- Email: [email protected]
Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection
G Model EA 26249 No. of Pages 7
Electrochimica Acta xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection Atsumu Odaa,b , Dai Katoa,* , Kyoko Yoshiokaa , Mutsuo Tanakaa , Tomoyuki Kamataa,c , Masami Todokorod , Osamu Niwaa,b,* a
National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1Higashi, Tsukuba, Ibaraki 305-8566, Japan Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8573, Japan Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan d JNC Corporation, 5-1, Okawa, Kanazawa-ku, Yokohama 236-8605, Japan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 June 2015 Received in revised form 14 December 2015 Accepted 14 December 2015 Available online xxx
We describe a current amplification system that employs a fluorinated nanocarbon (F-nanocarbon) film electrode formed by unbalanced magnetron (UBM) sputtering with a short CF4 plasma treatment. The Fnanocarbon film exhibited the typical electrochemical reaction of a ferrocene-based mediator while strongly suppressing the electrochemical oxidation of Fe2+ ions. This selectivity provided the current amplification of ferrocene mediators with Fe2+ ions solely by using the F-nanocarbon film electrode without interference from the direct oxidation current of Fe2+ ions. The current amplification system was used to realize an electrochemical biosensor with superior performance for detecting lipopolysaccharides. A detection limit of 2 ng mL 1 with good reproducibility (RSD of 4.2%) was achieved thanks to the very low noise made by possible by the ultraflat and hydrophobic surface. ã 2015 Elsevier Ltd. All rights reserved.
Keywords: nanocarbon film surface fluorination signal amplification lipopolysaccharide electrochemical sensors
1. Introduction The electrochemical performance of carbon film electrodes can be widely controlled by controlling the surface termination/ doping/modification of other atoms [1]. Various surface terminations on carbon-based electrodes have been reported including hydrogen [2,3], oxygen [4], nitrogen [5], fluorine [6–15] and metals [16,17] for modulating electron transfer rates, which are dependent on analytes. Fluorine is a fascinating atom because it has high hydrophobicity and the highest electro-negativity in the periodic table. Fluorination has been reported for various carbon electrodes including graphite, glassy carbon (GC), carbon nanotubes (CNT), carbon nanofiber, graphene and boron-doped diamond (BDD) [6– 15]. Although these fluorinated carbon electrodes provide unique characteristics such as improved hydrophobicity and a different electron transfer rate from those of original carbon electrodes, the property of a fluorinated GC electrode is frequently lost because fluorine atoms are often removed from the GC electrode during electrochemical measurement [7,15]. Furthermore, the fluorinated GC electrode is unstable as regards high potential polarization or continuous measurement because its surface is easily oxidized
* Corresponding authors. E-mail addresses: [email protected] (D. Kato), [email protected] (O. Niwa).
[15,18]. In contrast, fluorinated BDD electrodes exhibit better longterm stability [12,14], suggesting that a fluorinated surface containing sp3 carbon exhibits less oxidization and damage under anodic polarization than GC. We previously reported electrochemically stable fluorinated nanocarbon (F-nanocarbon) film electrode formed by electron cyclotron resonance (ECR) sputtering with a short CF4 plasma treatment [15]. The nanocarbon film electrode has a nanocrystalline sp2 and sp3 mixed-bond structure with an atomically flat surface. The fluorinated surface is easily prepared without losing the surface conductivity and surface flatness of the nanocarbon film electrode. The F-nanocarbon film electrode also exhibits high electrochemical selectivity for some species. For example, the Fnanocarbon film electrode suppresses the electrochemical oxidation of hydrophilic and inner-sphere species such as Fe2+/3+ and Fe (CN)63 /4 [15], thanks to its hydrophobic surface. In contrast, the responses of hydrophilic and outer-sphere Ru(NH3)63+/2+ are almost unchanged. The F-nanocarbon film has a very stable surface compared with fluorinated GC in terms of continuous electrochemical measurements [15]. In fact, the slow electron transfer rates for Fe2+/3+ and Fe(CN)63 /4 at the F-nanocarbon film electrode still remain after 20–50 potential cycles, whereas these slow electron transfer rates are easily recovered for fluorinated GC under the same conditions owing to the desorption of fluorine containing groups from the surface. We also employed the
http://dx.doi.org/10.1016/j.electacta.2015.12.100 0013-4686/ ã 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
2
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx
F-nanocarbon film electrode to selectively detect hydrophobic antioxidants in foods and drinks [19]. The F-nanocarbon film electrode exhibited fast electron transfer for hydrophobic a-tocopherol (vitamin E). In contrast, the electrochemical responses for hydrophilic antioxidants such as ascorbic acid (vitamin C) were effectively suppressed at the F-nanocarbon film electrode [19]. These properties allowed us to achieve selective and quantitative measurements of hydrophobic antioxidants while suppressing the responses of hydrophilic antioxidants in the analyte solution. We expect this selectivity to enable us to construct a current amplification system in combination with a relatively hydrophobic and outer-sphere ferrocene mediator and hydrophilic and innersphere Fe2+/3+ as a reductant. Current amplification systems for electron transfer mediators have been widely studied by using an enzyme-modified electrode to improve the sensitivity and detection limit of various biomolecules [20,21]. If we achieve a current amplification system by redox cycling with an F-nanocarbon film electrode as shown Fig. 1(a), we can expect to use this system for bioelectroanalysis with a lower concentration and a high S/N ratio because the electrochemical inactivity of the Fnanocarbon film suppresses the direct oxidation of Fe2+ ions at a fluorinated surface. Here we describe a current amplification system that uses an Fnanocarbon film electrode, which is unlike enzymatic amplification. We employed F-nanocarbon film to obtain the selective electrochemical reaction of ferrocene-based mediator against an Fe2+/Fe3+ redox couple. Our aim is to apply this approach to an electrochemical biosensor for detecting lipopolysaccharides (LPS) thus achieving superior performance to that reported in our previous studies [22–24]. 2. Experimental 2.1. Carbon film preparation and CF4 plasma treatment In this study, nanocarbon film electrodes were deposited with the unbalanced magnetron (UBM) sputtering method [24,25]. The film has relatively good electrochemical properties similar to those of our previously reported nanocarbon film formed using ECR sputtering [26–33]. Briefly, the nanocarbon films were deposited on highly doped silicon (100) substrates with UBM sputtering equipment (Universal Systems, Japan) at room temperature (without substrate heating). The DC voltage applied to the carbon target was 480 V. The argon gas pressure used for the sputtering was 6.0 10 1 Pa. During deposition, the irradiation ion current density was 3.0 mA cm 2 and the ion acceleration voltage was 100 V. The nanocarbon films were obtained in about 50 min and were typically 40 nm thick. The carbon electrodes were fluorinated by using reactive ion etching (RIE) equipment (Model RIE-200L, SAMCO, Inc., Japan) in accordance with previous reports [15,19].
The radio frequency power was 40 W and the CF4 gas pressure and flow rate were 10 Pa and 10 sccm, respectively. The plasma treatment was performed for 30 s under the above conditions. 2.2. Film characterization X-ray photoelectron spectroscopy (XPS) was conducted with a Shimadzu/Kratos model AXIS Ultra (Al Ka 1486.6 eV) spectrometer to determine the elemental composition of the film surface. The F/ C and O/C ratios were calculated from the intensities of C1s, F1s and O1s (n = 2). The water contact angle was measured with a Drop Master DM 300 (Kyowa Interface Science Co., Ltd.). Milli-Q water droplets were used to characterize the surface hydrophobicity of the nanocarbon film electrode. 2.3. Chemicals Poly-e-lysine (e-PL) (see Fig. 3(a)) was supplied by JNC Corporation (Japan). Ferrocene-attached polymyxin B (FcPMB) (Fig. 3(b)) was synthesized as previously reported [23]. The LPS used in this study was Japanese pharmacopoeia reference standard endotoxin purchased from the Pharmaceutical and Medical Device Regulatory Science Society of Japan. BS3 (bis(sulfosuccinimidyl) suberate), a homobifunctionally water-soluble crosslinker for amines was purchased from Thermo Scientific Pierce (USA). All other chemicals were of analytical grade. 2.4. Electrochemical experiments All the electrochemical experiments were performed using an ALS/CHI 760B electrochemical analyzer (CH Instruments, Inc. USA). A platinum wire and an Ag/AgCl (3 M NaCl) electrode were used as auxiliary and reference electrodes, respectively. A nanocarbon film was used as the working electrode. After depositing the nanocarbon film on the Si wafer, we cut the film into rectangles, and then fixed masking tape with a 2 mm diameter hole in it onto these rectangles to form disk electrodes. A 50 mM acetate buffer (pH 5.0) was used as the electrolyte solution for the electrochemical measurements. For electrochemical LPS measurement, we fabricated, as outlined below, an F-nanocarbon film electrode modified with e-PL with a high affinity for LPS, which was similar to that described in our previous report [24]. A mixture solution (5 ml) containing 12.5 v/v% e-PL, 4 w/w% bovine serum albumin (BSA) and 2 wt% BS3 was placed on the electrodes, and then allowed to dry overnight. In this way, a crosslinked e-PL membrane was formed on the surface of the F-nanocarbon film electrode. We also measured the LPS concentration by using a conventional LAL measurement system (Endosafe1-PTSTM, Charles River Laboratories International (USA)).
Table 1 Surface properties of the nanocarbon film before and after fluorination. fluorination
C 1s (%)a F/Ca O/C a Contact angle/ b C0/mF cm 2
Fig. 1. Schematic illustration of a current amplification system using an Fnanocarbon film electrode (a) and the original nanocarbon film electrode as a comparison (b).
nanocarbon
sp2 content sp3 content
before
after
50.1 49.9 – 0.02 75.6 0.4 9.31
34.6 54.3 0.15 0.02 90.8 0.9 2.63
a The chemical components of C, F and O were obtained and analyzed using XPS analysis. b The contact angle was the average value (S.D.) obtained from 10 measurements.
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx
3
Fig. 2. CVs of (a) FcMeOH (0.1 mM) and (b) Fe2+ ions (1 and 5 mM) at the F-nanocarbon film electrode (solid) and the original nanocarbon film electrode (dotted). (c, d) CVs of FcMeOH (0.1 mM) with different Fe2+ ion concentrations (0 to 10 mM) at the F-nanocarbon film electrode (c) and the original nanocarbon film electrode (d). (e) Relationship between Fe2+ concentration and the amplified limiting current obtained from the result of (c). All the CVs were measured in 50 mM acetate buffer (pH 5.0) at a scan rate of 20 mV s 1.
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
4
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx
3. Results and discussion We first characterized the surface properties of the F-nanocarbon film (Table 1). The sp2/sp3 content estimated by XPS measurement was 50/50 and this was in good agreement with a previous report that used ECR sputtered nanocarbon films [25]. After fluorination, the sp2 content decreased from 50.1 to 34.6%. At the same time, the F/C ratio was 0.15, which is similar to the result of a previous study [15]. These results clearly indicate that the sp2 bond is selectively fluorinated with CF4 plasma. The contact angle of the film surface increased from 75.6 to 90.8 degrees after surface fluorination. We also determined the capacitance values (C0) of the F-nanocarbon film electrode by using cyclic voltammetry (CV) measurements at 0.25 V vs Ag/AgCl in 1 M KCl (scan rate of 10 mV s 1) [34]. The F-nanocarbon film electrode exhibited a C00 value of 2.63 mF cm 2, which was only one-fourth that of the original nanocarbon film (9.31 mF cm 2) electrode. The value was also comparable to that of an F-nanocarbon film electrode (3.32 mF cm 2) formed with ECR sputtering [15]. On the other hand, the GC electrode was treated with fluorination under the same condition, and exhibited a larger C00 value of 9.79 mF cm 2. We previously reported that the F-nanocarbon film electrode exhibited selective electrochemical responses [15,19]. In this study, we employed the F-nanocarbon film electrode to construct a current amplification system consisting of ferrocene methanol (FcMeOH) and Fe2+ ions whose concept is outlined in Fig. 1(a). Proof-of-concept experiments were performed with CVs of FcMeOH and Fe2+ as shown in Fig. 2. With FcMeOH, we observed clear redox couple peaks at 0.25 and 0.19 V vs. Ag/AgCl for Epa and Epc, respectively (Fig. 2(a), solid), which agreed with the result of a previous study [24]. We also measured the CV of FcMeOH at the original nanocarbon film electrode (Fig. 2(a), dotted), and observed no difference in the CV curves. This indicated that the electrochemical reaction of FcMeOH occurred regardless of surface fluorination because a ferrocene mediator is an outer-sphere system [35]. In contrast, the oxidation reaction of the Fe2+ ions was strongly suppressed at the F-nanocarbon film electrode (Fig. 2(b) solid lines). The F-nanocarbon film electrode showed a 97% reduction in the oxidation current of Fe2+ (1 mM) at 0.48 V compared with the ip value at the original nanocarbon film electrode (Fig. 2(b) dotted lines). Inner-sphere Fe2+/3+ is generally very sensitive to the presence of oxygen containing functionalities on an sp2 carbon electrode surface [15,35]. In this study, the O/C ratio (O/C = 0.02, Table 1) remained almost unchanged after fluorination. These results clearly demonstrated that surface fluorination contributed to the suppression of the direct oxidation
of Fe2+ ions unlike that of surface oxygen functionalities. These results indicate the possibility of constructing a current amplification system solely with an F-nanocarbon film electrode as shown in Fig. 1(a). To demonstrate this, we also carried out CVs for a mixture of FcMeOH and Fe2+ ions as shown in Fig. 2(c). As expected, the oxidation current of FcMeOH gradually increased with increasing Fe2+ ion concentration, and there was a change in the limiting current behavior from the peak current behavior. Meanwhile, the reduction peak and current of FcMeOH gradually decreased, resulting in the sigmoidal shapes of the voltammograms. This clearly demonstrated that a typical current amplification by redox-cycling system was achieved. That is, the Fc of FcMeOH is oxidized on the electrode to form Fc+, and regenerated by Fe2+. The consumption/regeneration cycle for FcMeOH resulted in the occurrence of redox cycling. It is worth noting that this current amplification was achieved solely by using the working electrode, which is unlike enzymatic amplification using enzyme modified electrodes. In contrast, current caused only by redox cycling was not observed at the original nanocarbon film electrode (Fig. 2(d)). Indeed, current amplification occurred even at the nanocarbon film electrode (around 0.25 V), but the direct oxidation current of Fe2+ (around 0.48 V) was also observed simultaneously. This resulted in the obtained current being larger than that of the F-nanocarbon film electrode because the direct oxidation current of the Fe2+ ions contributed to an increase in the current for the original nanocarbon film electrode, as shown in Fig. 1(b). Therefore, surface fluorination plays an important role in realizing current amplification caused solely by redox cycling. With the Fnanocarbon film electrode, the limiting current obtained at 0.35 V was gradually increased and saturated with the Fe2+ ion concentration as shown in Fig. 2(e). However, a closer inspection revealed that Fe2+ ions with a high concentration (5 mM) were subtly oxidized even at the F-nanocarbon film electrode (Fig. 2(b), solid line). From these results, the obtained current at the Fnanocarbon film electrode (with 5 mM Fe2+,Fig. 2(c)) also include the direct oxidation current of Fe2+ ions (approximately 5%). More efficient suppression of Fe2+ oxidation can be expected if we are to increase surface fluorine concentration of the nanocarbon film. However, in higher concentration region of Fe2+ ions, we confirmed that the produced Fe3+ ions with higher concentration were subtly precipitated overnight as a result of formation of Fe(OH)3 in the used buffer condition. Although the precipitation of Fe(OH)3 did not give an affect during our proposed measurement because the most important species was Fe2+ ions (rather than Fe3+ ions) that caused redox-cycling. But such precipitation is disadvantageous for the use of the repetitive measurement with long-term periods.
Fig. 3. Chemical structures of (a) poly-e-lysine (e-PL) and (b) ferrocene labeled PMB (FcPMB). (c) Schematic illustration of a measurement using a combination of FcPMB and the e-PL modified F-nanocarbon film electrode.
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx
Therefore, the addition of 1 mM of Fe2+ ions is sufficient for a selective current amplification system of ferrocene-based mediators at an F-nanocarbon film electrode. We employed this system to detect a biomolecule, lipopolysaccharide (LPS), also known as endotoxin, which is a major component of the outer membrane of Gram-negative bacteria. LPS must be removed from biological products that are administered intravenously (e.g. infusion fluids and injections including water precursor) [36,37], because LPS triggers a wide variety of biological effects such as septic shock in humans [38,39]. This makes it essential to measure the LPS content to ensure the quality of these cellular products. The Limulus amoebocyte lysate (LAL)based assay technique is now the gold standard as regards a conventional LPS assay [40]. This method offers extremely high sensitivity (or a low detection limit) and selectivity against LPS, however it is relatively expensive and time-consuming, especially for use in the lower LPS concentration region. On the other hand, an LAL reagent-free LPS analysis method has recently been developed [22–24,41–46]. Although this is an ideal assay technique thanks to its rapid analysis time and inexpensive reagents and an alternative to LAL-based assays, this detection system still requires improved sensitivity and selectivity. To achieve this, some studies have been reported that synthesized chemical probes for detecting LPS photometrically (colorimetrically and fluorometrically) [41,42], although the detection limits reported in these studies fall far short of the requirement. Recently, electrochemically based LPS detection systems have been reported that employ recognition molecules (mainly proteins) [43–46]. This is because the electrochemical technique is very simple and inexpensive, and so is expected to be used for bioanalysis. Inoue and co-workers developed an electrochemical LPS sensor using recombinant factor C zymogen that exhibited a highly sensitive and low detection limit) [44]. Recently we also developed an electrochemical system for detecting LPS that uses an original nanocarbon film electrode modified with poly-e-lysine (e-PL, Fig. 3(a)) with a high affinity and selectivity for LPS [24–48]. Fig. 3(c) outlines the electrochemical LPS detection protocol [24]. Briefly, we made the e-PL modified F-nanocarbon film as described in the Experimental section. The LPS was captured on the e-PL modified electrode surface, and then the second LPS-recognition molecule, ferrocene labeled polymyxin B (FcPMB, Fig. 3(b)), was captured on the LPS adsorbed electrode via the LPS-PMB affinity interaction. Finally, the FcPMB oxidation current was amplified by the addition of Fe2+ ions. In this proposed protocol, proof-of-concept experiments were performed with surface plasmon resonance (SPR) measurement using the e-PL modified SPR sensing chip (Fig. S1, Supplementary data). The e-PL surface was unambiguously captured LPS and PMB (Fig. S2). In addition, the SPR signal intensity was proportional to the LPS concentration. These results suggest that our proposed concept (part of Fig. 3a-b) will be successfully in progress. First we investigated the electrochemical response of the synthesized mediator FcPMB by using the F-nanocarbon film electrode. Fig. 4 shows CVs of the FcPMB at the F-nanocarbon film and the original nanocarbon film electrodes. In the measurements, both electrodes exhibited fine redox peaks at 0.510.54 V vs. Ag/AgCl. Compared with the result obtained for the original nanocarbon film electrode, the F-nanocarbon film exhibited sharper peaks as a result of the lower background current, even with the same Ep values. Therefore, we can also expect to realize an improvement in the measurement performance of LPS with low concentrations by using the F-nanocarbon film electrode. Fig. 5 shows CVs of the adsorbed FcPMB on the e-PL modified F-nanocarbon film electrodes with and without 200 ng mL 1 LPS. As previously reported, we observed that the current for FcPMB was different before (dotted blue line) and after (solid blue line) the addition of LPS. Moreover, the obtained currents were
5
Fig. 4. Voltammograms of FcPMB (20 mg mL 1) on the F-nanocarbon film electrode (red) and the original nanocarbon film electrode (blue) in 50 mM acetate buffer (pH 5.0). Scan rate = 100 mV s 1. (For interpretation of the references to color in this figure legend as well as in the text, the reader is referred to the web version of this article.)
Fig. 5. CVs of FcPMB adsorbed on the e-PL modified F-nanocarbon film electrode surface without (blue) and with (red) 1 mM Fe2+ ions in 50 mM acetate buffer (pH 5.0) solution. The dotted and solid lines, respectively, represent 0 and 200 ng mL 1 of LPS cast on the e-PL modified electrode. The scan rate was 100 mV s 1. 10 mL of FcPMB (200 mg mL 1) and 10 mL of LPS (200 ng mL 1) were cast on the surface. (For interpretation of the references to color in this figure legend as well as in the text, the reader is referred to the web version of this article.)
amplified in the presence of Fe2+ as shown by the red lines. As a result, the e-PL modified electrode provided a response for LPS that was amplified about 3.1 times compared with that in the Fe2+ ionfree solution. This amplification factor was low compared with that obtained at the original nanocarbon film electrode (4.1 times) [24]. This was presumably because the amplified current at the original nanocarbon film electrode contained the direct oxidation current of Fe2+ ions. Indeed, in a higher potential region (over 0.8 V vs Ag/ AgCl), the obtained current gradually increased as a result of the direct oxidation current of the Fe2+ ions [24], whereas there was no increase in the current with the F-nanocarbon film electrode. We estimated the quantitative performance of the LPS concentration on the e-PL modified F-nanocarbon film electrode. Fig. 6 shows the relationship between the LPS concentration and the current obtained by the e-PL modified fluorinated and the original nanocarbon film electrodes. With both electrodes, the current obtained from the adsorbed FcPMB was dependent on the adsorbed LPS concentration. The F-nanocarbon film electrode
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
6
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx
Fig. 6. Relationship between LPS concentration and obtained current at the e-PL modified F-nanocarbon film (red) and the original nanocarbon film (blue) electrodes. The red open square indicates the obtained current of LPS (200 ng mL 1) at the F-nanocarbon film electrode without Fe2+ ions. (For interpretation of the references to color in this figure legend as well as in the text, the reader is referred to the web version of this article.)
provided smaller current responses than those of the original nanocarbon film electrode because the signal amplification factor was smaller than at the original nanocarbon film electrode as described above. Meanwhile, the F-nanocarbon film electrode had a good linear relationship over a relatively wide concentration range (y = 13.1 7.4 x + 122.0 8.9 (R2 = 0.985), 0.02–200 ng mL 1 LPS), despite the fact that the current at the original nanocarbon film electrode had a narrower linear relationship (y = 21.8 11.2 x + 168.6 4.6 (R2 = 0.998), 2–200 ng mL 1 LPS). This is because the fluorinated surface exhibited a lower background current as described above. It is noteworthy that such good linear relationship was attributed to the amplified current in the presence of Fe2+ ions. In fact, in the absence of Fe2+ ions, the obtained current was 75.2 nA (red open square of Fig. 6), which was too small to be obtained the calibration curve. Moreover, we confirmed the accuracy of our results at the Fnanocarbon film electrode by comparison with conventional LAL method (with the range of 0.02–2 ng mL 1 LPS). The results exhibited a high correlation between them with a correlation coefficient of 0.999. However, the limit of detection (LOD) of the LPS was not improved (2.0 ng mL 1 (S/N = 3)), and was the same as that of our previous study [24]. Nevertheless, as regards the reproducibility of LPS measurements, the F-nanocarbon film electrode was more effective than the original nanocarbon film electrode. In fact, the relative standard deviation (RSD) value of 2 ng mL 1 LPS at the fluorinated surface was 4.2% (n = 3), which represents superior reproducibility to that of the original nanocarbon film electrode (9.4%, n = 4). This superior RSD value is comparable to that reported by Heras and co-workers (<4%) [45]. This is presumably because the F-nanocarbon film electrode exhibited a low background current and almost no reactivity of Fe2 + ions and this provided the relative superior reproducible response (small error bars). As regards selectivity performance, although we have yet to estimate the selectivity performance of the e-PL modified nanocarbon film electrode, one of the authors demonstrated that e-PL modified microparticle exhibited superior selectivity for LPS, compared with various proteins including ovalbumin, BSA, myoglobin, g-globulin, and lysozyme [47]. Therefore, we can expect that our developed electrode also demonstrate the good selectivity for LPS. Optimization of the crosslinked e-PL membrane will allow us to improve the quantitative performance as regards the low concentration of
LPS because we expect to obtain more efficient signal amplification while maintaining a low background current with superior RSD values. Moreover, covalent crosslinking between the e-PL membrane and the nanocarbon film surface is very essential to obtain more stable e-PL membrane capable of achieving repeatable measurements of LPS. Indeed, the repeatable experiment was not suitable for our electrode. Because our modified electrode was relatively weak against the washing process, due to the physical modification of the crosslinked e-PL membrane on the nanocarbon surface. The covalent crosslinking between the e-PL membrane and our F-nanocarbon film surface is very difficult because our nanocarbon film contains chemically stable sp3 bonds and the fluorine-functional groups. If we form this e-PL membrane with covalent crosslinking with the nanocarbon film surface, more stable membrane will be obtained, and more sufficient repeatability will be realized. Indeed, the SPR sensing exhibited the good repeatability of LPS measurement (Fig. S3(b)) because the e-PL was covalently crosslinked on the sensing surface. Therefore, we must undertake a further investigation to realize a quantitatively superior performance without the need for the conventional LAL reagents. This is because an LPS concentration limit is imposed by international regulations (e.g., around several pg mL 1-several tens of pg mL 1 [46,49]). 4. Conclusion We successfully used an F-nanocarbon film electrode to construct a current amplification system in combination with ferrocene-based mediators and Fe2+ ions as a reductant. The Fnanocarbon film exhibited a typical electrochemical reaction of ferrocene-based mediators while strongly suppressing the electrochemical oxidation of Fe2+ ions. This selectivity, which was realized solely by using the F-nanocarbon film electrode, provided fine current amplification without interference from the direct oxidation current of Fe2+ ions. The system was employed for the LPS detection using the FcPMB and e-PL modified F-nanocarbon film electrode. The obtained electrode system allowed us to detect LPS with an LOD of 2 ng mL 1 and high reproducibility (RSD value of 4.2%) without the conventional LAL reagents of expensive biological products. We consider that this current amplification system, which operates solely by using the F-nanocarbon film electrode, to be a very promising electrochemical sensing system for various biomolecules. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (D.K. No. 25288071) from the Ministry of Education, Culture, Science, Sports and Technology of Japan. This work was conducted in part at the Nano-Processing Facility, AIST, Japan. We thank Masumi Hirashima for help with the LAL experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.electacta.2015.12.100. References [1] R.L. McCreery, Advanced carbon electrode materials for molecular electrochemistry, Chem. Rev. 108 (2008) 2646–2687. [2] T.C. Kuo, R.L. McCreery, Surface chemistry and electron transfer kinetics of hydrogen-modified glassy carbon electrodes, Anal. Chem. 71 (1999) 1553– 1560. [3] Q.Y. Chen, G.M. Swain, Structural characterization, electrochemical reactivity, and response stability of hydrogenated glassy carbon electrodes, Langmuir 14 (1998) 7017–7026.
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx [4] M.C. Granger, G.M. Swain, The influence of surface interactions on the reversibility of ferri/ferrocyanide at boron-doped diamond thin-film electrodes, J. Electrochem. Soc. 146 (1999) 4551–4558. [5] A. Denisenko, A. Romanyuk, L.A. Kibler, E. Kohn, Surface structure and electrochemical characteristics of boron-doped diamond exposed to rf N-2plasma, J. Electroanal. Chem. 657 (2011) 164–171. [6] H. Touhara, F. Okino, Property control of carbon materials by fluorination, Carbon 38 (2000) 241–267. [7] T. Nakajima, M. Koh, R.N. Singh, M. Shimada, Electrochemical behavior of surface-fluorinated graphite, Electrochim. Acta 44 (1999) 2879–2888. [8] V.N. Khabashesku, W.E. Billups, J.L. Margrave, Fluorination of single-wall carbon nanotubes and subsequent derivatization reactions, Acc. Chem. Res. 35 (2002) 1087–1095. [9] D. Damien, P.M. Sudeep, T.N. Narayanan, M.R. Anantharaman, P.M. Ajayan, M. M. Shaijumon, Fluorinated graphene based electrodes for high performance primary lithium batteries, RSC Adv. 3 (2013) 25702–25706. [10] S. Boopathi, T.N. Narayanan, S.S. Kumar, Improved heterogeneous electron transfer kinetics of fluorinated graphene derivatives, Nanoscale 6 (2014) 10140–10146. [11] W. Zhang, M. Dubois, K. Guerin, P. Bonnet, H. Kharbache, F. Masin, P. Thomas, J. L. Mansot, A. Hamwi, Fluorinated nanocarbons using fluorinating agent: Strategies of fluorination and applications, Eur. Phys. J. B 75 (2010) 133–139. [12] S. Ferro, A. De Battisti, Physicochemical properties of fluorinated diamond electrodes, J. Phys. Chem. B 107 (2003) 7567–7573. [13] T. Kondo, H. Ito, K. Kusakabe, K. Ohkawa, K. Honda, Y. Einaga, A. Fujishima, T. Kawai, Characterization and electrochemical properties of CF4 plasma-treated boron-doped diamond surfaces, Diam. Relat. Mater. 17 (2008) 48–54. [14] E. Silva, A.C. Bastos, M. Neto, A.J. Fernandes, R. Silva, M.G. Silva Ferreira, M. Zheludkevich, F. Oliveira, New fluorinated diamond microelectrodes for localized detection of dissolved oxygen, Sens. Actuat. B 204 (2014) 544–551. [15] A. Ueda, D. Kato, N. Sekioka, T. Kamata, R. Kurita, H. Uetsuka, Y. Hattori, S. Hirono, S. Umemura, O. Niwa, Fabrication of electrochemically stable fluorinated nano-carbon film compared with other fluorinated carbon materials, Carbon 47 (2009) 1943–1952. [16] X. Dai, O. Nekrassova, M.E. Hyde, R.G. Compton, Anodic stripping voltammetry of arsenic(III) using gold nanoparticle-modified electrodesic stripping voltammetry of arsenic(III) using gold nanoparticle-modified electrodes, Anal. Chem. 76 (2004) 5924–5929. [17] R.H. Tian, T.N. Rao, Y. Einaga, J.F. Zhi, Construction of two-dimensional arrays gold nanoparticles monolayer onto boron-doped diamond electrode surfaces, Chem. Mater. 18 (2006) 939–945. [18] N. Sekioka, D. Kato, A. Ueda, T. Kamata, R. Kurita, S. Umemura, S. Hirono, O. Niwa, Controllable electrode activities of nano-carbon films while maintaining surface flatness by electrochemical pretreatment, Carbon 46 (2008) 1918– 1926. [19] E. Kuraya, S. Nagatomo, K. Sakata, D. Kato, O. Niwa, T. Nishimi, M. Kunitake, Simultaneous electrochemical analysis of hydrophilic and lipophilic antioxidants in bicontinuous microemulsion, Anal. Chem. 87 (2015) 1489– 1493. [20] F. Mizutani, S. Yabuki, Y. Sato, S. Iijima, Amperometric measurement of ds-DNA content using a peroxidase-modified electrode, Bioelectrochemistry 63 (2004) 257–259. [21] F. Mizutani, T. Yamanaka, Y. Tanabe, K. Tsuda, An enzyme electrode for Llactate with a chemically-amplified response, Anal. Chim. Acta 177 (1985) 153–166. [22] D. Kato, S. Iijima, R. Kurita, Y. Sato, J. Jia, S. Yabuki, F. Mizutani, O. Niwa, Electrochemically amplified detection for lipopolysaccharide using ferrocenylboronic acid, Biosens. Bioelectron. 22 (2007) 1527–1531. [23] S. Iijima, D. Kato, S. Yabuki, O. Niwa, F. Mizutani, Enzymatically amplified electrochemical detection for lipopolysaccharide using ferrocene-attached polymyxin B and its analogue, Biosens. Bioelectron. 26 (2011) 2080–2084. [24] D. Kato, A. Oda, M. Tanaka, S. Iijima, T. Kamata, M. Todokoro, Y. Yoshimi, O. Niwa, Poly-epsilon-lysine modified nanocarbon film electrodes for LPS detection, Electroanal 26 (2014) 618–624. [25] T. Kamata, D. Kato, H. Ida, O. Niwa, Structure and electrochemical characterization of carbon films formed by unbalanced magnetron (UBM) sputtering method, Diam. Relat. Mater. 49 (2014) 25–32. [26] O. Niwa, J. Jia, Y. Sato, D. Kato, R. Kurita, K. Maruyama, K. Suzuki, S. Hirono, Electrochemical performance of angstrom level flat sputtered carbon film consisting of sp(2) and sp(3) mixed bonds, J. Am. Chem. Soc. 128 (2006) 7144– 7145.
7
[27] J.B. Jia, D. Kato, R. Kurita, Y. Sato, K. Maruyama, K. Suzuki, S. Hirono, T. Ando, O. Niwa, Structure and electrochemical properties of carbon films prepared by a electron cyclotron resonance sputtering method, Anal. Chem. 79 (2007) 98– 105. [28] D. Kato, N. Sekioka, A. Ueda, R. Kurita, S. Hirono, K. Suzuki, O. Niwa, A nanocarbon film electrode as a platform for exploring DNA methylation, J. Am. Chem. Soc. 130 (2008) 3716–3717. [29] D. Kato, N. Sekioka, A. Ueda, R. Kurita, S. Hirono, K. Suzuki, O. Niwa, Nanohybrid carbon film for electrochemical detection of SNPs without hybridization or labeling, Angew. Chem. Int. Ed. 47 (2008) 6681–6684. [30] D. Kato, K. Goto, S. Fujii, A. Takatsu, S. Hirono, O. Niwa, Electrochemical DNA methylation detection for enzymatically digested CpG oligonucleotides, Anal. Chem. 83 (2011) 7595–7599. [31] D. Kato, M. Sumimoto, A. Ueda, S. Hirono, O. Niwa, Evaluation of electrokinetic parameters for all DNA bases with sputter deposited nanocarbon film electrode, Anal. Chem. 84 (2012) 10607–10613. [32] D. Kato, M. Komoriya, K. Nakamoto, R. Kurita, S. Hirono, O. Niwa, Electrochemical determination of oxidative damaged DNA with high sensitivity and stability using a nanocarbon film, Anal. Sci. 27 (2011) 703–707. [33] S. Yamamura, M. Hoshikawa, D. Kato, H. Saito, N. Suzuki, O. Niwa, M. Okada, ONO-2506 inhibits spike-wave discharges in a genetic animal model without affecting traditional convulsive tests via gliotransmission regulation, Br. J. Pharmacol. 168 (2013) 1088–1100. [34] A. Ueda, D. Kato, R. Kurita, T. Kamata, H. Inokuchi, S. Umemura, S. Hirono, O. Niwa, Efficient direct electron transfer with enzyme on a nanostructured carbon film fabricated with a maskless top-down UV/ozone process, J. Am. Chem. Soc. 133 (2011) 4840–4846. [35] H.V. Patten, S.C.S. Lai, J.V. Macpherson, P.R. Unwin, Active sites for outersphere, inner-sphere, and complex multistage electrochemical reactions at polycrystalline boron-doped diamond electrodes (pBDD) revealed with scanning electrochemical cell microscopy (SECCM), Anal. Chem. 84 (2012) 5427–5432. [36] J.M. Ahn, P. Wentworth, K.D. Janda, Probing lipase/esterase libraries for lipid A hydrolases - discovery of biocatalysts for the detoxification of bacteriallyexpressed recombinant protein, Chem. Commun. (2004) 364–365. [37] C. Hirayama, M. Sakata, Chromatographic removal of endotoxin from protein solutions by polymer particles, J. Chromatogr. B 781 (2002) 419–432. [38] S.M. Opal, P.J. Scannon, J.L. Vincent, M. White, S.F. Carroll, J.E. Palardy, N.A. Parejo, J.P. Pribble, J.H. Lemke, Relationship between plasma levels of lipopolysaccharide (LPS) and LPS-binding protein in patients with severe sepsis and septic shock, J. Infect. Dis. 180 (1999) 1584–1589. [39] C. Alexander, E.T. Rietschel, Bacterial lipopolysaccharides and innate immunity, J. Endotoxin Res. 7 (2001) 167–202. [40] J.L. Ding, B. Ho, A new era in pyrogen testing, Trends Biotechnol. 19 (2001) 277– 281. [41] J.C. Wu, A. Zawistowski, M. Ehrmann, T. Yi, C. Schmuck, Peptide functionalized polydiacetylene liposomes act as a fluorescent turn-on sensor for bacterial lipopolysaccharide, J. Am. Chem. Soc. 133 (2011) 9720–9723. [42] M.H. Lan, J.S. Wu, W.M. Liu, W.J. Zhang, J.C. Ge, H.Y. Zhang, J.Y. Sun, W.W. Zhao, P.F. Wang, Copolythiophene-derived colorimetric and fluorometric sensor for visually supersensitive determination of lipopolysaccharide, J. Am. Chem. Soc. 134 (2012) 6685–6694. [43] P. Miao, Electrochemical sensing strategies for the detection of endotoxin: a review, RSC Adv. 3 (2013) 9606–9617. [44] K.Y. Inoue, K. Ino, H. Shiku, T. Matsue, Electrochemical detection of endotoxin using recombinant factor C zymogen, Electrochem. Comm. 12 (2010) 1066– 1069. [45] J.Y. Heras, D. Pallarola, F. Battaglini, Electronic tongue for simultaneous detection of endotoxins and other contaminants of microbiological origin, Biosens. Bioelectron. 25 (2010) 2470–2476. [46] W. Su, M. Cho, J.D. Nam, W.S. Choe, Y. Lee, Aptamer-assisted gold nanoparticles/PEDOT platform for ultrasensitive detection of LPS, Electroanal 25 (2013) 380–386. [47] M. Sakata, T. Inoue, M. Todokoro, M. Kunitake, Limulus amebocyte lysate assay for endotoxins by an adsorption method with polycation-immobilized cellulose beads, Anal. Sci. 26 (2010) 291–296. [48] M. Sakata, Y. Fukuma, M. Todokoro, M. Kunitake, Selective assay for endotoxin using poly(epsilon-lysine)-immobilized Cellufine and Limulus amoebocyte lysate (LAL), Anal. Biochem. 385 (2009) 368–370. [49] Pyrogen and Endotoxins Testing: Questions and Answers, U.S. FDA, 2012
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
Electrochimica Acta xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection Atsumu Odaa,b , Dai Katoa,* , Kyoko Yoshiokaa , Mutsuo Tanakaa , Tomoyuki Kamataa,c , Masami Todokorod , Osamu Niwaa,b,* a
National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1Higashi, Tsukuba, Ibaraki 305-8566, Japan Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8573, Japan Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan d JNC Corporation, 5-1, Okawa, Kanazawa-ku, Yokohama 236-8605, Japan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 June 2015 Received in revised form 14 December 2015 Accepted 14 December 2015 Available online xxx
We describe a current amplification system that employs a fluorinated nanocarbon (F-nanocarbon) film electrode formed by unbalanced magnetron (UBM) sputtering with a short CF4 plasma treatment. The Fnanocarbon film exhibited the typical electrochemical reaction of a ferrocene-based mediator while strongly suppressing the electrochemical oxidation of Fe2+ ions. This selectivity provided the current amplification of ferrocene mediators with Fe2+ ions solely by using the F-nanocarbon film electrode without interference from the direct oxidation current of Fe2+ ions. The current amplification system was used to realize an electrochemical biosensor with superior performance for detecting lipopolysaccharides. A detection limit of 2 ng mL 1 with good reproducibility (RSD of 4.2%) was achieved thanks to the very low noise made by possible by the ultraflat and hydrophobic surface. ã 2015 Elsevier Ltd. All rights reserved.
Keywords: nanocarbon film surface fluorination signal amplification lipopolysaccharide electrochemical sensors
1. Introduction The electrochemical performance of carbon film electrodes can be widely controlled by controlling the surface termination/ doping/modification of other atoms [1]. Various surface terminations on carbon-based electrodes have been reported including hydrogen [2,3], oxygen [4], nitrogen [5], fluorine [6–15] and metals [16,17] for modulating electron transfer rates, which are dependent on analytes. Fluorine is a fascinating atom because it has high hydrophobicity and the highest electro-negativity in the periodic table. Fluorination has been reported for various carbon electrodes including graphite, glassy carbon (GC), carbon nanotubes (CNT), carbon nanofiber, graphene and boron-doped diamond (BDD) [6– 15]. Although these fluorinated carbon electrodes provide unique characteristics such as improved hydrophobicity and a different electron transfer rate from those of original carbon electrodes, the property of a fluorinated GC electrode is frequently lost because fluorine atoms are often removed from the GC electrode during electrochemical measurement [7,15]. Furthermore, the fluorinated GC electrode is unstable as regards high potential polarization or continuous measurement because its surface is easily oxidized
* Corresponding authors. E-mail addresses: [email protected] (D. Kato), [email protected] (O. Niwa).
[15,18]. In contrast, fluorinated BDD electrodes exhibit better longterm stability [12,14], suggesting that a fluorinated surface containing sp3 carbon exhibits less oxidization and damage under anodic polarization than GC. We previously reported electrochemically stable fluorinated nanocarbon (F-nanocarbon) film electrode formed by electron cyclotron resonance (ECR) sputtering with a short CF4 plasma treatment [15]. The nanocarbon film electrode has a nanocrystalline sp2 and sp3 mixed-bond structure with an atomically flat surface. The fluorinated surface is easily prepared without losing the surface conductivity and surface flatness of the nanocarbon film electrode. The F-nanocarbon film electrode also exhibits high electrochemical selectivity for some species. For example, the Fnanocarbon film electrode suppresses the electrochemical oxidation of hydrophilic and inner-sphere species such as Fe2+/3+ and Fe (CN)63 /4 [15], thanks to its hydrophobic surface. In contrast, the responses of hydrophilic and outer-sphere Ru(NH3)63+/2+ are almost unchanged. The F-nanocarbon film has a very stable surface compared with fluorinated GC in terms of continuous electrochemical measurements [15]. In fact, the slow electron transfer rates for Fe2+/3+ and Fe(CN)63 /4 at the F-nanocarbon film electrode still remain after 20–50 potential cycles, whereas these slow electron transfer rates are easily recovered for fluorinated GC under the same conditions owing to the desorption of fluorine containing groups from the surface. We also employed the
http://dx.doi.org/10.1016/j.electacta.2015.12.100 0013-4686/ ã 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
2
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx
F-nanocarbon film electrode to selectively detect hydrophobic antioxidants in foods and drinks [19]. The F-nanocarbon film electrode exhibited fast electron transfer for hydrophobic a-tocopherol (vitamin E). In contrast, the electrochemical responses for hydrophilic antioxidants such as ascorbic acid (vitamin C) were effectively suppressed at the F-nanocarbon film electrode [19]. These properties allowed us to achieve selective and quantitative measurements of hydrophobic antioxidants while suppressing the responses of hydrophilic antioxidants in the analyte solution. We expect this selectivity to enable us to construct a current amplification system in combination with a relatively hydrophobic and outer-sphere ferrocene mediator and hydrophilic and innersphere Fe2+/3+ as a reductant. Current amplification systems for electron transfer mediators have been widely studied by using an enzyme-modified electrode to improve the sensitivity and detection limit of various biomolecules [20,21]. If we achieve a current amplification system by redox cycling with an F-nanocarbon film electrode as shown Fig. 1(a), we can expect to use this system for bioelectroanalysis with a lower concentration and a high S/N ratio because the electrochemical inactivity of the Fnanocarbon film suppresses the direct oxidation of Fe2+ ions at a fluorinated surface. Here we describe a current amplification system that uses an Fnanocarbon film electrode, which is unlike enzymatic amplification. We employed F-nanocarbon film to obtain the selective electrochemical reaction of ferrocene-based mediator against an Fe2+/Fe3+ redox couple. Our aim is to apply this approach to an electrochemical biosensor for detecting lipopolysaccharides (LPS) thus achieving superior performance to that reported in our previous studies [22–24]. 2. Experimental 2.1. Carbon film preparation and CF4 plasma treatment In this study, nanocarbon film electrodes were deposited with the unbalanced magnetron (UBM) sputtering method [24,25]. The film has relatively good electrochemical properties similar to those of our previously reported nanocarbon film formed using ECR sputtering [26–33]. Briefly, the nanocarbon films were deposited on highly doped silicon (100) substrates with UBM sputtering equipment (Universal Systems, Japan) at room temperature (without substrate heating). The DC voltage applied to the carbon target was 480 V. The argon gas pressure used for the sputtering was 6.0 10 1 Pa. During deposition, the irradiation ion current density was 3.0 mA cm 2 and the ion acceleration voltage was 100 V. The nanocarbon films were obtained in about 50 min and were typically 40 nm thick. The carbon electrodes were fluorinated by using reactive ion etching (RIE) equipment (Model RIE-200L, SAMCO, Inc., Japan) in accordance with previous reports [15,19].
The radio frequency power was 40 W and the CF4 gas pressure and flow rate were 10 Pa and 10 sccm, respectively. The plasma treatment was performed for 30 s under the above conditions. 2.2. Film characterization X-ray photoelectron spectroscopy (XPS) was conducted with a Shimadzu/Kratos model AXIS Ultra (Al Ka 1486.6 eV) spectrometer to determine the elemental composition of the film surface. The F/ C and O/C ratios were calculated from the intensities of C1s, F1s and O1s (n = 2). The water contact angle was measured with a Drop Master DM 300 (Kyowa Interface Science Co., Ltd.). Milli-Q water droplets were used to characterize the surface hydrophobicity of the nanocarbon film electrode. 2.3. Chemicals Poly-e-lysine (e-PL) (see Fig. 3(a)) was supplied by JNC Corporation (Japan). Ferrocene-attached polymyxin B (FcPMB) (Fig. 3(b)) was synthesized as previously reported [23]. The LPS used in this study was Japanese pharmacopoeia reference standard endotoxin purchased from the Pharmaceutical and Medical Device Regulatory Science Society of Japan. BS3 (bis(sulfosuccinimidyl) suberate), a homobifunctionally water-soluble crosslinker for amines was purchased from Thermo Scientific Pierce (USA). All other chemicals were of analytical grade. 2.4. Electrochemical experiments All the electrochemical experiments were performed using an ALS/CHI 760B electrochemical analyzer (CH Instruments, Inc. USA). A platinum wire and an Ag/AgCl (3 M NaCl) electrode were used as auxiliary and reference electrodes, respectively. A nanocarbon film was used as the working electrode. After depositing the nanocarbon film on the Si wafer, we cut the film into rectangles, and then fixed masking tape with a 2 mm diameter hole in it onto these rectangles to form disk electrodes. A 50 mM acetate buffer (pH 5.0) was used as the electrolyte solution for the electrochemical measurements. For electrochemical LPS measurement, we fabricated, as outlined below, an F-nanocarbon film electrode modified with e-PL with a high affinity for LPS, which was similar to that described in our previous report [24]. A mixture solution (5 ml) containing 12.5 v/v% e-PL, 4 w/w% bovine serum albumin (BSA) and 2 wt% BS3 was placed on the electrodes, and then allowed to dry overnight. In this way, a crosslinked e-PL membrane was formed on the surface of the F-nanocarbon film electrode. We also measured the LPS concentration by using a conventional LAL measurement system (Endosafe1-PTSTM, Charles River Laboratories International (USA)).
Table 1 Surface properties of the nanocarbon film before and after fluorination. fluorination
C 1s (%)a F/Ca O/C a Contact angle/ b C0/mF cm 2
Fig. 1. Schematic illustration of a current amplification system using an Fnanocarbon film electrode (a) and the original nanocarbon film electrode as a comparison (b).
nanocarbon
sp2 content sp3 content
before
after
50.1 49.9 – 0.02 75.6 0.4 9.31
34.6 54.3 0.15 0.02 90.8 0.9 2.63
a The chemical components of C, F and O were obtained and analyzed using XPS analysis. b The contact angle was the average value (S.D.) obtained from 10 measurements.
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx
3
Fig. 2. CVs of (a) FcMeOH (0.1 mM) and (b) Fe2+ ions (1 and 5 mM) at the F-nanocarbon film electrode (solid) and the original nanocarbon film electrode (dotted). (c, d) CVs of FcMeOH (0.1 mM) with different Fe2+ ion concentrations (0 to 10 mM) at the F-nanocarbon film electrode (c) and the original nanocarbon film electrode (d). (e) Relationship between Fe2+ concentration and the amplified limiting current obtained from the result of (c). All the CVs were measured in 50 mM acetate buffer (pH 5.0) at a scan rate of 20 mV s 1.
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
4
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx
3. Results and discussion We first characterized the surface properties of the F-nanocarbon film (Table 1). The sp2/sp3 content estimated by XPS measurement was 50/50 and this was in good agreement with a previous report that used ECR sputtered nanocarbon films [25]. After fluorination, the sp2 content decreased from 50.1 to 34.6%. At the same time, the F/C ratio was 0.15, which is similar to the result of a previous study [15]. These results clearly indicate that the sp2 bond is selectively fluorinated with CF4 plasma. The contact angle of the film surface increased from 75.6 to 90.8 degrees after surface fluorination. We also determined the capacitance values (C0) of the F-nanocarbon film electrode by using cyclic voltammetry (CV) measurements at 0.25 V vs Ag/AgCl in 1 M KCl (scan rate of 10 mV s 1) [34]. The F-nanocarbon film electrode exhibited a C00 value of 2.63 mF cm 2, which was only one-fourth that of the original nanocarbon film (9.31 mF cm 2) electrode. The value was also comparable to that of an F-nanocarbon film electrode (3.32 mF cm 2) formed with ECR sputtering [15]. On the other hand, the GC electrode was treated with fluorination under the same condition, and exhibited a larger C00 value of 9.79 mF cm 2. We previously reported that the F-nanocarbon film electrode exhibited selective electrochemical responses [15,19]. In this study, we employed the F-nanocarbon film electrode to construct a current amplification system consisting of ferrocene methanol (FcMeOH) and Fe2+ ions whose concept is outlined in Fig. 1(a). Proof-of-concept experiments were performed with CVs of FcMeOH and Fe2+ as shown in Fig. 2. With FcMeOH, we observed clear redox couple peaks at 0.25 and 0.19 V vs. Ag/AgCl for Epa and Epc, respectively (Fig. 2(a), solid), which agreed with the result of a previous study [24]. We also measured the CV of FcMeOH at the original nanocarbon film electrode (Fig. 2(a), dotted), and observed no difference in the CV curves. This indicated that the electrochemical reaction of FcMeOH occurred regardless of surface fluorination because a ferrocene mediator is an outer-sphere system [35]. In contrast, the oxidation reaction of the Fe2+ ions was strongly suppressed at the F-nanocarbon film electrode (Fig. 2(b) solid lines). The F-nanocarbon film electrode showed a 97% reduction in the oxidation current of Fe2+ (1 mM) at 0.48 V compared with the ip value at the original nanocarbon film electrode (Fig. 2(b) dotted lines). Inner-sphere Fe2+/3+ is generally very sensitive to the presence of oxygen containing functionalities on an sp2 carbon electrode surface [15,35]. In this study, the O/C ratio (O/C = 0.02, Table 1) remained almost unchanged after fluorination. These results clearly demonstrated that surface fluorination contributed to the suppression of the direct oxidation
of Fe2+ ions unlike that of surface oxygen functionalities. These results indicate the possibility of constructing a current amplification system solely with an F-nanocarbon film electrode as shown in Fig. 1(a). To demonstrate this, we also carried out CVs for a mixture of FcMeOH and Fe2+ ions as shown in Fig. 2(c). As expected, the oxidation current of FcMeOH gradually increased with increasing Fe2+ ion concentration, and there was a change in the limiting current behavior from the peak current behavior. Meanwhile, the reduction peak and current of FcMeOH gradually decreased, resulting in the sigmoidal shapes of the voltammograms. This clearly demonstrated that a typical current amplification by redox-cycling system was achieved. That is, the Fc of FcMeOH is oxidized on the electrode to form Fc+, and regenerated by Fe2+. The consumption/regeneration cycle for FcMeOH resulted in the occurrence of redox cycling. It is worth noting that this current amplification was achieved solely by using the working electrode, which is unlike enzymatic amplification using enzyme modified electrodes. In contrast, current caused only by redox cycling was not observed at the original nanocarbon film electrode (Fig. 2(d)). Indeed, current amplification occurred even at the nanocarbon film electrode (around 0.25 V), but the direct oxidation current of Fe2+ (around 0.48 V) was also observed simultaneously. This resulted in the obtained current being larger than that of the F-nanocarbon film electrode because the direct oxidation current of the Fe2+ ions contributed to an increase in the current for the original nanocarbon film electrode, as shown in Fig. 1(b). Therefore, surface fluorination plays an important role in realizing current amplification caused solely by redox cycling. With the Fnanocarbon film electrode, the limiting current obtained at 0.35 V was gradually increased and saturated with the Fe2+ ion concentration as shown in Fig. 2(e). However, a closer inspection revealed that Fe2+ ions with a high concentration (5 mM) were subtly oxidized even at the F-nanocarbon film electrode (Fig. 2(b), solid line). From these results, the obtained current at the Fnanocarbon film electrode (with 5 mM Fe2+,Fig. 2(c)) also include the direct oxidation current of Fe2+ ions (approximately 5%). More efficient suppression of Fe2+ oxidation can be expected if we are to increase surface fluorine concentration of the nanocarbon film. However, in higher concentration region of Fe2+ ions, we confirmed that the produced Fe3+ ions with higher concentration were subtly precipitated overnight as a result of formation of Fe(OH)3 in the used buffer condition. Although the precipitation of Fe(OH)3 did not give an affect during our proposed measurement because the most important species was Fe2+ ions (rather than Fe3+ ions) that caused redox-cycling. But such precipitation is disadvantageous for the use of the repetitive measurement with long-term periods.
Fig. 3. Chemical structures of (a) poly-e-lysine (e-PL) and (b) ferrocene labeled PMB (FcPMB). (c) Schematic illustration of a measurement using a combination of FcPMB and the e-PL modified F-nanocarbon film electrode.
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx
Therefore, the addition of 1 mM of Fe2+ ions is sufficient for a selective current amplification system of ferrocene-based mediators at an F-nanocarbon film electrode. We employed this system to detect a biomolecule, lipopolysaccharide (LPS), also known as endotoxin, which is a major component of the outer membrane of Gram-negative bacteria. LPS must be removed from biological products that are administered intravenously (e.g. infusion fluids and injections including water precursor) [36,37], because LPS triggers a wide variety of biological effects such as septic shock in humans [38,39]. This makes it essential to measure the LPS content to ensure the quality of these cellular products. The Limulus amoebocyte lysate (LAL)based assay technique is now the gold standard as regards a conventional LPS assay [40]. This method offers extremely high sensitivity (or a low detection limit) and selectivity against LPS, however it is relatively expensive and time-consuming, especially for use in the lower LPS concentration region. On the other hand, an LAL reagent-free LPS analysis method has recently been developed [22–24,41–46]. Although this is an ideal assay technique thanks to its rapid analysis time and inexpensive reagents and an alternative to LAL-based assays, this detection system still requires improved sensitivity and selectivity. To achieve this, some studies have been reported that synthesized chemical probes for detecting LPS photometrically (colorimetrically and fluorometrically) [41,42], although the detection limits reported in these studies fall far short of the requirement. Recently, electrochemically based LPS detection systems have been reported that employ recognition molecules (mainly proteins) [43–46]. This is because the electrochemical technique is very simple and inexpensive, and so is expected to be used for bioanalysis. Inoue and co-workers developed an electrochemical LPS sensor using recombinant factor C zymogen that exhibited a highly sensitive and low detection limit) [44]. Recently we also developed an electrochemical system for detecting LPS that uses an original nanocarbon film electrode modified with poly-e-lysine (e-PL, Fig. 3(a)) with a high affinity and selectivity for LPS [24–48]. Fig. 3(c) outlines the electrochemical LPS detection protocol [24]. Briefly, we made the e-PL modified F-nanocarbon film as described in the Experimental section. The LPS was captured on the e-PL modified electrode surface, and then the second LPS-recognition molecule, ferrocene labeled polymyxin B (FcPMB, Fig. 3(b)), was captured on the LPS adsorbed electrode via the LPS-PMB affinity interaction. Finally, the FcPMB oxidation current was amplified by the addition of Fe2+ ions. In this proposed protocol, proof-of-concept experiments were performed with surface plasmon resonance (SPR) measurement using the e-PL modified SPR sensing chip (Fig. S1, Supplementary data). The e-PL surface was unambiguously captured LPS and PMB (Fig. S2). In addition, the SPR signal intensity was proportional to the LPS concentration. These results suggest that our proposed concept (part of Fig. 3a-b) will be successfully in progress. First we investigated the electrochemical response of the synthesized mediator FcPMB by using the F-nanocarbon film electrode. Fig. 4 shows CVs of the FcPMB at the F-nanocarbon film and the original nanocarbon film electrodes. In the measurements, both electrodes exhibited fine redox peaks at 0.510.54 V vs. Ag/AgCl. Compared with the result obtained for the original nanocarbon film electrode, the F-nanocarbon film exhibited sharper peaks as a result of the lower background current, even with the same Ep values. Therefore, we can also expect to realize an improvement in the measurement performance of LPS with low concentrations by using the F-nanocarbon film electrode. Fig. 5 shows CVs of the adsorbed FcPMB on the e-PL modified F-nanocarbon film electrodes with and without 200 ng mL 1 LPS. As previously reported, we observed that the current for FcPMB was different before (dotted blue line) and after (solid blue line) the addition of LPS. Moreover, the obtained currents were
5
Fig. 4. Voltammograms of FcPMB (20 mg mL 1) on the F-nanocarbon film electrode (red) and the original nanocarbon film electrode (blue) in 50 mM acetate buffer (pH 5.0). Scan rate = 100 mV s 1. (For interpretation of the references to color in this figure legend as well as in the text, the reader is referred to the web version of this article.)
Fig. 5. CVs of FcPMB adsorbed on the e-PL modified F-nanocarbon film electrode surface without (blue) and with (red) 1 mM Fe2+ ions in 50 mM acetate buffer (pH 5.0) solution. The dotted and solid lines, respectively, represent 0 and 200 ng mL 1 of LPS cast on the e-PL modified electrode. The scan rate was 100 mV s 1. 10 mL of FcPMB (200 mg mL 1) and 10 mL of LPS (200 ng mL 1) were cast on the surface. (For interpretation of the references to color in this figure legend as well as in the text, the reader is referred to the web version of this article.)
amplified in the presence of Fe2+ as shown by the red lines. As a result, the e-PL modified electrode provided a response for LPS that was amplified about 3.1 times compared with that in the Fe2+ ionfree solution. This amplification factor was low compared with that obtained at the original nanocarbon film electrode (4.1 times) [24]. This was presumably because the amplified current at the original nanocarbon film electrode contained the direct oxidation current of Fe2+ ions. Indeed, in a higher potential region (over 0.8 V vs Ag/ AgCl), the obtained current gradually increased as a result of the direct oxidation current of the Fe2+ ions [24], whereas there was no increase in the current with the F-nanocarbon film electrode. We estimated the quantitative performance of the LPS concentration on the e-PL modified F-nanocarbon film electrode. Fig. 6 shows the relationship between the LPS concentration and the current obtained by the e-PL modified fluorinated and the original nanocarbon film electrodes. With both electrodes, the current obtained from the adsorbed FcPMB was dependent on the adsorbed LPS concentration. The F-nanocarbon film electrode
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
6
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx
Fig. 6. Relationship between LPS concentration and obtained current at the e-PL modified F-nanocarbon film (red) and the original nanocarbon film (blue) electrodes. The red open square indicates the obtained current of LPS (200 ng mL 1) at the F-nanocarbon film electrode without Fe2+ ions. (For interpretation of the references to color in this figure legend as well as in the text, the reader is referred to the web version of this article.)
provided smaller current responses than those of the original nanocarbon film electrode because the signal amplification factor was smaller than at the original nanocarbon film electrode as described above. Meanwhile, the F-nanocarbon film electrode had a good linear relationship over a relatively wide concentration range (y = 13.1 7.4 x + 122.0 8.9 (R2 = 0.985), 0.02–200 ng mL 1 LPS), despite the fact that the current at the original nanocarbon film electrode had a narrower linear relationship (y = 21.8 11.2 x + 168.6 4.6 (R2 = 0.998), 2–200 ng mL 1 LPS). This is because the fluorinated surface exhibited a lower background current as described above. It is noteworthy that such good linear relationship was attributed to the amplified current in the presence of Fe2+ ions. In fact, in the absence of Fe2+ ions, the obtained current was 75.2 nA (red open square of Fig. 6), which was too small to be obtained the calibration curve. Moreover, we confirmed the accuracy of our results at the Fnanocarbon film electrode by comparison with conventional LAL method (with the range of 0.02–2 ng mL 1 LPS). The results exhibited a high correlation between them with a correlation coefficient of 0.999. However, the limit of detection (LOD) of the LPS was not improved (2.0 ng mL 1 (S/N = 3)), and was the same as that of our previous study [24]. Nevertheless, as regards the reproducibility of LPS measurements, the F-nanocarbon film electrode was more effective than the original nanocarbon film electrode. In fact, the relative standard deviation (RSD) value of 2 ng mL 1 LPS at the fluorinated surface was 4.2% (n = 3), which represents superior reproducibility to that of the original nanocarbon film electrode (9.4%, n = 4). This superior RSD value is comparable to that reported by Heras and co-workers (<4%) [45]. This is presumably because the F-nanocarbon film electrode exhibited a low background current and almost no reactivity of Fe2 + ions and this provided the relative superior reproducible response (small error bars). As regards selectivity performance, although we have yet to estimate the selectivity performance of the e-PL modified nanocarbon film electrode, one of the authors demonstrated that e-PL modified microparticle exhibited superior selectivity for LPS, compared with various proteins including ovalbumin, BSA, myoglobin, g-globulin, and lysozyme [47]. Therefore, we can expect that our developed electrode also demonstrate the good selectivity for LPS. Optimization of the crosslinked e-PL membrane will allow us to improve the quantitative performance as regards the low concentration of
LPS because we expect to obtain more efficient signal amplification while maintaining a low background current with superior RSD values. Moreover, covalent crosslinking between the e-PL membrane and the nanocarbon film surface is very essential to obtain more stable e-PL membrane capable of achieving repeatable measurements of LPS. Indeed, the repeatable experiment was not suitable for our electrode. Because our modified electrode was relatively weak against the washing process, due to the physical modification of the crosslinked e-PL membrane on the nanocarbon surface. The covalent crosslinking between the e-PL membrane and our F-nanocarbon film surface is very difficult because our nanocarbon film contains chemically stable sp3 bonds and the fluorine-functional groups. If we form this e-PL membrane with covalent crosslinking with the nanocarbon film surface, more stable membrane will be obtained, and more sufficient repeatability will be realized. Indeed, the SPR sensing exhibited the good repeatability of LPS measurement (Fig. S3(b)) because the e-PL was covalently crosslinked on the sensing surface. Therefore, we must undertake a further investigation to realize a quantitatively superior performance without the need for the conventional LAL reagents. This is because an LPS concentration limit is imposed by international regulations (e.g., around several pg mL 1-several tens of pg mL 1 [46,49]). 4. Conclusion We successfully used an F-nanocarbon film electrode to construct a current amplification system in combination with ferrocene-based mediators and Fe2+ ions as a reductant. The Fnanocarbon film exhibited a typical electrochemical reaction of ferrocene-based mediators while strongly suppressing the electrochemical oxidation of Fe2+ ions. This selectivity, which was realized solely by using the F-nanocarbon film electrode, provided fine current amplification without interference from the direct oxidation current of Fe2+ ions. The system was employed for the LPS detection using the FcPMB and e-PL modified F-nanocarbon film electrode. The obtained electrode system allowed us to detect LPS with an LOD of 2 ng mL 1 and high reproducibility (RSD value of 4.2%) without the conventional LAL reagents of expensive biological products. We consider that this current amplification system, which operates solely by using the F-nanocarbon film electrode, to be a very promising electrochemical sensing system for various biomolecules. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (D.K. No. 25288071) from the Ministry of Education, Culture, Science, Sports and Technology of Japan. This work was conducted in part at the Nano-Processing Facility, AIST, Japan. We thank Masumi Hirashima for help with the LAL experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.electacta.2015.12.100. References [1] R.L. McCreery, Advanced carbon electrode materials for molecular electrochemistry, Chem. Rev. 108 (2008) 2646–2687. [2] T.C. Kuo, R.L. McCreery, Surface chemistry and electron transfer kinetics of hydrogen-modified glassy carbon electrodes, Anal. Chem. 71 (1999) 1553– 1560. [3] Q.Y. Chen, G.M. Swain, Structural characterization, electrochemical reactivity, and response stability of hydrogenated glassy carbon electrodes, Langmuir 14 (1998) 7017–7026.
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100
G Model EA 26249 No. of Pages 7
A. Oda et al. / Electrochimica Acta xxx (2015) xxx–xxx [4] M.C. Granger, G.M. Swain, The influence of surface interactions on the reversibility of ferri/ferrocyanide at boron-doped diamond thin-film electrodes, J. Electrochem. Soc. 146 (1999) 4551–4558. [5] A. Denisenko, A. Romanyuk, L.A. Kibler, E. Kohn, Surface structure and electrochemical characteristics of boron-doped diamond exposed to rf N-2plasma, J. Electroanal. Chem. 657 (2011) 164–171. [6] H. Touhara, F. Okino, Property control of carbon materials by fluorination, Carbon 38 (2000) 241–267. [7] T. Nakajima, M. Koh, R.N. Singh, M. Shimada, Electrochemical behavior of surface-fluorinated graphite, Electrochim. Acta 44 (1999) 2879–2888. [8] V.N. Khabashesku, W.E. Billups, J.L. Margrave, Fluorination of single-wall carbon nanotubes and subsequent derivatization reactions, Acc. Chem. Res. 35 (2002) 1087–1095. [9] D. Damien, P.M. Sudeep, T.N. Narayanan, M.R. Anantharaman, P.M. Ajayan, M. M. Shaijumon, Fluorinated graphene based electrodes for high performance primary lithium batteries, RSC Adv. 3 (2013) 25702–25706. [10] S. Boopathi, T.N. Narayanan, S.S. Kumar, Improved heterogeneous electron transfer kinetics of fluorinated graphene derivatives, Nanoscale 6 (2014) 10140–10146. [11] W. Zhang, M. Dubois, K. Guerin, P. Bonnet, H. Kharbache, F. Masin, P. Thomas, J. L. Mansot, A. Hamwi, Fluorinated nanocarbons using fluorinating agent: Strategies of fluorination and applications, Eur. Phys. J. B 75 (2010) 133–139. [12] S. Ferro, A. De Battisti, Physicochemical properties of fluorinated diamond electrodes, J. Phys. Chem. B 107 (2003) 7567–7573. [13] T. Kondo, H. Ito, K. Kusakabe, K. Ohkawa, K. Honda, Y. Einaga, A. Fujishima, T. Kawai, Characterization and electrochemical properties of CF4 plasma-treated boron-doped diamond surfaces, Diam. Relat. Mater. 17 (2008) 48–54. [14] E. Silva, A.C. Bastos, M. Neto, A.J. Fernandes, R. Silva, M.G. Silva Ferreira, M. Zheludkevich, F. Oliveira, New fluorinated diamond microelectrodes for localized detection of dissolved oxygen, Sens. Actuat. B 204 (2014) 544–551. [15] A. Ueda, D. Kato, N. Sekioka, T. Kamata, R. Kurita, H. Uetsuka, Y. Hattori, S. Hirono, S. Umemura, O. Niwa, Fabrication of electrochemically stable fluorinated nano-carbon film compared with other fluorinated carbon materials, Carbon 47 (2009) 1943–1952. [16] X. Dai, O. Nekrassova, M.E. Hyde, R.G. Compton, Anodic stripping voltammetry of arsenic(III) using gold nanoparticle-modified electrodesic stripping voltammetry of arsenic(III) using gold nanoparticle-modified electrodes, Anal. Chem. 76 (2004) 5924–5929. [17] R.H. Tian, T.N. Rao, Y. Einaga, J.F. Zhi, Construction of two-dimensional arrays gold nanoparticles monolayer onto boron-doped diamond electrode surfaces, Chem. Mater. 18 (2006) 939–945. [18] N. Sekioka, D. Kato, A. Ueda, T. Kamata, R. Kurita, S. Umemura, S. Hirono, O. Niwa, Controllable electrode activities of nano-carbon films while maintaining surface flatness by electrochemical pretreatment, Carbon 46 (2008) 1918– 1926. [19] E. Kuraya, S. Nagatomo, K. Sakata, D. Kato, O. Niwa, T. Nishimi, M. Kunitake, Simultaneous electrochemical analysis of hydrophilic and lipophilic antioxidants in bicontinuous microemulsion, Anal. Chem. 87 (2015) 1489– 1493. [20] F. Mizutani, S. Yabuki, Y. Sato, S. Iijima, Amperometric measurement of ds-DNA content using a peroxidase-modified electrode, Bioelectrochemistry 63 (2004) 257–259. [21] F. Mizutani, T. Yamanaka, Y. Tanabe, K. Tsuda, An enzyme electrode for Llactate with a chemically-amplified response, Anal. Chim. Acta 177 (1985) 153–166. [22] D. Kato, S. Iijima, R. Kurita, Y. Sato, J. Jia, S. Yabuki, F. Mizutani, O. Niwa, Electrochemically amplified detection for lipopolysaccharide using ferrocenylboronic acid, Biosens. Bioelectron. 22 (2007) 1527–1531. [23] S. Iijima, D. Kato, S. Yabuki, O. Niwa, F. Mizutani, Enzymatically amplified electrochemical detection for lipopolysaccharide using ferrocene-attached polymyxin B and its analogue, Biosens. Bioelectron. 26 (2011) 2080–2084. [24] D. Kato, A. Oda, M. Tanaka, S. Iijima, T. Kamata, M. Todokoro, Y. Yoshimi, O. Niwa, Poly-epsilon-lysine modified nanocarbon film electrodes for LPS detection, Electroanal 26 (2014) 618–624. [25] T. Kamata, D. Kato, H. Ida, O. Niwa, Structure and electrochemical characterization of carbon films formed by unbalanced magnetron (UBM) sputtering method, Diam. Relat. Mater. 49 (2014) 25–32. [26] O. Niwa, J. Jia, Y. Sato, D. Kato, R. Kurita, K. Maruyama, K. Suzuki, S. Hirono, Electrochemical performance of angstrom level flat sputtered carbon film consisting of sp(2) and sp(3) mixed bonds, J. Am. Chem. Soc. 128 (2006) 7144– 7145.
7
[27] J.B. Jia, D. Kato, R. Kurita, Y. Sato, K. Maruyama, K. Suzuki, S. Hirono, T. Ando, O. Niwa, Structure and electrochemical properties of carbon films prepared by a electron cyclotron resonance sputtering method, Anal. Chem. 79 (2007) 98– 105. [28] D. Kato, N. Sekioka, A. Ueda, R. Kurita, S. Hirono, K. Suzuki, O. Niwa, A nanocarbon film electrode as a platform for exploring DNA methylation, J. Am. Chem. Soc. 130 (2008) 3716–3717. [29] D. Kato, N. Sekioka, A. Ueda, R. Kurita, S. Hirono, K. Suzuki, O. Niwa, Nanohybrid carbon film for electrochemical detection of SNPs without hybridization or labeling, Angew. Chem. Int. Ed. 47 (2008) 6681–6684. [30] D. Kato, K. Goto, S. Fujii, A. Takatsu, S. Hirono, O. Niwa, Electrochemical DNA methylation detection for enzymatically digested CpG oligonucleotides, Anal. Chem. 83 (2011) 7595–7599. [31] D. Kato, M. Sumimoto, A. Ueda, S. Hirono, O. Niwa, Evaluation of electrokinetic parameters for all DNA bases with sputter deposited nanocarbon film electrode, Anal. Chem. 84 (2012) 10607–10613. [32] D. Kato, M. Komoriya, K. Nakamoto, R. Kurita, S. Hirono, O. Niwa, Electrochemical determination of oxidative damaged DNA with high sensitivity and stability using a nanocarbon film, Anal. Sci. 27 (2011) 703–707. [33] S. Yamamura, M. Hoshikawa, D. Kato, H. Saito, N. Suzuki, O. Niwa, M. Okada, ONO-2506 inhibits spike-wave discharges in a genetic animal model without affecting traditional convulsive tests via gliotransmission regulation, Br. J. Pharmacol. 168 (2013) 1088–1100. [34] A. Ueda, D. Kato, R. Kurita, T. Kamata, H. Inokuchi, S. Umemura, S. Hirono, O. Niwa, Efficient direct electron transfer with enzyme on a nanostructured carbon film fabricated with a maskless top-down UV/ozone process, J. Am. Chem. Soc. 133 (2011) 4840–4846. [35] H.V. Patten, S.C.S. Lai, J.V. Macpherson, P.R. Unwin, Active sites for outersphere, inner-sphere, and complex multistage electrochemical reactions at polycrystalline boron-doped diamond electrodes (pBDD) revealed with scanning electrochemical cell microscopy (SECCM), Anal. Chem. 84 (2012) 5427–5432. [36] J.M. Ahn, P. Wentworth, K.D. Janda, Probing lipase/esterase libraries for lipid A hydrolases - discovery of biocatalysts for the detoxification of bacteriallyexpressed recombinant protein, Chem. Commun. (2004) 364–365. [37] C. Hirayama, M. Sakata, Chromatographic removal of endotoxin from protein solutions by polymer particles, J. Chromatogr. B 781 (2002) 419–432. [38] S.M. Opal, P.J. Scannon, J.L. Vincent, M. White, S.F. Carroll, J.E. Palardy, N.A. Parejo, J.P. Pribble, J.H. Lemke, Relationship between plasma levels of lipopolysaccharide (LPS) and LPS-binding protein in patients with severe sepsis and septic shock, J. Infect. Dis. 180 (1999) 1584–1589. [39] C. Alexander, E.T. Rietschel, Bacterial lipopolysaccharides and innate immunity, J. Endotoxin Res. 7 (2001) 167–202. [40] J.L. Ding, B. Ho, A new era in pyrogen testing, Trends Biotechnol. 19 (2001) 277– 281. [41] J.C. Wu, A. Zawistowski, M. Ehrmann, T. Yi, C. Schmuck, Peptide functionalized polydiacetylene liposomes act as a fluorescent turn-on sensor for bacterial lipopolysaccharide, J. Am. Chem. Soc. 133 (2011) 9720–9723. [42] M.H. Lan, J.S. Wu, W.M. Liu, W.J. Zhang, J.C. Ge, H.Y. Zhang, J.Y. Sun, W.W. Zhao, P.F. Wang, Copolythiophene-derived colorimetric and fluorometric sensor for visually supersensitive determination of lipopolysaccharide, J. Am. Chem. Soc. 134 (2012) 6685–6694. [43] P. Miao, Electrochemical sensing strategies for the detection of endotoxin: a review, RSC Adv. 3 (2013) 9606–9617. [44] K.Y. Inoue, K. Ino, H. Shiku, T. Matsue, Electrochemical detection of endotoxin using recombinant factor C zymogen, Electrochem. Comm. 12 (2010) 1066– 1069. [45] J.Y. Heras, D. Pallarola, F. Battaglini, Electronic tongue for simultaneous detection of endotoxins and other contaminants of microbiological origin, Biosens. Bioelectron. 25 (2010) 2470–2476. [46] W. Su, M. Cho, J.D. Nam, W.S. Choe, Y. Lee, Aptamer-assisted gold nanoparticles/PEDOT platform for ultrasensitive detection of LPS, Electroanal 25 (2013) 380–386. [47] M. Sakata, T. Inoue, M. Todokoro, M. Kunitake, Limulus amebocyte lysate assay for endotoxins by an adsorption method with polycation-immobilized cellulose beads, Anal. Sci. 26 (2010) 291–296. [48] M. Sakata, Y. Fukuma, M. Todokoro, M. Kunitake, Selective assay for endotoxin using poly(epsilon-lysine)-immobilized Cellufine and Limulus amoebocyte lysate (LAL), Anal. Biochem. 385 (2009) 368–370. [49] Pyrogen and Endotoxins Testing: Questions and Answers, U.S. FDA, 2012
Please cite this article in press as: A. Oda, et al., Fluorinated Nanocarbon Film Electrode Capable of Signal Amplification for Lipopolysaccharide Detection, Electrochim. Acta (2015), http://dx.doi.org/10.1016/j.electacta.2015.12.100