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Label-free amperometric biosensor for Escherichia coli O157:H7 detection
Applied Surface Science 495 (2019) 143548
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Label-free amperometric biosensor for Escherichia coli O157:H7 detection a
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Nidhi Dhull , Gurpreet Kaur , Prateek Jain , Priyanka Mishra , Divya Singh , Lilly Ganju , ⁎ Vinay Guptaa, Monika Tomard, a
Department of Physics and Astrophysics, University of Delhi, Delhi, India Department of Biology, University of North Carolina at Chapel Hill, USA Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi, India d Department of Physics, Miranda House, University of Delhi, Delhi, India b c
A R T I C LE I N FO
A B S T R A C T
Keywords: E. coli O157:H7 Label-free Amperometric Nickel oxide
Foodborne pathogens pose a major threat of infectious life-threatening diseases all around the globe. Escherichia coli (E. coli) O157 is one such bacterial species that has caused several major outbreaks in the past. Advancement of a rapid sensing mechanism with high sensitivity is vital to avoid such epidemics by timely recognition of the infectious foodborne pathogens. The present work proposes the development of an amperometric biosensing platform for the detection of the pathogenic E. coli O157:H7. The developed biosensor works on the principle of antibody-antigen interactions, where the antibody is covalently bound to the surface of the nickel oxide thin film matrix prepared using sputtering technique. Accelerated charge transfer through NiO, an exceptional support provided to the antibody, and thus enhanced target capture efficiency leads to the direct detection of E. coli O157:H7 without the assistance of label. The electrochemical sensing results exhibit a wide linear range of 101 to 107 cells/mL and a low limit of detection of 100 i.e. 1 cell/mL along with high selectivity and specificity against other bacterial species.
1. Introduction One of the major risks to the food chain is posed by foodborne pathogens. A large number of infectious diseases caused by a wide range of pathogens have been enumerated till date that pose a serious threat of contamination. These infections have the tendency to breed through various environmental means such as air, water and food and can be highly communicable [1]. The concept of bioterrorism is not new and poses a serious threat to every country around the world. Various pathogenic microorganisms can be used as biological weapons by the terrorists to cause wide spread contaminations [2]. Escherichia coli, Salmonella typhimurium, Campylobacter jejuni, Legionella pneumophila and Staphylococcus aureus are a few bacterial strains that cause serious health hazards. Among these, Escherichia coli (E. coli) has been reported to cause large scale loss of life [3]. E. coli is a naturally occurring bacteria found in the intestinal tracts of human beings and other warm-blooded organisms to assist their bodies in synthesis of essential vitamins. However, not all strains of E. coli are non-pathogenic. E. coli O157 is a pathogenic strain that produces toxins having the capability to destruct the inner lining of the intestines and can cause illness ranging from stomach cramps and mild
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diarrhea to lethal complications like hemorrhagic colitis (bloody diarrhea), hemolytic uremic syndrome (HUS, leads to kidney failure), and thrombotic thrombocytopenic purpura (TTP, leads to cardiac and neurological manifestations) [4,5]. Major outbreaks of diseases instigated by E. coli O157 have been reported by the World Health Organization (WHO) recently in the most developed countries of the world such as UK, Germany and Canada where the source of the pathogen has been attributed to contaminated food or water [6]. Global Health Observatory (GHO) data of 2016 lists diarrheal diseases caused by communicable, maternal, perinatal and nutritional conditions as one of the top 10 causes of death world wide which accounts for 1.5 million deaths annually [7]. Among these, E. coli has a great share of 5.6%, high enough for it to make to the list of major infectious pathogens [8]. Such wide spread outbreaks have resulted in adverse socioeconomic effects. A high-throughput mechanism for bacterial detection is vital keeping in mind the exponential rate of growth of the bacterial cells. The recognition of foodborne pathogens in natural food resources, packaged foods, processing and assembling units, drinking water sources and hospitals persist to be based on the traditional techniques of cell culturing, evaluation of the cell morphology or their ability to
Corresponding author. E-mail address: [email protected] (M. Tomar).
https://doi.org/10.1016/j.apsusc.2019.143548 Received 28 February 2019; Received in revised form 18 July 2019; Accepted 30 July 2019 Available online 31 July 2019 0169-4332/ © 2019 Elsevier B.V. All rights reserved.
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bioassay strain of E. coli O157:H7 (ATCC #700728) was purchased from BCCM/LMG (Belgium-Europe). The cells were cultured in Luria Bertini (LB) broth and then reconstituted in Phosphate Buffer Saline (PBS) for electrochemical measurements. Cultured samples of S. aureus ATCC #25923 and E. coli ATCC #25922 (each with a concentration of 108 cells/mL) were obtained from Defence Institute of Physiological and Allied Research (DIPAS), a laboratory of the Defence Research and Development Organization (DRDO), India. Powdered form of EDC (N(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide) were acquired from Sigma-Aldrich. (3Aminopropyl) triethoxysilane (APTES) was procured from Alfa-Aesar.
grow. Plate counting, polymerase chain reaction (PCR), bioluminescence, fluorescent bacteriophage assay, chemiluminescence, enzymelinked immunosorbent assay (ELISA), etc. are few such widely used techniques that require lengthy enrichment procedure, isolation, morphological examination and large sample volumes to positively identify food pathogens [9,10]. Such techniques are reliable but labor-intensive and highly time-consuming to arrive at a quantitative conclusion. Lately, numerous configurations of biosensors have technologically advanced as more rapid alternatives to such traditional techniques for the identification of food borne pathogens. These are generally based on either optical system, piezoelectric platforms, pathogen specific DNA or to some extent, electrochemical methods. Among these, antibody based amperometric immunosensors have drawn significant attention because of their uncomplicated preparation steps, high-throughput, possibility of miniaturization, higher selectivity and specificity [11]. However, most of the biosensors for the recognition of bacterial species use labels such as green fluorescent protein or enzymes fused into antibodies to facilitate the detection. The additional steps of labelling may lead to a compromise in sensitivity of the antibody to the extent where they fail to distinguish between viable and nonviable cells [12]. Thus, development of a label-free sensing mechanism is very significant, in addition to the above mentioned attributes. Looking at the critical importance of timely and accurate detection of such pathogens, the detection platform requires to be free from complex fabrication process, apart from being rapid, highly sensitive and selective. Nanocomposite materials have been greatly exploited for the advancement of electrochemical biosensors not only because of their biocompatibility but also due to their tunable conduction characteristics and large effective surface area for adsorption of desired biomolecules [13,14]. Recently, there has been a substantial increase in the development of metal oxide thin film based amperometric biosensors due to their exceptional stability. Nickel oxide (NiO), in particular, is an appropriate material of matrix because of its outstanding charge transfer characteristics [15]. It has been reported to be used in chemical and biological sensors as a functional layer due to its exceptional chemical stability and charge conduction. The performance of a biosensor in terms of sensing response and stability largely depends on the choice of matrix. The chemical stability of NiO permits the surface modification for covalent binding of biomolecules that enhances the biosensing response characteristics along with a larger shelf-life. Thus, in the present work NiO has been chosen as the matrix in the form of rf sputtered thin film. ITO coated corning glass has been employed as the substrate as it is electrically conducting (can be directly used as the working electrode in the electrochemical studies) and low cost as compared to stable metals such as gold [16]. This paper reports an amperometric biosensing mechanism for label-free recognition of pathogenic E. coli O157:H7 using nanostructured NiO thin film. The biosensor exploits the notion of standard antibody-antigen interactions, antibody bound covalently to the surface of nanostructured NiO matrix. The interactions of the antibody with the matrix surface and with the E. coli O157:H7 cells have been confirmed using FTIR spectroscopy. Cyclic voltammetry technique has been exploited to study the amperometric response of developed bioelectrode against E. coli O157:H7 concentration varying between 107 cells/ mL and 101 cells/mL. The selectivity of the prepared biosensor has been tested against a gram-positive bacterium (gram stain opposite to that of E. coli) Staphylococcus aureus (S. aureus) and a non-pathogenic strain of E. coli.
2.2. Bacterial cell culturing and surface plating Frozen stocks of E. coli O157:H7 were maintained at a temperature of −80 °C in 50% glycerol. Cell culturing was carried out by reconstituting the cells in nutrient broth and also surface plating on Luria agar to observe the cell growth. The bacterial suspension was kept in an incubator and shaker at 37 °C for 24 h for enrichment. The concentration of the cells was then estimated using UV–Visible spectrophotometer (PerkinElmer Lambda 35). For biosensing measurements, the cells were centrifuged at 5000 rpm for 5 min and re-suspend in Phosphate Buffer Saline (PBS) of pH 7.5. A series of eight 10-fold serial dilutions varying in the range 108 cells/mL to 101 cells/mL was prepared from the fresh stock culture using PBS for testing with the prepared bioelectrode. For studying the selectivity and sensitivity of the proposed biosensing mechanism in the co-existence of a non-target bacterial species, a mixed culture containing equal amounts of each organism (E. coli O157:H7, S. aureus ATCC #25923 and E. coli ATCC #25922) from its respective stock culture were harvested by centrifuging at 5000 rpm for 5 min and resuspending in PBS. 2.3. Preparation of bioelectrode Rf magnetron sputtering technique was exploited to prepare a nanostructured NiO thin film matrix on a conducting ITO coated glass substrates (NiO/ITO) using previously optimized parameters [16]. The surface of the as-deposited NiO matrix was functionalized by hydroxylation using a mixed solution of hydrogen peroxide and liquid ammonia followed by silanization using 1% solution of APTES diluted in toluene. An optimized concentration of antibody (Ab) E. coli O157:H7 (1 μg/mL) was immobilized covalently on the functionalized NiO matrix surface using 0.4 M EDC and 0.1 M NHS as coupling agents. Functionalization of the matrix surface using APTES provided free amino groups on it. EDC being a carboxyl and amine-reactive crosslinker forms amide bonds with these amino groups present on the surface of NiO matrix and further binds to the carboxyl groups on the antibodies. NHS stabilized the unstable reactive intermediate resulting from the crosslinking reaction of EDC with the carboxyl groups on the antibodies [17]. Various concentrations of E. coli O157:H7 were drop casted uniformly on the electrode surface (Ab/NiO/ITO) and incubated at 37 °C to study the amperometric response of the developed bioelectrode. The volume of the Ab E. coli O157:H7 and the various concentrations of E. coli O157:H7 samples utilized in each measurement was kept constant at 20 μL. 2.4. Apparatus Gamry Interface 1000 Potentiostat was used to perform cyclic voltammetry (CV) measurements via the three-electrode set-up. The working electrode being the developed NiO thin film based bioelectrode (Ab/NiO/ITO), Ag wire enclosed in saturated KCl solution as the reference electrode and platinum foil as counter electrode. All the measurements were recorded 50 mM PBS solution comprising of 0.9% NaCl and 5 mM [Fe(CN)6]3−/4− as external mediator. The cyclic voltammetry studies were performed in the voltage range of −0.3 V to 0.8 V
2. Experimental 2.1. Materials and reagents E. coli O157 (1.B.250B), a mouse monoclonal antibody (Ab E. coli) raised against E. coli whole cells was procured from Santa Cruz Biotechnology, Inc. with a concentration of 100 μg/mL. A nontoxigenic 2
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Fig. 1. UV–Visible spectra of the freshly cultured E. coli O157:H7 cells. Inset shows growth of the bacterial colonies on the agar plate after 24 h of incubation.
Fig. 3. FTIR spectra of as prepared NiO thin film based bioelectrode for the recognition of E. coli O157:H7. Table 1 Assignment of peaks identified in the FTIR spectra. Peak positions (cm−1) NiO
Ab/NiO
E.coli/Ab/NiO
532 611 742 870
532 611 742 870
1100 1240
1100
503 611 742 890 1100
1294 1648 1660
Possible peak assignment
Stretching vibration of NieO bond [20] Shifted NieO band [22] Ni-O-H stretching bond [27] C=O stretching vibrations [21] NH2 wagging and twisting [24] Bending of SieO bond [23] Stretching of SieO bond [23] C-N bond stretching [25] Protein components of Amide III band [26] N-H bending of Amide II [25] Protein components of Amide I band [26]
Fig. 2. Cyclic voltammetry studies of the as prepared NiO thin film based bioelectrode for the recognition of E. coli O157:H7.
with a scan rate of 100 mV/s. PerkinElmer Lambda 35 UV/Vis Spectrometer was used to determine the concentration of E. coli O157:H7 cells in the freshly cultured stock. Fourier-transform Infrared (FTIR) spectroscopic analysis was carried with PerkinElmer FTIR Spectrometer Frontier to verify the successful immobilization of Ab E. coli O157:H7 and E. coli O157:H7 on the surface of the NiO matrix.
3. Results and discussion 3.1. UV–visible spectroscopic analysis In the present work, freshly cultured E. coli O157:H7 were reconstituted in PBS from the growth media for the electrochemical studies. Hence, to verify the concentration of viable cells in the PBS, UV–Visible spectroscopic analysis was performed and the same was confirmed by plating the sample on agar plate. The absorbance spectra of the freshly cultured stock of E. coli O157:H7 and a 10-fold dilution of the same in
Fig. 4. CV curves for E. coli O157:H7 with varying incubation time for the Ab/ NiO/ITO electrode. Inset shows the corresponding variation in the bioelectrode response.
3
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Fig. 6. Calibration curve for the response of Ab/NiO/ITO bioelectrode towards E. coli O157:H7. Inset shows variation in oxidation peaks with varying cell concentrations.
corresponding to the external mediator [Fe(CN)6]3−/4− arising due to transition between Fe2+ and Fe3+ states. The intense redox peaks of NiO/ITO indicate the high charge conduction characteristics of the NiO matrix. The peak oxidation current (Ipo) reduces significantly from 1.27 to 1.03 mA on covalent immobilization of the E. coli O157:H7 antibody over the NiO matrix (Ab/NiO/ITO). This suggests that the protein molecules act as a barrier to the conduction of the matrix by hindering the electron exchange through the working electrode [19].
3.3. FTIR spectroscopic analysis The NiO thin film was deposited on Si substrates for FTIR studies and same steps as defined above for antibody immobilization and E. coli O157:H7 adsorption were performed. The resulting transmission spectra for NiO thin film, Ab/NiO and E.coli/Ab/NiO in the wavenumber range of 400 cm−1 to 2500 cm−1 are represented in Fig. 3. The typical band due to OeH centered about 3420 cm−1 indicating moisture content in the structure was observed in each case, with greater intensity in case of Ab/NiO and E.coli/Ab/NiO. However, the band has not been shown here to highlight other essential bands. The absorption modes at 503 cm−1 and 611 cm−1, in case of bare NiO thin film may be attributed to the stretching vibrations of NieO and Ni-O-H bonds respectively [20] [21]. The surface modification of NiO due to immobilization of the E. coli O157:H7 antibodies might cause variation in intensity of the mode and also wavenumber shift which was evident for the NieO peak in the FTIR spectra of Ab/NiO and E.coli/Ab/NiO [22]. The absorption peak at 742 cm−1 may be attributed to C]O stretching vibrations occurring due to traces of atmospheric carbon [21]. The additional peaks at 890 cm−1 and 1100 cm−1 correspond to the bending and stretching of SieO bond respectively [23]. The peak occurring at 870 cm−1 after immobilization of E. coli O157:H7 antibody on the surface of NiO thin film may be assigned to NH2 wagging and twisting resulting from surface modification using APTES [24]. The peaks at 1240 cm−1 and 1648 cm−1 indicate the stretching of CeN bond and NeH bending of Amide II bond due to peptide linkages respectively [25]. After interaction of E.coli cells with the antibody molecules, protein components of amide III and amide I bands appeared at 1294 cm−1 and 1648 cm−1 respectively [26]. All the bands with possible peak assignment are summarized in Table 1. The assigned peaks match very well with the reports available indicating proper bonding of antibody E. coli O157:H7 and E. coli O157:H7 cells on the surface of NiO
Fig. 5. (a): Amperometric response of the prepared bioelectrode (Ab/NiO/ITO) towards E. coli O157:H7. (b): EIS studies of the prepared bioelectrode for the detection of E. coli O157:H7.
the UV–visible wavelength range of 250 to 1100 nm is shown in Fig. 1. The optical density of approximately 0.2 and 0.04 corresponding to the wavelength of 600 nm (OD600) estimated the cell concentration to be 108 cells/mL and 107 cells/mL respectively [18]. The cell concentrations tend to show maximum absorbance in the UV region around 300 nm. The absorbance of further dilutions was too low to distinguish from one another and hence, not represented. Colonies of E. coli O157:H7 bacteria could be clearly visualized on the agar plate after incubation of 24 h (Inset of Fig. 1). Thus, the analysis confirmed the presence of specified concentration of viable E. coli O157:H7 cells in the PBS solution.
3.2. Bioelectrode characterization The cyclic voltammetry (CV) curves of the prepared bioelectrode with the applied potential varying from −0.3 V to 0.8 V at each step can be observed from Fig. 2. The redox peaks of the ITO coated glass substrates are negligible as compared to the intense peaks of the NiO thin film (NiO/ITO) at the potential values of 0.2 V and 0.4 V. These can be attributed to the respective reduction and oxidation peaks 4
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Fig. 8. Amperometric response of the prepared bioelectrode (Ab/NiO/ITO) towards varying E. coli O157:H7 concentrations inoculated in real milk samples.
Bioelectrode Response (%) =
Ipo (Ab)–Ipo (Cells) × 100. Ipo (Ab)
It is evident from Fig. 4 that the bioelectrode yields limited response when incubated with the cells for 5 to 30 min since the time is insufficient for efficient interaction between the cells and the antibody. Most of the cells remain unbound and are removed from the surface in the intermediate washing steps. The response increased with further increasing the incubation time and maximum response of 26% was obtained when cells were left undisturbed on the bioelectrode for 1 h. Beyond that, the response starts decreasing again. This can be reasoned on the basis of high rate of bacterial growth in ambient temperature and media. With increasing incubation time, the number of cells becomes excess to the extent that individual cells are not allowed to interact with antibody terminations present on the surface. A biofilm of the E. coli O157:H7 cells is formed on the surface of the electrode that gets carried away when washed with buffer. Thus, an optimum incubation time of 1 h was kept constant for further studies to obtain a maximum sensing response.
Fig. 7. (a): Selectivity and specificity studies of the Ab/NiO/ITO bioelectrode towards E. coli O157:H7. Inset shows the bioelectrode response against the nontarget bacterial species. (b): Reproducibility studies of the Ab/NiO/ITO bioelectrode towards E. coli O157:H7.
3.5. Amperometric response of the bioelectrode Fig. 5(a) shows the amperometric response of the prepared bioelectrode (Ab/NiO/ITO) towards the E. coli O157:H7 concentrations varying in the range 101 cells/mL to 107 cells/mL. It can be observed that the values of peak redox current decrease with increasing cell concentrations. This is due to the antibody-antigen immuno-complex formed on interaction of the E. coli O157:H7 cells with the antibody molecules on the matrix surface. The protein molecules present on the surface of E. coli O157:H7 cells bind with the antibody molecules present on the electrode surface. The immuno-complex being insulating in nature, obstructs the charge transfer from the surface of NiO matrix. Fig. 5(b) presents the Nyquist plots obtained from the electrochemical impedance spectroscopic (EIS) response of the prepared bioelectrode in the frequency range of 0.1–104 Hz. The equivalent Randles circuit shown in the inset of Fig. 5(b) has been used to model the EIS data. The circuit consists of the capacitance of dielectric layer at the electrode-electrolyte interface (Cdl) which is connected in a series with the electrolyte resistance (RS), the charge transfer resistance at the electrolyte-electrode interface (RCT) and the Warburg impedance of the bulk (ZW) [19]. It can be observed that the RCT (semicircular region of the Nyquist plot) increased with increasing concentration of E. coli O157:H7 cells that were made to interact with the antibody molecules
thin film matrix. 3.4. Optimization of incubation time for bacterial cells For biosensing response studies, E. coli O157:H7 cells of concentrations varying between 101 cells/mL and 107 cells/mL cells were incubated on the prepared bioelectrode (AB/NiO/ITO) for varying incubation time. The CV curves for an optimum cell concentration of 106 cells/mL with varying incubation time from 5 min (0.08 h) to 3 h are shown in Fig. 4. It can be observed that the change in peak oxidation current (Ipo) in the presence of bacterial cells with respect to Ab/NiO/ ITO depends upon the incubation time to a great extent. The variation of bioelectrode response with varying incubation time of cells for binding with the antibody molecules is represented in the inset of Fig. 4. Here, the bioelectrode response has been defined as the difference in peak oxidation current (Ipo (Cells)) upon interaction of the bacterial cells with the antibody molecules immobilized on the electrode surface with respect to the Ipo of antibody immobilized electrode in absence of the cells (Ipo (Ab)) i.e. 5
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Table 2 Analysis of E. coli O157:H7 concentrations estimated in real milk samples. E. coli concentration inoculated (cells/mL) E. coli concentration estimated (cells/mL)
1 × 101
1 × 102
1 × 103
1 × 104
1 × 105
1 × 106
1 × 107
1.03 × 101
0.95 × 102
1.01 × 103
1.09 × 104
1.07 × 105
0.98 × 106
0.91 × 107
present on the surface of NiO matrix. Hence, the results of EIS studies corroborate with the cyclic voltammetry analysis. The calibration curve corresponding to the amperometric response of the developed bioelectrode towards E. coli O157:H7 is illustrated in Fig. 6. A linear variation in the Ipo values is observed with increasing cell concentrations. The limit of detection (LOD) of the proposed biosensing mechanism turns out to be as low as 100 i.e. 1 cell/mL. The experiment was repeated multiple times and the calibration curve shows a variation of less than 3% which is within the acceptable limits. The response time of the Ab/NiO/ITO bioelectrode after incubation with the target cells was less than 5 s. Recent reports for electrochemical detection of E. coli O157:H7 have demonstrated a limit of detection of up to 12 CFU/mL using immunomagnetic separation and urease catalysis [28]; and a limit of detection of 48 CFU/mL using a self-assembled layer of gold nanoparticles [29]. However, the use of label, complex intermediate steps and several chemical reagents limit the application of such techniques in the practical field. In the present assay, the unique properties of NiO thin film not only accelerate charge transfer, but also provide an exceptional support to the antibody that considerably enhances the target capture efficiency to an extent that direct detection (without the requirement of a label) can be performed. As expected, the nanostructured NiO thin film facilitates effective and conformational loading of the antibody molecules on its surface and thus efficient binding with the target E. coli O157:H7 cells. Thus, a label-free and rapid biosensing mechanism has been successfully demonstrated in the present work that has a great deal of potential for being elemental in the development of field-deployable biosensor for foodborne pathogens.
O157:H7 cells ranging from 10 to 107 cells/mL were inoculated into milk samples from the growth media. Fig. 8 shows the cyclic voltammetric studies for the milk samples with varying E. coli O157:H7 concentrations along with a milk sample without E. coli O157:H7 as a control measurement (0 cells/mL). It can be observed from Fig. 8 that the value of peak oxidation current remained same as that of the Ab/ NiO/ITO in the case of the control sample (0 cells/mL) and decreased linearly with increasing concentration of E. coli O157:H7. The accuracy of the results was estimated by comparing the analysis of milk samples with the calibration curve drawn from the E. coli O157:H7 cells inoculated in PBS buffer (Fig. 6) and also by the plate count method. The results summarized in Table 2 show that the concentrations of E. coli O157:H7 estimated in milk samples deviated from the known concentrations within a range of 10%. Thus, the devised bioelectrode presents reliable practical application for the detection of E. coli O157:H7.
3.6. Selectivity, specificity and reproducibility of the bioelectrode
Acknowledgment
Selectivity of the proposed amperometric biosensor for E. coli O157:H7 has been demonstrated in Fig. 7(a) by testing the biosensor against non-target antigens that include the non-pathogenic E. coli ATCC #25922, S. aureus (bacterium with gram stain opposite to that of E. coli) and a mixed culture containing equal proportions of the three species. The cell concentration of each species was kept to be 105 cells/ mL. It can be clearly observed from the inset of Fig. 7(a) that the bioelectrode shows the maximum response towards the target strain of E. coli O157:H7 with negligible response towards other bacterial cells. In case of mixed culture, the response has decreased because of the reduced number of target species. Thus, the biosensor proves to be highly specific and selective towards the target bacterial strain that can be attributed to high binding affinity of the antibody with regard to its target antigen. To study the reproducibility of the bioelectrode, three different sets of bioelectrodes were prepared and their response was studied against E. coli O157:H7. The values of peak oxidation current obtained for each set of measurements for different concentrations of E. coli O157:H7 are shown in Fig. 7(b). It is evident that the response for any particular concentration of the cells varies within the ± 5% range. Thus, the sensing response of the proposed amperometric biosensor is found to be reproducible.
The authors are grateful to the Department of Physics and Astrophysics, University of Delhi for providing the characterization facilities. Financial support from the Department of Science & Technology (DST), Ministry of Science and Technology, Government of India is gratefully acknowledged.
5. Conclusion A label-free amperometric sensing platform based on NiO thin film has been successfully devised for rapid recognition of the target pathogenic Escherichia coli O157:H7. The senor exhibits a wide linear range of 107 to 101 cells/mL with a significantly low limit of detection i.e. 1 cell/mL. The biosensor has proved to be highly specific and selective towards the target species even in the samples comprising of multiple bacterial species (mixed culture). Results also support the versatility of the prepared biosensor for possible application in the fields of bio-defense, food security and medical diagnosis.
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[13] P. Arora, A. Sindhu, N. Dilbaghi, A. Chaudhury, Biosensors as innovative tools for the detection of food borne pathogens, Biosens. Bioelectron. 28 (1) (2011) 1–12 Oct. [14] Y. Wang, Z. Ye, Y. Ying, Y. Wang, Z. Ye, Y. Ying, New trends in impedimetric biosensors for the detection of foodborne pathogenic bacteria, Sensors 12 (3) (2012) 3449–3471 Mar. [15] P.R. Solanki, A. Kaushik, V.V. Agrawal, B.D. Malhotra, Nanostructured metal oxidebased biosensors, NPG Asia Mater 3 (1) (2011) 17–24 Jan. [16] N. Dhull, G. Kaur, V. Gupta, M. Tomar, Development of nanostructured nickel oxide thin film matrix by rf sputtering technique for the realization of efficient bioelectrode, Vacuum 158 (2018) 68–74 Dec. [17] S.K. Vashist, Comparison of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide based strategies to crosslink antibodies on amine-functionalized platforms for immunodiagnostic applications, Diagnostics 2 (3) (2012) 23–33. Aug. [18] Agilent Genomics: Tools - Bio Calculators, [Online]. Available https://www. genomics.agilent.com/biocalculators/calcODBacterial.jsp , Accessed date: 17 August 2018. [19] G. Kaur, M. Tomar, V. Gupta, Realization of a label-free electrochemical immunosensor for detection of low density lipoprotein using NiO thin film, Biosens. Bioelectron. 80 (2016) 294–299. [20] X.H. Xia, J.P. Tu, J. Zhang, X.L. Wang, W.K. Zhang, H. Huang, Electrochromic properties of porous NiO thin films prepared by a chemical bath deposition, Sol. Energy Mater. Sol. Cells 92 (6) (2008) 628–633. [21] H. Qiao, Z. Wei, H. Yang, L. Zhu, X. Yan, Preparation and characterization of NiO nanoparticles by anodic arc plasma method, J. Nanomater. (2009) 1–5, 2009.
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Full length article
Label-free amperometric biosensor for Escherichia coli O157:H7 detection a
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Nidhi Dhull , Gurpreet Kaur , Prateek Jain , Priyanka Mishra , Divya Singh , Lilly Ganju , ⁎ Vinay Guptaa, Monika Tomard, a
Department of Physics and Astrophysics, University of Delhi, Delhi, India Department of Biology, University of North Carolina at Chapel Hill, USA Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi, India d Department of Physics, Miranda House, University of Delhi, Delhi, India b c
A R T I C LE I N FO
A B S T R A C T
Keywords: E. coli O157:H7 Label-free Amperometric Nickel oxide
Foodborne pathogens pose a major threat of infectious life-threatening diseases all around the globe. Escherichia coli (E. coli) O157 is one such bacterial species that has caused several major outbreaks in the past. Advancement of a rapid sensing mechanism with high sensitivity is vital to avoid such epidemics by timely recognition of the infectious foodborne pathogens. The present work proposes the development of an amperometric biosensing platform for the detection of the pathogenic E. coli O157:H7. The developed biosensor works on the principle of antibody-antigen interactions, where the antibody is covalently bound to the surface of the nickel oxide thin film matrix prepared using sputtering technique. Accelerated charge transfer through NiO, an exceptional support provided to the antibody, and thus enhanced target capture efficiency leads to the direct detection of E. coli O157:H7 without the assistance of label. The electrochemical sensing results exhibit a wide linear range of 101 to 107 cells/mL and a low limit of detection of 100 i.e. 1 cell/mL along with high selectivity and specificity against other bacterial species.
1. Introduction One of the major risks to the food chain is posed by foodborne pathogens. A large number of infectious diseases caused by a wide range of pathogens have been enumerated till date that pose a serious threat of contamination. These infections have the tendency to breed through various environmental means such as air, water and food and can be highly communicable [1]. The concept of bioterrorism is not new and poses a serious threat to every country around the world. Various pathogenic microorganisms can be used as biological weapons by the terrorists to cause wide spread contaminations [2]. Escherichia coli, Salmonella typhimurium, Campylobacter jejuni, Legionella pneumophila and Staphylococcus aureus are a few bacterial strains that cause serious health hazards. Among these, Escherichia coli (E. coli) has been reported to cause large scale loss of life [3]. E. coli is a naturally occurring bacteria found in the intestinal tracts of human beings and other warm-blooded organisms to assist their bodies in synthesis of essential vitamins. However, not all strains of E. coli are non-pathogenic. E. coli O157 is a pathogenic strain that produces toxins having the capability to destruct the inner lining of the intestines and can cause illness ranging from stomach cramps and mild
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diarrhea to lethal complications like hemorrhagic colitis (bloody diarrhea), hemolytic uremic syndrome (HUS, leads to kidney failure), and thrombotic thrombocytopenic purpura (TTP, leads to cardiac and neurological manifestations) [4,5]. Major outbreaks of diseases instigated by E. coli O157 have been reported by the World Health Organization (WHO) recently in the most developed countries of the world such as UK, Germany and Canada where the source of the pathogen has been attributed to contaminated food or water [6]. Global Health Observatory (GHO) data of 2016 lists diarrheal diseases caused by communicable, maternal, perinatal and nutritional conditions as one of the top 10 causes of death world wide which accounts for 1.5 million deaths annually [7]. Among these, E. coli has a great share of 5.6%, high enough for it to make to the list of major infectious pathogens [8]. Such wide spread outbreaks have resulted in adverse socioeconomic effects. A high-throughput mechanism for bacterial detection is vital keeping in mind the exponential rate of growth of the bacterial cells. The recognition of foodborne pathogens in natural food resources, packaged foods, processing and assembling units, drinking water sources and hospitals persist to be based on the traditional techniques of cell culturing, evaluation of the cell morphology or their ability to
Corresponding author. E-mail address: [email protected] (M. Tomar).
https://doi.org/10.1016/j.apsusc.2019.143548 Received 28 February 2019; Received in revised form 18 July 2019; Accepted 30 July 2019 Available online 31 July 2019 0169-4332/ © 2019 Elsevier B.V. All rights reserved.
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bioassay strain of E. coli O157:H7 (ATCC #700728) was purchased from BCCM/LMG (Belgium-Europe). The cells were cultured in Luria Bertini (LB) broth and then reconstituted in Phosphate Buffer Saline (PBS) for electrochemical measurements. Cultured samples of S. aureus ATCC #25923 and E. coli ATCC #25922 (each with a concentration of 108 cells/mL) were obtained from Defence Institute of Physiological and Allied Research (DIPAS), a laboratory of the Defence Research and Development Organization (DRDO), India. Powdered form of EDC (N(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide) were acquired from Sigma-Aldrich. (3Aminopropyl) triethoxysilane (APTES) was procured from Alfa-Aesar.
grow. Plate counting, polymerase chain reaction (PCR), bioluminescence, fluorescent bacteriophage assay, chemiluminescence, enzymelinked immunosorbent assay (ELISA), etc. are few such widely used techniques that require lengthy enrichment procedure, isolation, morphological examination and large sample volumes to positively identify food pathogens [9,10]. Such techniques are reliable but labor-intensive and highly time-consuming to arrive at a quantitative conclusion. Lately, numerous configurations of biosensors have technologically advanced as more rapid alternatives to such traditional techniques for the identification of food borne pathogens. These are generally based on either optical system, piezoelectric platforms, pathogen specific DNA or to some extent, electrochemical methods. Among these, antibody based amperometric immunosensors have drawn significant attention because of their uncomplicated preparation steps, high-throughput, possibility of miniaturization, higher selectivity and specificity [11]. However, most of the biosensors for the recognition of bacterial species use labels such as green fluorescent protein or enzymes fused into antibodies to facilitate the detection. The additional steps of labelling may lead to a compromise in sensitivity of the antibody to the extent where they fail to distinguish between viable and nonviable cells [12]. Thus, development of a label-free sensing mechanism is very significant, in addition to the above mentioned attributes. Looking at the critical importance of timely and accurate detection of such pathogens, the detection platform requires to be free from complex fabrication process, apart from being rapid, highly sensitive and selective. Nanocomposite materials have been greatly exploited for the advancement of electrochemical biosensors not only because of their biocompatibility but also due to their tunable conduction characteristics and large effective surface area for adsorption of desired biomolecules [13,14]. Recently, there has been a substantial increase in the development of metal oxide thin film based amperometric biosensors due to their exceptional stability. Nickel oxide (NiO), in particular, is an appropriate material of matrix because of its outstanding charge transfer characteristics [15]. It has been reported to be used in chemical and biological sensors as a functional layer due to its exceptional chemical stability and charge conduction. The performance of a biosensor in terms of sensing response and stability largely depends on the choice of matrix. The chemical stability of NiO permits the surface modification for covalent binding of biomolecules that enhances the biosensing response characteristics along with a larger shelf-life. Thus, in the present work NiO has been chosen as the matrix in the form of rf sputtered thin film. ITO coated corning glass has been employed as the substrate as it is electrically conducting (can be directly used as the working electrode in the electrochemical studies) and low cost as compared to stable metals such as gold [16]. This paper reports an amperometric biosensing mechanism for label-free recognition of pathogenic E. coli O157:H7 using nanostructured NiO thin film. The biosensor exploits the notion of standard antibody-antigen interactions, antibody bound covalently to the surface of nanostructured NiO matrix. The interactions of the antibody with the matrix surface and with the E. coli O157:H7 cells have been confirmed using FTIR spectroscopy. Cyclic voltammetry technique has been exploited to study the amperometric response of developed bioelectrode against E. coli O157:H7 concentration varying between 107 cells/ mL and 101 cells/mL. The selectivity of the prepared biosensor has been tested against a gram-positive bacterium (gram stain opposite to that of E. coli) Staphylococcus aureus (S. aureus) and a non-pathogenic strain of E. coli.
2.2. Bacterial cell culturing and surface plating Frozen stocks of E. coli O157:H7 were maintained at a temperature of −80 °C in 50% glycerol. Cell culturing was carried out by reconstituting the cells in nutrient broth and also surface plating on Luria agar to observe the cell growth. The bacterial suspension was kept in an incubator and shaker at 37 °C for 24 h for enrichment. The concentration of the cells was then estimated using UV–Visible spectrophotometer (PerkinElmer Lambda 35). For biosensing measurements, the cells were centrifuged at 5000 rpm for 5 min and re-suspend in Phosphate Buffer Saline (PBS) of pH 7.5. A series of eight 10-fold serial dilutions varying in the range 108 cells/mL to 101 cells/mL was prepared from the fresh stock culture using PBS for testing with the prepared bioelectrode. For studying the selectivity and sensitivity of the proposed biosensing mechanism in the co-existence of a non-target bacterial species, a mixed culture containing equal amounts of each organism (E. coli O157:H7, S. aureus ATCC #25923 and E. coli ATCC #25922) from its respective stock culture were harvested by centrifuging at 5000 rpm for 5 min and resuspending in PBS. 2.3. Preparation of bioelectrode Rf magnetron sputtering technique was exploited to prepare a nanostructured NiO thin film matrix on a conducting ITO coated glass substrates (NiO/ITO) using previously optimized parameters [16]. The surface of the as-deposited NiO matrix was functionalized by hydroxylation using a mixed solution of hydrogen peroxide and liquid ammonia followed by silanization using 1% solution of APTES diluted in toluene. An optimized concentration of antibody (Ab) E. coli O157:H7 (1 μg/mL) was immobilized covalently on the functionalized NiO matrix surface using 0.4 M EDC and 0.1 M NHS as coupling agents. Functionalization of the matrix surface using APTES provided free amino groups on it. EDC being a carboxyl and amine-reactive crosslinker forms amide bonds with these amino groups present on the surface of NiO matrix and further binds to the carboxyl groups on the antibodies. NHS stabilized the unstable reactive intermediate resulting from the crosslinking reaction of EDC with the carboxyl groups on the antibodies [17]. Various concentrations of E. coli O157:H7 were drop casted uniformly on the electrode surface (Ab/NiO/ITO) and incubated at 37 °C to study the amperometric response of the developed bioelectrode. The volume of the Ab E. coli O157:H7 and the various concentrations of E. coli O157:H7 samples utilized in each measurement was kept constant at 20 μL. 2.4. Apparatus Gamry Interface 1000 Potentiostat was used to perform cyclic voltammetry (CV) measurements via the three-electrode set-up. The working electrode being the developed NiO thin film based bioelectrode (Ab/NiO/ITO), Ag wire enclosed in saturated KCl solution as the reference electrode and platinum foil as counter electrode. All the measurements were recorded 50 mM PBS solution comprising of 0.9% NaCl and 5 mM [Fe(CN)6]3−/4− as external mediator. The cyclic voltammetry studies were performed in the voltage range of −0.3 V to 0.8 V
2. Experimental 2.1. Materials and reagents E. coli O157 (1.B.250B), a mouse monoclonal antibody (Ab E. coli) raised against E. coli whole cells was procured from Santa Cruz Biotechnology, Inc. with a concentration of 100 μg/mL. A nontoxigenic 2
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Fig. 1. UV–Visible spectra of the freshly cultured E. coli O157:H7 cells. Inset shows growth of the bacterial colonies on the agar plate after 24 h of incubation.
Fig. 3. FTIR spectra of as prepared NiO thin film based bioelectrode for the recognition of E. coli O157:H7. Table 1 Assignment of peaks identified in the FTIR spectra. Peak positions (cm−1) NiO
Ab/NiO
E.coli/Ab/NiO
532 611 742 870
532 611 742 870
1100 1240
1100
503 611 742 890 1100
1294 1648 1660
Possible peak assignment
Stretching vibration of NieO bond [20] Shifted NieO band [22] Ni-O-H stretching bond [27] C=O stretching vibrations [21] NH2 wagging and twisting [24] Bending of SieO bond [23] Stretching of SieO bond [23] C-N bond stretching [25] Protein components of Amide III band [26] N-H bending of Amide II [25] Protein components of Amide I band [26]
Fig. 2. Cyclic voltammetry studies of the as prepared NiO thin film based bioelectrode for the recognition of E. coli O157:H7.
with a scan rate of 100 mV/s. PerkinElmer Lambda 35 UV/Vis Spectrometer was used to determine the concentration of E. coli O157:H7 cells in the freshly cultured stock. Fourier-transform Infrared (FTIR) spectroscopic analysis was carried with PerkinElmer FTIR Spectrometer Frontier to verify the successful immobilization of Ab E. coli O157:H7 and E. coli O157:H7 on the surface of the NiO matrix.
3. Results and discussion 3.1. UV–visible spectroscopic analysis In the present work, freshly cultured E. coli O157:H7 were reconstituted in PBS from the growth media for the electrochemical studies. Hence, to verify the concentration of viable cells in the PBS, UV–Visible spectroscopic analysis was performed and the same was confirmed by plating the sample on agar plate. The absorbance spectra of the freshly cultured stock of E. coli O157:H7 and a 10-fold dilution of the same in
Fig. 4. CV curves for E. coli O157:H7 with varying incubation time for the Ab/ NiO/ITO electrode. Inset shows the corresponding variation in the bioelectrode response.
3
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Fig. 6. Calibration curve for the response of Ab/NiO/ITO bioelectrode towards E. coli O157:H7. Inset shows variation in oxidation peaks with varying cell concentrations.
corresponding to the external mediator [Fe(CN)6]3−/4− arising due to transition between Fe2+ and Fe3+ states. The intense redox peaks of NiO/ITO indicate the high charge conduction characteristics of the NiO matrix. The peak oxidation current (Ipo) reduces significantly from 1.27 to 1.03 mA on covalent immobilization of the E. coli O157:H7 antibody over the NiO matrix (Ab/NiO/ITO). This suggests that the protein molecules act as a barrier to the conduction of the matrix by hindering the electron exchange through the working electrode [19].
3.3. FTIR spectroscopic analysis The NiO thin film was deposited on Si substrates for FTIR studies and same steps as defined above for antibody immobilization and E. coli O157:H7 adsorption were performed. The resulting transmission spectra for NiO thin film, Ab/NiO and E.coli/Ab/NiO in the wavenumber range of 400 cm−1 to 2500 cm−1 are represented in Fig. 3. The typical band due to OeH centered about 3420 cm−1 indicating moisture content in the structure was observed in each case, with greater intensity in case of Ab/NiO and E.coli/Ab/NiO. However, the band has not been shown here to highlight other essential bands. The absorption modes at 503 cm−1 and 611 cm−1, in case of bare NiO thin film may be attributed to the stretching vibrations of NieO and Ni-O-H bonds respectively [20] [21]. The surface modification of NiO due to immobilization of the E. coli O157:H7 antibodies might cause variation in intensity of the mode and also wavenumber shift which was evident for the NieO peak in the FTIR spectra of Ab/NiO and E.coli/Ab/NiO [22]. The absorption peak at 742 cm−1 may be attributed to C]O stretching vibrations occurring due to traces of atmospheric carbon [21]. The additional peaks at 890 cm−1 and 1100 cm−1 correspond to the bending and stretching of SieO bond respectively [23]. The peak occurring at 870 cm−1 after immobilization of E. coli O157:H7 antibody on the surface of NiO thin film may be assigned to NH2 wagging and twisting resulting from surface modification using APTES [24]. The peaks at 1240 cm−1 and 1648 cm−1 indicate the stretching of CeN bond and NeH bending of Amide II bond due to peptide linkages respectively [25]. After interaction of E.coli cells with the antibody molecules, protein components of amide III and amide I bands appeared at 1294 cm−1 and 1648 cm−1 respectively [26]. All the bands with possible peak assignment are summarized in Table 1. The assigned peaks match very well with the reports available indicating proper bonding of antibody E. coli O157:H7 and E. coli O157:H7 cells on the surface of NiO
Fig. 5. (a): Amperometric response of the prepared bioelectrode (Ab/NiO/ITO) towards E. coli O157:H7. (b): EIS studies of the prepared bioelectrode for the detection of E. coli O157:H7.
the UV–visible wavelength range of 250 to 1100 nm is shown in Fig. 1. The optical density of approximately 0.2 and 0.04 corresponding to the wavelength of 600 nm (OD600) estimated the cell concentration to be 108 cells/mL and 107 cells/mL respectively [18]. The cell concentrations tend to show maximum absorbance in the UV region around 300 nm. The absorbance of further dilutions was too low to distinguish from one another and hence, not represented. Colonies of E. coli O157:H7 bacteria could be clearly visualized on the agar plate after incubation of 24 h (Inset of Fig. 1). Thus, the analysis confirmed the presence of specified concentration of viable E. coli O157:H7 cells in the PBS solution.
3.2. Bioelectrode characterization The cyclic voltammetry (CV) curves of the prepared bioelectrode with the applied potential varying from −0.3 V to 0.8 V at each step can be observed from Fig. 2. The redox peaks of the ITO coated glass substrates are negligible as compared to the intense peaks of the NiO thin film (NiO/ITO) at the potential values of 0.2 V and 0.4 V. These can be attributed to the respective reduction and oxidation peaks 4
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Fig. 8. Amperometric response of the prepared bioelectrode (Ab/NiO/ITO) towards varying E. coli O157:H7 concentrations inoculated in real milk samples.
Bioelectrode Response (%) =
Ipo (Ab)–Ipo (Cells) × 100. Ipo (Ab)
It is evident from Fig. 4 that the bioelectrode yields limited response when incubated with the cells for 5 to 30 min since the time is insufficient for efficient interaction between the cells and the antibody. Most of the cells remain unbound and are removed from the surface in the intermediate washing steps. The response increased with further increasing the incubation time and maximum response of 26% was obtained when cells were left undisturbed on the bioelectrode for 1 h. Beyond that, the response starts decreasing again. This can be reasoned on the basis of high rate of bacterial growth in ambient temperature and media. With increasing incubation time, the number of cells becomes excess to the extent that individual cells are not allowed to interact with antibody terminations present on the surface. A biofilm of the E. coli O157:H7 cells is formed on the surface of the electrode that gets carried away when washed with buffer. Thus, an optimum incubation time of 1 h was kept constant for further studies to obtain a maximum sensing response.
Fig. 7. (a): Selectivity and specificity studies of the Ab/NiO/ITO bioelectrode towards E. coli O157:H7. Inset shows the bioelectrode response against the nontarget bacterial species. (b): Reproducibility studies of the Ab/NiO/ITO bioelectrode towards E. coli O157:H7.
3.5. Amperometric response of the bioelectrode Fig. 5(a) shows the amperometric response of the prepared bioelectrode (Ab/NiO/ITO) towards the E. coli O157:H7 concentrations varying in the range 101 cells/mL to 107 cells/mL. It can be observed that the values of peak redox current decrease with increasing cell concentrations. This is due to the antibody-antigen immuno-complex formed on interaction of the E. coli O157:H7 cells with the antibody molecules on the matrix surface. The protein molecules present on the surface of E. coli O157:H7 cells bind with the antibody molecules present on the electrode surface. The immuno-complex being insulating in nature, obstructs the charge transfer from the surface of NiO matrix. Fig. 5(b) presents the Nyquist plots obtained from the electrochemical impedance spectroscopic (EIS) response of the prepared bioelectrode in the frequency range of 0.1–104 Hz. The equivalent Randles circuit shown in the inset of Fig. 5(b) has been used to model the EIS data. The circuit consists of the capacitance of dielectric layer at the electrode-electrolyte interface (Cdl) which is connected in a series with the electrolyte resistance (RS), the charge transfer resistance at the electrolyte-electrode interface (RCT) and the Warburg impedance of the bulk (ZW) [19]. It can be observed that the RCT (semicircular region of the Nyquist plot) increased with increasing concentration of E. coli O157:H7 cells that were made to interact with the antibody molecules
thin film matrix. 3.4. Optimization of incubation time for bacterial cells For biosensing response studies, E. coli O157:H7 cells of concentrations varying between 101 cells/mL and 107 cells/mL cells were incubated on the prepared bioelectrode (AB/NiO/ITO) for varying incubation time. The CV curves for an optimum cell concentration of 106 cells/mL with varying incubation time from 5 min (0.08 h) to 3 h are shown in Fig. 4. It can be observed that the change in peak oxidation current (Ipo) in the presence of bacterial cells with respect to Ab/NiO/ ITO depends upon the incubation time to a great extent. The variation of bioelectrode response with varying incubation time of cells for binding with the antibody molecules is represented in the inset of Fig. 4. Here, the bioelectrode response has been defined as the difference in peak oxidation current (Ipo (Cells)) upon interaction of the bacterial cells with the antibody molecules immobilized on the electrode surface with respect to the Ipo of antibody immobilized electrode in absence of the cells (Ipo (Ab)) i.e. 5
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Table 2 Analysis of E. coli O157:H7 concentrations estimated in real milk samples. E. coli concentration inoculated (cells/mL) E. coli concentration estimated (cells/mL)
1 × 101
1 × 102
1 × 103
1 × 104
1 × 105
1 × 106
1 × 107
1.03 × 101
0.95 × 102
1.01 × 103
1.09 × 104
1.07 × 105
0.98 × 106
0.91 × 107
present on the surface of NiO matrix. Hence, the results of EIS studies corroborate with the cyclic voltammetry analysis. The calibration curve corresponding to the amperometric response of the developed bioelectrode towards E. coli O157:H7 is illustrated in Fig. 6. A linear variation in the Ipo values is observed with increasing cell concentrations. The limit of detection (LOD) of the proposed biosensing mechanism turns out to be as low as 100 i.e. 1 cell/mL. The experiment was repeated multiple times and the calibration curve shows a variation of less than 3% which is within the acceptable limits. The response time of the Ab/NiO/ITO bioelectrode after incubation with the target cells was less than 5 s. Recent reports for electrochemical detection of E. coli O157:H7 have demonstrated a limit of detection of up to 12 CFU/mL using immunomagnetic separation and urease catalysis [28]; and a limit of detection of 48 CFU/mL using a self-assembled layer of gold nanoparticles [29]. However, the use of label, complex intermediate steps and several chemical reagents limit the application of such techniques in the practical field. In the present assay, the unique properties of NiO thin film not only accelerate charge transfer, but also provide an exceptional support to the antibody that considerably enhances the target capture efficiency to an extent that direct detection (without the requirement of a label) can be performed. As expected, the nanostructured NiO thin film facilitates effective and conformational loading of the antibody molecules on its surface and thus efficient binding with the target E. coli O157:H7 cells. Thus, a label-free and rapid biosensing mechanism has been successfully demonstrated in the present work that has a great deal of potential for being elemental in the development of field-deployable biosensor for foodborne pathogens.
O157:H7 cells ranging from 10 to 107 cells/mL were inoculated into milk samples from the growth media. Fig. 8 shows the cyclic voltammetric studies for the milk samples with varying E. coli O157:H7 concentrations along with a milk sample without E. coli O157:H7 as a control measurement (0 cells/mL). It can be observed from Fig. 8 that the value of peak oxidation current remained same as that of the Ab/ NiO/ITO in the case of the control sample (0 cells/mL) and decreased linearly with increasing concentration of E. coli O157:H7. The accuracy of the results was estimated by comparing the analysis of milk samples with the calibration curve drawn from the E. coli O157:H7 cells inoculated in PBS buffer (Fig. 6) and also by the plate count method. The results summarized in Table 2 show that the concentrations of E. coli O157:H7 estimated in milk samples deviated from the known concentrations within a range of 10%. Thus, the devised bioelectrode presents reliable practical application for the detection of E. coli O157:H7.
3.6. Selectivity, specificity and reproducibility of the bioelectrode
Acknowledgment
Selectivity of the proposed amperometric biosensor for E. coli O157:H7 has been demonstrated in Fig. 7(a) by testing the biosensor against non-target antigens that include the non-pathogenic E. coli ATCC #25922, S. aureus (bacterium with gram stain opposite to that of E. coli) and a mixed culture containing equal proportions of the three species. The cell concentration of each species was kept to be 105 cells/ mL. It can be clearly observed from the inset of Fig. 7(a) that the bioelectrode shows the maximum response towards the target strain of E. coli O157:H7 with negligible response towards other bacterial cells. In case of mixed culture, the response has decreased because of the reduced number of target species. Thus, the biosensor proves to be highly specific and selective towards the target bacterial strain that can be attributed to high binding affinity of the antibody with regard to its target antigen. To study the reproducibility of the bioelectrode, three different sets of bioelectrodes were prepared and their response was studied against E. coli O157:H7. The values of peak oxidation current obtained for each set of measurements for different concentrations of E. coli O157:H7 are shown in Fig. 7(b). It is evident that the response for any particular concentration of the cells varies within the ± 5% range. Thus, the sensing response of the proposed amperometric biosensor is found to be reproducible.
The authors are grateful to the Department of Physics and Astrophysics, University of Delhi for providing the characterization facilities. Financial support from the Department of Science & Technology (DST), Ministry of Science and Technology, Government of India is gratefully acknowledged.
5. Conclusion A label-free amperometric sensing platform based on NiO thin film has been successfully devised for rapid recognition of the target pathogenic Escherichia coli O157:H7. The senor exhibits a wide linear range of 107 to 101 cells/mL with a significantly low limit of detection i.e. 1 cell/mL. The biosensor has proved to be highly specific and selective towards the target species even in the samples comprising of multiple bacterial species (mixed culture). Results also support the versatility of the prepared biosensor for possible application in the fields of bio-defense, food security and medical diagnosis.
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