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Sensitive electrochemical detection of ciprofloxacin at screen-printed diamond electrodes
Carbon 159 (2020) 247e254
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Sensitive electrochemical detection of ciprofloxacin at screen-printed diamond electrodes Tomohiro Matsunaga a, Takeshi Kondo a, b, *, Takahiro Osasa c, Akihiro Kotsugai c, Isao Shitanda a, b, Yoshinao Hoshi a, Masayuki Itagaki a, b, Tatsuo Aikawa a, Toshifumi Tojo a, Makoto Yuasa a, b a b c
Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan Ricoh Co., Ltd, 2-7-1 Izumi, Ebina, Kanagawa, 243-0460, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 October 2019 Received in revised form 19 December 2019 Accepted 20 December 2019 Available online 23 December 2019
This study investigates the effect of chemical surface termination on the electrochemical characteristics of boron-doped diamond powder (BDDP). The aim is to realize highly sensitive electrochemical detection of ciprofloxacin (CIP) on BDDP-printed electrodes. To this end, we prepared oxygen-terminated BDDP (OBDDP) and hydrogen-terminated BDDP (H-BDDP), and mixed them with an insulating polyester (PES) resin binder to obtain BDDP ink for the printed electrode. Scanning electron microscopy of the BDDPprinted electrodes revealed that the O-BDDPs were partially covered with PES resin, while the HBDDPs were entirely covered with resin. This structural difference might explain the lower chargeredox reaction at the O-BDDP-printed electrode than at the Htransfer resistance of the Ru(NH3)2þ/3þ 6 BDDP-printed electrode. The slope of the calibration curve of the linear sweep voltammogram of CIP was steeper at the O-BDDP-printed electrode than at the H-BDDP-printed electrode and the O-BDD thin-film electrode, and was similar to that at the H-BDD thin-film electrode. Using the O-BDDP-printed electrode, we determined the CIP in artificial buffer-diluted urine in the concentration range 1e30 mM with a recovery of 107%. We conclude that the O-BDDP-printed electrodes provide a highly sensitive and disposable electrochemical sensor for CIP detection. © 2019 Elsevier Ltd. All rights reserved.
1. Introduction Most medicated antibiotics are known to be excreted from the body through urine and feces, and are eventually discharged into environmental water, exacerbating the rise of antibiotic-resistant bacteria. In 2017, the United Nations announced that discharging antibiotics into environmental water might spur the development of ferocious superbugs that are extensively or totally drug resistant; moreover, this problem is global and threatening and requires an
* Corresponding author. Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan. E-mail address: [email protected] (T. Kondo). 1 United Nations. Careless disposal of antibiotics could produce ‘ferocious superbugs,’ UN environment experts warn. Available online: https://news.un.org/ en/story/2017/12/638352-careless-disposal-antibiotics-could-produce-ferocioussuperbugs-un-environment (accessed on 29 September 2019). https://doi.org/10.1016/j.carbon.2019.12.051 0008-6223/© 2019 Elsevier Ltd. All rights reserved.
urgent solution.1 Many recent studies on this problem have developed technologies that determine antibiotic concentrations in environmental water and soil [1e3]. Ciprofloxacin (CIP) is a fluoroquinolone antibiotic used in the treatment of various infections such as urinary tract infections, respiratory infections, and gastrointestinal diseases. Being active against both gram-negative and gram-positive bacteria, CIP is broadly prescribed to humans and livestock [4e6]. CIP is also detected at high concentrations in river waters [7,8]. Against this background, researchers have measured CIP concentrations by various analytical techniques such as highperformance liquid chromatography, mass spectrometry, fluorescence, and ultravioletevisible absorption spectroscopy. Electrochemical measurements are popular as they are simple and quick, and CIP has been electrochemically detected by various types of electrodes [6,9e11]. The boron-doped diamond (BDD) electrode possesses superior characteristics such as extreme physical/chemical stability, small background current, and a wide potential window. Additionally, BDD cannot be fouled by adsorption of protein or
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Fig. 1. XPS patterns of (a) H-BDD (b) O-BDD (c) H-BDDP (c) O-BDDP.
other matter, so is suitable for electrochemical measurements in reported that body fluids such as blood and urine [12e14]. Radi cova a BDD thin-film electrode detects CIP at extremely low concentrations (limit of detection (LOD) ¼ 0.05 mM) [15]. BDD is typically deposited on silicon wafer substrate via chemical vapor deposition (CVD), a high-cost production method that is unsuitable for mass electrode production. As an alternative method, we investigated screen-printed diamond electrodes coated with BDD powder (BDDP). This approach is expected to realize a sensitive and disposable electrode for practical use, because of the superior characteristics of BDD and inexpensive substrate used [16e18]. The electrochemical characteristics of BDD thin-film electrodes are known to depend on the type of surface termination (hydrogen or oxygen) [19e21]. Therefore, we investigated how surface termination of the BDDP influences the electrochemical properties of screen-printed diamond electrodes, with a special focus on CIP detection. 2. Experimental Commercial diamond powder (DP) (Micron þ MDA 3-6, Element Six, nominal particle size 3e6 mm) was used as the core of BDDP. The BDDP was prepared as described in our previous report [16e18,22]. Briefly, 0.8 g of DP was subjected to microwave plasmaassisted chemical vapor deposition (MPCVD), which grew a BDD layer on the DP surface under 1300 W of plasma power and a hydrogen gas flow rate of 400 sccm at 800 C for 8 h. After the MPCVD process, the sample was heated in air at 425 C for 5 h to reduce the sp2 carbon impurities by oxidation [22]. This process resulted in oxidation of the BDDP surface (O-BDDP). The O-BDDP surface can be converted to the hydrogen-terminated form by hydrogen-plasma treatment of the MPCVD system under 500 W plasma power and a hydrogen gas flow rate of 100 sccm at 800 C for 1 h (H-BDDP). The electrical conductivity of the BDDP was measured as reported previously [22]. Briefly, the BDDP was packed into a glass tube (of diameter 1.0 mm) and its conductivity was calculated from the slope of the currentevoltage (IeV) curve. Here, the IeV measurements were taken between both ends of the packed BDDP [23]. BDDP ink containing BDDP and polyester resin (PES) (PES/BDDP ¼ 0.3 w/w) was prepared in a solvent of mixed isophorone and methyl ethyl ketone. The BDDP-printed electrode
was screen-printed onto a polyimide film substrate. The configuration of the BDDP-printed electrode was identical to that of an earlier reported electrode, with a geometric electrode area of 0.28 cm2 (diameter ¼ 6 mm) [17]. BDD thin-film electrode was also prepared by MPCVD. Heat treatment in air and hydrogen plasma treatment mentioned above were employed for oxygen and hydrogen termination of BDD thin-film (O- and H-BDD) electrodes, respectively. The surface terminations of O- and H-BDDP were analyzed by Xray photoelectron spectroscopy (XPS, Axis-Nova, Kratos, province, country). The surfaces of the BDDP-printed electrodes were observed by scanning electron microscopy (SEM, JSM-7600F, Jeol, province, country) and the cross sections of the BDDP-printed electrodes were observed by three-dimensional laser scanning confocal microscope (SCM, VK-X200, Keyence). Electrochemical measurements of the BDDP-printed electrodes were taken by a potentiostat (HZ-7000, Hokuto Denko). As CIP (FUJIFILM Wako) is poorly soluble in neutral solution, we first prepared a 10 mM stock solution of CIP in 0.5 M H2SO4. The stock solution was diluted by 66.7 mM (1/15 M) phosphate buffer solution (PBS) to obtain sample solutions of various concentrations at pH 7.0. Artificial urine containing urea, sodium chloride, ammonium chloride, and other urine constituents, as defined by Japanese industry standard (JIS T3214), was purchased from Isekyu (Aichi, Japan). 3. Result and discussion 3.1. Characterization of O- and H-BDDPs XPS wide spectra of H-BDD, O-BDD, H-BDDP, and O-BDDP are shown in Fig. 1, and the O/C atomic concentration ratios calculated from the spectra are listed in Table 1. When O- and H-BDDP (0.03 g
Table 1 O/C atomic concentration ratios evaluated by XPS. O/C ratio H-BDD O-BDD H-BDDP O-BDDP
0.016 0.098 0.031 0.083
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Fig. 2. SEM images of (a) H-BDDP, (b) O-BDDP, (c) H-BDDP-printed, and (d) O-BDDP-printed electrode surfaces. Cross-sectional images of (e) H- and (f) O-BDDP-printed electrodes. (i), (ii), and (iii) indicate polyimide film, carbon layer, and BDDP layer, respectively. (A colour version of this figure can be viewed online.)
each) were added to water (1.0 mL), the O-BDDP was easily dispersed whereas the H-BDDP floated on the water surface and was hardly dispersed. This result confirmed that the surface termination (oxygen or hydrogen) greatly affected the
hydrophilicity of BDDP. In contrast, the type of surface termination little affected the electrical conductivity of BDDP, which was measured as 0.2e0.7 S cm1 with some dispersion. We conclude that both O- and H-BDDP are sufficiently conductive for electrochemistry, and are equally suitable electrode materials with different hydrophilicities.
3.2. Properties of the BDDP-printed electrodes
Fig. 3. CVs of 1 mM Ru(NH3)6Cl3 in 0.1 M HClO4 at the H-BDDP-printed (dotted line) and O-BDDP-printed electrode (solid line). Potential sweep rate was 100 mV/s. (A colour version of this figure can be viewed online.)
SEM images of the BDDPs and the surface of BDDP-printed electrodes, and SCM images of the cross section of BDDP-printed electrodes are shown in Fig. 2. The particle sizes (~3.5 mm) of the H- and O-BDDP were not obviously different (Figs. 2a and b). In surface images of the BDDP-printed electrodes (Figs. 2c and d), the BDDP appeared to be packed uniformly and densely. However, the PES coverage differed on the O- and H-BDDP surfaces. The O-BDDP was partially covered by PES resin, with deep voids between the particles and the rough surface morphology of BDDP. On the other hand, H-BDDP was entirely covered by PES resin, with shallow voids between the particles and a smooth BDDP surface morphology. This difference likely originates from the different miscibilities of the BDDP surfaces in hydrophobic PES resin. As
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measured in the cross-sectional SCM images, the BDDP layer of the printed electrode was 50 mm thick at least (Figs. 2e and f). The sufficient thickness should prevent deformation of the electrode layer and/or penetration of the electrolyte solution through the layer. The electrochemical properties of the BDDP-printed electrodes were characterized by cyclic voltammetry (CV) in a solution of 1 mM Ru(NH3)6Cl3 in 0.1 M HClO4 (Fig. 3). The peak separation (DEP) was smaller in the CV of the O-BDDP-printed electrode than in the CV of the H-BDDP-printed electrode (96 mV vs. 126 mV), indicating that the electron-transfer rate was faster at the O-BDDPprinted electrode than at the H-BDDP-printed one. The CV results were corroborated by the results of electrochemical impedance spectroscopy. Fig. 4 shows the Nyquist plots for Ru(NH3)2þ/3þ at the 6 redox potential at the O- and H-BDDP-printed electrodes. The charge-transfer resistance (Rct), evaluated as the diameter of a semicircle in the high-frequency region, was evaluated as 20.9 U cm2 at the O-BDDP-printed electrode, and as 112 U cm2 at the HBDDP-printed electrode. As is already known, the electron-transfer rate of Ru(NH3)2þ/3þ at a BDD electrode is insensitive to the type 6 (hydrogen or oxygen) of surface termination [24]. Therefore, the faster electron-transfer rate at the O-BDDP-printed electrode might be explained by the incomplete PES resin coverage of the O-BDDP. The uncovered parts would be more exposed to the electrolyte solution than the H-BDDP-printed electrode. On the other hand, the series resistances (RS), evaluated as the Re (Z) in the high-frequency limit, were very similar in the O- and H-BDDP-printed electrodes (19.3 and 24.2 U cm2, respectively). Therefore, it is considered that the difference in electrical resistivities between the two electrodes negligibly affects the CV properties for Ru(NH3)2þ/3þ . 6 CV for 1 mM Ru(NH3)6Cl3 in 0.1 M HClO4 was performed in the potential range from 0.5 to þ1.3 V vs. Ag/AgCl to test the stability of the BDDP-printed electrodes to high anodic potentials. The CV showed almost no change in the shape or current after 100 cycles, indicating sufficient stability of the electrode (not shown). In addition, no change in the morphology was shown on the electrode surface by SEM observation.
3.3. Electrochemical detection of CIP at the BDD thin-film and BDDP-printed electrodes In a previous study, the CV curve of CIP at BDD thin-film electrodes presented one anodic peak at þ1.15 V vs. Ag/AgCl [15]. This peak was attributed to a two-electron reaction that oxidized the hydrogen of the piperazine skeleton to a hydroxyl group [15,25]. Fig. 5 shows linear sweep voltammograms (LSVs) of CIP at the BDD thin-film and BDDP-printed electrodes. The anodic peak was more intense at the H-BDD thin-film electrode than at the O-BDD thinfilm electrode. This feature is consistent with Garbellini et al. who prepared O-BDD by an anodic treatment [25]. In the present study, the anodic peak current density was larger at the H-BDD thin-film electrode than at the O-BDD thin-film electrode; consequently, the calibration curve was steeper at the H-BDD thin-film electrode (0.222 mA cm2 mM1) than at the O-BDD thin-film electrode (0.131 mA cm2 mM1). However, surface-terminating the BDDPprinted electrodes exerted the opposite effect on the electrochemical detection of CIP to that observed on the BDD thin-film electrodes: the anodic peak was sharper and the current density larger at the O-BDDP-printed electrode than at the H-BDDP-printed electrode. The slope of the calibration curve at the O-BDDP-printed electrode (0.215 mA cm2 mM1) was approximately twice that at the H-BDDP-printed electrode (0.110 mA cm2 mM1), and almost matched that of the H-BDD thin-film electrode. The anodic peak potential for CIP was more positive at the H-BDDP-printed electrode than at the O-BDDP-printed electrode, suggesting a slower charge-transfer rate of CIP oxidation at the former electrode. In summary, the O-BDDP-printed electrode detected CIP more sensitively than the H-BDDP-printed electrode, but the O-BDD thin-film electrode detected CIP less sensitively than the H-BDD thin-film electrode. We attribute this reversal to the low miscibility of OBDDP and PES resin binder, which would increase the apparent charge-transfer rate as discussed above for Ru(NH3)2þ/3þ . Thus, we 6 decided that the O-BDDP-printed electrode is more suitable for sensitive CIP detection than the H-BDDP-printed electrode. Peak current density in the LSV for CIP at BDD thin-film and BDDPprinted electrodes was found to decrease by successive scans possibly due to fouling by the CIP oxidation products. However, peak current density was recovered to the initial one after rinsing the electrode surface simply with deionized water. 3.4. Effect of PES/BDDP ratio
Fig. 4. Nyquist plots in a solution of 1 mM Ru(NH3)6Cl3 in 0.1 M HClO4 at the H-BDDPprinted (triangles) and O-BDDP-printed electrodes (circles). Inset indicates the equivalent circuit model. (A colour version of this figure can be viewed online.)
In our earlier study, we reported that the diffusion mode of BDDP-printed electrodes can be controlled by adjusting the insulating resin (PES)-to-BDDP ratio [18]. When the PES/BDDP ratio is large, a random microelectrode array effect appears at the BDDPprinted electrode, yielding microelectrode-like voltammograms with highly sensitive detection of electroactive analytes. Thus, we here investigated the effect of varying the PES/BDDP ratio on the CIP detection sensitivity of the O-BDDP-printed electrode. Fig. 6 compares the LSVs for CIP at O-BDDP-printed electrodes with PES/BDDP ratios of 0.5, 1.0 and 2.0. The calibration curves for CIP detection at these electrodes are also plotted. The random microelectrode array effect was confirmed by the remarkably decreased anodic current, and the improved signal-to-background ratio of CIP detection, as the PES/BDDP ratio increased (Table 2). However, the anodic peak for CIP shifted to highly positive potentials and became unclear, suggesting that a PES/BDDP ratio of 0.3 is useful for CIP detection in practice. The effect of the PES/BDDP ratio for the HBDDP-printed electrode was also investigated. Although the current density increased as the PES/BDDP ratio decreased from 0.3 to 0.1, however, it was still smaller than that obtained at the O-BDDPprinted electrode (PES/BDDP ratio of 0.3).
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Fig. 5. LSVs of CIP in 66.7 mM PBS at (a) H-BDD thin-film, (b) O-BDD thin-film, (c) H-BDDP-printed and (d) O-BDDP-printed electrodes. Potential sweep rate was 10 mV/s. Calibration curves of CIP detection by LSV at (e) H-BDD thin-film (dotted line) and O-BDD thin-film electrodes (solid line), (f) O-BDDP-printed (solid line), and H-BDDP-printed electrodes (dotted line). (A colour version of this figure can be viewed online.)
The calibration curve for CIP detection at the O-BDDP-printed electrode with a PES/BDDP ratio of 0.3 (the optimal electrode in this study), was linear in the 1e30 mM range. The relative standard deviation (RSD) of the curve was 2.74% and the LOD was 0.588 mM. To find the LOD, we multiplied the standard deviation by three and divided the result by the slope of the calibration curve. Other groups have achieved extremely low LODs for CIP detection at electrodes modified by an appropriate catalyst (such as graphene, or multi-walled film of carbon nanotubes) [9,10], or in enzymebased systems (horseradish peroxidase) [11]. Therefore, we consider that CIP could be even more sensitively detected by modifying our BDDP-printed electrodes with appropriate catalysts and enzymes. 3.5. Examination using artificial urine To examine the applicability of the O-BDDP-printed electrode to
CIP detection in human urine, we detected CIP in an artificial urine matrix. Human urine constitutes water (98%), urea produced by protein metabolism (2%) and trace amounts of other substances (such as sodium and ammonium chlorides) [26,27]. These constituents might interfere with or inhibit the CIP detection at the BDDP-printed electrode. Artificial urine samples containing various concentrations of CIP were prepared, and diluted 100 times by 66.7 mM PBS before recording the LSV at the O-BDDP-printed electrode (Fig. 7). The anodic peak for CIP in the diluted artificial urine was similar to that observed in PBS. The LSV was linear in the 1e30 mM range, and the slope of the calibration curve was 0.225 mA cm2 mM1, with an RSD of 2.36%, an LOD of 0.898 mM, and a recovery of 107%. As the linear concentration range was 100 times lower than that assumed in the actual urine of patients taking CIP [5], we inferred that the CIP concentration was determinable in the urine samples diluted by 100 times. In addition, the final CIP concentration in the 1e30 mM range was measurable even in artificial
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Fig. 6. LSVs of CIP in 66.7 mM PBS at O-BDDP-printed electrodes with different PES/BDDP ratios: (a) 0.5, (b) 1.0 and (c) 2.0. Potential sweep rate was 10 mV/s. (d) Calibration curves of CIP detection at O-BDDP-printed electrodes with different PES/BDDP ratios: (A) 0.3, (B) 0.5, (C) 1.0 and (D) 2.0. Plots for PES/BDDP ratio of 0.3 were created from the data in Fig. 5d. Straight lines indicate regression lines in the linear range of the calibration curves. (A colour version of this figure can be viewed online.)
Table 2 IP and IBG of CIP detection by LSV at the O-BDDP-printed electrode.a Electrode
O-BDDP O-BDDP O-BDDP O-BDDP a
0.3 0.5 1.0 2.0
Potential
IP
IBG
Slope
Linearity range
[V vs. Ag/AgCl]
[mA/cm2]
[mA/cm2]
[(mA/cm2)/mM]
[mM]
1.07 1.12 1.12 1.73
8.25 10.2 3.06 0.826
1.54 4.14 0.549 0.110
0.219 0.293 0.164 0.0217
1e30 1e10 1e10 1e30
S/B ratio
4.35 1.46 4.58 6.50
CIP concentration was 30 mM.
Fig. 7. (a) LSVs of CIP in artificial urine diluted 100 times in 66.7 mM PBS at the O-BDDP-printed electrode. Potential sweep rate was 10 mV/s. (b) Calibration curve created from the data in panel a. Final concentrations (after dilution) are indicated in (a). (A colour version of this figure can be viewed online.)
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Table 3 Analytical figures of merit in CIP detection by LSV at the BDD thin-film and BDDP-printed electrodes. Electrode
Sensitivity
R2
[(mA/cm2)/mM] H-BDD O-BDD H-BDDP O-BDDP O-BDDP in diluted artificial urine
0.222 0.131 0.110 0.215 0.225
urine diluted 10 times. We conclude that CIP in urine can be determined in the concentration range of 10e3000 mM by changing the dilution ratio. Table 3 summarizes the analytical figures of merit in CIP detection in this study, showing that the O-BDDP-printed electrodes should be useful for sensitive detection of CIP even from a urine matrix. 4. Conclusion We fabricated H- and O-BDDP-printed electrodes and investigated their electrochemical CIP-detection properties. Observing the SEM images, the surface of the printed O-BDDP electrode was found to be partially covered with PES resin, whereas the surface of the HBDDP electrode was almost completely covered with resin. From this result, we inferred that the total area of the BDDP/electrolyte solution interface was larger at the O-BDDP-printed electrode than at the H-BDDP-printed electrode, enabling a faster apparent electron-transfer rate for Ru(NH3)2þ/3þ and CIP at the O-BDDP 6 electrode. The O-BDDP-printed electrode exhibited a sharp LSV peak and a steep calibration curve of CIP detection. Under the optimal condition (PES/BDDP ¼ 0.3), the LOD for CIP detection was evaluated (by LSV) as 0.588 mM at the O-BDDP-printed electrode. CIP in artificial urine diluted with 66.7 mM PBS was detected over a wide concentration range (10e3000 mM) by changing the dilution ratio. The O-BDDP-printed electrode showed similar sensitivity to conventional BDD thin-film electrodes, but is more suitable for mass production. Therefore, we conclude that the O-BDDP-printed electrodes can provide a highly sensitive and disposable electrochemical sensor for CIP detection, especially in clinical situations. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Tomohiro Matsunaga: Investigation, Writing - original draft, Visualization. Takeshi Kondo: Conceptualization, Writing - review & editing, Supervision. Takahiro Osasa: Conceptualization. Akihiro Kotsugai: Conceptualization. Isao Shitanda: Resources. Yoshinao Hoshi: Resources. Masayuki Itagaki: Resources. Tatsuo Aikawa: Supervision. Toshifumi Tojo: Supervision. Makoto Yuasa: Project administration. Acknowledgments This research was partly supported by the Joint Usage/Research Program of the Photocatalysis International Research Center, Research Institute for Science and Technology, Tokyo University of Science and by Tokyo Ohka Foundation for the Promotion of Science and Technology.
0.996 0.996 0.975 0.999 0.998
Linearity range
RSD
LOD
[mM]
[%]
[mM]
1e30 1e30 1e30 1e30 1e30
2.11 2.74 2.30 2.74 2.36
0.281 0.715 0.0671 0.588 0.898
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Sensitive electrochemical detection of ciprofloxacin at screen-printed diamond electrodes Tomohiro Matsunaga a, Takeshi Kondo a, b, *, Takahiro Osasa c, Akihiro Kotsugai c, Isao Shitanda a, b, Yoshinao Hoshi a, Masayuki Itagaki a, b, Tatsuo Aikawa a, Toshifumi Tojo a, Makoto Yuasa a, b a b c
Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan Ricoh Co., Ltd, 2-7-1 Izumi, Ebina, Kanagawa, 243-0460, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 October 2019 Received in revised form 19 December 2019 Accepted 20 December 2019 Available online 23 December 2019
This study investigates the effect of chemical surface termination on the electrochemical characteristics of boron-doped diamond powder (BDDP). The aim is to realize highly sensitive electrochemical detection of ciprofloxacin (CIP) on BDDP-printed electrodes. To this end, we prepared oxygen-terminated BDDP (OBDDP) and hydrogen-terminated BDDP (H-BDDP), and mixed them with an insulating polyester (PES) resin binder to obtain BDDP ink for the printed electrode. Scanning electron microscopy of the BDDPprinted electrodes revealed that the O-BDDPs were partially covered with PES resin, while the HBDDPs were entirely covered with resin. This structural difference might explain the lower chargeredox reaction at the O-BDDP-printed electrode than at the Htransfer resistance of the Ru(NH3)2þ/3þ 6 BDDP-printed electrode. The slope of the calibration curve of the linear sweep voltammogram of CIP was steeper at the O-BDDP-printed electrode than at the H-BDDP-printed electrode and the O-BDD thin-film electrode, and was similar to that at the H-BDD thin-film electrode. Using the O-BDDP-printed electrode, we determined the CIP in artificial buffer-diluted urine in the concentration range 1e30 mM with a recovery of 107%. We conclude that the O-BDDP-printed electrodes provide a highly sensitive and disposable electrochemical sensor for CIP detection. © 2019 Elsevier Ltd. All rights reserved.
1. Introduction Most medicated antibiotics are known to be excreted from the body through urine and feces, and are eventually discharged into environmental water, exacerbating the rise of antibiotic-resistant bacteria. In 2017, the United Nations announced that discharging antibiotics into environmental water might spur the development of ferocious superbugs that are extensively or totally drug resistant; moreover, this problem is global and threatening and requires an
* Corresponding author. Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan. E-mail address: [email protected] (T. Kondo). 1 United Nations. Careless disposal of antibiotics could produce ‘ferocious superbugs,’ UN environment experts warn. Available online: https://news.un.org/ en/story/2017/12/638352-careless-disposal-antibiotics-could-produce-ferocioussuperbugs-un-environment (accessed on 29 September 2019). https://doi.org/10.1016/j.carbon.2019.12.051 0008-6223/© 2019 Elsevier Ltd. All rights reserved.
urgent solution.1 Many recent studies on this problem have developed technologies that determine antibiotic concentrations in environmental water and soil [1e3]. Ciprofloxacin (CIP) is a fluoroquinolone antibiotic used in the treatment of various infections such as urinary tract infections, respiratory infections, and gastrointestinal diseases. Being active against both gram-negative and gram-positive bacteria, CIP is broadly prescribed to humans and livestock [4e6]. CIP is also detected at high concentrations in river waters [7,8]. Against this background, researchers have measured CIP concentrations by various analytical techniques such as highperformance liquid chromatography, mass spectrometry, fluorescence, and ultravioletevisible absorption spectroscopy. Electrochemical measurements are popular as they are simple and quick, and CIP has been electrochemically detected by various types of electrodes [6,9e11]. The boron-doped diamond (BDD) electrode possesses superior characteristics such as extreme physical/chemical stability, small background current, and a wide potential window. Additionally, BDD cannot be fouled by adsorption of protein or
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Fig. 1. XPS patterns of (a) H-BDD (b) O-BDD (c) H-BDDP (c) O-BDDP.
other matter, so is suitable for electrochemical measurements in reported that body fluids such as blood and urine [12e14]. Radi cova a BDD thin-film electrode detects CIP at extremely low concentrations (limit of detection (LOD) ¼ 0.05 mM) [15]. BDD is typically deposited on silicon wafer substrate via chemical vapor deposition (CVD), a high-cost production method that is unsuitable for mass electrode production. As an alternative method, we investigated screen-printed diamond electrodes coated with BDD powder (BDDP). This approach is expected to realize a sensitive and disposable electrode for practical use, because of the superior characteristics of BDD and inexpensive substrate used [16e18]. The electrochemical characteristics of BDD thin-film electrodes are known to depend on the type of surface termination (hydrogen or oxygen) [19e21]. Therefore, we investigated how surface termination of the BDDP influences the electrochemical properties of screen-printed diamond electrodes, with a special focus on CIP detection. 2. Experimental Commercial diamond powder (DP) (Micron þ MDA 3-6, Element Six, nominal particle size 3e6 mm) was used as the core of BDDP. The BDDP was prepared as described in our previous report [16e18,22]. Briefly, 0.8 g of DP was subjected to microwave plasmaassisted chemical vapor deposition (MPCVD), which grew a BDD layer on the DP surface under 1300 W of plasma power and a hydrogen gas flow rate of 400 sccm at 800 C for 8 h. After the MPCVD process, the sample was heated in air at 425 C for 5 h to reduce the sp2 carbon impurities by oxidation [22]. This process resulted in oxidation of the BDDP surface (O-BDDP). The O-BDDP surface can be converted to the hydrogen-terminated form by hydrogen-plasma treatment of the MPCVD system under 500 W plasma power and a hydrogen gas flow rate of 100 sccm at 800 C for 1 h (H-BDDP). The electrical conductivity of the BDDP was measured as reported previously [22]. Briefly, the BDDP was packed into a glass tube (of diameter 1.0 mm) and its conductivity was calculated from the slope of the currentevoltage (IeV) curve. Here, the IeV measurements were taken between both ends of the packed BDDP [23]. BDDP ink containing BDDP and polyester resin (PES) (PES/BDDP ¼ 0.3 w/w) was prepared in a solvent of mixed isophorone and methyl ethyl ketone. The BDDP-printed electrode
was screen-printed onto a polyimide film substrate. The configuration of the BDDP-printed electrode was identical to that of an earlier reported electrode, with a geometric electrode area of 0.28 cm2 (diameter ¼ 6 mm) [17]. BDD thin-film electrode was also prepared by MPCVD. Heat treatment in air and hydrogen plasma treatment mentioned above were employed for oxygen and hydrogen termination of BDD thin-film (O- and H-BDD) electrodes, respectively. The surface terminations of O- and H-BDDP were analyzed by Xray photoelectron spectroscopy (XPS, Axis-Nova, Kratos, province, country). The surfaces of the BDDP-printed electrodes were observed by scanning electron microscopy (SEM, JSM-7600F, Jeol, province, country) and the cross sections of the BDDP-printed electrodes were observed by three-dimensional laser scanning confocal microscope (SCM, VK-X200, Keyence). Electrochemical measurements of the BDDP-printed electrodes were taken by a potentiostat (HZ-7000, Hokuto Denko). As CIP (FUJIFILM Wako) is poorly soluble in neutral solution, we first prepared a 10 mM stock solution of CIP in 0.5 M H2SO4. The stock solution was diluted by 66.7 mM (1/15 M) phosphate buffer solution (PBS) to obtain sample solutions of various concentrations at pH 7.0. Artificial urine containing urea, sodium chloride, ammonium chloride, and other urine constituents, as defined by Japanese industry standard (JIS T3214), was purchased from Isekyu (Aichi, Japan). 3. Result and discussion 3.1. Characterization of O- and H-BDDPs XPS wide spectra of H-BDD, O-BDD, H-BDDP, and O-BDDP are shown in Fig. 1, and the O/C atomic concentration ratios calculated from the spectra are listed in Table 1. When O- and H-BDDP (0.03 g
Table 1 O/C atomic concentration ratios evaluated by XPS. O/C ratio H-BDD O-BDD H-BDDP O-BDDP
0.016 0.098 0.031 0.083
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Fig. 2. SEM images of (a) H-BDDP, (b) O-BDDP, (c) H-BDDP-printed, and (d) O-BDDP-printed electrode surfaces. Cross-sectional images of (e) H- and (f) O-BDDP-printed electrodes. (i), (ii), and (iii) indicate polyimide film, carbon layer, and BDDP layer, respectively. (A colour version of this figure can be viewed online.)
each) were added to water (1.0 mL), the O-BDDP was easily dispersed whereas the H-BDDP floated on the water surface and was hardly dispersed. This result confirmed that the surface termination (oxygen or hydrogen) greatly affected the
hydrophilicity of BDDP. In contrast, the type of surface termination little affected the electrical conductivity of BDDP, which was measured as 0.2e0.7 S cm1 with some dispersion. We conclude that both O- and H-BDDP are sufficiently conductive for electrochemistry, and are equally suitable electrode materials with different hydrophilicities.
3.2. Properties of the BDDP-printed electrodes
Fig. 3. CVs of 1 mM Ru(NH3)6Cl3 in 0.1 M HClO4 at the H-BDDP-printed (dotted line) and O-BDDP-printed electrode (solid line). Potential sweep rate was 100 mV/s. (A colour version of this figure can be viewed online.)
SEM images of the BDDPs and the surface of BDDP-printed electrodes, and SCM images of the cross section of BDDP-printed electrodes are shown in Fig. 2. The particle sizes (~3.5 mm) of the H- and O-BDDP were not obviously different (Figs. 2a and b). In surface images of the BDDP-printed electrodes (Figs. 2c and d), the BDDP appeared to be packed uniformly and densely. However, the PES coverage differed on the O- and H-BDDP surfaces. The O-BDDP was partially covered by PES resin, with deep voids between the particles and the rough surface morphology of BDDP. On the other hand, H-BDDP was entirely covered by PES resin, with shallow voids between the particles and a smooth BDDP surface morphology. This difference likely originates from the different miscibilities of the BDDP surfaces in hydrophobic PES resin. As
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measured in the cross-sectional SCM images, the BDDP layer of the printed electrode was 50 mm thick at least (Figs. 2e and f). The sufficient thickness should prevent deformation of the electrode layer and/or penetration of the electrolyte solution through the layer. The electrochemical properties of the BDDP-printed electrodes were characterized by cyclic voltammetry (CV) in a solution of 1 mM Ru(NH3)6Cl3 in 0.1 M HClO4 (Fig. 3). The peak separation (DEP) was smaller in the CV of the O-BDDP-printed electrode than in the CV of the H-BDDP-printed electrode (96 mV vs. 126 mV), indicating that the electron-transfer rate was faster at the O-BDDPprinted electrode than at the H-BDDP-printed one. The CV results were corroborated by the results of electrochemical impedance spectroscopy. Fig. 4 shows the Nyquist plots for Ru(NH3)2þ/3þ at the 6 redox potential at the O- and H-BDDP-printed electrodes. The charge-transfer resistance (Rct), evaluated as the diameter of a semicircle in the high-frequency region, was evaluated as 20.9 U cm2 at the O-BDDP-printed electrode, and as 112 U cm2 at the HBDDP-printed electrode. As is already known, the electron-transfer rate of Ru(NH3)2þ/3þ at a BDD electrode is insensitive to the type 6 (hydrogen or oxygen) of surface termination [24]. Therefore, the faster electron-transfer rate at the O-BDDP-printed electrode might be explained by the incomplete PES resin coverage of the O-BDDP. The uncovered parts would be more exposed to the electrolyte solution than the H-BDDP-printed electrode. On the other hand, the series resistances (RS), evaluated as the Re (Z) in the high-frequency limit, were very similar in the O- and H-BDDP-printed electrodes (19.3 and 24.2 U cm2, respectively). Therefore, it is considered that the difference in electrical resistivities between the two electrodes negligibly affects the CV properties for Ru(NH3)2þ/3þ . 6 CV for 1 mM Ru(NH3)6Cl3 in 0.1 M HClO4 was performed in the potential range from 0.5 to þ1.3 V vs. Ag/AgCl to test the stability of the BDDP-printed electrodes to high anodic potentials. The CV showed almost no change in the shape or current after 100 cycles, indicating sufficient stability of the electrode (not shown). In addition, no change in the morphology was shown on the electrode surface by SEM observation.
3.3. Electrochemical detection of CIP at the BDD thin-film and BDDP-printed electrodes In a previous study, the CV curve of CIP at BDD thin-film electrodes presented one anodic peak at þ1.15 V vs. Ag/AgCl [15]. This peak was attributed to a two-electron reaction that oxidized the hydrogen of the piperazine skeleton to a hydroxyl group [15,25]. Fig. 5 shows linear sweep voltammograms (LSVs) of CIP at the BDD thin-film and BDDP-printed electrodes. The anodic peak was more intense at the H-BDD thin-film electrode than at the O-BDD thinfilm electrode. This feature is consistent with Garbellini et al. who prepared O-BDD by an anodic treatment [25]. In the present study, the anodic peak current density was larger at the H-BDD thin-film electrode than at the O-BDD thin-film electrode; consequently, the calibration curve was steeper at the H-BDD thin-film electrode (0.222 mA cm2 mM1) than at the O-BDD thin-film electrode (0.131 mA cm2 mM1). However, surface-terminating the BDDPprinted electrodes exerted the opposite effect on the electrochemical detection of CIP to that observed on the BDD thin-film electrodes: the anodic peak was sharper and the current density larger at the O-BDDP-printed electrode than at the H-BDDP-printed electrode. The slope of the calibration curve at the O-BDDP-printed electrode (0.215 mA cm2 mM1) was approximately twice that at the H-BDDP-printed electrode (0.110 mA cm2 mM1), and almost matched that of the H-BDD thin-film electrode. The anodic peak potential for CIP was more positive at the H-BDDP-printed electrode than at the O-BDDP-printed electrode, suggesting a slower charge-transfer rate of CIP oxidation at the former electrode. In summary, the O-BDDP-printed electrode detected CIP more sensitively than the H-BDDP-printed electrode, but the O-BDD thin-film electrode detected CIP less sensitively than the H-BDD thin-film electrode. We attribute this reversal to the low miscibility of OBDDP and PES resin binder, which would increase the apparent charge-transfer rate as discussed above for Ru(NH3)2þ/3þ . Thus, we 6 decided that the O-BDDP-printed electrode is more suitable for sensitive CIP detection than the H-BDDP-printed electrode. Peak current density in the LSV for CIP at BDD thin-film and BDDPprinted electrodes was found to decrease by successive scans possibly due to fouling by the CIP oxidation products. However, peak current density was recovered to the initial one after rinsing the electrode surface simply with deionized water. 3.4. Effect of PES/BDDP ratio
Fig. 4. Nyquist plots in a solution of 1 mM Ru(NH3)6Cl3 in 0.1 M HClO4 at the H-BDDPprinted (triangles) and O-BDDP-printed electrodes (circles). Inset indicates the equivalent circuit model. (A colour version of this figure can be viewed online.)
In our earlier study, we reported that the diffusion mode of BDDP-printed electrodes can be controlled by adjusting the insulating resin (PES)-to-BDDP ratio [18]. When the PES/BDDP ratio is large, a random microelectrode array effect appears at the BDDPprinted electrode, yielding microelectrode-like voltammograms with highly sensitive detection of electroactive analytes. Thus, we here investigated the effect of varying the PES/BDDP ratio on the CIP detection sensitivity of the O-BDDP-printed electrode. Fig. 6 compares the LSVs for CIP at O-BDDP-printed electrodes with PES/BDDP ratios of 0.5, 1.0 and 2.0. The calibration curves for CIP detection at these electrodes are also plotted. The random microelectrode array effect was confirmed by the remarkably decreased anodic current, and the improved signal-to-background ratio of CIP detection, as the PES/BDDP ratio increased (Table 2). However, the anodic peak for CIP shifted to highly positive potentials and became unclear, suggesting that a PES/BDDP ratio of 0.3 is useful for CIP detection in practice. The effect of the PES/BDDP ratio for the HBDDP-printed electrode was also investigated. Although the current density increased as the PES/BDDP ratio decreased from 0.3 to 0.1, however, it was still smaller than that obtained at the O-BDDPprinted electrode (PES/BDDP ratio of 0.3).
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Fig. 5. LSVs of CIP in 66.7 mM PBS at (a) H-BDD thin-film, (b) O-BDD thin-film, (c) H-BDDP-printed and (d) O-BDDP-printed electrodes. Potential sweep rate was 10 mV/s. Calibration curves of CIP detection by LSV at (e) H-BDD thin-film (dotted line) and O-BDD thin-film electrodes (solid line), (f) O-BDDP-printed (solid line), and H-BDDP-printed electrodes (dotted line). (A colour version of this figure can be viewed online.)
The calibration curve for CIP detection at the O-BDDP-printed electrode with a PES/BDDP ratio of 0.3 (the optimal electrode in this study), was linear in the 1e30 mM range. The relative standard deviation (RSD) of the curve was 2.74% and the LOD was 0.588 mM. To find the LOD, we multiplied the standard deviation by three and divided the result by the slope of the calibration curve. Other groups have achieved extremely low LODs for CIP detection at electrodes modified by an appropriate catalyst (such as graphene, or multi-walled film of carbon nanotubes) [9,10], or in enzymebased systems (horseradish peroxidase) [11]. Therefore, we consider that CIP could be even more sensitively detected by modifying our BDDP-printed electrodes with appropriate catalysts and enzymes. 3.5. Examination using artificial urine To examine the applicability of the O-BDDP-printed electrode to
CIP detection in human urine, we detected CIP in an artificial urine matrix. Human urine constitutes water (98%), urea produced by protein metabolism (2%) and trace amounts of other substances (such as sodium and ammonium chlorides) [26,27]. These constituents might interfere with or inhibit the CIP detection at the BDDP-printed electrode. Artificial urine samples containing various concentrations of CIP were prepared, and diluted 100 times by 66.7 mM PBS before recording the LSV at the O-BDDP-printed electrode (Fig. 7). The anodic peak for CIP in the diluted artificial urine was similar to that observed in PBS. The LSV was linear in the 1e30 mM range, and the slope of the calibration curve was 0.225 mA cm2 mM1, with an RSD of 2.36%, an LOD of 0.898 mM, and a recovery of 107%. As the linear concentration range was 100 times lower than that assumed in the actual urine of patients taking CIP [5], we inferred that the CIP concentration was determinable in the urine samples diluted by 100 times. In addition, the final CIP concentration in the 1e30 mM range was measurable even in artificial
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Fig. 6. LSVs of CIP in 66.7 mM PBS at O-BDDP-printed electrodes with different PES/BDDP ratios: (a) 0.5, (b) 1.0 and (c) 2.0. Potential sweep rate was 10 mV/s. (d) Calibration curves of CIP detection at O-BDDP-printed electrodes with different PES/BDDP ratios: (A) 0.3, (B) 0.5, (C) 1.0 and (D) 2.0. Plots for PES/BDDP ratio of 0.3 were created from the data in Fig. 5d. Straight lines indicate regression lines in the linear range of the calibration curves. (A colour version of this figure can be viewed online.)
Table 2 IP and IBG of CIP detection by LSV at the O-BDDP-printed electrode.a Electrode
O-BDDP O-BDDP O-BDDP O-BDDP a
0.3 0.5 1.0 2.0
Potential
IP
IBG
Slope
Linearity range
[V vs. Ag/AgCl]
[mA/cm2]
[mA/cm2]
[(mA/cm2)/mM]
[mM]
1.07 1.12 1.12 1.73
8.25 10.2 3.06 0.826
1.54 4.14 0.549 0.110
0.219 0.293 0.164 0.0217
1e30 1e10 1e10 1e30
S/B ratio
4.35 1.46 4.58 6.50
CIP concentration was 30 mM.
Fig. 7. (a) LSVs of CIP in artificial urine diluted 100 times in 66.7 mM PBS at the O-BDDP-printed electrode. Potential sweep rate was 10 mV/s. (b) Calibration curve created from the data in panel a. Final concentrations (after dilution) are indicated in (a). (A colour version of this figure can be viewed online.)
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Table 3 Analytical figures of merit in CIP detection by LSV at the BDD thin-film and BDDP-printed electrodes. Electrode
Sensitivity
R2
[(mA/cm2)/mM] H-BDD O-BDD H-BDDP O-BDDP O-BDDP in diluted artificial urine
0.222 0.131 0.110 0.215 0.225
urine diluted 10 times. We conclude that CIP in urine can be determined in the concentration range of 10e3000 mM by changing the dilution ratio. Table 3 summarizes the analytical figures of merit in CIP detection in this study, showing that the O-BDDP-printed electrodes should be useful for sensitive detection of CIP even from a urine matrix. 4. Conclusion We fabricated H- and O-BDDP-printed electrodes and investigated their electrochemical CIP-detection properties. Observing the SEM images, the surface of the printed O-BDDP electrode was found to be partially covered with PES resin, whereas the surface of the HBDDP electrode was almost completely covered with resin. From this result, we inferred that the total area of the BDDP/electrolyte solution interface was larger at the O-BDDP-printed electrode than at the H-BDDP-printed electrode, enabling a faster apparent electron-transfer rate for Ru(NH3)2þ/3þ and CIP at the O-BDDP 6 electrode. The O-BDDP-printed electrode exhibited a sharp LSV peak and a steep calibration curve of CIP detection. Under the optimal condition (PES/BDDP ¼ 0.3), the LOD for CIP detection was evaluated (by LSV) as 0.588 mM at the O-BDDP-printed electrode. CIP in artificial urine diluted with 66.7 mM PBS was detected over a wide concentration range (10e3000 mM) by changing the dilution ratio. The O-BDDP-printed electrode showed similar sensitivity to conventional BDD thin-film electrodes, but is more suitable for mass production. Therefore, we conclude that the O-BDDP-printed electrodes can provide a highly sensitive and disposable electrochemical sensor for CIP detection, especially in clinical situations. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Tomohiro Matsunaga: Investigation, Writing - original draft, Visualization. Takeshi Kondo: Conceptualization, Writing - review & editing, Supervision. Takahiro Osasa: Conceptualization. Akihiro Kotsugai: Conceptualization. Isao Shitanda: Resources. Yoshinao Hoshi: Resources. Masayuki Itagaki: Resources. Tatsuo Aikawa: Supervision. Toshifumi Tojo: Supervision. Makoto Yuasa: Project administration. Acknowledgments This research was partly supported by the Joint Usage/Research Program of the Photocatalysis International Research Center, Research Institute for Science and Technology, Tokyo University of Science and by Tokyo Ohka Foundation for the Promotion of Science and Technology.
0.996 0.996 0.975 0.999 0.998
Linearity range
RSD
LOD
[mM]
[%]
[mM]
1e30 1e30 1e30 1e30 1e30
2.11 2.74 2.30 2.74 2.36
0.281 0.715 0.0671 0.588 0.898
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