Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 5(12), 88-97, December (2015) Res. J. Chem. Sci. International Science Congress Association 88 Development of Electrochemical Biosensor for the Detection of Klebsiella pneumoniae as Biological Weapon Harish Kumar and BhawanaMaterial Science and Electrochemistry Lab., Dept. of Chemistry, Ch. Devi Lal University, Sirsa, Haryana, 125 055, INDIAAvailable online at: www.isca.in, www.isca.me Received 7th December 2015, revised 11th December 2015, accepted 17th December 2015 AbstractRapid, explicit detection and identification of Biological Warfare Agents (BWAs) with early warning signals for detecting possible biological attack is a main challenge for health, military and other government defence agencies. The current research is focussed on development of electrochemical biosensors for the detection of biological warfare agent. For this purpose, a carbon based (Graphene) working electrode containing enzyme alkaline phosphatase, cellulose acetate and Poly Vinyl Pyrolidone (PVP), Ferrocene, Horseradish peroxidase, aq. KOH was fabricated. It is then combined with Ag/AgCl reference and a platinum electrode (auxiliary) to form a three electrode based electrochemical biosensor for the electrochemical detection of Klebsiella pneumoniae as biological warfare agent in the presence and absence of Fenanoparticles. Fe nanoparticles were produced by sol-gel technique and were characterized by UV-visible, FTIR, TEM and XRD techniques. Change in current response and OCP values helps in the detection of biological warfare agent in presence and absence of Fe4 nanoparticles. Effects of temperature, stirring and Fe nanoparticles on the BWA have also been investigated. Keywords: Biological warfare agents, biosensors, Sol-gel method, Fe4 nanoparticles, Klebsiella, pneumoniae.Introduction The term Bioterrorism is defined as the use of a biological weapon (bacteria, virus or spores) on human life as a weapon of mass infection. It ultimately proves more powerful than a chemical or a nuclear weapon because it works silently and its effects can be extensive and out of control. List of pathogenic/harmful bacteria that may act as biological warfare agents is unending. Highly dangerous include Botulinum toxin, Francisellatularensis, Salmonella typhimurium, Staphylococcus epidermis, Klebsiella pneumoniae and Yersinia pestis. Other bio-agents, like Venezuelan equine encephalitis, Marburg, Ebola, and influenza viruses are of lesser importance, despite the fact, that infections with these viruses are serious and mortality is relatively high, but due to the difficulty in their preparation, their position on the list of Biological Warfare Agents (BWA) is lower. First evidence of bioterrorism came in to existence in 1979, when doctors presented a report of mass civilian death due to B. anthracis pneumonia i.e. due to inhalation of anthrax. A person exposed to B. Anthracis died immediately. The bacilli of anthracis multiply rapidly in the body and produce a harmful toxin that stops the process of breathing. On the other hand, Klebsiella pneumoniae can also be used as BWA. K. pneumoniae causes infection in lungs, where they cause necrosis, inflammation, and hemorrhage within the lung tissue. In comparison with chemical warfare agents (CWA), BWA production is much cheaper and terrorist or military attack with BWA is more effective in the range of hazard area and in the number of expected casualties. The infectious dose (ID) (amount of organism needed for infection outbreak) is different for every agent. Usually the intake of aerosol (particles 1 - 10 µm) through lung is able to evocate disease with a lower ID for the given BWA. Different types of electrochemical biosensors which are in common practice are: Potentiometric, conductometric, amperometric, and impedimetric2-5. Out of these, amperometric biosensors are more common in practice and are typically based on the use of ion-selective working electrodes. These are three electrode based electrochemical systems which are attached with electrochemical detectors which measure the changes in analyte concentration during the course of reaction taking place at the surface of bio-recognition layer. The advantage of Amperometric biosensors over other biosensors is that they are highly sensitive, rapid, linear concentration dependent and inexpensive. Amperometric biosensors aimed at microbial analysis have been reported by different researchers7-11. First biosensors used pH glass electrode with enzymes captured in a suitable membrane. In potentiometric immune-sensor electrochemical biosensor, enzyme-labelled antibodies are used. The most common labelling enzymes are urease, glucoseoxidase or alkaline phosphatase, which are able to change either pH or ionic strength in the course of the detection12. Very popular semiconductor-based biosensors are light-addressable potentiometric sensors (LAPS). Due to their small size and possible multichannel arrangement, these devices seem to be very convenient for simultaneous analysis of several analytes13. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(12), 88-97, December (2015) Res. J. Chem. Sci. International Science Congress Association 89 The LAPS immune-sensors were used to detect Francisella tularensis14with a limit of detection (LOD) at 3.4 × 10 cells/ml and Bacillus melitensis with LOD equal to 6 × 10 cells/ml during the 1h incubation time15. A better LOD was achieved for Escherichia coli DH5 a strain16; the secondary antibody specific against Escherichia coli labelled with urease was used and LOD of 10 cells/ml for1.5 h assay time was claimed. In continuation to our earlier study17, in this paper, we have focussed on the fabrication of electrochemical biosensor for the detection of Klebsiella pneumoniae as biological warfare agent. Material and Methods Samples of disease causing bacteria i.e. Klebsiella pneumonia (MTCC No. 3384) were collected on request from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, situated at Chandigarh, India. Different steps used for the electrochemical determination of disease causing bacteria are: Preparation of Bacterial Strain: The bacterial test organism Klebsiella pneumonia was grown in nutrient broth for 24.0 hours at 37.0 °C. Sodium phosphate buffer solution (pH 7.0) was selected to hold these disease causing bacteria at a very low temperature i.e. 4.0 ºC. Synthesis of Fe4 Nanoparticles and Graphene: Fe4 nanoparticles were synthesized by Sol-gel technique. In this technique, metal salt solution having Ferric chloride is added drop wise in a mixture of tetraethyl orthosilicate in ethanol. Reduced graphene was synthesized by well known Hummers method. Characterization of Fe4 Nanoparticles and Graphene: Characterization of Fe4 nanoparticles and Graphene were carried out by using UV-visible spectroscopy, Fourier Transform Infra-Red (FT-IR) technique, X-ray diffraction (XRD) study and Transmission Electron Microscopy (TEM) techniques. Fabrication of Working Test Electrode: A carbon paste (Graphene) working electrode was fabricated for the electrochemical determination of biological weapon. A slurry was prepared by mixing reduced graphene, alkaline phosphatase, cellulose acetate, Ferrocene, Horseradish peroxidase, aq. KOH and Poly vinyl pyrolidone (PVP). This slurry was filled in working test electrode with the help of luggin capillary. A copper wire is dipped from outside in the slurry for making electrical connections. Fabrication of Three electrodes based Electrochemical Cell: A three electrodes based electrochemical cell was fabricated having three electrodes i.e. a working test electrode (cellulose acetate, PVP bound carbon paste electrode), Ag/AgCl reference electrode and a platinum electrode acting as an auxiliary electrode for the electrochemical determination of disease causing pathogen (figure-1). Figure-1 A three electrodes based electrochemical biosensor for the detection of biological warfare agent Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(12), 88-97, December (2015) Res. J. Chem. Sci. International Science Congress Association 90 Electrochemical Characterization: Three electrodes based electrochemical cell is connected to the instrument electrochemical workstation PGSTAT 128N, Metrohm Autolab attached to a PC and digital controlled water bath to maintain constant temperature. Electrochemical measurement experiments were performed on a pathogenic bacteria Klebsiella pneumoniae and change in Open Circuit Potential (OCP), current (nA) and potential values were recorded at different conditions. For the electrochemical characterization, 3 × 10 CFU of Klebsiella pneumoniae in 50 ml of PBS buffer solution was used. Four different samples were prepared i.e. pure PBS buffer solution as sample 1, PBS buffer solution with 3 × 10 CFU of Klebsiella pneumoniae assample 2, PBS buffer solution with Fe nanoparticles assample 3 and PBS buffer solution with Fe nanoparticles and 3 × 10 CFU of Klebsiella pneumoniaeassample-4. The above samples were kept under following two observations: Heating at a constant temperature of 70.0 ºC without stirring for 6.0 hours. Continuous stirring for 6.0 hours at room temperature. After 6.0 hours, we have recorded the current and potential values with the help of three electrodes based electrochemical cell connected to the instrument PGSTAT 128N, Metrohm Autolab, Netherland. Results and Discussion Result of electrochemical characterization on Klebsiella pneumoniae is recorded in the form of tables-1 and 2 and figures-2 to 8. Table-1 shows OCP values of unstirred and after 6 hours of continuous stirring. Table-2 shows the OCP values of four heated samples at initial time and after 6.0 hours. Figure-1 shows electrochemical biosensor for the detection of biological warfare agent. Figure-2 shows current and potential values of pure PBS buffer solution at 0.0 and 6.0 hours. Figure-3 shows current and potential behaviour of PBS buffer solution in presence of Klebsiella pneumoniae at 0.0 and 6.0 hours. Figure-4 shows current and potential behaviour of PBS buffer solution in presence of Fe4 nanoparticles (50 µl/ ml) at 0.0 and 6.0 hours. Figure-5 shows current and potential behaviour of PBS buffer solution in presence of Klebsiella pneumoniaeand Fe4 nanoparticles (50 µl/ ml) at 0.0 and 6.0 hours. Figure-6 shows current and potential values of pure PBS buffer solution at 0.0 and after 6.0 hours of heating at 70.0C. Figure-7 shows current and potential behaviour of PBS buffer solution in presence of Klebsiella pneumoniae at 0.0 and after 6.0 hours of heating at 70.0C. Figure-8 shows current and potential behaviour of PBS buffer solution in presence of Klebsiella pneumoniae and Fe4 nanoparticles (50 µl/ ml) at 0.0 and after 6.0 hours of heating at 70.0C. Figure-9 shows UV-visible spectra of Fe metal nanoparticles synthesized by sol-gel technique as a function of wavelength. Figure-10 shows Fourier Transform Infra-Red (FT-IR) spectra (Thermo-USA, FTIR-3800) in the wavelength range of 400 - 4000 cm-1 of Fe nanoparticles produced by sol-gel technique. Figure-11 shows XRD pattern of Fe nanoparticles produced by sol-gel technique. Figure-12 shows TEM images of Femetal nanoparticles produced by sol-gel technique. -0.8-0.6-0.4-0.20.00.20.40.60.81.01.21.41.51.61.71.81.92.02.12.22.32.42.5 Current (nAmp)Potential (Volt) at 0 hrs after 6 hrsFigure-2 Current versus potential behaviour of pure PBS buffer solution at 0.0 and after 6.0 hours of stirring Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(12), 88-97, December (2015) Res. J. Chem. Sci. International Science Congress Association 91 -0.8-0.6-0.4-0.20.00.20.40.60.81.01.20.51.01.52.02.5 Current (nAmp)Potential (Volt) at 0 hrs after 6 hrsFigure-3 Current versus potential behaviour of PBS buffer solution in presence of Fe4 nanoparticles (50 µl/ ml) at 0.0 and after 6.0 hours of stirring-0.8-0.6-0.4-0.20.00.20.40.60.81.01.21.61.82.02.22.42.6 Current (nAmp)Potential (Volt) at 0 hrs after 6 hrsFigure-4 Current versus potential behaviour of PBS buffer solution with Klebsiella Pneumoniae at 0.0 and 6.0 hours of stirring Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(12), 88-97, December (2015) Res. J. Chem. Sci. International Science Congress Association 92 -0.8-0.6-0.4-0.20.00.20.40.60.81.01.21.61.71.81.92.02.12.22.32.42.52.62.7 Current (nAmp)Potential (Volt) at 0 hrs after 6 hrsFigure-5 Current versus potential behaviour of PBS buffer solution in presence of Klebsiella Pneumoniae and Fe4 nanoparticles (50 µl/ ml) at 0.0 and 6.0 hours of stirring-0.8-0.6-0.4-0.20.00.20.40.60.81.01.21.01.21.41.61.82.02.22.42.6 Current (nAmp)Potential (Volt) at 0 hrs after 6 hrsFigure-6 Current versus potential behaviour of pure PBS buffer solution at 0.0 and after 6.0 hours of heating Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(12), 88-97, December (2015) Res. J. Chem. Sci. International Science Congress Association 93 -0.8-0.6-0.4-0.20.00.20.40.60.81.01.21.21.41.61.82.02.22.42.6 Current (nAmp)Potential (Volt) at 0 hrs after 6 hrsFigure-7 Current versus potential behaviour of PBS buffer solution in presence of Fe4 nanoparticles (50 µl/ ml) at 0.0 and after 6.0 hours of heating-0.8-0.6-0.4-0.20.00.20.40.60.81.01.21.41.61.82.02.22.42.62.8 Current (nAmp)Potential (Volt) at 0 hrs after 6 hrsFigure-8 Current versus potential behaviour of PBS buffer solution in presence of Klebsiella Pneumoniae and Fe4 nanoparticles (50 µl/ ml) at 0.0 and after 6.0 hours of heating Research Journal of Chemical Sciences ___ ______________________________ Vol. 5(12), 88-97, December (2015) International Science Congress Association UV Discussion: The UV absorption band of Fe nanoparticles (figure- 9) was observed in the wavelength range of 330 which may be due to the absorption and scattering of light by iron nanoparticles18-19 . The low absorption band at a of 410 nm may be due to the formation of least agglomerated Fe nanoparticles. Absence of additional peaks corresponding to alcohol further indicates that the metal nanoparticles were not surrounded by ethanol and they only acted as a soft tem FT- IR spectroscopic technique was carried out in order to ascertain the purity and nature of ferrite metal nanoparticles synthesized by sol-gel technique. A peak at 3,440 cm FTIR spectrum of iron oxide nanoparticles (f traces of ferric hydroxide in Fe20-21 . The peaks at 565 and 421 cm-1 are may be due to the vibrations of Fe 2- respectively22 . Another peak (sharp and high intensity) at 565 cm-1 indicates the presence of crystallinity in the metal ______________________________ ___________________ International Science Congress Association Figure-9 UV -Visible spectra of Fe4 nanoparticles The UV absorption band of Fe nanoparticles 9) was observed in the wavelength range of 330 –450 nm which may be due to the absorption and scattering of light by . The low absorption band at a wavelength of 410 nm may be due to the formation of least agglomerated nanoparticles. Absence of additional peaks corresponding to alcohol further indicates that the metal nanoparticles were not surrounded by ethanol and they only acted as a soft tem plate. IR spectroscopic technique was carried out in order to ascertain the purity and nature of ferrite metal nanoparticles A peak at 3,440 cm -1 in the (f igure 10), proves . The peaks at 565 and are may be due to the vibrations of Fe 2+–O2- and Fe3+ . Another peak (sharp and high intensity) at indicates the presence of crystallinity in the metal nanopar ticles. The characteristic absorption bands at 565 and 421 cm-1 confirms the presence of spinel structure in Fe nanoparticles. The reflection peak at 2 = 35.60 ferrite (Fe ) nanoparticles (JCPDS, PDF cards 3 1086) (figure- 11). The diffractions peaks of the ferrite nanoparticles were observed at 2 nm), 35.61 (d spacing = 0.253 nm), 43.27 nm), 53.56 (d spacing = 0.171 nm) and 57.11 0.162 nm)23. The peaks at an angle of 30.18 (311), 43.278 (400), 53.569 (422), 57.118 (440) correspond to Fe. The average particle size of ferrite nanoparticles has been calculated using well known Scherrer equation24 and was found t o be 31.0 nm peak broadening confirms the formation of the fine Fe nanoparticles. Figure-10 FT-IR spectra of Fe4 nanoparticles ___________________ _______ ISSN 2231-606X Res. J. Chem. Sci. 94 ticles. The characteristic absorption bands at 565 and confirms the presence of spinel structure in Fe 4 = 35.60 confirm spinel phase of ) nanoparticles (JCPDS, PDF cards 3 -864 and 22- 11). The diffractions peaks of the ferrite = 30.18 (d spacing = 0.297 (d spacing = 0.253 nm), 43.27 (d spacing = 0.209 (d spacing = 0.171 nm) and 57.11 (d spacing = peaks at an angle of 30.18 (220), 35.612 (422), 57.118 (511) and 62.655 The average particle size of ferrite nanoparticles has been calculated using well known Scherrer o be 31.0 nm . Further, diffraction peak broadening confirms the formation of the fine Fe 4 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(12), 88-97, December (2015) Res. J. Chem. Sci. International Science Congress Association 95 Figure-11 XRD diffraction pattern of Fe4 nanoparticlesFigure-12 (a) TEM images of Fe4 nanoparticles (b) Diffraction pattern of Fe4 nanoparticlesStructural and optical properties of Fe nanoparticles were determined by using Transmission Electron Microscope (TEM) of made Morgagni 268 D, FEI Philips at a resolution of 2 A0 from Electron Microscope Facility (SAIF), AIIMS, New Delhi (figure-12). The TEM images reveals self-organized network like morphology of ferrite (Fe) nanoparticles which are almost identical in shape and found to be uniformly dispersed. The average particles size is in close agreement with both the technique i.e. as observed in TEM and the crystallite size calculated by the Scherrer equation (~31.0 nm) with the help of XRD technique. Further, the TEM diffraction ring (Figure 12 b) confirms that the ferrite nanoparticles are in a well crystalline state25. Purpose of addition of Horseradish peroxidase (HRP) in the slurry of working electrode is because HRP enhance the intensity of weak electrochemical signal. Ferrocene act as stimulator. Grapheme was added in to the slurry of working electrode in order to increase its conductivity and surface area of the electrode. Table-1 OCP (Open Circuit Potential) values of stirred samples of Klebsiella pneumoniaSamples OCP Values (Volt) at 0.0 and 6.0 hours Pure PBS solution 0.524 0.545 PBS solution with Fe nanoparticles 0.747 0.753 PBS solution with 3.0 ×10 7 CFU of Klebsiella Pneumoniae0.238 0.288 PBS solution with Fe nanoparticles and 3.0 ×10 CFU of Klebsiella Pneumoniae0.247 0.264 15202530354045505560657080010001200140016001800 Intensity (cps)2 Theta (degree) B Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(12), 88-97, December (2015) Res. J. Chem. Sci. International Science Congress Association 96 Table-2 OCP (Open Circuit Potential) values of heated samples of Klebsiella PneumoniaeSamples OCP Values (Volt) at 0.0 and 6.0 hours Pure PBS solution 0.514 0.553 PBS solution with Fenanoparticles 0.782 0.550 PBS solution with 3.0 ×10 7 CFU of Klebsiella Pneumoniae0.247 0.231 From the tables-1 and 2, it is observed that OCP value of PBS buffer solution decreases on addition of 3.0 × 10 CFU of Klebsiella pneumoniae. OCP value of PBS buffer solution increases on addition of Fe nanoparticles. OCP value of Klebsiella pneumoniae increases (0.238 to 0.247 V) on addition of Fe nanoparticles (50 µl/ ml). As Fe nanoparticles are electro active species and hence increase in OCP value is due to increase in electrical conductivity of the medium. Heating of sample no. 1 to 70.0C leads to increase in the OCP value from 0.514 to 0.553 V. Heating of sample no. 3 to 70.0 C makes Klebsiella pneumoniae inactive which lowers its OCP value. It is observed from the table-1 that the addition of Fenanoparticles (50 µl/ml) at room temperature in the sample containing 3.0 × 10 CFU of Klebsiella pneumoniae results into slight increase in OCP valueIt is observed from table-2 that heating of sample no. 3 to 70.0 C for 6.0 hours results in decrease in the OCP value from 0.247 to 0.231 V. A constant OCP value of 0.247 V in phosphate buffer solution indicates the presence of Klebsiella pneumoniae pathogenic bacteria in the sample. Heating of the samples up to 70.0C for a definite period of time leads to decrease in OCP value. Addition of Fe nanoparticles leads to slight increase in OCP value i.e. heating of sample makes bacteria inactive which results in the decrease in OCP value. It is observed from figure-2 that the current and potential behaviour of pure PBS solution remains almost same at 0.0 and 6.0 hours of continuous stirring. The current value first decreases and then increases with increase in potential value. It is observed from figure-3 that there is deviation in current value at smaller value of potential of PBS buffer solution in presence of Fe4 nanoparticles (50 µl/ ml) at 0.0 and after 6.0 hours of continuous stirring. But at higher potential value current and potential behaviour of pure PBS solution remains almost same. Current value increases on addition of Fe4 nanoparticles in phosphate buffer solution. Similar types of results were observed in case of samples containing Klebsiella pneumoniabut deviation is small in comparison to Fe4 nanoparticles(figure-4). The current and potential behaviour of samples containing both Fe4 nanoparticles (50 µl/ ml) and 3.0 × 10CFU of Klebsiella pneumonia are almost same at initial time and after 6.0 hours of continuous stirring(figure-5). It is observed from the figure-6 that the value of current first decreases and then increases continuously with increase in potential value at initial time and after 6.0 hours of continuous stirring. It is observed from the figure-7 that the value of current first increase and then decrease and then increase with increase in potential value in presence of Fe4 nanoparticles at initial time and after 6.0 hours of heating at 70C. Figure 8 shows that value of current initially remains constant and then decrease and then continuously increase with increase in potential value in presence of 3.0 × 10 CFU of Klebsiella pneumonia in PBS solution at initial time and after 6.0 hours at a constant temperature of 70.0 C. The current value increases slightly after six hours of heating at high value of potential. Conclusion A carbon (Graphene) based working electrode having alkaline phosphatase, cellulose acetate, Ferrocene, Horseradish peroxidase, aq. KOH and PVP was fabricated which when combined with Ag/AgCl reference and a platinum auxiliary electrode to form a three electrode based electrochemical cell for the electrochemical detection of Klebsiella pneumoniae as biological warfare agent. Fe4 nanoparticles were synthesized by sol-gel method. Characterization of Fe4 nanoparticles was carried out by using UV-visible, FT-IR, XRD and TEM techniques. An UV–visible absorption band at the wavelength of 410 nm and a sharp absorption band at 600 cm-1 in FTIR spectra confirm the formation of ferrite nanoparticles. OCP value of PBS buffer solution decreases on addition of 3.0 × 10CFU of Klebsiella pneumoniae. OCP value of PBS buffer solution increases on addition of Fe nanoparticles. Current value increases on addition of Fe4 nanoparticles in phosphate buffer solution. A constant OCP value of 0.247 V in phosphate buffer solution indicates the presence of Klebsiella pneumoniae pathogenic bacteria in the sample. Acknowledgement Many-many thanks to Defence Research and Development Organisation (DRDO), New Delhi for providing us financial support for carrying out this research work. 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