Development of a Biochemical Oxygen Demand Sensor Using Gold

The sensor was applied to BOD measurements of the water from a lake at the University of Indonesia in Jakarta, Indonesia, with results comparable to t...
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Development of a Biochemical Oxygen Demand Sensor Using GoldModified Boron Doped Diamond Electrodes Tribidasari A. Ivandini,*,†,‡ Endang Saepudin,† Habibah Wardah,† Harmesa,† Netra Dewangga,† and Yasuaki Einaga*,‡,§ †

Department of Chemistry, Faculty of Mathematics and Science, University of Indonesia, Kampus UI Depok, Jakarta 16424, Indonesia ‡ Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Yokohama 223-8522, Japan § CREST, JST, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan ABSTRACT: Gold-modified boron doped diamond (BDD) electrodes were examined for the amperometric detection of oxygen as well as a detector for measuring biochemical oxygen demand (BOD) using Rhodotorula mucilaginosa UICC Y-181. An optimum potential of −0.5 V (vs Ag/AgCl) was applied, and the optimum waiting time was observed to be 20 min. A linear calibration curve for oxygen reduction was achieved with a sensitivity of 1.4 μA mg−1 L oxygen. Furthermore, a linear calibration curve in the glucose concentration range of 0.1−0.5 mM (equivalent to 10−50 mg L−1 BOD) was obtained with an estimated detection limit of 4 mg L−1 BOD. Excellent reproducibility of the BOD sensor was shown with an RSD of 0.9%. Moreover, the BOD sensor showed good tolerance against the presence of copper ions up to a maximum concentration of 0.80 μM (equivalent to 50 ppb). The sensor was applied to BOD measurements of the water from a lake at the University of Indonesia in Jakarta, Indonesia, with results comparable to those made using a standard method for BOD measurement.

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material in water can be considered as the BOD value. The method was reported to shorten the BOD measurement time from 5 days to 30 s.3 Since the operation of the sensor is based on the concept of respiration, development of the sensors has been explored with many different kinds of aerobic microorganisms.2−8 On the other hand, nanotechnology is rapidly growing due to the advantages provided by the large surface areas that can be obtained. Modifying solid electrodes with nanometal particles was reported to have enlarged the signal to background noise ratio, resulting in a very low detection limit.9,10 Moreover, better stability of the current responses was reported with the decreasing of the size of modifying nanoparticles.11 Typical behavior was also reported for the oxygen reduction reaction at gold nanoparticle modified electrode carbon electrodes.12 The gold electrode had been established as an oxygen electrode since the introduction of the Clark electrode in the mid1950s.13,14 However, its limitation due to large overpotential for oxygen reduction had driven the development of gold nanoparticle modified electrodes to be extensively explored.15 Furthermore, recently the toxic effect of nanomaterials on living cells has been reported,16,17 which will probably have an influence on the application of nano-gold-modified electrodes for BOD sensors.

he presence of pollutants, such as heavy metal ions or organic material, in water decreases its quality. One of the parameters generally monitored in controlling water quality is the oxygen content, such as the dissolved oxygen (DO), the chemical oxygen demand (COD), and the biochemical oxygen demand (BOD).1 The DO value indicates the oxygen concentration in water, whereas BOD and COD describe the amount of oxygen required to oxidize organic materials in water. While a strong chemical agent is applied to oxidize the organic material for COD measurements, microorganisms are used for BOD measurements. High DO values show better quality of the water, whereas high values of COD and BOD indicate high concentrations of organic material in the water or low quality of the water. The established method for BOD measurements needs 5 days to grow the microorganisms in the water sample before a measurement of the amount of consuming oxygen can be performed. Therefore, the method is inadequate for industrial use since the data can only obtained after 5 days. The use of microbial BOD sensors has been reported as an alternative method for conventional BOD measurements.2−8 The BOD sensor was first introduced by Karube et al., who applied a microbial electrode consisting of immobilized bacteria in a collagen membrane and a gold electrode as a transducer.2 Basically, the BOD sensor uses an optimum amount of microorganisms (approximately equivalent to 5 days growth) as the biosensing agent. Therefore, the amount of oxygen required by the microorganisms to decompose the organic © 2012 American Chemical Society

Received: July 24, 2012 Accepted: October 22, 2012 Published: October 22, 2012 9825

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investigate the nanoparticle dimensions. Modification of the BDD was conducted by a procedure adapted from Tian et al.22 The BDD films were immersed in allylamine and irradiated by UV light (λ = 254 nm) for about 6 h. After cleaning and drying, the materials were immersed in a colloid of gold nanoparticles for 20 min followed by drying. Then, the materials were characterized and used as working electrodes. Fabrication of the Electrochemical Cell. An electrochemical cell with a cover and with a total volume of 5 mL was used for the BOD sensor. A gold-modified BDD working electrode was positioned at the bottom of the cell. The contact area of the working electrode was estimated to be 0.26 cm2. A spiral platinum wire and an Ag/AgCl (saturated KCl) system were used as counter and reference electrodes, respectively. Before being used as a BOD sensor, the cell was examined as an oxygen sensor. The cell was filled with 5 mL of 0.1 M phosphate buffer solution (PBS) with pH 6.8 and then purged with nitrogen for 5 min to remove all oxygen. Then, oxygen aeration was carried out and cyclic voltammetry (CV) was conducted. The concentration of the dissolved oxygen was confirmed using a dissolved oxygen meter (DO meter, Hana). Preparation of the Yeast. The R. mucilaginosa UICC Y181 culture was incubated in a shaking incubator (120 rpm) at 30 °C for 24 h, followed by 10 min centrifugation to separate the medium. The biomass was then washed with distilled water and stored in PBS, pH 7. The growth curve was determined by incubating the yeast in several conical flasks with different incubation times from 0 to 48 h. Each flask contained 25 mL of liquid yeast media broth (YMB) as a fermentation medium and a 1.25 mL cell suspension. The growth of the cells was monitored by observation of the turbidity using a UV spectrophotometer at 600 nm. The number of yeast cells was counted using the counting chamber method. Briefly, 1 mL cell suspensions were diluted by 10, 100, 1000, and 10 000 times and the cell number in each sample was counted using a microscope in a counting chamber. In addition, for confirmation, the optical density was also measured. It was concluded that 1 mL of yeast suspension contained 2.56 × 108 yeast cells. Electrochemical Measurements of BOD Sensors. The electrochemical cell was filled with 3.5 mL of 0.1 M PBS, pH 6.8. Then, 0.5 mL of standard glucose solution was added to make a total glucose concentration of 0.1 mM. After that, 1 mL of yeast suspension was added followed by oxygen aeration for 5 min before an amperometric measurement was made. The current immediately measured after oxygen aeration was labeled I0. The optimum waiting time was found by varying the waiting time from 5 to 25 min. After a waiting time of 5 min for the assimilation process, the current was measured and labeled I5. Using the same method, I10−I25 were measured. The optimum waiting time was then used for subsequent measurements. A linear calibration curve for BOD was constructed from the various glucose concentrations (0.1−0.5 mM). The influence of heavy metals on the system was determined by adding various concentrations of copper ions. Application to a real sample analysis was performed using water samples from a lake at the University of Indonesia, Jakarta, Indonesia. Sampling was done on water from three different areas of the lake. Analysis was performed within 2 h of collection. Before analysis, the samples were filtered using filter paper. A total standard addition of 0.5 mM was added in the sample solution. The results were compared to those of analysis results using a standard method for BOD measurement.1 Briefly, a 10 mL

In this research, a BOD sensor based on yeast with nanogold-modified boron doped diamond electrodes as the oxygen sensor was developed. Highly boron doped diamond (BDD) was selected for gold deposition due to its outstanding properties compared to other solid electrode materials. BDD has a wide electrochemical potential window, low background current, and high physical and chemical stability.18 Moreover, the biocompatibility and stability of the modified electrodes have been reported.10,18 However, although various method were developed to prepare gold-modified BDD electrodes,12,15,17−22 including those for oxygen reduction applications,12,15,17−21 to the best of our knowledge there was a very limited number of reports of the stability of the current responses. A remarkable result was reported for N-terminated BDD modified with gold nanoparticles, which was claimed to provide a strong interaction between the BDD and the gold nanoparticles.22 Therefore, this method was selected for this research. As the biosensing agent, Rhodotorula mucilaginosa UICC Y-181, a species of yeast isolated from Jakarta Bay, Jakarta, Indonesia, was used. As Jakarta Bay is known to have high levels of pollution, R. mucilaginosa UICC Y-181 was expected to be resistant to applications in extreme conditions. R. mucilaginosa can be found in air, soil, lakes, ocean water, and dairy products, colonized with plants, humans, and other mammals.23 Its applications to the degradation of nitrobenzene,24,25 to the decolorization of azo dyes,26 and as a copper biosensor,27 have been studied. Glucose was used as the model for the organic compounds. The interference of copper ions was also investigated. Furthermore, an application to a real sample was demonstrated by measuring the BOD value of water from a lake at the University of Indonesia in Jakarta, Indonesia. The data were compared with and validated by the standard conventional method for measuring BOD. The results show that nano-gold-modified N-terminated BDD (Au-BDD) electrodes can be applied for oxygen and as BOD sensors.

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EXPERIMENTAL SECTION Chemicals. Glucose, peptone, HAuCl4, K2HPO4, KH2PO4, and other chemicals were of analytical grade and supplied by Merck. Yeast extract of R. mucilaginosa UICC Y-181, isolated from Jakarta Bay, Jakarta, Indonesia, was obtained from the University of Indonesia Culture Collection (UICC), Laboratory of Microbiology, Department of Biology, Faculty of Mathematics and Science, University of Indonesia. The yeast extracts were supplied in the form of culture in solutions of yeast glucose peptone broth (YGPB) containing 0.5% yeast extract, 0.5% peptone, and 4% glucose. Preparation of Gold-Modified BDD Electrodes. BDD electrodes were deposited on Si(100) wafers in a microwave plasma-assisted chemical vapor deposition (MPCVD) system (ASTeX Corp.). Details of the preparation are described elsewhere.18 Acetone was used as the carbon source and B(OCH3)3 was used as the boron source in a B/C atomic ratio of 1:100. Before being used, the BDD film was pretreated by ultrasonication in 2-propanol for 10 min followed by rinsing with high purity water. Colloidal gold nanoparticles were prepared by adding 0.5 mL of 0.01 M HAuCl4 solution to 18.5 mL of water and stirring for 5 min at room temperature. Into that solution, 0.5 mL of 0.1 M sodium citrate was added and stirred for 5 min. Then, 0.5 mL of 0.1 M freshly prepared NaBH4 solution was added. Characterization was done by UV− visible spectroscopy at the adsorption band at ∼519 nm. Transmission electron microscopy (TEM) was utilized to 9826

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sample was prepared in a flask. Then, 1 mL of 0.25 M PBS, pH 6.8, 1 mL of MgSO4·7H2O solution (22.5 mg L−1), 1 mL of CaCl2 solution (27.5 mg L−1), and 1 mL of FeCl3 solution (0.25 mg L−1) were added. The solution was then saturated with dissolved oxygen by shaking the flask for 1 h before dilution by water into 25 mL. The dissolved oxygen was determined by using a DO meter immediately after dilution by water (D1), and after incubation at 20 °C for 5 days (D5). The BOD5 was calculated as follows: BOD5 (mg L−1) = (D1 − D5)/P

where P is the diluting factor (in this case P = 5).

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RESULTS AND DISCUSSION Gold nanoparticles were synthesized using citrate ions as a capping agent. After a reduction reaction using NaBH4 solution, the gold solution changed from yellow to ruby red (λ = 518 nm, data not shown), indicating that colloidal gold nanoparticles with an average dimension of 2−5 nm had been formed.27 Characterization using TEM confirmed that the average dimension of the gold nanoparticles was ∼3 nm (Figure 1). The gold nanoparticles were then used to modify the BDD surface.

Figure 2. XPS results of (a) as-deposited, (b) allylamine-modified, and (c) gold-modified BDD. The insets show magnifications of the N 1s peaks for each BDD.

indicates that the surface of the BDD had been partly oxidized in the ambient air. It is well-known that BDD is initially Hterminated due to its fabrication process in a H2 atmosphere.18 The H-functional groups on the BDD can be oxidized gradually during storage in an oxygen ambient.29 Figure 2b shows the XPS result of the BDD surface after pretreatment with allylamine. A new peak of N 1s at a binding energy of 399 eV was observed. Calculation of the O/C ratio before and after pretreatment showed a decrease from 0.3 to 0.1, while an N/C ratio of 0.01 was observed. The results suggest that part of the O-termination on the BDD changed to N-termination. Modification of the BDD surface with gold nanoparticles was then performed by immersion of the allylamine-modified BDD in the colloid of gold nanoparticles. Unfortunately, the evidence of gold adlayer at the surface of BDD could not be seen through SEM imaging since the size of gold nanoparticles used was very small (2−5 nm). However, a previous report using bigger gold nanoparticles (15−40 nm) showed an increase of the affinity of gold nanoparticles to the BDD surface in the sequence H-termination < O-termination < N-termination.22 XPS characterization after gold nanoparticle modification (Figure 2c) shows that the N 1s peak disappeared, while Au 4f7/2 and 4f5/2 at binding energies of 84 and 88 eV, respectively, could clearly be detected with an Au/C ratio of 0.289. The result indicates that the N-functional groups of the allylamine-

Figure 1. TEM image of gold nanoparticles.

Before being modified with gold nanoparticles, the BDD surface was pretreated to N-terminate to facilitate deposition of the nanoparticles. It has been documented that nitrogenmodified BDD has a better affinity for gold nanoparticles than BDD modified with oxygen or hydrogen.22 In order to acquire nitrogen termination, pretreatment was conducted by a photochemistry reaction under UV light (λ = 254 nm) in concentrated allylamine. It was expected that the double bonds in the allylamine would be destroyed and new covalent bonds between the C ions in allylamine and the C ions in BDD would be formed.22 X-ray photoelectron spectroscopic (XPS) characterization of the as-deposited BDD (Figure 2a) shows the bulk diamond (C 1s) peak at a binding energy of 284.5 eV as well as a couple of peaks corresponding to the C−OH and CO functional groups at binding energies of 534 and 550 eV, respectively,22,28 with an O/C ratio of 0.3. The XPS result 9827

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modified BDD surface had already been covered with gold nanoparticles. Tian et al. have proposed that self-assembly of a monolayer of gold nanoparticles could be performed due to the electrostatic interaction between the negatively charged citratecapped gold nanoparticles and the positively charged protonated allylamine-BDD.22 In such a case, the N-functional groups of BDD should remain with the presence of gold. However, the evidence of the XPS data showed that the Nfunctional groups disappeared, suggesting formation of a bond between the gold nanoparticles and the N-functional groups of BDD. The formation of covalent bonds between the gold and nitrogen could be possible since nitrogen has a lone pair of electrons, which can contribute to forming coordination covalent bonds with the free f orbital of gold. The chemical bonds between gold and the BDD surface are of considerable importance since very limited information about the stability of oxygen reduction responses was reported at gold-modified BDD. Various methods studied to prepare gold-modified BDD electrodes for oxygen reduction, such as electrodeposition,12,19 vacuum evaporation,15 thermal deposition,20 and sputter deposition,21 have shown high catalytic activity without any record of the stability. Indeed, lack of data on the stability of metal-modified diamond is reasonable because those methods cannot provide chemical bonds with the sp3 coordination of the diamond surface. A special case, when the stability of goldmodified BDD was observed, was for arsenic ion detection, which proposed a formation of As−Au alloy in anodic stripping voltammetry mechanisms.30 The formation seemed to stabilize the deposition of gold particles at the surface of the BDD, while the behavior is not found in the mechanism of oxygen reduction. The electrochemical behavior of the electrodes for oxygen reduction was examined using cyclic voltammetry. Figure 3 shows a cyclic voltammogram (CV) of a 0.1 M PBS, pH 6.8, saturated by oxygen for an Au-BDD electrode. The CV shows a couple of gold reduction oxidation peaks at +0.22 and +0.32 V (vs Ag/AgCl), respectively. In addition, two other reduction peaks at −0.20 and −0.5 V (Figure 3a) were observed. The linear increase of the current of those two peaks with the change of O2 concentration (Figure 3b and its inset) indicated that those peaks were attributable to oxygen reduction. A sensitivity of 1.4 μA mg−1 L oxygen could be achieved. Comparison to the CV at the bare gold electrode (inset of Figure 3a) shows a higher oxygen reduction current was observed at Au-BDD, indicating higher catalytic activity of the electrodes, which is confirmed by the previous reports of goldmodified BDD electrodes.12,15,19−22 The mechanism of oxygen reduction at the gold surface is critically dependent on the pH of the medium.31−33 Generally, it was concluded that two-electron reduction of O2 to H2O2 occurs in acidic media,31,32 while a four-direct electron process, which leads to the formation of H2O (or OH−), was reported for basic media.33 As two reduction peaks were observed in the CVs, it can be proposed that the mechanism of two steps of a four-electron process occurred at Au-BDD electrodes. However, further experiments were required for conclusions. Furthermore, although both Figure 3a and the inset display similar peaks in the CVs, it can be seen that the first oxygen reduction peak at Au-BDD appeared at the more negative potential compared to that at the bare gold electrode. Slower kinetics of oxygen reduction at Au-BDD indicated that reaction was strongly affected by the BDD surface. As the XPS data in Figure 2 show that the modified BDD surface was mainly

Figure 3. (a) Cyclic voltammograms of 0.1 M PBS, pH 6.8, in saturated dissolved oxygen (8.2 ppm) at an Au-BDD electrode (inset shows CV at bare gold electrode), and (b) magnification of the related peaks at −0.5 V at in various concentrations of oxygen (inset shows the linear dependence of the current response on the oxygen concentration). The scan rate was 100 mV/s. An Ag/AgCl (saturated KCl) electrode was used as the reference electrode.

terminated by gold and oxygen, such behavior was reasonable due to the repulsion effect of O-termination to oxygen molecules. The behavior was in agreement with previous reports, which showed that the kinetics of direct oxygen reduction at the BDD surface was affected by its termination.12,19 In order to apply the Au-BDD as a BOD sensor, the yeast was prepared. Figure 4 shows observation of the growth of the

Figure 4. Growth curve of R. mucilaginosa UICC Y-181, measured through the change in turbidity.

R. mucilaginosa UICC Y-181 cells monitored by UV spectroscopy through the change of turbidity. It can be seen from Figure 4 that the lag phase was not observed, indicating that there was no adaptation time of R. mucilaginosa cells required in its new environment (medium). The cells entered a logarithmic phase during 0−24 h shown by an elevated number of the cells. 9828

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measurements of oxygen concentration currents in PBS, pH 6.8, in the presence of various volumes of a free cell suspension of R. mucilaginosa UICC Y-181. It can be seen that the current decreased with the volume as increasing the amount of R. mucilaginosa UICC Y-181 would consume more oxygen. Extracted data in the inset of Figure 5b shows that the decreasing of the current was saturated with a suspension volume of 1 mL. Based on the result, 1 mL of suspension volume of R. mucilaginosa UICC Y-181 (or equivalent to 2.56 × 108 yeast cells) was fixed for the optimum condition. The dependence of the oxygen requirement of the system on the glucose concentration was then investigated. The oxygen requirement was observed as a change of oxygen concentration after the optimum waiting time (20 min). The current of the solution was measured immediately after oxygen aeration (I0). Then after a waiting time of 20 min the measurement was repeated to perform I20. Figure 6 shows the chronoamperograms of the system at 0 and 20 min after oxygen aeration in different concentrations of glucose in the concentration range 0.1−0.5 mM, which is equivalent to 10−50 mg L−1 BOD.3,34 Figure 6a shows that different concentrations of glucose resulted in a linear correlation to the change of oxygen concentration with an R2 of 0.98. The results indicate that the system with Au-BDD can be used in BOD sensors. The limit of detection (LOD) was estimated from yLOD = a + 3Sy. With a value of Sy of 0.1 μA in the linear equation y = 8.3x + 0.41, an LOD of 0.04 mM glucose or equivalent to 4 mg L−1 BOD was estimated. The reproducibility of the signal current was also investigated. Figure 7 shows 15 consecutive measurements of oxygen content in a solution of 0.1 mM glucose in 0.1 M PBS, pH 6.8, in the presence of free cells of R. mucilaginosa 20 min after the solution had been saturated with oxygen. Excellent reproducibility of the current with a relative standard deviation (RSD) of 0.9% is displayed in the inset. The high reproducibility of the results indicated the endurance of R. mucilaginosa in the system and the stability of the gold nanoparticles on the BDD surface. Moreover, the influence of gold nanoparticles on R. mucilaginosa can be ignored. The results also suggest that the stable interaction between the gold nanoparticles and the N-terminated BDD could occur due to the chemical bonding between them. If only an electrostatic interaction occurs, the interaction could be easily reversed by the change of the pH or the potential. Furthermore, as the measurement of oxygen reduction was performed in the presence of glucose and the oxygen measurement included the enzymatic oxidation of glucose by the yeast, there was a possibility that the adsorption of glucose and its oxidation results at the electrode surface were inhibited the electroreduction of oxygen. However, a very low RSD value indicated the fouling was very small. The probable reason was because of the very small size of the electroactive site (gold nanoparticle with average size of 3−5 nm) resulted in a less blocking site due to the adsorption of glucose. The behavior was consistent with the results of Tominaga et al. for glucose oxidation at gold nanoparticle modified carbon electrodes.11 The influence of the presence of copper ions was also observed. Various concentrations of copper (0−1.60 μM) were presented in solutions of 0.1 mM glucose in 0.1 M PBS, pH 6.8, with the presence of 1 mL of free cells of R. mucilaginosa. Figure 8 shows that the presence of copper can lead to an increase in signal current. The presence of 0.08−0.80 μM Cu in the system increased the signal current by 10−11%. However, when the

Then yeast entered a stationary phase followed by the death phase after 30 h. As a result, 24 h was fixed as an optimum time to harvest the yeast. Amperometry at an applied potential of −0.5 V (vs Ag/ AgCl) was used for the oxygen concentration measurements in the presence of the yeasts. Basically, R. mucilaginosa is an aerobic microorganism, with the respiration and metabolic functions of the microorganism performed by oxygen consumption.24−26 When an organic substrate is added to the solution, R. mucilaginosa will try to transport the substrate into the inner cells. This activity can be detected since the respiration rate increases due to the energy requirement for the activity.3 The more the substrate is transported, the more oxygen is required. This activity needs time so that the optimum waiting time for the system to reach equilibrium was examined. Oxygen concentrations after various waiting times were measured in 0.1 M PBS, pH 6.8, in the presence of 0.1 mM glucose with a 1 mL suspension of free cells of R. mucilaginosa UICC Y-181. The solution was saturated with oxygen before the experiments were started. Figure 5 shows

Figure 5. Amperometric voltammograms of oxygen in 0.1 M PBS, pH 6.8, at Au-BDD (a) for various waiting times in the presence of 1 mL of free cells of R. mucilaginosa UICC Y-181 and (b) for various volumes of free cells of R. mucilaginosa UICC Y-181 with waiting time of 20 min. The applied potential was −0.5 V (vs Ag/AgCl). The insets show the dependence of the current response on (a) the waiting time and (b) the yeast volume. All data in the insets were extracted at 120 s.

that the current of the chronoamperogram decreased with waiting times due to oxygen consumption by the microorganisms. Figure 5 shows that the decreasing current saturated after 20 min, indicating that the oxidation process of the microorganisms had terminated and the oxygen concentration was in equilibrium. Based on this result, the optimum waiting time was fixed as 20 min and used for subsequent experiments. The amount of R. mucilaginosa UICC Y-181 cells required by the system was also optimized. Figure 5b shows the 9829

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Figure 6. Amperometric voltammograms of oxygen in (a)−(e) 0.1−0.5 mM glucose in 0.1 M PBS, pH 6.8, in the presence of 2.56 × 108 free cells of R. mucilaginosa. The measurements started immediately 0 min (I0, dashed line) and 20 min (I20, solid line) after the oxygen aeration. The applied potential was −0.5 V (vs Ag/AgCl). A linear calibration curve of the difference in current response (I0 − I20) vs glucose concentration is displayed in (f). All data in (f) were extracted from the related amperometric voltammogram at 30 s.

Figure 7. Amperometric voltammograms of oxygen in 0.1 mM glucose in 0.1 M PBS, pH 6.8, in the presence of 2.56 × 108 free cells of R. mucilaginosa. The measurements started 20 min (I20) after oxygen aeration. The applied potential was −0.5 V (vs Ag/AgCl). The inset shows the current response at 120 s.

Figure 8. Amperometric voltammograms of oxygen in 0.1 mM glucose in 0.1 M PBS, pH 6.8, in the presence of 2.56 × 108 free cells of R. mucilaginosa with various concentrations of copper ions. The amperometric measurements started 20 min after oxygen aeration. The applied potential was −0.5 V (vs Ag/AgCl).

copper concentration was increased to 1.60 μM, the signal current increased to ∼30%. The increase in signal current might be possible due to a contribution from copper ion reduction as well as the death of some microorganism cells causing a reduction in the use of oxygen. Since the measurements were performed at pH 6.8, the contribution to the signal current from the copper ions may be negligible due to the precipitation of a large quantity of copper ions at pH levels greater than 6 (KSP[Cu(OH)2] = 10−19.32).35 On the other hand, although R. mucilaginosa has been reported to have high resistance to heavy metals, including copper,36 it seems a tolerance limit is required. In this case, the results indicate that the sensor can only be used in the presence of less than 0.08 μM (equivalent to 50 ppb) copper ions. The BOD sensor was then examined in a real sample analysis. Samples were taken from three different parts of a lake at the University of Indonesia in Jakarta on Nov 7, 2011, at ∼10:00 a.m. Characterization of the samples showed that they

had an average pH 7.3, total dissolved solids (TDS) of 524− 540 mg/L, and COD values of 45−54 mg L−1. No significant amount of heavy metal ions was found. A standard addition of 0.05 mM glucose was added into the sample before dilution in 0.1 M PBS in order to normalize the sample as it was expected that the sample contained some other organic matter. Typical sample measurement is shown in Figure 9. The measurement resulted in an average I0−I20 of 0.78 μA. By using the diluting factor (P) of 5, the current could be converted to a glucose concentration of 0.18 mM and was equivalent to BOD values of ∼18 mg L−1.3,34 Measurements for the same sample three times showed an RSD value of 5.5%, which is relatively higher than that of the RSD value of standard glucose measurements as shown in Figure 7. This difference was reasonable since the lake water sample was expected to contain more organic matter than the standard glucose solution. All measurement results are 9830

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ACKNOWLEDGMENTS

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REFERENCES

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This work was granted by the New Energy and Industrial Technology Development Organization (NEDO), a Research Grant for the Graduate School from the Directorate of Higher Education, Department of Education, Republic of Indonesia (HPTP 2011), and a Grant for Advanced Research of the University of Indonesia (RUUI 2011). We also are indebted to the University of Indonesia Culture Collection for discussions on the microorganisms.

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Figure 9. Amperometric voltammograms of oxygen of the University of Indonesia water lake sample diluted in 0.1 M PBS, pH 6.8 (dilution factor was 5 times), in the presence of 2.56 × 108 free cells of R. mucilaginosa. A total concentration of 0.05 mM glucose was added as a standard addition in the sample. The measurements started immediately 0 min (I0, dashed line) and 20 min (I20, solid line) after oxygen aeration. The applied potential was −0.5 V (vs Ag/AgCl).

shown in Table 1. Comparable results from standard conventional BOD measurements indicate that the developed sensor is a promising BOD sensor. Table 1. Comparison of the BOD Values of Water Samples from a Lake at the University of Indonesia in Jakarta Measured by the Standard Method for BOD Measurements1 and Using the Developed BOD Sensor stand. method for BOD measurements (mg L−1)

developed BOD sensor (mg L−1)

20 ± 2 17 ± 1 17 ± 2

19 ± 2 18 ± 2 17 ± 1

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CONCLUSION Modification of boron doped diamond (BDD) electrodes can be performed by a photochemical reaction using allylamine and a chemical reaction using gold nanoparticles. Application of the gold nanoparticle modified BDD showed good linearity for oxygen and BOD measurements using free cells of R. mucilaginosa UICC Y-181 as the biosensing agent. A linear calibration curve in the concentration range 0.1−0.5 mM glucose, which is equivalent to 10−50 mg L−1 BOD, was achieved with an estimated detection limit of 4 mg L−1 BOD. Excellent reproducibility of the current response (RSD 0.9%) indicated that the modified electrodes were highly stable. The sensors were used for a successful BOD analysis of water from a lake at the University of Indonesia in Jakarta, Indonesia, with results comparable to those of measurements made using a standard conventional BOD method.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Y.E.). Notes

The authors declare no competing financial interest. 9831

dx.doi.org/10.1021/ac302090y | Anal. Chem. 2012, 84, 9825−9832

Analytical Chemistry

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