating in Vivo−Methods, Applications and Perspectives−Feature Article. Energy Environ. Sci. 2013, 6, 2791−2803. (20) Cracknell, J. A.; Vincent, K. A.; Armstrong, F. A. Enzymes as Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis. Chem. Rev. 2008, 108, 2439−2461. (21) Hou, C. T.; Yang, D. P.; Liang, B.; Liu, A. H. Enhanced Performance of a Glucose/O2 Biofuel Cell Assembled with LaccaseCovalently Immobilized Three-Dimensional Macroporous Gold FilmBased Biocathode and Bacterial Surface Displayed Glucose Dehydrogenase-Based Bioanode. Anal. Chem. 2014, 86, 6057−6063. (22) de la Garza, L.; Jeong, G.; Liddell, P. A.; Sotomura, T.; Moore, T. A.; Moore, A. L.; Gust, D. Enzyme-Based Photoelectrochemical Biofuel Cell. J. Phys. Chem. B 2003, 107, 10252−10260. (23) Ramanaviciene, A.; Nastajute, G.; Snitka, V.; Kausaite, A.; German, N.; Barauskas-Memenas, D.; Ramanavicius, A. Spectrophotometric Evaluation of Gold Nanoparticles as Redox Mediator for Glucose Oxidase. Sens. Actuators, B 2009, 137, 483−489. (24) Zebda, A.; Gondran, C.; Le Goff, A.; Holzinger, M.; Cinquin, P.; Cosnier, S. Mediatorless High-Power Glucose Biofuel Cells Based on 24336
DOI: 10.1021/acsami.5b07698 ACS Appl. Mater. Interfaces 2015, 7, 24330−24337
Research Article
ACS Applied Materials & Interfaces (42) Chang, C. C.; Chiu, N. F.; Lin, D. S.; Chu-Su, Y.; Liang, Y. H.; Lin, C. W. High-Sensitivity Detection of Carbohydrate Antigen 15−3 Using a Gold/Zinc Oxide Thin Film Surface Plasmon ResonanceBased Biosensor. Anal. Chem. 2010, 82, 1207−1212. (43) Jiang, X. Y.; Wang, H. J.; Yuan, R.; Chai, Y. Q. Sensitive Electrochemiluminescence Detection for CA15−3 Based on Immobilizing Luminol on Dendrimer Functionalized ZnO Nanorods. Biosens. Bioelectron. 2015, 63, 33−38.
24337
DOI: 10.1021/acsami.5b07698 ACS Appl. Mater. Interfaces 2015, 7, 24330−24337
