Novel surface antigen based impedimetric immunosensor for detection of Salmonella typhimurium in water and juice samples

Author’s Accepted Manuscript Novel surface antigen based impedimetric immunosensor for detection of Salmonella typhimurium in water samples Ruchi Mutreja, Monu Jariyal, Preeti Pathania, Arunima Sharma, D.K. Sahoo, C. Raman Suri www.elsevier.com/locate/bios

PII: DOI: Reference:

S0956-5663(16)30509-7 http://dx.doi.org/10.1016/j.bios.2016.05.079 BIOS8769

To appear in: Biosensors and Bioelectronic Received date: 2 March 2016 Revised date: 5 May 2016 Accepted date: 23 May 2016 Cite this article as: Ruchi Mutreja, Monu Jariyal, Preeti Pathania, Arunima Sharma, D.K. Sahoo and C. Raman Suri, Novel surface antigen based impedimetric immunosensor for detection of Salmonella typhimurium in water s a m p l e s , Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.05.079 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Novel surface antigen based impedimetric immunosensor for detection of Salmonella typhimurium in water samples Ruchi Mutreja, Monu Jariyal, Preeti Pathania, Arunima Sharma, D.K. Sahoo and C. Raman Suri* CSIR-Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India *Corresponding author. Tel.: +911726665225. [email protected]

Abstract A specific surface antigen, OmpD has been reported first time as a surface biomarker in the development of selective and sensitive immunosensor for detecting Salmonella typhimurium species. The OmpD surface antigen extraction was done from Salmonella typhimurium serovars, under the optimized growth conditions for its expression. Anti-OmpD antibodies were generated and used as detector probe in immunoassay format on graphene-graphene oxide (G-GO) modified screen printed carbon electrodes. The water samples were spiked with standard Salmonella typhimurium cells, and detection was done by measuring the change in impedimetric response of developed immunosensor to know the concentration of serovar Salmonella typhimurium. The developed immunosensor was able to specifically detect S. typhimurium in spiked water samples with a sensitivity of 101 CFU mL-1, with high selectivity and very low cross-reactivity with other strains. This is the first report on the detection Salmonella typhimurum species using a specific biomarker, OmpD. The developed technique could be very useful for the detection of nontyphoidal Salmonellosis and is also important from an epidemiological point of view.

Key words: OmpD; graphene-graphene oxide nanocomposite; Immunosensor; Impedimetric; Salmonella typhimurium.

1. Introduction: Salmonella is a leading human foodborne pathogen (Poirier et al., 2007) which causes more deaths than any other known human foodborne pathogen such as E. Coli, Listeria, Vibrio, Yersinia etc. (Centers for Disease Control and Prevention, 2015). Salmonellosis, caused by

Salmonella may result in small outbreaks in the general population to large outbreaks in hospitals. According to the Centers for Disease Control and Prevention (CDC) food safety report, 1.2 million cases of Salmonellosis were reported in the United States, with 19,000 hospital cases and around 380 deaths annually (Scallan, 2011). Salmonella is Gram-negative, motile, non-spore forming facultative anaerobes belonging to Enterobacteriaceae family. Salmonella enterica species have 2,463 serovars (Popoff et al., 2000) that include both typhoidal and nontyphoidal strains of Salmonella. Typhoid fever caused by Salmonella typhi (S. typhi), characterized by fever and abdominal pain is human-restricted pathogen whereas Salmonella typhimurium (S. typhimurium), that causes acute gastroenteritis in humans, swine, cattle, and poultry is a broad host range pathogen (Pegues and Miller, 2009). If timely treatment is not provided, Salmonellosis can be potentially fatal (La Belle, 2008). Infection mainly occurs by the oral ingestion of contaminated water or food which potentially makes it a grave bioterrorism threat. Salmonella has been listed as a Category B bioterrorism agent by CDC, and a few studies also show the use of Salmonella as a bioterrorism agent in the past (Tucker, 1999). According to USDA food safety news 2015, Salmonella is the costliest foodborne pathogens accounting maximum deaths incurred from food poisoning. Since symptoms of Salmonellosis overlaps with a wide spectrum of other diseases and infection can result in diverse clinical manifestations, its detection is challenging. Therefore, there is an urgent need to develop fast, specific, sensitive as well as reliable immunosensors to detect Salmonella at pre-infectious levels. Many kits are available in the market for the detection of S. typhi (Bange et al., 2005; Kawano et al., 2007), but very few are available for the detection of S. typhimurium. A number of immunoassay methods, including enzyme-linked immunosorbent assays ELISA (Kumar et al., 2008), amperometric immunosensors (Singh et al., 2005), metallo-immunoassays (Dungchai et al., 2008), Faradic Impedimetric Immunosensor (Mantzila et al., 2008) have been used for the detection and diagnosis of Salmonella. Chai has reported detection of S. typhimurium using wireless biosensors on eggshells with detection limits of 1.6 × 102 CFU cm-2 (Chai et al., 2012). Wen showed the detection of S. typhimurium with the detection limit of 105 to 107 CFU mL-1 (Wen et al., 2013). Visualization of S. typhimurium cells using aptamer recognition by nanogold labelling (Yuan et al., 2014) with the detection limit of 7 CFU mL-1 has also been reported. Taitt and his colleague used array based immunosensor for the detection of S. typhimurium species with the detection limit of 104 CFU mL-1 (Taitt et al., 2004). Salmonella detection in milk was

done by Liebana and colleagues using electrochemical magneto-immunosensing (Liébana et al., 2009) with a detection limit of 5 × 103 and 7.5 × 103 CFU mL-1. Studies also shows the detection of S. typhimurium using monoclonal antibodies against S. typhimurium in electrochemical immunosensor format (Salam and Tothill, 2009) with the detection of 20 cells mL-1. La Belle deviced a methodology for detected S. typhimurium with detection limit of 500 CFU mL-1 using label-free electrochemical impedance spectroscopy (La Belle, 2008). However, most of the methods available for the detection of Salmonella are time consuming (Jenïkovâ et al., 2000), require higher pathogen concentration and show high cross-reactivity with other closely related food and water borne pathogens. The present study reports the screening of a novel specific outer membrane antigen (OmpD) which has been used first time for the detection of S. typhimurium in food and water samples. The screened biomarker shows very low cross reactivity with S. typhi and other closely related food and water borne pathogens. The outer membrane of S. typhimurium contains many proteins of almost similar molecular weight such as OmpC, OmpD, OmpF and OmpR. The expression of OmpC and OmpF are known to be regulated by OmpR (Nikaido, 2003). However, the expression of OmpD which is not regulated by OmpR, changes with pH and anaerobiosis (Ipinza et al., 2014). Also, it has been reported that the protein OmpD, a key target for the B cell antibody for nontyphoidal Salmonellosis (Gil-cruz et al., 2009) is one of the specific biomarker in the outer membrane of S. typhimurium and constitutes about half of the porin molecules per cell under optimum growth conditions. Several studies have been reported in the literature that showed the presence of OmpD in S. typhimurium to be used as a negative marker vaccine (Selke et al., 2007) but no reports show the use of OmpD as a specific biomarker for detection of S. typhimurium. In the present study, specific biomarker screening was done by running SDSPAGE. The whole cell lysate protein of Salmonella serovars has been previously reported to be evaluated using SDS-PAGE (Nakamura et al., 2002; Ngwai et al., 2005). With this novel OmpD antigen, we have developed a sensitive biosensing platform in an Electrochemical impedance spectroscopy format (Yang et al., 2004; Radke and Alocilja, 2005; Kim et al., 2013) using graphene-graphene oxide (G-GO) nanocomposite as a transducing element (Scheme 1). This new strategy provides both qualitative as well as quantitative information about the specific electrochemical event and is particularly advantageous in the field of biosensors to detect the

bimolecular interactions occurring at the surface (Salam and Tothill, 2009) with high degree of sensitivity and specificity. LOCATION OF SCHEME 1 2. Materials and methods 2.1. Materials 3-Cyclohexylaamino-1-propane sulfonic acid (CAPS), Tween 20, Amido black, Ammonium sulphate, Ammonium bicarbonate (NH4HCO3), Iodoacetamide, Formic acid, Nitric acid

(HNO3),

Poly-L-lysine,

Dithiothreitol

(DTT),

1-Ethyl-3-(3-dimethylaminopropyl)

carbodiimide (EDC), N-Hydroxysuccinimide (NHS), Skim milk, Trypsin (sequencing grade), Urea, Thiourea, Sodium dodecyl sulphate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS), Sarcosyl, Phenylmethylsulfonyl fluoride (PMSF), Brilliant Blue R250, Formic acid, Acetonitrile, Sodium carbonate, Sodium meta periodate, Silver nitrate (AgNO3), Potassium ferrocyanide, Tris-Cl were purchased from Sigma (USA). Nutrient agar (NA) and Nutrient broth (NB) were purchased from Himedia. Screen printed carbon electrodes (SPE) were products of CH Instruments (USA, Model TE 100). Polyvinylidene fluoride (PVDF) and nitrocellulose membranes were purchased from Advanced Microdevices Pvt. Ltd. (Ambala, India). HRP conjugated goat anti-rabbit IgG, 3,3',5,5'-Tetramethylbenzidine (TMB), Protein A sepharose column were procured from Bangalore Genei, India. Double distilled water was used for preparing all buffers. S. typhi was procured from Central Research Institute (CRI), Kasauli and all other strains such as S. typhimurium, Escherichia coli (E.coli) etc. were procured from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh. The microorganism was grown on nutrient agar and subsequently cultured in nutrient broth at 37ºC with constant shaking at 200 rpm for 18 h. 2.2. Extraction and identification of outer membrane protein Crude membrane Protein (CMP) was extracted from S. typhimurium, S. typhi and E. coli as reported by Krishnaswamy (Arockiasamy and Krishnaswamy, 2000) with modifications. Briefly, cells were grown at 37ºC with constant shaking at 200 rpm for 18 h, harvested at 8000 g for 15 min, washed with 0.85% NaCl twice, sonicated in 10 mM Tris-Cl for 30 min (optimized), and were centrifuged at 14000 rpm for 30 min. The supernatant obtained was ultracentrifuged at

35000 rpm for 1 h to separate the crude membrane protein from the cytosolic fraction. CMP was further solubilized in different solubilization buffers (Table ST1) at a concentration of 20 mg mL-1 containing 1 mM PMSF at 37ºC, 100 rpm overnight. Solubilized CMP was ultracentrifuged at 25000 rpm for 30 min. Following ultracentrifugation, the inner (supernatant), as well as pellet (outer membrane fraction) dissolved in 10 mM Tris-Cl, pH 7.4, was separated and analyzed by 15%

sodium

dodecyl

sulfate

polyacrylamide

gel

electrophoresis (SDS-PAGE).

Lipopolysaccharides (LPS) contamination was further checked by periodate staining, which oxidized glycols present in the LPS to aldehydes by periodate with little modifications (Zhu et al., 2012). Briefly, gel bands were fixed using 40 % ethanol, 5 % acetic acid for 1 hr, oxidized with 0.7% periodate, rinsed, stained with silver nitrate, developed using sodium carbonate and reaction was stopped using 1% acetic acid. For LC-MS/MS analysis, the putative protein bands were cut from the gel, washed with ddH2O, in-gel reduced, S-alkylated and digested with trypsin at 37°C overnight (Shevchenko et al., 2007). Briefly, dehydration of the gel pieces containing protein bands of interest was done with acetonitrile and then rehydrated with rehydration buffer (10 mM DTT in 100 mM NH4HCO3, incubated at 56ºC for 30 min, and then treated with 100 mM NH4HCO3 containing 100 mM iodoacetamide. Destaining was done with 1:1 mixture of acetonitrile/ammonium bicarbonate, incubated with 20 ng/μl trypsin in 10 mM NH4HCO3 containing 10% (v/v) acetonitrile, overnight at 37°C. Extraction of peptides was done using 1:2 (v/v) 5% Formic acid/acetonitrile, concentrated in speed vac. LC-MS/MS analysis was carried out using Agilent 6550 iFunnel Q-TOF LC/MS instrument equipped with Dual Agilent Jet Stream Electrospray Ionization (Dual AJS-ESI). Mass spectrometric data was analyzed using Mascot software and by searching at the National Centre for Biotechnology Information (NCBI) complete database. Nterminal analysis was also carried out using sequencer (Procise, Pulsed-Liquid PVDF Clc, 491). For this, putative Omp’s fraction dissolved in 10 mM Tris-Cl, pH 7.4 was separated on SDSPAGE and transferred to 0.45μ PVDF membranes. The transfer was done in western blot transfer buffer (10 mM CAPS, 10% methanol, 0.037% SDS, pH 11.0) at 350 mA for 35 min. Protein bands on the PVDF membranes were staining with 0.1 % amido black and then destained with 50% methanol. The stained membrane containing the protein band of interest was excised, rinsed with 50% methanol to remove the stain and was sequenced. The density of the protein was measured using ImageJ 1.49 software (Image processing and Analysis in Java).

2.3. Antibody generation and purification Young white Newzealand rabbit was immunized with OmpD protein band for generating specific polyclonal antibodies against OmpD using standard immunization protocol (Sharma et al., 2012). The gel band containing protein of interest was cut, homogenized in pestle motor in 10 mM phosphate buffer saline (PBS). Pre-immunized sera were collected and rabbits were immunized with approximately 300 μg of OmpD emulsified in Complete Freund's adjuvant and in Freund's incomplete adjuvant for subsequent 4 booster doses repeated on every 21 days post immunization and blood was collected on every 5th/ 7th day after booster dose. Blood was centrifuged at 10,000 rpm for 25 min, followed by decomplementation of sera at 56ºC for 20 min. 0.85% NaCl was added at a ratio of 1:2 (saline), followed by precipitating antibodies using 45% saturated ammonium sulphate at 4°C for 1 h, centrifuged at 10,000 rpm for 15 min, the pellet was resuspended in 1/10 (v/v) of 10 mM PBS and was extensively dialyzed against 10 mM PBS (pH 7.4) at 4°C. IgG fraction was further purified from Immunoglobulin fractions using protein A sepharose column. Elution of IgG was then done with 0.1 M glycine-HCl buffer (pH 2.5) followed by its neutralization immediately with 1 M Tris (pH 8.0) and dialyzed in PBS pH 7.4 at 4°C, and stored at -20oC with 0.05% sodium azide.

2.4. Specificity of OmpD with S. typhimurium cells For ELISA, Nunc plates were coated with 100 µL 0.01% poly-L-lysine (Sigma), and incubated overnight at 4ºC, washed with 10 mM PBS. 100 µL of cells in PBS was added, incubated for 2 h at RT, washed, blocked with 5% Skim milk, washed with PBST (PBS with 0.05% Tween 20). Anti-OmpD antibodies were diluted in PBSM (PBS with 0.1% skim milk) and 100 µL was added in each well, incubated for 1 h at 37ºC, washed with PBST, followed by addition of HRP conjugated goat anti-rabbit IgG diluted (1:20 000) in PBSM, incubated for 45 min at 37ºC. The plates were washed again, and 100 µL of TMB was added to each well, incubated at 37ºC for 15 min. The reaction was stopped by the addition of 50 µL of 2N H2SO4 and absorbance was measured at 450 nm using BioTek synergy 2 ELISA plate reader. Specificity was also checked at different antibody dilutions (1:2K to 1:64K) with different cell dilutions (107 to 101 CFU mL-1). For western blot study, proteins from SDS-PAGE were transferred to 0.45μ nitrocellulose membrane at 150 mA for 2 h at RT. Following transfer, the

membrane was washed with PBST and blocking was done with 5% skim milk in PBST for 2 h at RT, washed, incubated with primary antibody at 1:8000 dilution in PBSM for 2 h, washed, incubated with 1:20000 dilution of anti rabbit IgG HRP labeled for 45 min, developed with TMB. 2.5. Development of biosensing platform for detection of S. typhimurium cells A highly sensitive biosensing platform using graphene-graphene oxide (G-GO) nanocomposite embedded on screen printed carbon electrodes (SPE) was developed. SPE having 3 mm diameter carbon paste working electrode, a counter electrode and Ag/AgCl reference electrode were functionalized with G-GO nanocomposite as described by Sharma et al., 2013. The carboxilated (-COOH) G-GO was prepared by adding 10 mg of graphene in 100 ml of HNO3 (nitric acid) in a round bottom flask, followed by refluxing for 24 h at 70°C. The solution was filtered through 0.2 µm polycarbonate membrane filter. The obtained graphene nanosheets were vacuum dried and used for further experiment. An impedance spectrum of bare SPE was taken in 50 ul of 5 mM potassium ferrocyanide, washed with dH2O, and subsequently dropcasting 10 µL of G-GO (2.5 µg mL-1) on the working area of SPE. Modified electrodes were incubated for 30 min in oven, after which reduction in 10 mM PBS was done and subsequently reduction scans were taken. For molecular surface characterization, Field emission scanning electronic microscopic studies at an accelerating voltage of 10 kV (FESEM-EDX, Hitachi S4300 SE/N, Japan) were done. Further surface morphology was investigated Transmission Electron Microscope (TEM, JEOL 2100F, USA; operating at 200 kV). Spectroscopic analysis was carried out by Raman spectroscopy (InviaRenishaw, UK; Laser: 1.58 eV 785- HP-NIR) to carry out raman area scans on SPE (grating: 1200 lines mm-1). Selected area on SPE was located randomly by imaging with the optical microscope. Cyclic voltammetry (CV) scans were carried out in 5 mM potassium ferrocyanide solution using CH Instrument 600D electrochemical workstation. Reductive scans were recorded between 0 and -1.5 V at a scan rate of 50 mV s-1.

2.6. Electroimpedance spectroscopy based assay development For

the

functionalization

of

anti-OmpD

antibodies

on

rG-GO

SPE,

carbodiimide crosslinker Chemistry was used as per standard protocol ( Sharma et al., 2013). The carboxyl groups of rG-GO were activated by adding 10 μL of 50 mM of 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and 10 μL of 50 mM of N-Hydroxysuccinimide

(NHS), incubated for 1 h at 37ºC, washed with dH2O to remove excess EDC and NHS. Antibody was then drop cast at a concentration of 10 μg mL-1 to the activated rG-GO nanocomposite on the working area of SPE, incubated for 2 h at 37ºC, washed with dH2O to remove unbound antibody. Cells were then drop casted to the bound antibody for 10 min, washed with dH2O and impedimetric scans were taken. Cells were added starting from the lowest dilution of 101 to 106 CFU mL-1 and impedance was measured in 5 mM potassium ferrocyanide at each step of immunosensor development. In order to test the robustness of the methodology, all the experiments were performed with three different SPE.

3. Results and discussion 3.1 Screening and identification of specific biomarker for S. typhimurium To identify specific surface exposed, immunogenic protein for S. typhimurium, CMP was separated from cytosolic protein by ultracentrifugation for different pathogens and was confirmed by running 15% SDS-PAGE (Fig S1; Supplementary data). CMP extracted from S. typhimurium, S. typhi and E. coli was solubilized in buffers containing different detergents. The composition of different solubilisation buffers is given in Table ST1 (Supplementary data). The collected fractions were screened for the specific biomarker for S. typhimurium. The pellet containing outer membrane proteins collected after solubilization in buffer 2 shows few highly resolved protein bands as compared to the pellet of other solubilization buffers (Fig 1A). A significant protein band of 39.6 kDa was detected only in S. typhimurium and was found to be absent from S. typhi and E. coli (Fig 1B), clearly depicting the specificity of this isolated protein band for S. typhimurium. The specific protein band was further characterized by LC-MS/MS by trypsinization of peptides (Fig S2, Supplementary data), and was found to be OmpD reported to be present only in S. typhimurium (Singh et al., 1996). N-terminal sequencing of this 39.6 kDa protein band provided 10 mers of A-E-V-Y-N-K-D-G-N-K. Comparison of this N-terminal sequence with NCBI database shows 100% sequence identity with 1-10 amino acid residues of OmpD of S. typhimurium strain LT2. ImageJ software shows that the protein is present at a concentration of 5 μg/band (data not shown). When the purpose is to immunize the outer membrane protein from crude fraction, removal of LPS contamination is critical, since lipid A component of the LPS is toxic to the animal. In the present study, LPS contamination was checked in the outer membrane protein fractions of S. typhimurum and LPS was found to be

absent from the outer membrane protein fraction (to be immunized) as shown in Fig S3A (Supplementary data). Silver staining of the fraction also confirms the absence of other contaminating proteins (Fig S3B, Supplementary data). Therefore, Omp fraction is free of contaminating LPS and protein impurities. Hence, it can be used for immunization to generate specific antibodies. LOCATION FIGURE 1 3.2. Antibody generation and binding assay Significantly high titers of anti-OmpD antisera were observed for rabbit immunized against OmpD after the fourth booster dose of the immunogen as shown in Fig S4, Supplementary data. The serum was purified using protein A sepharose column to obtain specific anti-OmpD antibodies. Antibody concentration was determined by taking absorbance at 280 nm (~4 mg mL-1). The generated antibodies show very good specificity towards S. typhimurium, showing very less percentage cross-reactivity towards S. typhi cells. Fig 2A presents the crossreactivity pattern of OmpD against other enteric pathogens viz. S. typhi, E. coli, S. paratyphi, K. pneumonia, Bacillus at different antibody dilutions, clearly indicating very low cross-reactivity because of the presence of OmpD only in S. typhimurium (Singh et al., 1996). Cross-reactivity was also checked at different cell dilution (Fig 2B) clearly indicating the high specificity of generated antibodies towards S. typhimurium and detection upto 101 CFU mL-1 is possible. Cross-reactivity analyzed by western blot (Fig S3C, Supplementary data) also indicates its absence in S. typhi (Ipinza et al., 2014).

LOCATION FIGURE 2

3.3. rG-GO functionalized SPE immunosensor A highly sensitive biosensing platform using reduced graphene-graphene oxide (rG-GO) nanocomposite embedded on SPE was developed. Structural aspects of rG-GO functionalized SPE immunosensor were investigated by using FESEM, TEM, and Raman spectroscopy for confirming the deposition of rG-GO on SPE’s (Fig S5 Supplementary data). The sheet-like structure of the graphene was preserved after chemical modification as confirmed by FESEM and TEM (Fig S5A, B Supplementary data). Further, first-order Raman scattering (D and G peaks) were observed around 1350 cm-1 and 1600 cm-1 respectively in Raman spectra which

clearly shows a shift in the G and D bands (Fig S5C, Supplementary data). The shift in the D peak can be attributed to the presence of defects formed due to the reduction of G-GO nanocomposite. Also, the number of graphene layers can be depicted from the width and shape of 2D peak which shows a decrease after reduction of G-GO nanocomposite.

3.4. Electroimpedance spectroscopy based immunosensor development Electrochemical impedance spectroscopy (EIS) is one of the very sensitive techniques used for analyzing surface modifications on the transducers devices and also for detecting bimolecular interactions occurring at the surface/electrolyte interface. The appearance of a semicircle at higher frequencies (used to compute charge transfer resistance (Rct) and a straight line at relatively low frequencies in impedance spectrum generally correspond to the charge transfer and diffusion processes, respectively. A low value of Rct commonly indicates the fast rate of charge transfer between redox probe and electrode. Fig 3A shows the impedance spectra recorded during different development stages of immunosensor and corresponding CV scans are shown in Fig S6 (Supplementary data). The values of Rct, Rs, CdL were deduced by modelling and fitting the obtained impedance data into Randle’s equivalent circuit (Fig 3A, inset) during different stages of immunosensor development. The initial modification of the bare SPE electrode with rGO resulted in an enhanced conductance value as indicated by a lower Rct. The rGO are well-known as the conducting tunnel enabling an improved kinetics of charge transfer between the electroactive surface and the redox probe whereas Rct values show an increase after the immobilization of anti-OmpD antibody and successive exposure of the bioelectrode to the S. typhimurium cells at increasing concentration levels. The observed Nyquist plots for the tested cells at different concentrations are shown in Fig 3B. A linear increase in the Rct value as a function of analyte concentration (Fig 3B, inset) can be attributed to the presence of additional kinetic barrier molecules (cells) as well as an increasing steric hinderance which limits the access of the redox couple to the electrode surface. The proposed Ab/rGO/SPE immunosensor is capable of detecting 101 S. typhimurium CFU mL-1. We observed a significant increase in impedance with the increase in the number of cells. The experiment was repeated with three times with three different SPE and similarity in the impedance spectra at a different dilution of the cell was observed. The similarity in the plots proved the uniformity in the sensor’s response. LOCATION FIGURE 3

To demonstrate the applicability of the proposed immunosensor to real samples analysis, recovery studies on spiked lichi and orange juices were also performed. The Rct values obtained were then fitted to the calibration curve, in order to calculate the recovered cells from the spiked samples. After fitting into standard curve, the developed immunosensor was able to specifically detect S. typhimurium in spiked water samples with a sensitivity of 101 CFU mL-1 and 1.04x101 CFU mL-1 and 1.07x101 CFU mL-1 in lichi and orange juices respectively (Fig 4A, 4B). Comparison of the performance of the developed immunosensor with those of the available immunosensors indicates that the developed immunosensor is more sensitive and specific towards the detection of S. typhimurium (Dastider et al., 2015; Fei et al., 2015).

LOCATION FIGURE 4

3.5. Specificity of the developed immunosensor The Ab/rGO/SPE Immunosensor proposed in this study has also been tested for its specificity by conducting some negative control experiments with S. typhi, E.coli, K. pneumoniea, S. paratyphi and Bacillus species. These ancillary experiments were conducted in an identical manner as with those for the S. typhimurium cells. Cross-reactivity studies were carried out at 105 CFU mL-1. The results of these experiments are shown in Fig 5A. The computed Rct values of the immunosensor during the above non-specific tests differed only insignificantly from the baseline value (Fig 5B), thereby confirming the high specificity of the present immunosensing method toward the target S. typhimurium cells. In order to observe the change in impedance with increase in cell no. for non specific cells, S. typhi concentration was varied from 101 to 105 CFU mL-1 (Fig S7, Supplementary data) and not much change (insignificant) in impedance was observed. LOCATION FIGURE 5

Conclusion OmpD has been used for the first time in the detection of S. typhimurium. In this study, we have screened OmpD as a specific biomarker from SDS-PAGE and detected S. typhimurium

using antibodies against this specific surface antigen in an electrochemical impedance spectroscopy immunoassay format with rG-GO modified SPE as a sensitive biosensing platform. The developed immunoassay format shows the detection limit of ~10 CFU mL-1 in spiked water samples which is highly selective, sensitive and shows very less cross-reactivity with S. typhi and other pathogens. Further, the developed immunosensor was able to specifically detect S. typhimurium in spiked juices samples (lichi and orange) with a limit of detection 1.04x101 CFU mL-1 and 1.07x101 CFU mL-1 respectively. No pre-enrichment step is required before sample analysis. The developed technique could be useful for the detection of non-typhoidal diseases and is important from the epidemiologic point of view.

Acknowledgements The

authors

thank

Department

of

Biotechnology,

India

(Grant

No.

BT/IN/FINNISH/09/CRS/2013) for providing the financial support for this project. R.M is thankful to CSIR-UGC, India for Sr. Research fellowship. Authors also thank Dr. Priyanka Sharma and Dr. Neeraj Maheshwari for their kind technical support.

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Scheme 1: Schematic representation of S. typhimurium cells detection using specific OmpD antibody on rG-GO modified SPE.

Fig 1: 15% SDS-PAGE. (A) showing outer membrane proteins fraction comparision between S. typhi and S. typhimurium. Lane 1: S. typhi Omp fraction in 4th solubilization buffer, Lane 2: S. typhimurium Omp fraction in 4th solubilization buffer pellet, Lane 4: S. typhi Omp fraction in 1st solubilization buffer pellet, Lane 5: S. typhimurium Omp fraction in 1st solubilization buffer pellet, Lane 7: Marker, Lane 8: S. typhi Omp fraction in 2nd solubilization buffer pellet, Lane 9: S. typhimurium Omp fraction in 2nd solubilization buffer pellet, (B) showing the presence of OmpD only in S. typhimurium. Lane 1: S. typhimurium Omp fraction in 2nd solubilizaton buffer pellet, Lane 3: Marker, Lane 5: E.coli Omp fraction in 2nd solubilization buffer pellet, Lane 7: S. typhi Omp fraction in 2nd solubilization buffer pellet. Fig 2: Cross-reactivity studies of anti-OmpD antibody with different pathogens (107 CFU mL-1) at varying antibody dilutions (A) at different cell concentration with 1:4000 dilution of antibody (B). Fig 3: Impedance spectra on SPE surface using randles model. (A) An optimum concentration of GO (2.5 µg mL-1) was drop-casted on SPE, antibody was attached to the rG-GO by EDC/NHS chemistry, inset shows Randle’s equivalent circuit. (B) Impedance spectra on SPE surface that correspond to specific detection of S.typhimurium, inset shows increase in Rct with increase in cell concentration. Fig 4: Impedance spectra on SPE surface for S. typhimurium detection in spiked lichi juice (A), spiked orange juice (B). Inset shows the recovery results for the detection of S. typhimurium. Fig 5: Specificity of developed immunosensor for the detection of S. typhimurium by impedance spectra (A). Rct values (B).

Highlights     

A specific surface antigen, OmpD is reported first time as a surface biomarker for detecting Salmonella typhimurium species. The OmpD extraction was done under the optimised growth conditions. Anti-OmpD antibodies were generated, and were used as detector probe Graphene-graphene oxide (G-GO) nanocomposites modified screen printed carbon electrodes were used for the developed immunosensor. The immunosensor was able to specifically detect S. typhimurium in standard water and juices samples with limit of detection upto 10 CFU mL-1.

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Scheme1