A Rapid Low Power Ultra-Violet Light-Assisted Bacterial Sensor for Coliform Determination

Jia Shin Ho, Chee-Seng Toh

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore.

DOI: 10.4236/ajac.2013.410A1001                

Abstract

Titanium dioxide (TiO₂) particle-incorporated Prussian blue (PB) sensor for the detection and inactivation of Escherichia coli (E. coli) is developed in this study. The system requires low power ultra-violet (UV) light to photoactivate TiO₂ particles and change of signal response is measured immediately upon irradiation using cyclic voltammetry. The generation of free radical species (OH^.) and H₂O₂ from the oxidation of water by the hole (h⁺) are the main components which cause destruction of cell membrane and eventually result in the inactivation of cell. Our study also shows direct oxidation of cells by h⁺ as one of the mechanisms for cell inactivation due to the close contact between TiO₂ particles and E. coli cells. Highly attractive features of this unique sensor include its ability to be regenerated and reused for at least three times without the use of harsh chemicals, good reproducibility and its specificity in bacteria sensing when tested against organic contaminants, which potentially reduce the operation cost when incorporated into water disinfection system. Its superior performance in detection of total coliform without additional steps of sample treatment is also demonstrated in river water. TiO₂ particle-incorporated PB membrane sensor exhibits signal response with higher current output compared to PB-TiO₂ coated screen printed carbon electrode (SPCE) due to its porous structure and higher surface area, suggesting its potential development into a powerful and low cost contamination monitoring tool.

Keywords

Bacterial Sensor; Low Power; Cell Inactivation; E. coli; Coliform; Photocatalysis

1. Introduction

Water contamination is one of the most problematic issues where millions of people die from water borne diseases due to the consumption of unsafe or contaminated water. Escherichia coli (E. coli) has been identified as one of the agents for waterborne diseases and its presence in water represents water quality deterioration and contamination by human or animal wastes [1]. This poses serious health risks, such as diarrhea, nausea and other symptoms to consumers, especially infants and those with severely compromised immune system. The contamination of water source by bacteria and pathogen also leads to limited sustainable water supply where one-fifth of world’s population has no clean access to water and this scenario is more prominent in developing countries [2]. Hence, there is an urgent need to develop rapid, low cost and simple bacterial sensor with early warning capability and of low power [3,4] in order to address these problems. Electrochemical sensing which is portable and inexpensive has shown its superior performances in the detection of cells, viruses and biological samples [5-8]. However, real-time detection and regeneration of sensing materials have always been the limitations for biosensing [9]. An electrochemical sensor which exhibits immediate response upon detection and the ability to be reused is expected to be highly useful due to the potential enhanced performance for on-site analysis.

TiO₂ photocatalyst has been used extensively for the photocatalytic degradation of organic compounds for water purification [10,11]. However, the development of photocatalytic reactors remains a challenge due to the high recombination rates of holes and electrons [12] and the lack of ability to regenerate and reuse the material. On the contrary, a polymer incorporated with TiO₂ would be expected to partly resolve the problem of recombination because the polymer serves as the electron acceptor in the conduction band. To develop a low power bacterial sensor, we choose iron hexacyanoferrate polymer which is also known as Prussian blue (PB), incorporated with titanium dioxide (TiO₂) particles as the sensing material. Two electrode configurations, namely porous alumina membrane and screen printed carbon electrode (SPCE) have been employed in this study to investigate the performance of sensor. The polymer incorporated TiO₂ is deposited onto the platinum-coated porous alumina membrane template and SPCE, which act as the working electrode in this study. The setup of polymer-coated membrane sensor operates like a conventional two-electrode electrochemical system but requires only a very low power ultra-violet (UV) lamp to function while SPCE functions as a conventional three-electrode system.

Ultrathin PB film is employed in this work because it can significantly improve the signal response and improve the sensitivity owing to the fast mass transfer of analytes [13,14]. In this study, the sensing of bacteria is carried out without the labeling of antibody as well as with small volume which significantly simplifies the procedures for operation. To further enhance the sensing performance of sensor, large TiO₂ particle size is chosen owing to its good mechanical adhesion [15] and to maximize interaction between particles and bacteria. The two-electrode system used in this work is simple and potentially useful in the application of field measurement conditions. Herein, we utilize a TiO₂ particles incorporated PB film to construct a novel membrane sensor with bacterial sensing capability in small volume of analytes with the aid of low power UV light (Scheme 1). E. coli sensing and detection of total coliform in river water are performed by the sensor using cyclic voltammetry. To further evaluate the application of TiO₂ incorporated PB sensor on E. coli detection, the performance of SPCE coated with PB and TiO₂ is also investigated in this study.

2. Experimental

2.1. Materials and Instruments

Nanoporous alumina membrane (Anodisc^TM, 13 mm diameter, 0.02 µm pore size) was purchased from Whatman (Maidstone, Kent, UK). SPCE was obtained from

Scheme 1. Fabrication of the membrane-based bacterial sensor by sputtering ~50 nm thick platinum layer on both sides of 60 µm thick nanoporous alumina membrane, followed by coating of TiO₂ incorporated PB film galvanostatically onto the Pt electrode.

Dropsens. The working electrode (4 mm diameter) of SPCE was carbon (C) while the counter and reference electrodes were platinum (Pt) and silver (Ag) respectively. Hydrochloric acid (HCl, 37%) was obtained from P. P. Chemical. Potassium chloride (KCl) was purchased from Sinopharm Chemical Reagent Co. Ltd. Potassium hexacyanoferrate (III) (K₃Fe(CN)₆) and Nafion perfluorinated ion-exchange resin were obtained from SigmaAldrich. Anhydrous iron (III) chloride (FeCl₃) was purchased from Merck. Titanium (IV) oxide (TiO₂, −325 mesh powder, anatase, 99.6%) was obtained from Alfa Aesar. Methanol (MeOH, ≥99.9%) was purchased from Tedia Company, Inc., toluene (C₆H₅OH, 99.5%) was obtained from RCI Labscan Limited and 1,4-dioxane (C₄H₈O₂, ≥99.9%) was purchased from Merck. E. coli K12 was obtained from ATCC and it was prepared in 0.01 M phosphate buffered saline (PBS, pH 7.4). All chemicals and solvents were used as received. Ultrapure water (Sartorius Ultrapure Water System) was used for all solution preparation, unless otherwise stated.

Sputter coating of Pt onto the nanoporous alumina membrane was performed by JEOL Auto Fine Coater (JFC-1600). Coating of TiO₂ incorporated PB onto the electrodes and electrochemical measurements were carried out by e-corder 401 (eDaQ) and potentiostat (eDaQ EA161) controlled by a PC.

2.2. E. coli Culturing

E. coli K12 was purchased from ATCC. Luria Broth (LB) containing 10.0 g tryptone, 5.0 g yeast extract and 10.0 g sodium chloride was used to grow the pure culture of E. coli. The culture was grown on an orbital shaker at 37˚C for 18 h and it was subsequently diluted to10 cfu∙mL⁻¹ with 0.01 M PBS, pH 7.4. E. coli cell number was enumerated by spread plate method where 0.1 mL of diluted solution was spread evenly on LB agar plate and incubated at 37˚C for 24 h. E. coli colonies on the plates were counted for determination of the number of viable cell in colony-forming units per milliliter (cfu∙mL⁻¹).

2.3. River Water Spiking

One-liter of water sample was collected from Kallang river, Singapore on 16 Jan 2013. The water sample was divided into three equal volumes and two of the solutions were spiked with different volumes of 30 cfu∙mL⁻¹ E. coli solution to make up the desired spiked concentration. All solutions were finally made up to equal volumes using PBS. The electrochemical measurement was then carried out according to the procedures stated below.

2.4. Sensor Fabrication

Fabrication of TiO₂ incorporated PB membrane sensor was based on the procedure described elsewhere [16]. Both sides of the nanoporous alumina membrane were sputtered with conductive Pt layers and it was subsequently electrodeposited with PB (Scheme 1). The active side of the sputtered membrane served as the working electrode while the passive side of the membrane as counter electrode and silver/silver chloride (Ag/AgCl) in 1 M KCl was used as the reference electrode during the coating process. The membrane was coated using galvanostat for 1 h in an aqueous solution of 20 mM K₃Fe(CN)₆ and 20 mM FeCl₃ with pH adjusted to be around 2.0 using HCl. TiO₂ particles were suspended in the same solution with a concentration of 15 g∙dm⁻³. The solution was stirred and dropped onto the surface of membrane every 10 min to ensure that TiO₂ was coated together with PB. The current density was 20 µA∙cm⁻² and total electrolysis charge passed was 72.2 mC∙cm⁻². The TiO₂ incorporated PB membrane was then rinsed with ultrapure water and dried overnight at room temperature. The passive side of the membrane was coated with Nafion before it was used for sensing. The coating of SPCE was accomplished by the same procedures except Pt and Ag were used as the counter and reference electrodes respectively.

2.5. Sensing and Disinfection of E. coli

E. coli sensing was carried out by spreading 20 µL of E. coli solution on both sides of the membrane and the working electrode of SPCE prior to irradiation and signal response was obtained from cyclic voltammogram (CV). The sensor was then subjected to UV light irradiation which the source was a 4 W money detector (MD401, Khind) before the determination of electrochemical response. After the first measurement, the sensor was rinsed with copious amount of PBS to remove bacteria from the previous scan. Higher concentration of E. coli solution was applied onto the sensor and the signal response was determined again. The electrochemical measurement system comprised an integrated two-electrode setup or SPCE as well as e-corder 401 (eDaQ) and potentiostat (eDaQ EA161) controlled by a PC.

3. Results and Discussion

3.1. Model for Bacterial Sensor Signal Response

The TiO₂ incorporated PB membrane sensor presents a large signal response toward E. coli solution upon irradiation in contrast to the signal response before exposure of light (Figure 1(a)). This remarkably high electroactivity of bacterial sensor cannot be observed on sensor in the absence of PB films (Figure 2(a)) because electron transfer is harder to occur in TiO₂ due to its larger band gap of 3.2 eV [17]. The change of peak current is insignificant when PB sensor without TiO₂ is used (Figure 2(b)). This clearly indicates that bacteria sensing happens only when there are photogenerated electrons and highly reactive radical species from TiO₂ particles. Further evaluation of the sensor in differentiating its signal response from organic compound, methanol reveals insignificant change of signal response (Figure 1(b)). The difference in electrochemical behaviors can be explained by the chemical reaction of PB and methanol [18] causing the PB film to dissolve away thereby, reducing electrochemical activity.

The significantly high oxidative current of bacterial sensor upon irradiation is owing to the formation of Prussian white (PW) as a result of injection of photoexcited electron into PB as shown in Scheme 2. A hole (h⁺) and electron (e⁻) pair is generated in the valence and conduction bands respectively when TiO₂ is illuminated with light of appropriate wavelength (300 - 400 nm). The valence band h⁺ reacts with water molecule to produce hydroxyl radical (OH·) and the recombination of OH· produces hydrogen peroxide (H₂O₂). To prevent the ex-

(a)(b)

Figure 1. Cyclic voltammogram of TiO₂ incorporated PB membrane sensor with (a) 20 µL 10 cfu∙mL⁻¹ of E. coli before () and after () UV light irradiation (b) 20 µL of methanol before () and after () UV light irradiation. Conditions: scan rate = 20 mV∙s⁻¹, potential range = −0.4 to 0.4 V, E. coli concentration = 10 cfu∙mL⁻¹, methanol concentration = 20% (v/v).

(a)(b)

Figure 2. Cyclic voltammogram of (a) TiO₂ sensor without PB before () and after () irradiation; (b) PB sensor without TiO₂ before () and after () irradiation. Conditions: scan rate = 20 mV∙s⁻¹, potential range = −0.4 to 0.4 V, E. coli concentration = 10 cfu∙mL⁻¹.

tremely deleterious e⁻ − h⁺ recombination reaction, PB with compatible band gap energy [19] was chosen to TiO₂ particles as the bacteria sensing material. In this study, the bacteria sensing is proposed to be coupled with disinfection of bacteria, where highly reactive OH· and H₂O₂ are responsible for cell wall decomposition, subsequently change the cell membrane permeability [20-24]. Hence, the disinfection of E. coli is mainly due to the destruction of cell wall and followed by cell membrane which permeability change allows the penetration of highly reactive species into the cytoplasmic membrane. Direct oxidation of cells by h⁺ has also been proposed previously but the detailed mechanism has not been discussed extensively [24,25].

3.2. Analytical Performance of Bacterial Sensor

Figure 3 illustrates the correlation between oxidative current with increasing concentration of E. coli upon irradiation. The bacterial sensor shows significant change of response upon irradiation and the short analysis time outperforms the conventional methods for detection of E. coli in water, including multiple-tube fermentation and membrane filter techniques which require time-consuming and labor-intensive procedures [26]. The correlation between logarithm concentration of E. coli and oxidative peak current is somewhat linear between 10 to 10⁵ cfu∙mL⁻¹ (Figure 3) and it can be established using the equation:

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	P. R. Hunter, J. M. Colford, M. W. LeChevallier, S. Binder and P. S. Berger, “Waterborne Diseases,” Emerging Infectious Disease, Vol. 7, No. 7, 2001, p. 544. http://dx.doi.org/10.3201/eid0707.017723
[2]	T. F. Clasen and H. Laurence, “Water Quality Interventions to Prevent Diarrhoea,” World Health Organization, Geneva, 2008.
[3]	D. Battistel, F. Baldi, D. Marchetto, M. Gallo and S. Daniele, “A Rapid Electrochemical Procedure for the Detection of Hg(0) Produced by Mercuric-Reductase: Application for Monitoring Hg-Resistant Bacteria Activity,” Environmental Science & Technology, Vol. 46, No. 19, 2012, pp. 10675-10681. http://dx.doi.org/10.1021/es301444a
[4]	E. Neumann, K. Tonsing and P. Siemens, “Perspectives for Microelectrode Arrays for Biosensing and Membrane Electroporation,” Bioelectrochemistry, Vol. 51, No. 2, 2000, pp. 125-132. http://dx.doi.org/10.1016/S0302-4598(99)00084-7
[5]	M. S. Cheng, S. H. Lau, V. T. Chow and C. S. Toh, “Membrane-Based Electrochemical Nanobiosensor for Escherichia coli Detection and Analysis of Cells Viability,” Environmental Science & Technology, Vol. 45, No. 15, 2011, pp. 6453-6459. http://dx.doi.org/10.1021/es200884a
[6]	M. S. Cheng, J. S. Ho, C. H. Tan, J. P. S. Wong, L. C. Ng and C. S. Toh, “Development of an Electrochemical Membrane-Based Nanobiosensor for Ultrasensitive Detection of Dengue Virus,” Analytica Chimica Acta, Vol. 725, 2012, pp. 74-80. http://dx.doi.org/10.1016/j.aca.2012.03.017
[7]	S. K. Yadav, P. Chandra, R. N. Goyal and Y.-B. Shim, “A Review on Determination of Steroids in Biological Samples Exploiting Nanobio-Electroanalytical Methods,” Analytica Chimica Acta, Vol. 762, 2013, pp. 14-24. http://dx.doi.org/10.1016/j.aca.2012.11.037
[8]	V. C. Diculescu, S. Kumbhat and A. M. Oliveira-Brett, “Electrochemical Behaviour of Isatin at a Glassy Carbon Electrode,” Analytica Chimica Acta, Vol. 575, No. 2, 2006, pp. 190-197. http://dx.doi.org/10.1016/j.aca.2006.05.091
[9]	C. M. A. Brett and A. M. Oliveira-Brett, “Electrochemical Sensing in Solution-Origins, Applications and Future Perspectives,” Journal of Solid State Electrochemistry, Vol. 15, No. 7-8, 2011, pp. 1487-1494. http://dx.doi.org/10.1007/s10008-011-1447-z
[10]	T. Matsunaga, R. Tomoda, T. Nakajima and H. Wake, “Photoelectrochemical Sterilization of Microbial Cells by Semiconductor Powders,” FEMS Microbiology Letters, Vol. 29, No. 1-2, 1985, pp. 211-214. http://dx.doi.org/10.1111/j.1574-6968.1985.tb00864.x
[11]	J.-M. Herrmann, “Heterogeneous Photocatalysis: Fundamentals and Applications to the Removal of Various Types of Aqueous Pollutants,” Catalysis Today, Vol. 53, No. 1, 1999, pp. 115-129. http://dx.doi.org/10.1016/S0920-5861(99)00107-8
[12]	L. Sun and J. R. Bolton, “Determination of the Quantum Yield for the Photochemical Generation of Hydroxyl Radicals in TiO2 Suspensions,” The Journal of Physical Chemistry, Vol. 100, No. 10, 1996, pp. 4127-4134. http://dx.doi.org/10.1021/jp9505800
[13]	B. T. T. Nguyen, J. Q. Ang and C. S. Toh, “Sensitive Detection of Potassium Ion Using Prussian Blue Nanotube Sensor,” Electrochemistry Communications, Vol. 11, No. 10, 2009, pp. 1861-1864. http://dx.doi.org/10.1016/j.elecom.2009.08.003
[14]	M. C. Henstridge and R. G. Compton, “Mass Transport to Micro-and Nanoelectrodes and Their Arrays: A Review,” The Chemical Record, Vol. 12, No. 1, 2012, pp. 63-71. http://dx.doi.org/10.1002/tcr.201100032
[15]	N. Baram, D. Starosvetsky, J. Starosvetsky, M. Epshtein, R. Armon and Y. Ein-Eli, “Enhanced Inactivation of E. coli Bacteria Using Immobilized Porous TiO2 Photoelectrocatalysis,” Electrochimica Acta, Vol. 54, No. 12, 2009, pp. 3381-3386. http://dx.doi.org/10.1016/j.electacta.2008.12.033
[16]	M. Nishizawa, S. Kuwabata and H. Yoneyama, “Photoimage Formation in a TiO2 Particle-Incorporated Prussian Blue Film,” Journal of the Electrochemical Society, Vol. 143, No. 11, 1996, pp. 3462-3465. http://dx.doi.org/10.1149/1.1837237
[17]	W. Jin, Z. Chu and Y. Zhang, “Design and Preparation of Nanostructured Prussian Blue Modified Electrode for Glucose Detection,” In: P. A. Serra, Ed., New Perspectives in Biosensors Technology and Applications, InTech, 2011, pp. 329-342. http://dx.doi.org/10.5772/17798
[18]	A. Goel, R. Bhatt and N. Rani, “Removal of Methyl Orange, an Azo Dye, Using Oxidative Degradation by Hexacyanoferrate(III) Ions,” Discovery Science, Vol. 2, No. 4, 2012, pp. 32-36. discovery.org.in/ds/A201210_07
[19]	H. Yu, J. Ma, Y. Zhang, X. Zhang and W. Shi, “Cyclic Voltammetry Studies of TiO2 Nanotube Arrays Electrode: Conductivity and Reactivity in the Presence of H+ and Aqueous Redox Systems,” Electrochimica Acta, Vol. 56, No. 18, 2011, pp. 6498-6502. http://dx.doi.org/10.1016/j.electacta.2011.05.004
[20]	K. Sunada, T. Watanabe and K. Hashimoto, “Studies on Photokilling of Bacteria on TiO2 Thin Film,” Journal of Photochemistry and Photobiology A: Chemistry, Vol. 156, No. 1, 2003, pp. 227-233. http://dx.doi.org/10.1016/S1010-6030(02)00434-3
[21]	P. C. Maness, S. Smolinski, D. M. Blake, Z. Huang, E. J. Wolfrum and W. A. Jacoby, “Bactericidal Activity of Photocatalytic TiO2 Reaction: Toward an Understanding of Its Killing Mechanism,” Applied and Environmental Microbiology, Vol. 65, No. 9, 1999, pp. 4094-4098.
[22]	K. Sunada, Y. Kikuchi, K. Hashimoto and A. Fujishima, “Bactericidal and Detoxification Effects of TiO2 Thin Film Photocatalysts,” Environmental Science & Technology, Vol. 32, No. 5, 1998, pp. 726-728. http://dx.doi.org/10.1021/es970860o
[23]	Z.-X. Lu, L. Zhou, Z.-L. Zhang, W.-L. Shi, Z.-X. Xie, H.-Y. Xie, D.-W. Pang and P. Shen, “Cell Damage Induced by Photocatalysis of TiO2 Thin Films,” Langmuir, Vol. 19, No. 21, 2003, pp. 8765-8768. http://dx.doi.org/10.1021/la034807r
[24]	H. Foster, I. Ditta, S. Varghese and A. Steele, “Photocatalytic Disinfection Using Titanium Dioxide: Spectrum and Mechanism of Antimicrobial Activity,” Applied Microbiology and Biotechnology, Vol. 90, No. 6, 2011, pp. 1847-1868. http://dx.doi.org/10.1007/s00253-011-3213-7
[25]	W. Wang, Y. Yu, T. An, G. Li, H. Y. Yip, J. C. Yu and P. K. Wong, “Visible-Light-Driven Photocatalytic Inactivation of E. coli K-12 by Bismuth Vanadate Nanotubes: Bactericidal Performance and Mechanism,” Environmental Science & Technology, Vol. 46, No. 8, 2012, pp. 4599-4606. http://dx.doi.org/10.1021/es2042977
[26]	A. Rompre, P. Servais, J. Baudart, M.-R. de-Roubin and P. Laurent, “Detection and Enumeration of Coliforms in Drinking Water: Current Methods and Emerging Approaches,” Journal of Microbiological Methods, Vol. 49, No. 1, 2002, pp. 31-54. http://dx.doi.org/10.1016/S0167-7012(01)00351-7
[27]	H. M. Coleman, V. Vimonses, G. Leslie and R. Amal, “Degradation of 1,4-Dioxane in Water Using TiO2 Based Photocatalytic and H2O2/UV Processes,” Journal of Hazardous Materials, Vol. 146, No. 3, 2007, pp. 496-501. http://dx.doi.org/10.1016/j.jhazmat.2007.04.049
[28]	World Health Organization, “Guidelines for Drinking-Water Quality,” Geneva, 2011.
[29]	N. D. Bedding, A. E. McIntyre, R. Perry and J. N. Lester, “Organic Contaminants in the Aquatic Environment I. Sources and Occurrence,” Science of the Total Environment, Vol. 25, No. 2, 1982, pp. 143-167. http://dx.doi.org/10.1016/0048-9697(82)90083-3
[30]	E. Diaz, A. Ferrandez, M. A. Prieto and J. L. Garcia, “Biodegradation of Aromatic Compounds by Escherichia coli,” Microbiology and Molecular Biology Reviews, Vol. 65, No. 4, 2001, pp. 523-569. http://dx.doi.org/10.1128/MMBR.65.4.523-569.2001
[31]	K. Yagi, “Applications of Whole-Cell Bacterial Sensors in Biotechnology and Environmental Science,” Applied Microbiology and Biotechnology, Vol. 73, No. 6, 2007, pp. 1251-1258. http://dx.doi.org/10.1007/s00253-006-0718-6
[32]	J. H. Yuan, K. Wang and X. H. Xia, “Highly Ordered Platinum-Nanotubule Arrays for Amperometric Glucose Sensing,” Advanced Functional Materials, Vol. 15, No. 5, 2005, pp. 803-809. http://dx.doi.org/10.1002/adfm.200400321
[33]	A. Curulli, F. Valentini, S. Orlanduci, M. L. Terranova and G. Palleschi, “Pt Based Enzyme Electrode Probes Assembled with Prussian Blue and Conducting Polymer Nanostructures,” Biosensors and Bioelectronics, Vol. 20, No. 6, 2004, pp. 1223-1232. http://dx.doi.org/10.1016/j.bios.2004.06.026