- Email: [email protected]
Amperometric microsensor for direct probing of ascorbic acid in human gastric juice
Analytica Chimica Acta 678 (2010) 176–182
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Amperometric microsensor for direct probing of ascorbic acid in human gastric juice Emily A. Hutton a , Rasa Pauliukaite˙ a,1 , Samo B. Hocevar a , Boˇzidar Ogorevc a,∗ , Malcolm R. Smyth b a b
Analytical Chemistry Laboratory, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia National Centre for Sensor Research, School of Chemical Sciences, Dublin City University, Dublin 9, Ireland
a r t i c l e
i n f o
Article history: Received 8 June 2010 Received in revised form 19 August 2010 Accepted 20 August 2010 Available online 18 September 2010 Keywords: Ascorbic acid Gastric juice Amperometry Microsensor Electrode modification
a b s t r a c t This article reports on a novel microsensor for amperometric measurement of ascorbic acid (AA) under acidic conditions (pH 2) based on a carbon fiber microelectrode (CFME) modified with nickel oxide and ruthenium hexacyanoferrate (NiO–RuHCF). This sensing layer was deposited electrochemically in a twostep procedure involving an initial galvanostatic NiO deposition followed by a potentiodynamic RuHCF deposition from solutions containing the precursor salts. Several important parameters were examined to characterize and optimize the NiO–RuHCF sensing layer with respect to its current response to AA by using cyclic voltammetry, and scanning electron microscopy–energy dispersive X-ray spectroscopy methods. With the NiO–RuHCF coated CFME, the AA oxidation potential under acidic conditions was shifted to a less positive value for about 0.2 V (Ep of ca. 0.23 V vs. Ag/AgCl) as compared to a bare CFME, which greatly improves the electrochemical selectivity. Using the hydrodynamic amperometry mode, the current vs. AA concentration in 0.01 M HCl, at a selected operating potential of 0.30 V, was found to be linear over a wide range of 10–1610 M (n = 22, r = 0.999) with a calculated limit of detection of 1.0 M. The measurement repeatability was satisfactory with a relative standard deviation (r.s.d.) ranging from 4% to 5% (n = 6), depending on the AA concentration, and with a sensor-to-sensor reproducibility (r.s.d.) of 6.9% at 100 M AA. The long-term reproducibility, using the same microsensor for 112 consecutive measurements of 20 M AA over 11 h of periodic probing sets over 4 days, was 16.1% r.s.d., thus showing very good stability at low AA levels and suitability for use over a prolonged period of time. Moreover, using the proposed microsensor, additionally coated with a protective cellulose acetate membrane, the calibration plot obtained in the extremely complex matrix of real undiluted gastric juice was linear from 10 to 520 M (n = 14, r = 0.998). These results demonstrated the unique featuring of the proposed NiO–RuHCF microsensor under acidic conditions with enhanced sensitivity and stability and proved its promising potentiality for direct amperometric probing of AA at physiological levels in real gastric juice environments. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ascorbic acid (AA), also known as vitamin C (its l-enantiomer) or ascorbate (at physiological pH), is an essential component of the diet of a number of vertebrates including humans [1]. In the human body, its primary function is to act as an antioxidant, neutralizing toxic peroxides and stabilizing free radicals [2,3]. Evidence suggests that AA may play an important role in cancer and heart disease pre-
∗ Corresponding author at: Analytical Chemistry Laboratory, National Institute of Chemistry, P.O. Box 660, SI-1001 Ljubljana, Slovenia. Tel.: +386 14760230; fax: +386 14760300. E-mail address: [email protected] (B. Ogorevc). 1 Present address: Department of Chemistry, University of Coimbra, Coimbra, Portugal. 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.08.027
vention [4,5], alleviation of high blood pressure [6], and immune system boosting [7]. AA is thought to protect against gastric cancer, which is the fourth most common cancer and the second most common cause of cancer deaths worldwide [8], through its actions in scavenging nitrite and preventing the formation of nitrite-derived mutagens such as nitrosamines and nitrosamides [9–11]. The concentration of AA in gastric juice is therefore a potentially critical factor in the prevention of intragastric N-nitrosation [12], with significantly lower gastric AA concentrations found in patients infected with H. pylori compared to an uninfected control group [13]. Median gastric AA concentrations in both healthy and compromised individuals can be expected to range between approximately 16 and 154 M, with the variations in concentration due to differing subject gastric mucosal pathology [12]. Several interesting studies have been devoted to examining interactions between AA, nitrite and thiocyanate in benchtop models [11,14,15].
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
Due to its crucial role in the human body, numerous methods for the determination of AA in biological samples have been proposed, including spectrophotometry [16,17], chemiluminesence [18], capillary electrophoresis [19] and high performance liquid chromatography (HPLC) [20,21]. Although some of these methods are very sensitive, they can be very complex, time consuming and/or need sophisticated equipment. HPLC and related methods [14,22,23] are commonly employed in measurements of AA in gastric juice, but relatively complicated sampling and storage of the gastric juice samples is required. The oxidation of AA, as a basis of its electrochemical sensing, has been the subject of numerous studies in which electrodes composed of various modified or unmodified transducer materials, e.g. Au [24], Pt [25], glassy carbon [26] and carbon paste [27], have been employed. Within the field of electroanalytical measurement of AA and other biologically significant compounds, considerable focus has been placed on the use of microelectrodes which, due to their attractive inherent advantages, allow convenient probing in microenvironments [28–33]. Although various microprobes for direct measurement of AA in real biological samples have been proposed [28,30] this is, to the best of our knowledge, the first report on a microsensor suitable for the direct probing of gastric AA. Metal hexacyanoferrate (MeHCF) deposits as electrode modifiers have been extensively investigated to exploit their electrocatalytic properties for the detection of various organic and inorganic species, e.g. different cations [34], glucose [35], hydrazine [36], and AA [37,38]. Mixed metal HCFs as electrode coatings have also been studied and employed in improved electrochemical sensing, for example CoCuHCF in potentiometric detection of hydrazine [39] and FeCoHCF in amperometric sensing of hydrogen peroxide [40]. The most notable advantages of MeHCF electrode coatings are certainly their electrocatalytic activity and prolonged stability. This article reports on the development and attractive performance of a new microsensor, prepared by modifying a substrate carbon fiber microelectrode (CFME) with an electrochemically deposited composite nickel oxide and ruthenium hexacyanoferrate (NiO–RuHCF) layer. The proposed microsensor was found to be suitable for measuring AA under strong acidic conditions and in model gastric juice solution, exhibiting prolonged stability. The performance of the proposed microprobe was studied in detail, with particular emphasis on amperometric response, stability and selectivity. Upon the application of an additional protective membrane, its practical use for direct amperometric measurement of physiological concentrations of AA in undiluted real gastric juice is also demonstrated.
2. Experimental 2.1. Apparatus Cyclic volammetric (CV), amperometric and galvanostatic experiments were performed using a modular electrochemical workstation (Autolab PGSTAT12, Eco Chemie, Utrecht, The Netherlands), equipped with a low-current ECD module and driven by GPES 4.9 software (Eco Chemie). The three-electrode configuration employed in all experiments consisted of a modified carbon fiber microelectrode (CFME), a platinum wire and Ag/AgCl (satd. KCl) as the working, counter and the reference electrode, respectively. All potentials in this work are referred to this reference. All electrochemical measurements were carried out at room temperature (23 ± 2 ◦ C) in a standard voltammetric cell placed in a Faraday cage. Stirring conditions in hydrodynamic amperometric measurements were provided by a magnetic stirrer at 300 rpm. Scanning electron microscopy (SEM) including energy dispersive X-ray (EDX) analysis operations were performed using a
177
Hitachi S 3000N Scanning Electron Microscope (Hitachi, Japan) equipped with a Link Isis Energy Dispersive Spectrometer (Oxford Instruments, UK). 2.2. Chemicals and solutions All chemicals employed in this work were of analytical-grade purity and were used as received. Ascorbic acid (AA), nickel(II) sulfate hexahydrate and l-glucose were obtained from Kemika (Zagreb, Croatia). Ruthenium(III) chloride was obtained from Sigma, while potassium hexacyanoferrate(III) was purchased from Fluka. Protein standard, lyophilized (1.32 mg mL−1 protein) was provided by Bio-Rad (CA, USA). Water used to prepare all solutions was prepared via a water purification unit (Elix/Milli-Q Gradient, Millipore, Bedford, USA). Real gastric juice samples were obtained from a healthy volunteer (female, age 24) and stored in a freezer at −18 ◦ C and thawed before use. AA standard stock solutions (0.1 M) were prepared daily and were refrigerated when not in use. Modification solutions for applying (a) ruthenium hexacyanoferrate (RuHCF), (b) nickel hexacyanoferrate (NiHCF) or (c) nickel ruthenium hexacyanoferrate (NiRuHCF) coatings were composed of 0.1 M KCl, 4 mM HCl, 0.2 mM K3 [Fe(CN)6 ], and either (a) 0.1 mM RuCl3 or (b) 0.1 mM NiSO4 or (c) both a + b in 1:1 ratios in aqueous solution. 0.01 M HCl solution (pH 2) was used as supporting electrolyte for all measurements unless otherwise stated. The model gastric juice solution that was used in this work was composed of 0.01 M HCl, 1 mM NaCl, 2 mM glucose and 20% protein, the major components present in human gastric juice. 2.3. Microsensor preparation 2.3.1. Fabrication of CFMEs A cleaned single carbon fiber (7 m in diameter, 2–3 cm in length, Goodfellow Co., Oxford, UK) was first attached to a copper wire and then thermally sealed into a pulled glass capillary employing a microelectrode puller (PP-830, Narishige, Tokyo, Japan), with a final cut of the carbon fiber tip to a length of ca. 1 mm. The carbon fiber tip lengths of all fabricated and successfully tested CFMEs in this work were exactly measured using an inverted optical microscope and appropriate image analyzer programme. These tip length values were used to calculate corresponding geometric surface areas in order to obtain current densities where required. More details are given elsewhere [41]. 2.3.2. Modification of CFMEs Prior to modification, the CFME was pretreated electrochemically in a 0.1 M KCl solution by applying a continuous potential scan between 0.0 and +1.0 V at a scan rate of 100 mV s−1 until a steady-state current–voltage profile was reached, upon which it was thoroughly washed with Milli-Q water. In the two-step modification procedure, the first step, galvanostatic deposition of nickel oxide (NiO), was carried out by immersing the CFME into an aqueous solution containing 1.1 M NiSO4 and 1.2 M H3 BO3 and applying a constant current of +0.4 A for 10 s (whereupon the potential observed was ca. +2.3 V). In the second step, the RuHCF film was electrochemically grown onto the NiO-coated CFME in its respective modification solution (see Section 2.2 above) by cycling the potential between 0.0 and +1.0 V at a scan rate of 100 mV s−1 until a desired coverage was obtained (22 full cycles unless otherwise stated). The same potentiodynamic coating procedure was used for the deposition of the other metal hexacyanoferrate films onto bare CFMEs by using corresponding modification solutions (see Section 2.2 above). Following this step, the modified CFME was rinsed with water and was ready for use or was further coated with an additional protective membrane. This was achieved by dip-coating the
178
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
Fig. 1. The dependence of the cyclic voltammetric peak current response to AA at a NiO–RuHCF-modified CFME on the conditions of the two-step modification procedure: (A) on the galvanostatic treatment time in the first step (with RuHCF coverage fixed at 25 CV runs) and (B) on the RuHCF coverage in the second step, expressed in the number of CV runs (with NiO deposition time fixed at 10 s). Other modification conditions: (A) galvanostatic treatment of a bare CFME in the nickel modification solution at +0.4 A, (B) repetitive cycling of the potential (CV runs) between 0.0 and +1.0 V in the respective ruthenium modification solution at a scan rate of 100 mV s−1 . Measurement solution: 0.01 M HCl containing 1 mM AA; CV scan rate 100 mV s−1 . For easier comparison the current response is shown in a relative scale.
modified CFME in 1:1 (v/v) cyclohexanone–acetone solution containing 1% cellulose acetate, using a laboratory made dip-coating device with speed of dipping set to 0.34 mm s−1 . After each dipping (6 in total), the tip was allowed to dry in air at room temperature for approximately 5 min.
3. Results and discussion 3.1. Preparation, optimization and characterization of the modification layer In order to achieve the optimum electrochemical detection of AA under strong acidic conditions, we developed a two-step procedure for depositing a composite nickel oxide and ruthenium hexacyanoferrate (NiO–RuHCF) film onto a carbon fiber microelectrode (CFME). In the first step, the idea for which was adopted from the known surface-finishing process of plating with nickel [42], a clean bare CFME was galvanostatically treated in the corresponding nickel modification solution, while the RuHCF layer was subsequently deposited using a multiscan cyclic voltammetry protocol. As shown in Fig. 1, the formation of this sensing layer was studied, with respect to its voltammetric response to AA, by monitoring the effect of the galvanostatic treatment time (Fig. 1A) and the potentiodynamic coverage of the RuHCF film (Fig. 1B). As displayed in Fig. 1A, the voltammetric response to AA exhibited a maximum signal after a positive current of 0.4 A in the galvanostatic step was applied for 10 s and up to 15 s. While at shorter times the amount of NiO coverage was apparently inadequate, treatment times longer than 15 s resulted in a significant decrease of the signal, either due to lower conductivity of the gradually thicker NiO layer or because of anodic damage to the carbon fiber substrate electrode. From Fig. 1B, where the RuHCF coverage is defined in terms of the number of CV runs, it is well evident that the optimum response to AA was provided with 22 CV runs whereas thinner or thicker films yielded lower AA signals. Similar behavior has been reported for AA detection at a CoHCF film [37]. It was postulated that the thinner film does not provide a sufficient number of catalytic sites to handle the available supply of AA, whereas the thicker film may form a resistive barrier to the transfer of electrons. Based on the results from Fig. 1, 10 s of galvanostatic coating of NiO and 22 CV runs of potentiodynamic RuHCF deposition under the applied conditions were selected as optimum and employed in all subsequent experiments.
Deposits formed in each phase of the two-step coating procedure were inspected using optical and scanning electron microscopy (SEM) as well as energy dispersive X-ray spectroscopy (EDX). These characterizations proved that, as expected, upon application of positive current during the galvanostatic treatment, nickel oxide was deposited onto the CFME bare surface. Using an optical microscope it was observed that a green film was formed, which is the typical color of NiO [43]. Further evidence of NiO formation was provided upon SEM analysis of the film surface yielding images (not shown) with a strong similarity to those of a nickel oxide film reported elsewhere [44]. EDX spectra of this film revealed the presence of nickel (K␣ , L␣ and K lines) and oxygen, again indicating the presence of a nickel oxide layer. The SEM/EDX analysis of the final coating produced during the second step (using potentiodynamic treatment) revealed a markedly different surface from that obtained during the first step. While the SEM imaging (not shown) revealed that the surface was not perfectly uniform, the EDX spectra exhibited the presence of Ru(L␣ ), Ni(K␣ ) and Fe(K␣ ) lines. These data suggest the possibility that cycling the nickel oxide surface at positive potentials in the ruthenium(III) and hexacyanoferrate(III) solution can result in the formation of oxide bridging leading to the NiO–RuHCF composite coating. However, while our SEM/EDX surface analyses are sufficiently indicative for the purpose of this study, a much more dedicated investigation should be conducted in order to get a complete insight into the composition, structure and morphology of the NiO–RuHCF coating prepared by the proposed procedure. To verify the advantage of the proposed modification procedure, several experiments were conducted to compare different modified CFMEs, as illustrated in Fig. 2. First (Fig. 2A) we compared the NiO–RuHCF microsensor and a bare CFME. Evidently, at the NiO–RuHCF-modified CFME a large shift of AA oxidation potential to less positive value for ca. 210 mV (Ep of about +0.23 V) with a concurrent increase in signal height can be clearly observed. This apparent catalytic activity of the NiO–RuHCF surface significantly improves both the electrochemical selectivity and sensitivity of the proposed microsensor for AA detection. An apparent breakage of the voltammetric curve at potentials around +0.25 V might be attributed to a sudden re-arrangement of the modification layer and/or charging/discharging process. The observed behavior was very reproducible and, in addition, did not effect our further amperometric measurements of AA under constant potential regime. From Fig. 2A, b and d, which show cyclic voltammograms obtained at the modified CFME, it can be seen that there is an additional
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
179
Fig. 2. Cyclic voltammograms of AA at different modified and unmodified CFMEs: (A) comparison of CV responses at bare CFME (a, c) and NiO–RuHCF-modified CFME (b, d) for 0.01 M HCl, pH 2 (a, b) and 1 mM AA + 0.01 M HCl (c, d). (B) Comparison of CV responses to 1 mM AA in 0.01 M HCl, pH 2, of CFMEs modified with (a) NiHCF, (b) NiO, (c) RuHCF, and (d) NiRuHCF. CV scan rate: 100 mV s−1 . Current density is presented to allow comparison.
redox process at ca. 0.0 and 0.1 V, in both the 0.01 M HCl and 0.01 M HCl + 1 mM AA solutions, respectively. Examination of the current response for this process revealed that it is linearly dependent (r = 0.999) on the scan rate, indicating that it is a function of a redox system confined to the surface of the microsensor and not connected to AA detection. Further comparisons (Fig. 2B) of the NiO–RuHCF were made with various other modification layers consisting of one or both of its components: NiHCF deposited using a multiscan (CV) protocol, NiO alone prepared using the galvanostatic treatment, RuHCF alone deposited using a multiscan CV protocol, and NiRuHCF deposited from the solution containing respective precursor salts using the multiscan CV protocol as well. The NiHCF coating (Fig. 2B, a) exhibited a well-defined response to AA, which is in agreement with the literature data [38]. Although NiHCF was proposed to catalyze the oxidation of AA via surface layer mediated charge-transfer [45], it provided under our experimental conditions a negative shift of AA oxidation potential for only approximately 20 mV. The galvanostatically deposited NiO layer (Fig. 2B, b) also exhibited a well-defined signal, with an AA oxidation potential of about +0.30 V, which was considerably shifted from that obtained at a bare carbon fiber (+0.44 V). It is clearly evident that the responses of both the RuHCF and NiRuHCF coatings (Fig. 2B, c and d) are ill-defined in the potential range examined. Thus, it may be concluded that under the conditions applied, the NiO–RuHCF modification layer (Fig. 2A, d) provided the optimum response to AA, with the least positive oxidation potential and a clearly defined signal. An investigation was performed to characterize the electrochemical behavior of the NiO–RuHCF-modified CFME in the presence of AA at a pH of 2. Since the plots of ip vs. scan rate and ip vs. square root of scan rate did not show typical behavior, we plotted ip /Cv1/2 (current function vs. v1/2 , where C is the concentration of AA and v is the scan rate). This plot exhibited a negative slope, which is typical of a catalytic process involving a chemical reaction followed by an electron transfer process [46]. On the basis of these results it is confirmed that the electrocatalysis of AA at the NiO–RuHCF layer is a catalytic process which is consistent with the known electrochemical oxidation reaction of AA.
(OP) for conducting amperometric measurements of AA at the proposed NiO–RuHCF microsensor, the amperometric response to AA was plotted against the applied OP in the format of a pseudovoltammogram, displayed in Fig. 3. It can be seen that the current response increases sharply at OPs from +0.27 to +0.30 V. Because the signal increase beyond this OP is insignificant and, in particular, to avoid possible interferences from other electroactive species as well as to omit the increasing background current contribution at higher potentials, the optimum OP was selected to be +0.30 V. In addition, this OP also corresponds to an inflection point in the pseudo-voltammogram (Fig. 3), which is desirable for improved stability in the current measurements [46]. To obtain calibration data in 0.01 M HCl, the hydrodynamic amperometric mode at the OP of +0.30 V was employed and the current signal to AA concentration relationship was found linear over the range of 10–1610 M (n = 22), with a slope of 0.12 nA M−1 (or 0.57 A cm2 M−1 if the area of the microsensor is taken into consideration) and a correlation coefficient (r) of 0.999, as demonstrated in Fig. 4. From these results, it is evident that the NiO–RuHCF microsensor provides detection possibilities over the entire range of AA levels encountered in human gastric juice. The repeatability of
3.2. Amperometric AA detection and analytical performance of the NiO–RuHCF microsensor With the final goal in focus, from this stage of the work on, we employed the amperometric mode under hydrodynamic conditions. In order to determine the optimum operating potential
Fig. 3. A pseudo-voltammogram constructed from amperometric responses recorded at each operating potential separately, after addition of 50 M AA to a stirred 0.01 M HCl solution, using a NiO–RuHCF microsensor. Current density is presented to allow comparison of results.
180
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
Fig. 4. Hydrodynamic amperogram obtained during additions of AA to 0.01 M HCl solution at a NiO–RuHCF microsensor; AA increments: (a) 6 times 10 M, (b) 7 times 50 M, (c) 6 times 100 M, and (d) 3 times 200 M; operating potential: +0.30 V. Inset shows corresponding calibration plot.
amperometric AA measurements was found to be satisfactory with relative standard deviations (r.s.d.s) of 5.1% and 4.1% obtained for 6 consecutive additions of 10 and 100 M AA, respectively. The limit of detection (based on S/N = 2) was calculated to be 1.0 M, which is consistent with experiments (not shown) in which even 2 M AA elicited a discernable response at the proposed microsensor. The sensor-to-sensor reproducibility was also found to be satisfactory. For 10 and 100 M AA measured at 5 freshly prepared NiO–RuHCF microsensors, the r.s.d.s were 9.0% and 6.9%, respectively. These calibration data revealed the promising performance of the NiO–RuHCF microsensor and suggested its potential applicability in measurements of gastric juice AA levels. Interestingly, at a bare CFME, upon successive additions of 20 M AA to a 0.01 M HCl solution applying an OP of +0.50 V (not shown), the current signal was observed to deviated from linearity with an increasingly diminishing and tailing peak-shaped current response observed following each AA addition. We also examined the performance of the NiO–RuHCF microsensor for its use over a prolonged period of time. In this testing, a single microsensor was applied for continuous amperometric measurements (at an OP of +0.30 V) of 20 M AA increments, by periodically adding the corresponding amounts of AA to the stirred 0.01 M HCl solution, over 4 days (with intermediate overnight storing of the used microsensor in 0.01 M HCl) in the following pattern: first day over 6 h, second day over 2 h, third day over 2 h and the fourth day over 1 h. 20 M AA was chosen as a concentration which relates closely to the amount of AA secreted periodically into the human stomach [11]. This represented a total working time of 11 h with the total elapsed time from the first to the last measurement of 75 h. The r.s.d. for all 112 hydrodynamic amperometric measurements of 20 M AA increments over this entire testing time (i.e. 75 h) was 16.1%. For a relatively low concentration level of AA this long-term repeatability is considered to be very good. It clearly indicates that the proposed microsensor is well suited for direct amperometric probing of AA under strong acidic conditions such as those encountered in gastric juice over a prolonged period of time. 3.3. Direct amperometric AA probing in model solution and in real gastric juice In order to protect the NiO–RuHCF microsensor from inevitable fouling by macromolecular components present in the gastric
Fig. 5. Hydrodynamic amperometric recording of the response to the addition of (a) and (c) 4 times 20 M AA, and (b) and (d) 100 M NaNO2 , obtained at a cellulose acetate membrane coated NiO–RuHCF microsensor; medium: gastric juice model solution containing 1 mM NaCl, 2 mM glucose, 20% protein and 0.01 M HCl; operating potential: +0.30 V.
matrix, we considered the application of a cellulose acetate membrane (CAM) deposited onto the microsensor surface by employing a dip-coating procedure. Cellulose acetate was chosen due to its neutrality and its known applicability in protection of electrode surfaces from electrochemical fouling [47]. Employing a strategy of using several thin coatings instead of one thicker coating and based on a compromise between appropriate surface protection and “not affecting” the analyte signal, examination showed that six layers of the cellulose acetate polymer should be adequate for protection of the microsensor while still providing a strong AA signal. Several experiments (not shown) were also carried out using the proposed microsensor in order to determine if the components of the model gastric solution elicited a response at the employed operating potential of +0.3 V. First of all, individual solutions of thiocyanate (1 mM), sodium nitrite (1 mM) and glucose (2 mM) in 0.01 M HCl were cycled over the potential range of ca. −0.5 to +0.5 V. No response was observed at the oxidation potential of AA (+0.23 V). Secondly, hydrodynamic amperometry experiments were carried out by spiking, individually, the abovementioned solutions into a solution of 0.01 M HCl. No response was observed at the applied OP of +0.3 V. As the proposed microsensor is insensitive to nitrite, as evidenced from the experiments described above, it is certain that the response obtained in the solution containing AA and nitrite will solely be due to the oxidation of AA. We further investigated whether the proposed microsensor was capable of continuously monitoring the level of AA in the model solution, which is often required in studies of the interactions between AA and nitrite [14,15]. For this reason, hydrodynamic amperometric measurements were performed in the gastric juice model solution, to which four additions of 20 M AA were made, followed by the addition of 100 M NaNO2 , whereupon this sequence was repeated after 5 min, as displayed in Fig. 5. For the sake of figure clarity and time saving a new sequence was started before the current response to AA faded to the background level. It can be noted that even in the presence of a high protein concentration in the solution, the response of the protective CAM-coated microsensor to AA was well-defined, with the current remaining steady after each addition, indicating the absence of fouling of the microsensor surface. From Fig. 5, the existence of a disparity between the currents obtained for the AA additions in the absence (Fig. 5a) and in the presence (Fig. 5c) of nitrite can be
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
181
using a two-step galvanostatic/potentiodynamic protocol, with further application of a cellulose acetate membrane (CAM) protective coating. The signal to AA concentration relationship in acidic media (0.01 M HCl and gastric juice) was linear over a wide range corresponding to physiological AA concentrations commonly encountered in gastric juice. The proposed microsensor exhibited either satisfactory or excellent selectivity, sensitivity, repeatability and life-time properties, even in the combination with the extremely complex gastric juice matrix. Acknowledgements
Fig. 6. Hydrodynamic amperometric recording of five 50 M AA increments after spiking an appropriate amount of AA into a stirred sample (volume 2 mL) of undiluted real human gastric juice, using a cellulose acetate membrane coated NiO–RuHCF microsensor; operating potential: +0.30 V.
seen, i.e. the amperometric signals observed for AA in the presence of nitrite are lower than those observed in the nitrite-free solution, while in both cases linearly increasing with AA concentration. This can be explained by nitric oxide formation via the reaction of AA with nitrite, combining with oxygen present in the solution to reform nitrite, which can then react further with AA [11]. This recycling will continue until either the AA or oxygen has been depleted. These attractive results (Fig. 5) demonstrated that the proposed microsensor can be successfully applied for on-line real-time monitoring of AA levels in this type of in vitro investigations. To examine the suitability of the proposed NiO–RuHCF microsensor coated with a protective CAM for direct probing of AA in human gastric juice, amperometric measurements were conducted whereupon a stirred undiluted gastric juice sample was spiked with an appropriate amount of AA, as is illustrated in Fig. 6. As is evident, it is demonstrated that in an extremely complicated matrix such as pure gastric juice, the direct real-time probing of AA is possible: the CAM-coated NiO–RuHCF microsensor response to AA is linear, the response time is rapid enough and the observed background current contribution is satisfactorily low. Under the described in vitro experimental conditions, roughly simulating the in vivo periodic secretions of AA into gastric juice, the signal to AA concentration relationship was found to be linear over the range of 10–520 M, with a slope of 0.02 nA M−1 (or 0.12 A cm2 M−1 ) and a correlation coefficient of 0.998 (n = 14), with an estimated limit of detection (S/N = 2, n = 6) of 8.5 M. The lower sensitivity, as compared with that achieved in 0.01 M HCl, can be attributed to the lower conductivity of the gastric juice solution and also to the presence of the cellulose acetate membrane on the surface of the microsensor. These results indicate the practical applicability of directly measuring AA physiological levels in gastric juice and, after some further technological improvement of the proposed microsensor, also monitoring secretion of AA into the gastric cavity. 4. Conclusions The purpose of this work was to develop an all-solid microsensor for amperometric probing of ascorbic acid (AA) in gastric juice. This was achieved by modifying the surface of a substrate carbon fiber microelectrode with an electrochemically deposited nickel oxide and ruthenium hexacyanoferrate (NiO–RuHCF) sensing layer,
This work was supported by Slovenian Research Agency (Program P1-0034). E.A.H. thanks the National Centre for Sensor Research, Dublin City University for funding and the National Institute of Chemistry, Ljubljana, for partial support. The authors thank Prof. Kenneth McColl from Faculty of Medicine, University of Glasgow, Glasgow, UK, for helpful suggestions. References [1] G.L. Wheeler, M.A. Jones, N. Smirnoff, Nature 393 (1998) 365–369. [2] S.J. Padayatty, A. Katz, Y.H. Wang, P. Eck, O. Kwon, J.H. Lee, S.L. Chen, C. Corpe, A. Dutta, S.K. Dutta, M. Levine, J. Am. Coll. Nutr. 22 (2003) 18–35. [3] H. Sies, W. Stahl, Am. J. Clin. Nutr. 62 (1995) S1315–S1321. [4] A.C. Carr, B. Frei, Am. J. Clin. Nutr. 69 (1999) 1086–1107. [5] K.T. Khaw, S. Bingham, A. Welch, R. Luben, N. Wareham, S. Oakes, N. Day, Lancet 357 (2001) 657–663. [6] S.J. Duffy, N. Gokce, M. Holbrook, A. Huang, B. Frei, J.F. Keaney, J.A. Vita, Lancet 354 (1999) 2048–2049. [7] P.C. Calder, S. Kew, Brit. J. Nutr. 88 (2002) S165–S176. [8] P. Correa, M. Blanca Piazuela, M. Constanza Camargo, Gastric Cancer 7 (2004) 9–16. [9] C. Mowat, A. Carswell, A. Wirz, K.E.L. McColl, Gastroenterology 116 (1999) 813–822. [10] M. Hayat, S. Everett, Nutrition 15 (1999) 402–403. [11] A. Moriya, J. Grant, C. Mowat, C. Williams, A. Carswell, T. Preston, S. Anderson, K. Iijima, K.E.L. McColl, Scand. J. Gastroenterol. 37 (2002) 253–261. [12] C.J. Schorah, G.M. Sobala, M. Sanderson, N. Collis, J.N. Primrose, Am. J. Clin. Nutr. 53 (1991) S287–S293. [13] Z.W. Zhang, S.E. Patchett, D. Perrett, P.H. Katelaris, P. Domizio, M.P.G. Farthing, Gut 43 (1998) 322–326. [14] H. Suzuki, A. Moriya, K. Iijima, K. McElroy, V.E. Fyfe, K.E.L. McColl, Scand. J. Gastroenterol. 38 (2003) 856–863. [15] K. Iijima, J. Grant, K. McElroy, V. Fyfe, T. Preston, K.E.L. McColl, Carcinogenesis 24 (2003) 1951–1960. [16] K. Guclu, K. Sozgen, E. Tutem, M. Ozyurek, R. Apak, Talanta 65 (2005) 1226–1232. [17] K. Kodama, K. Sumii, M. Kawano, T. Kido, K. Nojima, M. Sumii, K. Haruma, M. Yoshihara, K. Chayama, Europ. J. Gastroenterol. Hepatol. 15 (2003) 987–993. [18] Y.J. Ma, M. Zhou, X.Y. Jin, B.Z. Zhang, H. Chen, N.Y. Guo, Anal. Chim. Acta 464 (2002) 289–293. [19] W.S. Kim, R.L. Dahlgren, L.L. Moroz, J.V. Sweedler, Anal. Chem. 74 (2002) 5614–5620. [20] J. Lykkesfeldt, Anal. Biochem. 282 (2000) 89–93. [21] M.A. Kall, C. Andersen, J. Chromatogr. B 730 (1999) 101–111. [22] M.J. Sanderson, C.J. Schorah, Biomed. Chromatogr. 2 (1987) 197–202. [23] A.G. Fraser, G.A. Woollard, J. Gastroenterol. Hepatol. 14 (1999) 1070–1073. [24] J.J. Fei, L.M. Luo, S.S. Hu, Z.Q. Gao, Electroanalysis 16 (2004) 319–323. [25] H. Ernst, M. Knoll, Anal. Chim. Acta 449 (2001) 129–134. [26] W. Hu, D. Sun, W. Ma, Electroanalysis 22 (2010) 584–589. [27] S. Shahrokhian, E. Asadian, Electrochim. Acta 55 (2010) 666–672. [28] P.S. Cahill, R.M. Wightman, Anal. Chem. 67 (1995) 2599–2605. [29] P. Janda, J. Weber, L. Dunsch, A.B.P. Lever, Anal. Chem. 68 (1996) 960–965. [30] T.R.L.C. Paixão, L.F. Barbosa, M.T. Carrì, M.H.G. Medeiros, M. Bertotti, Analyst 133 (2008) 1605–1610. [31] J.-W. Mo, B. Ogorevc, Anal. Chem. 73 (2001) 1196–1202. [32] C.A. Anastassiou, B.A. Patel, M. Arundell, M.S. Yeoman, K.H. Parker, D. O’Hare, Anal. Chem. 78 (2006) 6990–6998. [33] R.M. Wightman, Science 311 (2006) 1570–1574. [34] Q. Xu, S. Zhang, W. Zhang, L.T. Jin, K. Tanaka, H. Haraguchi, A. Itoh, Fresenius J. Anal. Chem. 367 (2000) 241–245. [35] J. Wang, X.J. Zhang, L. Chen, Electroanalysis 16 (2000) 1277–1281. [36] A. Salimi, K. Abdi, Talanta 63 (2004) 475–483. [37] C.-X. Cai, K.-H. Xue, S.-M. Xu, J. Electroanal. Chem. 486 (2000) 111–118. [38] M.H. Pournaghi-Azar, H. Razmi-Nerbin, B. Hafezi, Electroanalysis 14 (2002) 206–212. [39] J.-W. Mo, B. Ogorevc, X.J. Zhang, B. Pihlar, Electroanalysis 12 (2000) 48–54.
182
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
[40] W.Y. Tao, D.W. Pan, Y.J. Liu, L.H. Nie, S.Z. Yao, Anal. Biochem. 338 (2005) 332–340. [41] S.B. Hoˇcevar, B. Ogorevc, K. Schachl, K. Kalcher, Electroanalysis 16 (2004) 1711–1716. [42] G.A. DiBari, Met. Finish. 98 (2000) 270–288. [43] D.R. Lide (Ed.), Handbook of Chemistry and Physics, 80th ed., CRC Press, Boca Raton, 1999.
[44] P. Häring, R. Kötz, G. Repphun, O. Haas, H. Siegenthaler, Appl. Phys. A. Mater. Sci. Proc. 66 (1998) S481–S486. [45] M.H. Pournaghi-Azar, H. Razmi-Nerbin, J. Electroanal. Chem. 456 (1998) 83–90. [46] S.A. Wring, J.P. Hart, Anal. Chim. Acta 229 (1990) 63–70. [47] G. Roman, A.C. Pappas, D. Kovala-Demertzi, M.I. Prodromidis, Anal. Chim. Acta 456 (2004) 201–207.
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Amperometric microsensor for direct probing of ascorbic acid in human gastric juice Emily A. Hutton a , Rasa Pauliukaite˙ a,1 , Samo B. Hocevar a , Boˇzidar Ogorevc a,∗ , Malcolm R. Smyth b a b
Analytical Chemistry Laboratory, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia National Centre for Sensor Research, School of Chemical Sciences, Dublin City University, Dublin 9, Ireland
a r t i c l e
i n f o
Article history: Received 8 June 2010 Received in revised form 19 August 2010 Accepted 20 August 2010 Available online 18 September 2010 Keywords: Ascorbic acid Gastric juice Amperometry Microsensor Electrode modification
a b s t r a c t This article reports on a novel microsensor for amperometric measurement of ascorbic acid (AA) under acidic conditions (pH 2) based on a carbon fiber microelectrode (CFME) modified with nickel oxide and ruthenium hexacyanoferrate (NiO–RuHCF). This sensing layer was deposited electrochemically in a twostep procedure involving an initial galvanostatic NiO deposition followed by a potentiodynamic RuHCF deposition from solutions containing the precursor salts. Several important parameters were examined to characterize and optimize the NiO–RuHCF sensing layer with respect to its current response to AA by using cyclic voltammetry, and scanning electron microscopy–energy dispersive X-ray spectroscopy methods. With the NiO–RuHCF coated CFME, the AA oxidation potential under acidic conditions was shifted to a less positive value for about 0.2 V (Ep of ca. 0.23 V vs. Ag/AgCl) as compared to a bare CFME, which greatly improves the electrochemical selectivity. Using the hydrodynamic amperometry mode, the current vs. AA concentration in 0.01 M HCl, at a selected operating potential of 0.30 V, was found to be linear over a wide range of 10–1610 M (n = 22, r = 0.999) with a calculated limit of detection of 1.0 M. The measurement repeatability was satisfactory with a relative standard deviation (r.s.d.) ranging from 4% to 5% (n = 6), depending on the AA concentration, and with a sensor-to-sensor reproducibility (r.s.d.) of 6.9% at 100 M AA. The long-term reproducibility, using the same microsensor for 112 consecutive measurements of 20 M AA over 11 h of periodic probing sets over 4 days, was 16.1% r.s.d., thus showing very good stability at low AA levels and suitability for use over a prolonged period of time. Moreover, using the proposed microsensor, additionally coated with a protective cellulose acetate membrane, the calibration plot obtained in the extremely complex matrix of real undiluted gastric juice was linear from 10 to 520 M (n = 14, r = 0.998). These results demonstrated the unique featuring of the proposed NiO–RuHCF microsensor under acidic conditions with enhanced sensitivity and stability and proved its promising potentiality for direct amperometric probing of AA at physiological levels in real gastric juice environments. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ascorbic acid (AA), also known as vitamin C (its l-enantiomer) or ascorbate (at physiological pH), is an essential component of the diet of a number of vertebrates including humans [1]. In the human body, its primary function is to act as an antioxidant, neutralizing toxic peroxides and stabilizing free radicals [2,3]. Evidence suggests that AA may play an important role in cancer and heart disease pre-
∗ Corresponding author at: Analytical Chemistry Laboratory, National Institute of Chemistry, P.O. Box 660, SI-1001 Ljubljana, Slovenia. Tel.: +386 14760230; fax: +386 14760300. E-mail address: [email protected] (B. Ogorevc). 1 Present address: Department of Chemistry, University of Coimbra, Coimbra, Portugal. 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.08.027
vention [4,5], alleviation of high blood pressure [6], and immune system boosting [7]. AA is thought to protect against gastric cancer, which is the fourth most common cancer and the second most common cause of cancer deaths worldwide [8], through its actions in scavenging nitrite and preventing the formation of nitrite-derived mutagens such as nitrosamines and nitrosamides [9–11]. The concentration of AA in gastric juice is therefore a potentially critical factor in the prevention of intragastric N-nitrosation [12], with significantly lower gastric AA concentrations found in patients infected with H. pylori compared to an uninfected control group [13]. Median gastric AA concentrations in both healthy and compromised individuals can be expected to range between approximately 16 and 154 M, with the variations in concentration due to differing subject gastric mucosal pathology [12]. Several interesting studies have been devoted to examining interactions between AA, nitrite and thiocyanate in benchtop models [11,14,15].
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
Due to its crucial role in the human body, numerous methods for the determination of AA in biological samples have been proposed, including spectrophotometry [16,17], chemiluminesence [18], capillary electrophoresis [19] and high performance liquid chromatography (HPLC) [20,21]. Although some of these methods are very sensitive, they can be very complex, time consuming and/or need sophisticated equipment. HPLC and related methods [14,22,23] are commonly employed in measurements of AA in gastric juice, but relatively complicated sampling and storage of the gastric juice samples is required. The oxidation of AA, as a basis of its electrochemical sensing, has been the subject of numerous studies in which electrodes composed of various modified or unmodified transducer materials, e.g. Au [24], Pt [25], glassy carbon [26] and carbon paste [27], have been employed. Within the field of electroanalytical measurement of AA and other biologically significant compounds, considerable focus has been placed on the use of microelectrodes which, due to their attractive inherent advantages, allow convenient probing in microenvironments [28–33]. Although various microprobes for direct measurement of AA in real biological samples have been proposed [28,30] this is, to the best of our knowledge, the first report on a microsensor suitable for the direct probing of gastric AA. Metal hexacyanoferrate (MeHCF) deposits as electrode modifiers have been extensively investigated to exploit their electrocatalytic properties for the detection of various organic and inorganic species, e.g. different cations [34], glucose [35], hydrazine [36], and AA [37,38]. Mixed metal HCFs as electrode coatings have also been studied and employed in improved electrochemical sensing, for example CoCuHCF in potentiometric detection of hydrazine [39] and FeCoHCF in amperometric sensing of hydrogen peroxide [40]. The most notable advantages of MeHCF electrode coatings are certainly their electrocatalytic activity and prolonged stability. This article reports on the development and attractive performance of a new microsensor, prepared by modifying a substrate carbon fiber microelectrode (CFME) with an electrochemically deposited composite nickel oxide and ruthenium hexacyanoferrate (NiO–RuHCF) layer. The proposed microsensor was found to be suitable for measuring AA under strong acidic conditions and in model gastric juice solution, exhibiting prolonged stability. The performance of the proposed microprobe was studied in detail, with particular emphasis on amperometric response, stability and selectivity. Upon the application of an additional protective membrane, its practical use for direct amperometric measurement of physiological concentrations of AA in undiluted real gastric juice is also demonstrated.
2. Experimental 2.1. Apparatus Cyclic volammetric (CV), amperometric and galvanostatic experiments were performed using a modular electrochemical workstation (Autolab PGSTAT12, Eco Chemie, Utrecht, The Netherlands), equipped with a low-current ECD module and driven by GPES 4.9 software (Eco Chemie). The three-electrode configuration employed in all experiments consisted of a modified carbon fiber microelectrode (CFME), a platinum wire and Ag/AgCl (satd. KCl) as the working, counter and the reference electrode, respectively. All potentials in this work are referred to this reference. All electrochemical measurements were carried out at room temperature (23 ± 2 ◦ C) in a standard voltammetric cell placed in a Faraday cage. Stirring conditions in hydrodynamic amperometric measurements were provided by a magnetic stirrer at 300 rpm. Scanning electron microscopy (SEM) including energy dispersive X-ray (EDX) analysis operations were performed using a
177
Hitachi S 3000N Scanning Electron Microscope (Hitachi, Japan) equipped with a Link Isis Energy Dispersive Spectrometer (Oxford Instruments, UK). 2.2. Chemicals and solutions All chemicals employed in this work were of analytical-grade purity and were used as received. Ascorbic acid (AA), nickel(II) sulfate hexahydrate and l-glucose were obtained from Kemika (Zagreb, Croatia). Ruthenium(III) chloride was obtained from Sigma, while potassium hexacyanoferrate(III) was purchased from Fluka. Protein standard, lyophilized (1.32 mg mL−1 protein) was provided by Bio-Rad (CA, USA). Water used to prepare all solutions was prepared via a water purification unit (Elix/Milli-Q Gradient, Millipore, Bedford, USA). Real gastric juice samples were obtained from a healthy volunteer (female, age 24) and stored in a freezer at −18 ◦ C and thawed before use. AA standard stock solutions (0.1 M) were prepared daily and were refrigerated when not in use. Modification solutions for applying (a) ruthenium hexacyanoferrate (RuHCF), (b) nickel hexacyanoferrate (NiHCF) or (c) nickel ruthenium hexacyanoferrate (NiRuHCF) coatings were composed of 0.1 M KCl, 4 mM HCl, 0.2 mM K3 [Fe(CN)6 ], and either (a) 0.1 mM RuCl3 or (b) 0.1 mM NiSO4 or (c) both a + b in 1:1 ratios in aqueous solution. 0.01 M HCl solution (pH 2) was used as supporting electrolyte for all measurements unless otherwise stated. The model gastric juice solution that was used in this work was composed of 0.01 M HCl, 1 mM NaCl, 2 mM glucose and 20% protein, the major components present in human gastric juice. 2.3. Microsensor preparation 2.3.1. Fabrication of CFMEs A cleaned single carbon fiber (7 m in diameter, 2–3 cm in length, Goodfellow Co., Oxford, UK) was first attached to a copper wire and then thermally sealed into a pulled glass capillary employing a microelectrode puller (PP-830, Narishige, Tokyo, Japan), with a final cut of the carbon fiber tip to a length of ca. 1 mm. The carbon fiber tip lengths of all fabricated and successfully tested CFMEs in this work were exactly measured using an inverted optical microscope and appropriate image analyzer programme. These tip length values were used to calculate corresponding geometric surface areas in order to obtain current densities where required. More details are given elsewhere [41]. 2.3.2. Modification of CFMEs Prior to modification, the CFME was pretreated electrochemically in a 0.1 M KCl solution by applying a continuous potential scan between 0.0 and +1.0 V at a scan rate of 100 mV s−1 until a steady-state current–voltage profile was reached, upon which it was thoroughly washed with Milli-Q water. In the two-step modification procedure, the first step, galvanostatic deposition of nickel oxide (NiO), was carried out by immersing the CFME into an aqueous solution containing 1.1 M NiSO4 and 1.2 M H3 BO3 and applying a constant current of +0.4 A for 10 s (whereupon the potential observed was ca. +2.3 V). In the second step, the RuHCF film was electrochemically grown onto the NiO-coated CFME in its respective modification solution (see Section 2.2 above) by cycling the potential between 0.0 and +1.0 V at a scan rate of 100 mV s−1 until a desired coverage was obtained (22 full cycles unless otherwise stated). The same potentiodynamic coating procedure was used for the deposition of the other metal hexacyanoferrate films onto bare CFMEs by using corresponding modification solutions (see Section 2.2 above). Following this step, the modified CFME was rinsed with water and was ready for use or was further coated with an additional protective membrane. This was achieved by dip-coating the
178
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
Fig. 1. The dependence of the cyclic voltammetric peak current response to AA at a NiO–RuHCF-modified CFME on the conditions of the two-step modification procedure: (A) on the galvanostatic treatment time in the first step (with RuHCF coverage fixed at 25 CV runs) and (B) on the RuHCF coverage in the second step, expressed in the number of CV runs (with NiO deposition time fixed at 10 s). Other modification conditions: (A) galvanostatic treatment of a bare CFME in the nickel modification solution at +0.4 A, (B) repetitive cycling of the potential (CV runs) between 0.0 and +1.0 V in the respective ruthenium modification solution at a scan rate of 100 mV s−1 . Measurement solution: 0.01 M HCl containing 1 mM AA; CV scan rate 100 mV s−1 . For easier comparison the current response is shown in a relative scale.
modified CFME in 1:1 (v/v) cyclohexanone–acetone solution containing 1% cellulose acetate, using a laboratory made dip-coating device with speed of dipping set to 0.34 mm s−1 . After each dipping (6 in total), the tip was allowed to dry in air at room temperature for approximately 5 min.
3. Results and discussion 3.1. Preparation, optimization and characterization of the modification layer In order to achieve the optimum electrochemical detection of AA under strong acidic conditions, we developed a two-step procedure for depositing a composite nickel oxide and ruthenium hexacyanoferrate (NiO–RuHCF) film onto a carbon fiber microelectrode (CFME). In the first step, the idea for which was adopted from the known surface-finishing process of plating with nickel [42], a clean bare CFME was galvanostatically treated in the corresponding nickel modification solution, while the RuHCF layer was subsequently deposited using a multiscan cyclic voltammetry protocol. As shown in Fig. 1, the formation of this sensing layer was studied, with respect to its voltammetric response to AA, by monitoring the effect of the galvanostatic treatment time (Fig. 1A) and the potentiodynamic coverage of the RuHCF film (Fig. 1B). As displayed in Fig. 1A, the voltammetric response to AA exhibited a maximum signal after a positive current of 0.4 A in the galvanostatic step was applied for 10 s and up to 15 s. While at shorter times the amount of NiO coverage was apparently inadequate, treatment times longer than 15 s resulted in a significant decrease of the signal, either due to lower conductivity of the gradually thicker NiO layer or because of anodic damage to the carbon fiber substrate electrode. From Fig. 1B, where the RuHCF coverage is defined in terms of the number of CV runs, it is well evident that the optimum response to AA was provided with 22 CV runs whereas thinner or thicker films yielded lower AA signals. Similar behavior has been reported for AA detection at a CoHCF film [37]. It was postulated that the thinner film does not provide a sufficient number of catalytic sites to handle the available supply of AA, whereas the thicker film may form a resistive barrier to the transfer of electrons. Based on the results from Fig. 1, 10 s of galvanostatic coating of NiO and 22 CV runs of potentiodynamic RuHCF deposition under the applied conditions were selected as optimum and employed in all subsequent experiments.
Deposits formed in each phase of the two-step coating procedure were inspected using optical and scanning electron microscopy (SEM) as well as energy dispersive X-ray spectroscopy (EDX). These characterizations proved that, as expected, upon application of positive current during the galvanostatic treatment, nickel oxide was deposited onto the CFME bare surface. Using an optical microscope it was observed that a green film was formed, which is the typical color of NiO [43]. Further evidence of NiO formation was provided upon SEM analysis of the film surface yielding images (not shown) with a strong similarity to those of a nickel oxide film reported elsewhere [44]. EDX spectra of this film revealed the presence of nickel (K␣ , L␣ and K lines) and oxygen, again indicating the presence of a nickel oxide layer. The SEM/EDX analysis of the final coating produced during the second step (using potentiodynamic treatment) revealed a markedly different surface from that obtained during the first step. While the SEM imaging (not shown) revealed that the surface was not perfectly uniform, the EDX spectra exhibited the presence of Ru(L␣ ), Ni(K␣ ) and Fe(K␣ ) lines. These data suggest the possibility that cycling the nickel oxide surface at positive potentials in the ruthenium(III) and hexacyanoferrate(III) solution can result in the formation of oxide bridging leading to the NiO–RuHCF composite coating. However, while our SEM/EDX surface analyses are sufficiently indicative for the purpose of this study, a much more dedicated investigation should be conducted in order to get a complete insight into the composition, structure and morphology of the NiO–RuHCF coating prepared by the proposed procedure. To verify the advantage of the proposed modification procedure, several experiments were conducted to compare different modified CFMEs, as illustrated in Fig. 2. First (Fig. 2A) we compared the NiO–RuHCF microsensor and a bare CFME. Evidently, at the NiO–RuHCF-modified CFME a large shift of AA oxidation potential to less positive value for ca. 210 mV (Ep of about +0.23 V) with a concurrent increase in signal height can be clearly observed. This apparent catalytic activity of the NiO–RuHCF surface significantly improves both the electrochemical selectivity and sensitivity of the proposed microsensor for AA detection. An apparent breakage of the voltammetric curve at potentials around +0.25 V might be attributed to a sudden re-arrangement of the modification layer and/or charging/discharging process. The observed behavior was very reproducible and, in addition, did not effect our further amperometric measurements of AA under constant potential regime. From Fig. 2A, b and d, which show cyclic voltammograms obtained at the modified CFME, it can be seen that there is an additional
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
179
Fig. 2. Cyclic voltammograms of AA at different modified and unmodified CFMEs: (A) comparison of CV responses at bare CFME (a, c) and NiO–RuHCF-modified CFME (b, d) for 0.01 M HCl, pH 2 (a, b) and 1 mM AA + 0.01 M HCl (c, d). (B) Comparison of CV responses to 1 mM AA in 0.01 M HCl, pH 2, of CFMEs modified with (a) NiHCF, (b) NiO, (c) RuHCF, and (d) NiRuHCF. CV scan rate: 100 mV s−1 . Current density is presented to allow comparison.
redox process at ca. 0.0 and 0.1 V, in both the 0.01 M HCl and 0.01 M HCl + 1 mM AA solutions, respectively. Examination of the current response for this process revealed that it is linearly dependent (r = 0.999) on the scan rate, indicating that it is a function of a redox system confined to the surface of the microsensor and not connected to AA detection. Further comparisons (Fig. 2B) of the NiO–RuHCF were made with various other modification layers consisting of one or both of its components: NiHCF deposited using a multiscan (CV) protocol, NiO alone prepared using the galvanostatic treatment, RuHCF alone deposited using a multiscan CV protocol, and NiRuHCF deposited from the solution containing respective precursor salts using the multiscan CV protocol as well. The NiHCF coating (Fig. 2B, a) exhibited a well-defined response to AA, which is in agreement with the literature data [38]. Although NiHCF was proposed to catalyze the oxidation of AA via surface layer mediated charge-transfer [45], it provided under our experimental conditions a negative shift of AA oxidation potential for only approximately 20 mV. The galvanostatically deposited NiO layer (Fig. 2B, b) also exhibited a well-defined signal, with an AA oxidation potential of about +0.30 V, which was considerably shifted from that obtained at a bare carbon fiber (+0.44 V). It is clearly evident that the responses of both the RuHCF and NiRuHCF coatings (Fig. 2B, c and d) are ill-defined in the potential range examined. Thus, it may be concluded that under the conditions applied, the NiO–RuHCF modification layer (Fig. 2A, d) provided the optimum response to AA, with the least positive oxidation potential and a clearly defined signal. An investigation was performed to characterize the electrochemical behavior of the NiO–RuHCF-modified CFME in the presence of AA at a pH of 2. Since the plots of ip vs. scan rate and ip vs. square root of scan rate did not show typical behavior, we plotted ip /Cv1/2 (current function vs. v1/2 , where C is the concentration of AA and v is the scan rate). This plot exhibited a negative slope, which is typical of a catalytic process involving a chemical reaction followed by an electron transfer process [46]. On the basis of these results it is confirmed that the electrocatalysis of AA at the NiO–RuHCF layer is a catalytic process which is consistent with the known electrochemical oxidation reaction of AA.
(OP) for conducting amperometric measurements of AA at the proposed NiO–RuHCF microsensor, the amperometric response to AA was plotted against the applied OP in the format of a pseudovoltammogram, displayed in Fig. 3. It can be seen that the current response increases sharply at OPs from +0.27 to +0.30 V. Because the signal increase beyond this OP is insignificant and, in particular, to avoid possible interferences from other electroactive species as well as to omit the increasing background current contribution at higher potentials, the optimum OP was selected to be +0.30 V. In addition, this OP also corresponds to an inflection point in the pseudo-voltammogram (Fig. 3), which is desirable for improved stability in the current measurements [46]. To obtain calibration data in 0.01 M HCl, the hydrodynamic amperometric mode at the OP of +0.30 V was employed and the current signal to AA concentration relationship was found linear over the range of 10–1610 M (n = 22), with a slope of 0.12 nA M−1 (or 0.57 A cm2 M−1 if the area of the microsensor is taken into consideration) and a correlation coefficient (r) of 0.999, as demonstrated in Fig. 4. From these results, it is evident that the NiO–RuHCF microsensor provides detection possibilities over the entire range of AA levels encountered in human gastric juice. The repeatability of
3.2. Amperometric AA detection and analytical performance of the NiO–RuHCF microsensor With the final goal in focus, from this stage of the work on, we employed the amperometric mode under hydrodynamic conditions. In order to determine the optimum operating potential
Fig. 3. A pseudo-voltammogram constructed from amperometric responses recorded at each operating potential separately, after addition of 50 M AA to a stirred 0.01 M HCl solution, using a NiO–RuHCF microsensor. Current density is presented to allow comparison of results.
180
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
Fig. 4. Hydrodynamic amperogram obtained during additions of AA to 0.01 M HCl solution at a NiO–RuHCF microsensor; AA increments: (a) 6 times 10 M, (b) 7 times 50 M, (c) 6 times 100 M, and (d) 3 times 200 M; operating potential: +0.30 V. Inset shows corresponding calibration plot.
amperometric AA measurements was found to be satisfactory with relative standard deviations (r.s.d.s) of 5.1% and 4.1% obtained for 6 consecutive additions of 10 and 100 M AA, respectively. The limit of detection (based on S/N = 2) was calculated to be 1.0 M, which is consistent with experiments (not shown) in which even 2 M AA elicited a discernable response at the proposed microsensor. The sensor-to-sensor reproducibility was also found to be satisfactory. For 10 and 100 M AA measured at 5 freshly prepared NiO–RuHCF microsensors, the r.s.d.s were 9.0% and 6.9%, respectively. These calibration data revealed the promising performance of the NiO–RuHCF microsensor and suggested its potential applicability in measurements of gastric juice AA levels. Interestingly, at a bare CFME, upon successive additions of 20 M AA to a 0.01 M HCl solution applying an OP of +0.50 V (not shown), the current signal was observed to deviated from linearity with an increasingly diminishing and tailing peak-shaped current response observed following each AA addition. We also examined the performance of the NiO–RuHCF microsensor for its use over a prolonged period of time. In this testing, a single microsensor was applied for continuous amperometric measurements (at an OP of +0.30 V) of 20 M AA increments, by periodically adding the corresponding amounts of AA to the stirred 0.01 M HCl solution, over 4 days (with intermediate overnight storing of the used microsensor in 0.01 M HCl) in the following pattern: first day over 6 h, second day over 2 h, third day over 2 h and the fourth day over 1 h. 20 M AA was chosen as a concentration which relates closely to the amount of AA secreted periodically into the human stomach [11]. This represented a total working time of 11 h with the total elapsed time from the first to the last measurement of 75 h. The r.s.d. for all 112 hydrodynamic amperometric measurements of 20 M AA increments over this entire testing time (i.e. 75 h) was 16.1%. For a relatively low concentration level of AA this long-term repeatability is considered to be very good. It clearly indicates that the proposed microsensor is well suited for direct amperometric probing of AA under strong acidic conditions such as those encountered in gastric juice over a prolonged period of time. 3.3. Direct amperometric AA probing in model solution and in real gastric juice In order to protect the NiO–RuHCF microsensor from inevitable fouling by macromolecular components present in the gastric
Fig. 5. Hydrodynamic amperometric recording of the response to the addition of (a) and (c) 4 times 20 M AA, and (b) and (d) 100 M NaNO2 , obtained at a cellulose acetate membrane coated NiO–RuHCF microsensor; medium: gastric juice model solution containing 1 mM NaCl, 2 mM glucose, 20% protein and 0.01 M HCl; operating potential: +0.30 V.
matrix, we considered the application of a cellulose acetate membrane (CAM) deposited onto the microsensor surface by employing a dip-coating procedure. Cellulose acetate was chosen due to its neutrality and its known applicability in protection of electrode surfaces from electrochemical fouling [47]. Employing a strategy of using several thin coatings instead of one thicker coating and based on a compromise between appropriate surface protection and “not affecting” the analyte signal, examination showed that six layers of the cellulose acetate polymer should be adequate for protection of the microsensor while still providing a strong AA signal. Several experiments (not shown) were also carried out using the proposed microsensor in order to determine if the components of the model gastric solution elicited a response at the employed operating potential of +0.3 V. First of all, individual solutions of thiocyanate (1 mM), sodium nitrite (1 mM) and glucose (2 mM) in 0.01 M HCl were cycled over the potential range of ca. −0.5 to +0.5 V. No response was observed at the oxidation potential of AA (+0.23 V). Secondly, hydrodynamic amperometry experiments were carried out by spiking, individually, the abovementioned solutions into a solution of 0.01 M HCl. No response was observed at the applied OP of +0.3 V. As the proposed microsensor is insensitive to nitrite, as evidenced from the experiments described above, it is certain that the response obtained in the solution containing AA and nitrite will solely be due to the oxidation of AA. We further investigated whether the proposed microsensor was capable of continuously monitoring the level of AA in the model solution, which is often required in studies of the interactions between AA and nitrite [14,15]. For this reason, hydrodynamic amperometric measurements were performed in the gastric juice model solution, to which four additions of 20 M AA were made, followed by the addition of 100 M NaNO2 , whereupon this sequence was repeated after 5 min, as displayed in Fig. 5. For the sake of figure clarity and time saving a new sequence was started before the current response to AA faded to the background level. It can be noted that even in the presence of a high protein concentration in the solution, the response of the protective CAM-coated microsensor to AA was well-defined, with the current remaining steady after each addition, indicating the absence of fouling of the microsensor surface. From Fig. 5, the existence of a disparity between the currents obtained for the AA additions in the absence (Fig. 5a) and in the presence (Fig. 5c) of nitrite can be
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
181
using a two-step galvanostatic/potentiodynamic protocol, with further application of a cellulose acetate membrane (CAM) protective coating. The signal to AA concentration relationship in acidic media (0.01 M HCl and gastric juice) was linear over a wide range corresponding to physiological AA concentrations commonly encountered in gastric juice. The proposed microsensor exhibited either satisfactory or excellent selectivity, sensitivity, repeatability and life-time properties, even in the combination with the extremely complex gastric juice matrix. Acknowledgements
Fig. 6. Hydrodynamic amperometric recording of five 50 M AA increments after spiking an appropriate amount of AA into a stirred sample (volume 2 mL) of undiluted real human gastric juice, using a cellulose acetate membrane coated NiO–RuHCF microsensor; operating potential: +0.30 V.
seen, i.e. the amperometric signals observed for AA in the presence of nitrite are lower than those observed in the nitrite-free solution, while in both cases linearly increasing with AA concentration. This can be explained by nitric oxide formation via the reaction of AA with nitrite, combining with oxygen present in the solution to reform nitrite, which can then react further with AA [11]. This recycling will continue until either the AA or oxygen has been depleted. These attractive results (Fig. 5) demonstrated that the proposed microsensor can be successfully applied for on-line real-time monitoring of AA levels in this type of in vitro investigations. To examine the suitability of the proposed NiO–RuHCF microsensor coated with a protective CAM for direct probing of AA in human gastric juice, amperometric measurements were conducted whereupon a stirred undiluted gastric juice sample was spiked with an appropriate amount of AA, as is illustrated in Fig. 6. As is evident, it is demonstrated that in an extremely complicated matrix such as pure gastric juice, the direct real-time probing of AA is possible: the CAM-coated NiO–RuHCF microsensor response to AA is linear, the response time is rapid enough and the observed background current contribution is satisfactorily low. Under the described in vitro experimental conditions, roughly simulating the in vivo periodic secretions of AA into gastric juice, the signal to AA concentration relationship was found to be linear over the range of 10–520 M, with a slope of 0.02 nA M−1 (or 0.12 A cm2 M−1 ) and a correlation coefficient of 0.998 (n = 14), with an estimated limit of detection (S/N = 2, n = 6) of 8.5 M. The lower sensitivity, as compared with that achieved in 0.01 M HCl, can be attributed to the lower conductivity of the gastric juice solution and also to the presence of the cellulose acetate membrane on the surface of the microsensor. These results indicate the practical applicability of directly measuring AA physiological levels in gastric juice and, after some further technological improvement of the proposed microsensor, also monitoring secretion of AA into the gastric cavity. 4. Conclusions The purpose of this work was to develop an all-solid microsensor for amperometric probing of ascorbic acid (AA) in gastric juice. This was achieved by modifying the surface of a substrate carbon fiber microelectrode with an electrochemically deposited nickel oxide and ruthenium hexacyanoferrate (NiO–RuHCF) sensing layer,
This work was supported by Slovenian Research Agency (Program P1-0034). E.A.H. thanks the National Centre for Sensor Research, Dublin City University for funding and the National Institute of Chemistry, Ljubljana, for partial support. The authors thank Prof. Kenneth McColl from Faculty of Medicine, University of Glasgow, Glasgow, UK, for helpful suggestions. References [1] G.L. Wheeler, M.A. Jones, N. Smirnoff, Nature 393 (1998) 365–369. [2] S.J. Padayatty, A. Katz, Y.H. Wang, P. Eck, O. Kwon, J.H. Lee, S.L. Chen, C. Corpe, A. Dutta, S.K. Dutta, M. Levine, J. Am. Coll. Nutr. 22 (2003) 18–35. [3] H. Sies, W. Stahl, Am. J. Clin. Nutr. 62 (1995) S1315–S1321. [4] A.C. Carr, B. Frei, Am. J. Clin. Nutr. 69 (1999) 1086–1107. [5] K.T. Khaw, S. Bingham, A. Welch, R. Luben, N. Wareham, S. Oakes, N. Day, Lancet 357 (2001) 657–663. [6] S.J. Duffy, N. Gokce, M. Holbrook, A. Huang, B. Frei, J.F. Keaney, J.A. Vita, Lancet 354 (1999) 2048–2049. [7] P.C. Calder, S. Kew, Brit. J. Nutr. 88 (2002) S165–S176. [8] P. Correa, M. Blanca Piazuela, M. Constanza Camargo, Gastric Cancer 7 (2004) 9–16. [9] C. Mowat, A. Carswell, A. Wirz, K.E.L. McColl, Gastroenterology 116 (1999) 813–822. [10] M. Hayat, S. Everett, Nutrition 15 (1999) 402–403. [11] A. Moriya, J. Grant, C. Mowat, C. Williams, A. Carswell, T. Preston, S. Anderson, K. Iijima, K.E.L. McColl, Scand. J. Gastroenterol. 37 (2002) 253–261. [12] C.J. Schorah, G.M. Sobala, M. Sanderson, N. Collis, J.N. Primrose, Am. J. Clin. Nutr. 53 (1991) S287–S293. [13] Z.W. Zhang, S.E. Patchett, D. Perrett, P.H. Katelaris, P. Domizio, M.P.G. Farthing, Gut 43 (1998) 322–326. [14] H. Suzuki, A. Moriya, K. Iijima, K. McElroy, V.E. Fyfe, K.E.L. McColl, Scand. J. Gastroenterol. 38 (2003) 856–863. [15] K. Iijima, J. Grant, K. McElroy, V. Fyfe, T. Preston, K.E.L. McColl, Carcinogenesis 24 (2003) 1951–1960. [16] K. Guclu, K. Sozgen, E. Tutem, M. Ozyurek, R. Apak, Talanta 65 (2005) 1226–1232. [17] K. Kodama, K. Sumii, M. Kawano, T. Kido, K. Nojima, M. Sumii, K. Haruma, M. Yoshihara, K. Chayama, Europ. J. Gastroenterol. Hepatol. 15 (2003) 987–993. [18] Y.J. Ma, M. Zhou, X.Y. Jin, B.Z. Zhang, H. Chen, N.Y. Guo, Anal. Chim. Acta 464 (2002) 289–293. [19] W.S. Kim, R.L. Dahlgren, L.L. Moroz, J.V. Sweedler, Anal. Chem. 74 (2002) 5614–5620. [20] J. Lykkesfeldt, Anal. Biochem. 282 (2000) 89–93. [21] M.A. Kall, C. Andersen, J. Chromatogr. B 730 (1999) 101–111. [22] M.J. Sanderson, C.J. Schorah, Biomed. Chromatogr. 2 (1987) 197–202. [23] A.G. Fraser, G.A. Woollard, J. Gastroenterol. Hepatol. 14 (1999) 1070–1073. [24] J.J. Fei, L.M. Luo, S.S. Hu, Z.Q. Gao, Electroanalysis 16 (2004) 319–323. [25] H. Ernst, M. Knoll, Anal. Chim. Acta 449 (2001) 129–134. [26] W. Hu, D. Sun, W. Ma, Electroanalysis 22 (2010) 584–589. [27] S. Shahrokhian, E. Asadian, Electrochim. Acta 55 (2010) 666–672. [28] P.S. Cahill, R.M. Wightman, Anal. Chem. 67 (1995) 2599–2605. [29] P. Janda, J. Weber, L. Dunsch, A.B.P. Lever, Anal. Chem. 68 (1996) 960–965. [30] T.R.L.C. Paixão, L.F. Barbosa, M.T. Carrì, M.H.G. Medeiros, M. Bertotti, Analyst 133 (2008) 1605–1610. [31] J.-W. Mo, B. Ogorevc, Anal. Chem. 73 (2001) 1196–1202. [32] C.A. Anastassiou, B.A. Patel, M. Arundell, M.S. Yeoman, K.H. Parker, D. O’Hare, Anal. Chem. 78 (2006) 6990–6998. [33] R.M. Wightman, Science 311 (2006) 1570–1574. [34] Q. Xu, S. Zhang, W. Zhang, L.T. Jin, K. Tanaka, H. Haraguchi, A. Itoh, Fresenius J. Anal. Chem. 367 (2000) 241–245. [35] J. Wang, X.J. Zhang, L. Chen, Electroanalysis 16 (2000) 1277–1281. [36] A. Salimi, K. Abdi, Talanta 63 (2004) 475–483. [37] C.-X. Cai, K.-H. Xue, S.-M. Xu, J. Electroanal. Chem. 486 (2000) 111–118. [38] M.H. Pournaghi-Azar, H. Razmi-Nerbin, B. Hafezi, Electroanalysis 14 (2002) 206–212. [39] J.-W. Mo, B. Ogorevc, X.J. Zhang, B. Pihlar, Electroanalysis 12 (2000) 48–54.
182
E.A. Hutton et al. / Analytica Chimica Acta 678 (2010) 176–182
[40] W.Y. Tao, D.W. Pan, Y.J. Liu, L.H. Nie, S.Z. Yao, Anal. Biochem. 338 (2005) 332–340. [41] S.B. Hoˇcevar, B. Ogorevc, K. Schachl, K. Kalcher, Electroanalysis 16 (2004) 1711–1716. [42] G.A. DiBari, Met. Finish. 98 (2000) 270–288. [43] D.R. Lide (Ed.), Handbook of Chemistry and Physics, 80th ed., CRC Press, Boca Raton, 1999.
[44] P. Häring, R. Kötz, G. Repphun, O. Haas, H. Siegenthaler, Appl. Phys. A. Mater. Sci. Proc. 66 (1998) S481–S486. [45] M.H. Pournaghi-Azar, H. Razmi-Nerbin, J. Electroanal. Chem. 456 (1998) 83–90. [46] S.A. Wring, J.P. Hart, Anal. Chim. Acta 229 (1990) 63–70. [47] G. Roman, A.C. Pappas, D. Kovala-Demertzi, M.I. Prodromidis, Anal. Chim. Acta 456 (2004) 201–207.