An optical biochemical oxygen demand biosensor chip for environmental monitoring

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An optical biochemical oxygen demand biosensor chip for environmental monitoring Md. Abul Kashem a , Masayasu Suzuki a,b,∗ , Kazuki Kimoto b , Yasunori Iribe b a b

Graduate School of Innovative Life Science, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan Department of Electric and Electronic Engineering, Faculty of Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan

a r t i c l e

i n f o

Article history: Received 13 May 2015 Received in revised form 24 July 2015 Accepted 31 July 2015 Available online xxx Keywords: Biosensor chip Biofilm Polyethylene–polypropylene film Fluorescence intensity Environmental samples

a b s t r a c t An optical biochemical oxygen demand (BOD) biosensor chip has been developed by embedding a biofilm onto an oxygen sensing film (OSF) shielded with polyethylene–polypropylene (PE–PP) film for effect free detection of organic pollution in environmental samples. The biofilm was prepared by immobilizing baker’s yeasts (Saccharomyces cerevisiae) with polyvinyl alcohol–styrylpyridinium (PVA–SbQ) matrix and the OSF film was developed by coating the oxygen sensing ruthenium complex (dichloro tris (1,10phenanthroline)–ruthenium(II)) dye solution (solubilized in nafion fluoropolymer matrix) onto a SO3 glass slide. Silicone rubber (SR) sheet was used to control the biofilm thickness and make the sample injection cavity. Fluorescence intensity (FI) of the biosensor was recorded by an inverted microscope which varies with the concentration of dissolved oxygen (DO) in samples. The biosensor responses were drawn as the changing of FI due to microbial respiration with BOD standard solutions, glucose–glutamic acid soluton (GGA). A good linear relationship was observed between the maximum responses, It = 3 /I0 (It = 3 = intensity at 3 min time and I0 = intensity at 0 min time) and GGA concentrations (1 to 20 mg/L, R2 = 0.99, n = 3) either in phosphate buffer solution (PBS) or in environmental samples such as river water (RW). The effects of environmental samples upon the sensor performance were completely eliminated due to shielding with only oxygen permeable PE–PP film onto the OSF. In addition, the suitable biofilm type, effects of heavy metals ions as well as preservation and stability of the biosensor also have been investigated. Finally, the newly proposed approach offers a promising and prospective tool for frequent monitoring of organic pollution in environmental samples. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Excessive water pollution caused by organic pollutants in many countries of the world particularly in developing countries like Bangladesh has become an important issue and dictates to frequent monitoring of it in environmental samples [1–4]. Increasing organic pollution in environmental systems for an example in river water (RW) is the key concern because of diminishing fresh water and its resources which may threat not only to human beings but also ecosystem [5,6]. The common indicator of organic pollution identification is the biochemical oxygen demand (BOD) or usually termed as BOD5 (five days BOD test at 20 ◦ C) adopted by the American Public Health Association Standard Methods Committee

∗ Corresponding author at: Faculty of Engineering, University of Toyama, Gofuku 3190, Toyama, Toyama 930-8555, Japan. Tel.: +81 76 445 6707; fax: +81 76 445 6707. E-mail address: [email protected] (M. Suzuki).

in 1936. By the BOD, the amount of dissolved oxygen (DO) required by aerobic microorganisms under a specified standard conditions for assimilating of biodegradable substances is measured. But, the BOD5 method is time-consuming, required skilled personnel and carries less opportunity for frequent applications or instant feedback results for environmental monitoring [7–9]. In biosensors technology by replacing the conventional method (BOD5 test) a lot of efforts has been given as a result electrochemical and optical BOD biosensors have harnessed widely for rapid BOD monitoring but considering some important aspects such as low-cost, miniaturization or integrated system, electrochemical method is not always suite over optical method [10–14]. Others salient features such as easy to prepare, no oxygen consumption, robustness, no toxic chemicals discharge into the environment and even do not require any reference electrode of optical method have gained significant attention than other methods [15–17]. Principally in optical system, ruthenium complex dye e.g., dichloro tris (1,10-phenanthroline)–ruthenium(II) (abbriviated as Ru complex) has been using widely for preparing optical oxygen sensors

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where choice of matrix material for preparing oxygen sensing film (OSF) by Ru complex indicator dye is another important matter. Many studies have suggested different materials such as nafion, organically modified silica/silicate (ormosil), silicone rubber, polydimethylsiloxane (PDMS) etc. but, nafion has such property that can bind cationic fluorophore, ruthenium(II) ionically to the anionic sulphonic acid groups, leaching of dye is eliminated and obtained efficient regeneration capacity [18–20]. Microorganisms which have been applied to biosensing technology, baker’s yeasts (Saccharomyces cerevisiae) comprise numerous characteristics as a perfect organisms in environmental monitoring application. Basically, they are capable to assimilate broad spectrum of substances, easy to culture, resistant to negative environmental factors such as pH, temperature, osmolality etc. as well as have long-term stability [21,22]. Besides, polyvinyl alcohol–styrylpyridinium (PVA-SbQ) was evolved as the yeasts immobilizing material due to high oxygen diffusivity, good for oxygen dependent biosensors, adhesion and mechanical property [23,24]. As recent trend of BOD biosensor development demands for low-cost or disposable, integrated or miniaturized sensors chips as well as effect free monitoring in environmental samples [25,26], here in this study we demonstrated such a BOD sensing technique that may fulfill the major demands. The analytical principle of the method is fluorescence intensity (FI) quantification that varies depending on the DO concentrations in sample. The yeasts in embedded biofilm onto the OSF decreases the DO from the sample as of accelerating respiration activity in presence of organic substances thus the FI increases gradually. The amount of organic substances assimilated by the yeasts will be determined based on the FI changes. The another new addition in this biosensor was the polyethylene–polypropylene (PE–PP) film shielding onto the OSF film which permits only oxygen but no other substances that may make poison the surface of OSF. 2. Experimental

for at least 24 h at room temperature within a draft to make a thin film onto the glass substrate. The prepared dry dye film onto the glass substrate is termed as OSF throughout in this study. 2.3. PE–PP film shielding of OSF Almost an equal size of a dye coated glass slide, a PE–PP film (average thickness 10 ␮m and oxygen penetration rate 20000 mL/m2 .d.atm, Daiso, Japan) was attached upon the OSF by using few drops DW. Then, the PE–PP film shielded OSF was left in a dark environment for two days at room temperature to dry out the used water. Furthermore, it was also confirmed that there is no dampness present between the PE–PP film and OSF by incubating it at 35 ◦ C for one hour. After that, the whole arrangement was allowed for sufficient cooling at room temperature. Before testing the oxygen sensitivity, PE–PP shielded OSF was normalized by injecting PBS into the sample injection cavity that was made by silicone rubber (SR) (AS ONE Corp., Japan). 2.4. Yeasts culture and suspension preparation The baker’s yeasts (S. cerevisiae) containing vitamin C (Nisshin Foods Inc., Japan) were cultured at 35 ◦ C in an incubator on YPD (yeast-extract peptone dextrose) agar media [28]. The media were prepared by using 1 g yeast extract, 1 g bacto-peptone (Difco laboratories, USA), 2 g glucose and 2 g agar (Wako, Japan) in 100 mL RW and sterilized by autoclaving at 121 ◦ C for 20 min. After two days incubating the yeasts agar plates, cells were collected and suspended with PBS as well as sedimented by centrifugation at 4000 rpm for 10 min (Kubota-1720, Japan). Yeasts were washed at least twice with PBS before preparing the final suspension. Lastly, ninety milligram (90 mg) yeasts were suspended in 0.5 mL PBS and the same concentration was retained whenever it was required for further preparation.

2.1. Chemicals

2.5. Biofilms preparation

Ruthenium complex dye (dichloro tris(1,10-phenanthroline)–ruthenium(II) hydrate), 5% Nafion-perfluorinated resin solution, MOPS (3-(n-morpholino) propanesulfonic acid) were purchased from Sigma-Aldrich, USA. PVA–SbQ was kindly donated by Toyo Gosei Co., Ltd (Japan). PVA (polyvinyl alcohol), sodium sulfite, sodium dihydrogenphosphate dehydrate, disodium hydrogenphosphate, l-glutamic acid, d (+)-glucose were purchased from Wako pure chemical industries Ltd. Japan. Phosphate buffer solution (PBS), 50 mM, pH 7.0 was prepared by using sodium dihydrogenphosphate and disodium hydrogenphosphate with distilled water (DW). Other chemicals also used in this study were of analytical grade..

The ratios of PVA–SbQ and cells suspension (w/w) were used to prepare the five types biofilm. A space, 16 × 16 mm2 in the inside position of a 0.2 mm thickness SR was cut and attached onto polyvinylidene chloride film (Asahi kasei, Japan) wrapped glass slide. Then 0.12 g mixture from 0.40 g PVA–SbQ and 0.10 g cells suspension (4:1) were spread into the space of SR and stored in a refrigerator at 4 ◦ C for almost dry out the liquid portion used in mixture. After that, 5 min photo-cross linking was done with a lamp (6860 lx) sothat yeasts can be immobilized within PVA–SbQ matrix by simple entrapping process. The same procedure was also followed for preparing 3:2, 1:1, 2:3 and 1:4 types biofilm. Biofilms were separated from the SR spaces and reactivated individually through overnight incubating at 35 ◦ C dipping into the liquid YPD (0.3 mL) media with PBS (30 mL) and with the help of gentle rotating shaker. A microscopic image of dry state (1:1) type biofilm is shown in Fig. 1.

2.2. Preparation of OSF Oxygen sensing indicator dye, Ru-complex was solubilized in nafion-PVA solution with MOPS buffer. At first, PVA (0.6 g/L) was liquidized in 5 mM MOPS buffer, pH 7.0 by 4 h gentle magnetic stirring. Then Ru-complex dye (1 g/L) with the above solution and equal volume of nafion solution were completely mixed in a light proof container. The prepared composite Ru-complex dye solution (2 mL) was dropped onto a diamond like carbon (DLC) coated glass slide on which SO3 groups were introduced (generally known as SO3 glass slide) (Toyo Kohan Co., Ltd, Japan) to coat the whole surface area of the glass substrate [27]. After that, the coated glass slide was dried keeping in a dark environment

2.6. BOD biosensor chip formation In Fig. 2, a systematic development design of BOD biosensor chip is described. In which, a 0.5 mm thickness SR with an inside space (12 × 12 mm2 ) as well as a sample outing channel (width 1.5 mm, length 6 mm) from the inside space to the outside direction was attached onto the PE–PP film shielded OSF. Later, a biofilm (size 10 × 10 mm2 ) was embedded using few drops PBS into the space of the 0.5 mm thickness SR that is onto the OSF. Finally, above the

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Fig. 1. A microscopic image of a yeasts immobilized biofilm (1:1 type, within PVA–SbQ matrix and image was taken by Keyence VE-7800 microscope).

Fig. 2. Schematic design of the BOD biosensor chip and the components are (1) SO3 glass slide, (2) Ru-complex dye–OSF, (3) PE–PP film, (4) 0.5 mm thickness SR, (5) biofilm, (6) 1 mm thickness SR, (7) sample cavity or sample IN, (8) cover glass, and (9) sample OUT.

biofilm another 1 mm thickness SR (space 9 × 9 mm2 ) was attached. The space of upper 1 mm SR was used as the sample injection cavity.

3

RW as an oxygen free (DO, 0 mg/L) were run to check the oxygen sensing response of PE–PP shielded OSF. Before BOD standard; glucose–glutamic acid (GGA) solution injection, biosensor was normalized with PBS at least 30 min earlier. To avoid the phenomena such as reduction of DO in measuring sample due to increasing of temperature [9], room temperature (26 ◦ C) was always controlled at least 30 min before of every trial. The external room lights were switched off during the experimental period. The ON–OFF switching (electronic shutter) of the inverted microscope was applied to excite the dye of BOD biosensor as required. During measurement, at first, after injecting 0.2 mL PBS sample into the sensor cavity, it was made completely airtight by placing a cover glass onto the sample and allowed around 1–2 min to stabilize the sample. Then, using ON switch, the dye was excited and suddenly took an emission image. Then, the excitation light was kept off up to one min and again excited the dye as well as took the emission image just at one min time. By the same way, emission images were taken up to five min. For each emission image (from 0 to 5 min), ten FI measuring points (size 51 ␮m) as shown in Fig. 3 were selected and an average intensity of ten points were calculated (AquaCosmos software, Hamamatsu Photonics, Japan). The same procedure was also followed for different concentration of BOD standard solutions, GGA (150 mg/L glucose and 150 mg/L glutamic acid equals to 220 mg/L BOD, Japanese industrial standard-JIS) [29] with PBS and RW samples. The RW samples were collected from the adjacent channel near the Faculty of Engineering, University of Toyama and made organic matters free by several days incubating at 35 ◦ C. Before GGA solution preparation, both glucose and glutamic acid were dried at 100 ◦ C temperature for 10 min. The biosensor response curves of GGA solutions were made as FI ratios (It /I0 ) (It intensity at t min and I0 intensity at 0 min) against time profiles. From the response curves, maximum response (at 3 min time) for each BOD standard solution was evaluated and a calibration curve was drawn as (It = 3 /I0 ) against GGA concentrations. 3. Results and discussion

2.7. Instrumentation and analytical method 3.1. Characterization of PE–PP film shielded OSF An inverted fluorescent microscope (ECLIPSE, TE 2000-U, NIKON, Japan) was used to measure the FI in which dye of OSF was excited by a light at 450–490 nm wavelength and emission images were collected at above 520 nm wavelength by using a high sensitive camera (ORCA-ER C4742-80-12AG, Hamamatsu Photonics, Japan). The instrumental illustration of the inverted microscope is described in Fig. 3. PBS as an oxygen rich (DO, 7 mg/L) and 5% Na2 SO3 (w/v) solution in PBS or environmental samples such as

The Ru-complex dye solution was evenly swelled by coating process and made a thin film (thickness around 10 ␮m, measured by Keyence-7800 microscope, Japan) onto the SO3 glass slide. Intimate attachment of PE–PP film onto OSF film was noticed and no air bubbles were present between the two films. From the DO response results of samples, it was observed that the PE–PP shielded OSF has good FI dependence based on DO concentrations that means in highly oxygenated sample, the FI is always low whereas high in deoxygenated sample. Another important feature of PE–PP film shielded OSF was evaluated that is an equal response ability both in PBS and RW samples when DO was controlled by equal concentrations (Fig. 4). This results reveal that the PE–PP film shielding approach has completely overcome the effects or disturbances of heterogeneous components such as metal ions, biological materials, chemicals etc. of RW samples upon the surface of OSF which were previously reported by the study of Gillanders et al. [18]. The unique nature, only oxygen diffusion through the PE–PP film has restricted to direct contact of sample or its components to OSF which further increases the long term (several months) usability of sensing film preserving at dark and room temperature. 3.2. Yeasts biofilm with PVA–SbQ matrix

Fig. 3. Illustration of the key components of an inverted microscope where BOD biosensor chip is anchored with the XY stage.

Biofilm with high sensitivity and capable to close contact onto the PE–PP film shielded OSF should be used for developing BOD

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biosensor chip. PVA–SbQ matrix fulfilled the requirements because it is non-toxic, hydrophilic and high adhesion property bearing synthetic gel and the simple physical entrapment procedure of PVA–SbQ by photo-crosslinking does not damage the natural activity of yeasts [22,23]. Regarding high sensitive or suitable biofilm preparation, the ratio of PVA–SbQ and cells suspension (w/w) (1:1) was identified and the sensitivity results of different biofilms are presented in Fig. 5. The 1:1 ratio type biofilm guaranteed almost equal thickness (0.2 mm) and good cells distribution into the biofilm. High concentration of PVA–SbQ such as in 4:1, 3:2 ratios made unequal thicknesses, mixing bubbles and mostly irregular cells distribution into the film which were not apt for repeatable or reproducible responses. Besides, high cells density such as in 2:3, 1:4 ratios resembled very low thickness of biofilms (less than 0.2 mm) but inappropriate immobilization of the yeasts due to low concentration of PVA–SbQ, poor adhesive and also crumbling natures. It was also scrutinized that after preparing the biofilms, the sensing ability of the biofilms was not so good immediately; it is probably after completion of several steps such as (1) cells centrifugation, (2) cells mixing with PVA–SbQ, (3) overnight storage in refrigerator, and (4) photo-cross linking etc. may idle the cellular activeness. This low activeness of yeasts was overcome by improving the bioactivity through reactivation of biofilms in liquid YPD with PBS. The idea has increased the sensitivity by two folds of biofilm. It is obviously due to confirmation of by alive yeasts into the biofilm after reactivation process. Since, biofilm containing full of alive cells, the quick respiration activity of the yeasts has increased the oxygen consumption and thus resulted rapid FI increasing. The obtained sensitivity results of non-reactivated (NRA) and reactivated (RA) of 1:1 type biofilm measured in 9 mg/L GGA are shown in Fig. 6.

RA

Biofilm conditions Fig. 6. Sensitivity comparison of 1:1 type biofilm at non-reactivated (NRA) and reactivated (RA) conditions (measured in 9 mg/L GGA solution).

3.3. Responses and calibration In our developed BOD biosensor chip, the PE–PP film shielded OSF acts as a transducer and detects the amount of oxygen consumed by the yeasts during their respiration activity in presence of GGA solutions by increasing FI. We tested PBS (as a control) after stabilization the biosensor, basically no changing of FI was found from 0 to 5 min in control solution but when sample contained BOD standard solution i.e., GGA, the respiration activity of yeasts was accelerated and increased the rate of oxygen consumption as a result the FI was increased gradually. By assimilation of GGA, the rate of oxygen consumption and the rate of oxygen dissolution step in a balancing condition and after a while it reaches in a maximum response level and finally again into the steady state level. The biosensor response curves of several GGA solutions as It /I0 against time profile are presented in Fig. 7. The difference of increasing response of every GGA solution from the response of PBS solution is proportional to the amount of immediately assimilated GGA in sample. The experimental results showed within a complete sample air-tight situation, only five (5) min time are required to run an individual sample for reaching to the maximum response level followed by around 2 min initial sample stabilization time. The interval period between two samples measurement was found around 30 min. A calibration plot for the BOD biosensor using GGA standard solutions (up to 300 mg/L) as well as a linear plot (inset) (up to 20 mg/L) as It = 3 /I0 against GGA are presented in Fig. 8. In our present study, we applied and proposed an imaging system for quantifying the BOD but for acquiring the more accuracy level in our next stage, of course we have to concern some instrumental aspects such as excitation light (mercury lamp was used but it could be replaced by LED light), imaging tool (camera) and also FI

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counting and analytical softwares. Moreover, BOD biosensor is as the part of our future integrated biosensors chip, we have been trying to shape more improved system in the further study for more realistic BOD monitoring. 3.4. Application in environmental samples Since the components of environmental samples such as in RW differ greatly from GGA in PBS, the performance of our developed BOD biosensor chip was thus checked with GGA in RW and compared to the obtained results of GGA in PBS. Apparently, no significant differences were noticed either GGA in PBS or in RW (Fig. 9). It is the good evidence for effect free applicability of BOD biosensor in environmental samples. The application nature of sensor has also confirmed that environmental samples will not be the problem for our sensor as we are using PE–PP film for safeguarding the OSF. Moreover, for confirming the more robust application in real samples, the suitable species selection of microorganisms will be another important consideration in our next stage of study. A comparison results of with and without PE–PP film based biosensors and their sensing performance in real RW with GGA are also shown in Fig. 10. Without PE–PP film, the sensor showed always low performance in RW because effects of heterogeneous components onto OSF although the GGA concentrations were same that measured with PE–PP film based biosensor. 3.5. Effects of heavy metals ions Heavy metals may interfere to show the real respiration performance activity of yeasts. Thus, three common heavy metals in RW of Bangladesh (concentrations were prepared as RW standard in Bangladesh) [30] such as Cd2+ (0.5 mg/L), Cu2+ (0.5 mg/L), Zn2+

Fig. 10. Effects of RW onto OSF without PE–PP film compare to with PE–PP film.

(1 mg/L) in 15 mg/L GGA with 50 mM PBS were checked individually and the effects on the respiration activity were evaluated. No effects due to Cd2+ and Zn2+ ions were observed but, Cu2+ ion was increased the respiration activity as a result around 0.29% higher respiration performance was obtained compare to the performance in only 15 mg/L GGA (Fig. 11). The results confirmed that in standard concentrations of the three heavy metals, there will be no effect on the respiration activity of yeasts during the application in environmental samples. 3.6. Stability of biosensor Stability of the biosensor chip was characterized based on the sensing performance with the time dependent factors as well as preservation conditions. Some previously developed biosensors have been suggested to preserve with PBS or low concentrated GGA solution in refrigeration temperature [10,15]. It avoids the possibility of increasing cells into the biofilm. But, in case of our newly developed BOD biosensor with PBS in refrigeration (4 ◦ C) preservation showed low performances. The one possible cause maybe due to decreasing the activeness of cells in refrigeration temperature and another cause maybe in refrigeration temperature i.e., high oxygen concentration affects the base fluorescence of OSF which may delay the rapid response of BOD biosensor. Hence, instead of refrigerator preservation, room temperature (at 26 ◦ C) with PBS showed better choice for the biosensor preservation where the activeness of yeasts into biofilm remained almost same and no significant deviation (only 10–12%) of performance was observed after six days uses (Fig. 12). In addition, only the biofilms could be stored in refrigeration temperature for long term (several months) before reactivation or BOD biosensor chip development. Although this stability length is short but the simple BOD biosensor development system including easy reactivation process of biofilm have promoted the easy access to frequent development as well as the

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Heavy metals (in 15 mg/L GGA) Fig. 11. Respiration performance of biosensor (based on the performance in only 15 mg/L GGA solution and in addition of individual heavy metal in 15 mg/L GGA).

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Days Fig. 12. Stability of biosensor as of time dependent sensing performance (biofilm 1:1 type, measured in 9 mg/L GGA, preserved with PBS at room temperature (26 ◦ C) and refrigeration temperature (4 ◦ C).

application of the biosensor which are highly required in recent demands. In addition, as of low-cost or disposable manner of the biosensor chip, it is sufficient for week long application at present stage and also inspiring again of a new one preparation just by changing the biofilm only of the system. 4. Conclusion We developed a simple method in this study for fabricating FI based optical BOD biosensor chip and monitoring of BOD in RW samples. The PE–PP film shielded OSF showed effect free sensing capability of DO and BOD in RW samples. A suitable combination of PVA–SbQ and yeasts density (1:1) was identified for maximal sensitive biofilm preparation and from the calibrated results using BOD standard solutions (GGA) prepared with PBS or RW, the biosensor had no significant variation of performances. A good linear range was obtained from 1 to 20 mg/L GGA (having BOD 14.6 mg/L). For a single sample testing by this method only 200 ␮L sample and 5 min time are required. Some other factors e.g., oxygen saturation, stirring or raising temperature of samples are not necessary. Moreover, no effects onto the biosensor’s respiration performance were observed due to the co-existing heavy metals ions such as Cd2+ , Zn2+ except Cu2+ (only 0.29%). Since, the newly developed biosensor chip covers numerous advantages including low-cost or disposable nature which are the key concerns for regular environmental monitoring in developing countries, it could lead as a promising tool towards pollution surveying in real samples. Nevertheless, the accuracy and the long term stability of the sensor in the first stage of development were not in sufficient level and hopefully it will be overcome in the next stage of study. Acknowledgments The authors would like to thank Mr. Tanuma, Mr. Shida, and Mr. Dohama for their kind help during the laboratory experiments. References [1] Y. Tsuzuki, T. Koottatep, F. Ahmed, M.M. Rahman, Water quality and pollutant load in the ambient water and domestic wastewater pollutant discharges in the developing countries, J. Glob. Environ. Eng. 121 (2008) 121–133. [2] C. Chakraborty, M.M. Huq, S. Ahmed, T. Tabassum, M.R. Miah, Analysis of the causes and impacts of water pollution of Buriganga river, Int. J. Sci. Technol. Res. 2 (2013) 245–252. [3] M.K. Hasan, M.A. Happy, M.K. Nesha, K.H.R. Karim, Pollution status of Balu river due to industrial input at Dhaka, Bangladesh, Open J. Water Pollut. Treat. 1 (2014) 34–43.

[4] M. Akter, J. Sikder, A.K.M.A. Ullah, Water quality assessment of an industrial zone polluted aquatic body in Dhaka, Bangladesh, Am. J. Environ. Protect. 3 (2014) 232–237. [5] G. Chee, Biosensor for the determination of biochemical oxygen demand in rivers, in: V. Somerset (Ed.), Environmental Biosensors, InTech, Croatia, 2011, pp. 257–276. [6] X. Zhang, C. Chen, J. Ding, A. Hou, Y. Li, Z. Niu, X. Su, Y. Xu, E.A. Laws, The 2007 water crisis in Wuxi, China: analysis of the origin, J. Hazard. Mater. 182 (1–3) (2010) 130–135. [7] G. Chee, Development and characterization of microbial biosensors for evaluation low biochemical oxygen demand in rivers, Talanta 117 (2013) 366–370. [8] G. Chee, Y. Nomura, K. Ikebukuro, I. Karube, Optical fiber biosensor for the determination of low biochemical oxygen demand, Biosens. Bioelectron. 15 (2000) 371–375. [9] V. Arlyapov, S. Kamanin, O. Ponamoreva, A. Reshetilov, Biosensor analyzer for BOD index express control on the basis of the yeast microorganisms Candida maltosa, Candida, and Debaryomyces hansenii, Enzyme Microb. Technol. 50 (2012) 215–220. [10] N.-Y. Kwok, S. Dong, W. Lo, K.-Y. Wong, An optical biosensor for multi-sample determination of biochemical oxygen demand (BOD), Sens. Actuat., B: Chem. 110 (2005) 289–298. [11] A. Reshetilov, V. Arlyapov, V. Alfero, T. Reshetilova, BOD biosensors, application of novel technologies and prospects for the development, in: T. Rinken (Ed.), State of the art in Biosensors–Environmental and Medical Applications, Intech Open Publishing, Rijeka, Croatia, 2013, http://www. intechopen.com. [12] Y. Jiang, X. Lai-Long, Z. Li, C. Xi, W. Xiaoru, W. Kwok-yin, Optical biosensor for the determination of BOD in seawater, Talanta 70 (2006) 97–103. [13] S. Velling, A. Mashirin, K. Hellat, T. Tenno, Non-steady response of BOD biosensor for the determination of biochemical oxygen demand in wastewater, J. Environ. Monit. 13 (2011) 95–100. [14] I. Karube, M. Suzuki, Microbial biosensors, in: A.E.G. Cass (Ed.), Biosensors: A Practical Approach, Oxford University Press, Oxford, UK, 1990, pp. 155–170. [15] L. Lin, L. Xiao, S. Huang, L. Zhao, J. Cui, X. Wang, X. Chen, Novel BOD optical fiber biosensor based on co-immobilized microorganisms in ormosils matrix, Biosens. Bioelectron. 21 (2006) 1703–1709. [16] L. Guo, Q. Ni, J. Li, L. Zhang, X. Lin, Z. Xie, G. Chen, A novel sensor based on the porous plastic probe for determination of dissolved oxygen in seawater, Talanta 74 (2008) 1032–1037. [17] C. Chu, Y. Lo, T. Sung, Review on recent development of fluorescent oxygen and carbon dioxide optical fiber sensors, Photon. Sens. 1 (2011) 234–255. [18] R.N. Gillanders, M.C. Tedford, P.J. Crilly, R.T. Bailey, A composite thin film optical sensor for dissolved oxygen in contaminated aqueous environments, Anal. Chim. Acta 554 (2005) 189–194. [19] A.P. Leis, S. Schlicher, H. Franke, M. Strathmann, Optically transparent porous medium for nondestructive studies of microbial biofilm architecture and transport dynamics, Appl. Environ. Microbiol. 71 (2005) 4801–4808. [20] S.M. Grist, L. Chrostowski, K.C. Cheung, Optical oxygen sensors for applications in microfluidic cell culture, Sensors 10 (2010) 9286–9316. [21] K.H.R. Baronian, The use of yeasts and moulds as sensing elements in biosensors, Biosens. Bioelectron. 19 (2004) 953–962. [22] H. Nakamura, K. Suzuki, H. Ishikuro, S. Kinoshita, R. Koizumi, S. Okuma, M. Gotoh, I. Karube, A new BOD estimation method employing a double-mediator system by ferricyanide and menadione using the eukaryote Saccharomyces cerevisiae, Talanta 72 (2007) 210–216. [23] R. Renneberg, K. Sonomoto, S. Katoh, A. Tanaka, Oxygen diffusivity of synthetic gels derived from prepolymers, Appl. Microbiol. Biotechnol. 28 (1998) 1–7. [24] S.K. Yoo, J.H. Lee, S.S. Yun, M.B. Gu, J.H. Lee, Fabrication of a bio-MEMS based cell-chip for toxicity monitoring, Biosens. Bioelectron. 22 (2007) 1586–1592. [25] S. Rodriguez-Mozaz, M.J. Lopez de Alda, M.-P. Marco, D. Barcelo, Biosensors for environmental monitoring: a global perspective, Talanta 65 (2005) 291–297. [26] M. Suzuki, T. Ohshima, S. Hane, Y. Iribe, T. Tobita, Multiscale 2D-SPR biosensing for cell chips, J. Robot. Mechatron. 19 (2007) 519–524. [27] M. Suzuki, H. Tanaka, Y. Iribe, Detection and collection of target single cell based on pH and oxygen sensing, J. Robot. Mechatron. 22 (2010) 639–643. [28] D.A. King, M.W. Sheafor, J.K. Hurst, Comparative toxicities of putative phagocyte-generated oxidizing radicals towards a bacterium (Escherichia coli) and a yeast (Saccharomyces cerevisiae), Free Radic. Biol. Med. 41 (2006) 765–774. [29] Japanese Industrial Standard (JIS), Apparatus for the estimation of biochemical oxygen demand (BODs) with microbial sensor, in: JIS K 3602, Japanese Industrial Standard (JIS), Tokyo, Japan, 1990. [30] M.A.A. Mokaddes, B.S. Nahar, M.A. Baten, Status of heavy metal contaminations of river water of Dhaka metropolitan city, J. Environ. Sci. Nat. Resour. 5 (2) (2013) 349–353.

Biographies Md. Abul Kashem studied in Environmental Science (B. Sc. in 2006 and Master in 2009) from Khulna University, Bangladesh. He led several environmental quality monitoring and improvement research projects in his professional career. Currently,

Please cite this article in press as: Md.A. Kashem, et al., An optical biochemical oxygen demand biosensor chip for environmental monitoring, Sens. Actuators B: Chem. (2015), http://dx.doi.org/10.1016/j.snb.2015.07.119

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he is a doctoral student in University of Toyama, Japan. His main research interests comprise optical sensors/biosensors, chip technology and integrated system for environmental monitoring.

Kazuki Kimoto has been a master course student in the graduate school at University of Toyama. His research interest lies in the development of optical biosensors for environmental monitoring.

Masayasu Suzuki received his M. Eng. and Ph. D. degrees in electronic chemistry in 1984 and 1987, respectively, from Tokyo Institute of Technology. He has been a Professor at University of Toyama since 2000. His current interests include the miniaturization and integration of biosensors.

Yasunori Iribe received his M. Eng. degree in biochemical systems engineering in 1999 from Kyushu Institute of Technology. He has been a technical staff at University of Toyama since 2006. His research interest is the development of novel methodologies for biosensors.

Please cite this article in press as: Md.A. Kashem, et al., An optical biochemical oxygen demand biosensor chip for environmental monitoring, Sens. Actuators B: Chem. (2015), http://dx.doi.org/10.1016/j.snb.2015.07.119