- Email: [email protected]
Electrochemical piezoelectric reusable immunosensor for aflatoxin B1 detection
Accepted Manuscript Title: Electrochemical piezoelectric reusable immunosensor for aflatoxin B1 detection Author: Ruchika Chauhan Jay Singh Pratima R. Solanki T. Basu Richard O’Kennedy B.D. Malhotra PII: DOI: Reference:
S1369-703X(15)30010-3 http://dx.doi.org/doi:10.1016/j.bej.2015.07.002 BEJ 6243
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
4-3-2015 3-6-2015 4-7-2015
Please cite this article as: Ruchika Chauhan, Jay Singh, Pratima R.Solanki, T.Basu, Richard O’Kennedy, B.D.Malhotra, Electrochemical piezoelectric reusable immunosensor for aflatoxin B1 detection, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2015.07.002 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 proof before it is published in its final 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.
Electrochemical piezoelectric reusable immunosensor for AflatoxinB1 detection Ruchika Chauhan,1a Jay Singh,2b Pratima R. Solanki,3c T. Basu,4a* Richard O’Kennedy,d and B.D. Malhotra6e a
b
Amity Institute of Nanotechnology, Amity University Uttar Pradesh, NOIDA India
Department of Applied Chemistry & Polymer Technology, Delhi Technological University, Delhi-110042, India
c
Special Centre for Nano Sciences , Jawaharlal Nehru University, New Delhi-110067,India d
Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin 9,Ireland
e
Department of Biotechnology, Delhi Technological University, Delhi-110042 , India Email address: [email protected] , [email protected], 3
[email protected], [email protected], [email protected] 6
[email protected]
_________________________________________________________________________ *Corresponding author Email: [email protected] Highlights
•
Synthesis of gold coated iron oxide core shell nanoparticles (Au-Fe3O4 NPs).
•
Synthesis of antibody conjugates with nanoparticles (r-IgG-Cys-Au-Fe3O4 conjugate).
•
Iron oxide core shell are utilize for detection of aflatoxin B1
•
Fabricated reusable EQCM immunosensor.
•
Regeneration upto 15-16 times with negligible loss in activity.
Abstract A competitive, reusable, reliable and sensitive immunosensor based on electrochemical quartz crystal microbalance (EQCM) was developed for the detection of aflatoxin B1 (AFB1). The sensing platform was developed using a primary monoclonal anti-aflatoxin antibody (anti AFB1) covalently immobilized on a monolayer of 4- amino thiophenol self assembled on a gold coated quartz crystal electrode (anti AFB1/4-ATP/Au). The reusability was achieved by a sandwiched system using secondary rabbit-immunoglobulin antibodies (r-IgGs) tagged with core shell of gold coated iron oxide nanoparticles (r-IgG-Au-Fe3O4), which can be used to regenerate the bioelectrode. A competitive mode was employed, between free and coated AFB1 for fixed concentration of nanoparticle conjugate (r-IgG-Au-Fe3O4). After the competitive interaction, immunoelectrode was washed with phosphate saline buffer (PBS) at pH 7.4 and examined by electrochemical quartz crystal microbalance-cyclic voltammetry (EQCM-CV). Under the optimized conditions, the fabricated immunosensor can be used to detect AFB1 quantitatively with a linear range of 0.05 to 5 ngmL-1. For reliability, the fabricated immuno sensor was tested using cereal samples spiked with different concentrations of AFB1. In addition, this immuno electrode displays good reproducibility, and storage stability. The above immunosensor was regenerated with negligible loss in activity through removal of the r-IgG-Au-Fe3O4 conjugate using a strong magnet. It is shown that the r-IgG-Au-Fe3O4/AFB1/BSA/aAFB1/4-ATP/Au
immuno sensor is a new approach for developing sensitive reliable and reusable electrochemical piezoelectric immunosensors. Key words: Aflatoxin B1, immunosensor, EQCM, Fe3O4 NPs, Au-Fe3O4 NPs 1. Introduction Aflatoxin B1 (AFB1) is one of the most important mycotoxin, produced by strains of Aspergillus flavus and Aspergillus parasiticus and is a carcinogen. It is commonly present in various foods (e.g., corn, sorghum, peanuts, fruits, dried fruits, cocoa and spices) and causes human health disorders such as hepatocellular carcinoma, Reye’s syndrome and chronic hepatitis. The European Commission has set strict limit for the maximum level (2 µgkg−1) of AFB1 that can be allowed in food products [1-4]. Compared to conventional techniques [5-7] biosensors are a valuable alternative that can be used to quantify and detect the desired toxin molecules [8-11]. Some of the biosensing techniques that have been used for AFB1 detection include electrochemical biosensors [12-14], surface plasmon resonance based platforms [15,16], fluorescence based biosensors [17,18], and quartz crystal (piezoelectric) microbalance based sensors [19,20]. Among the signal detection techniques, quartz crystal microbalance (QCM) based detection systems are considered to be the most promising due to their affordable cost, real-time detection capability, label free format, compatibility with miniaturization, portability and high sensitivity [21]. The piezoelectric immunosensors are known to rely on interaction of an antibody with the antigen on the probe surface [22, 23]. However, self assembled monolayers (SAMs) have been predicted to play a potentially valuable role in the development of immunosensing surfaces in biosensing devices [24, 25].
Since AFB1 is a small molecule of low molecular weight its direct detection with a satisfactory detection limit and sensitivity is very difficult using QCM [26]. It was reported that small molecules such as TNT, ochratoxin A and aflatoxins could be detected with high sensitivity using a competitive immunoassay format with a signal enhancing label [27, 28]. This label may comprise of an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP) [29-30], a nano label (e.g. gold NPs) [31] or nanogold-attached carbon nanotubes [32]. It was found that some of these bionano labels are difficult to isolate and hence may cause interference during experiments. Several researchers are currently working on the reusability or regeneration of sensing elements functionalized with various biomolecules [33, 34]. To overcome these problem magnetic nanoparticles (MNPs) may be used for the signal amplification, rapid separation and purification of bioconjugates using an external magnet [35-37]. Iron oxide nanoparticles are being used in biomedical application due to their biocompatibility and magnetic properties [38,39]. Magnetic separation and concentration using nanoparticles was studied by Wu et. al. using antigen-modified magnetic nanoparticles as immunosensing probes and fluorescent bio labels [40]. Some of the current challenges include the synthesis of MNPs with well controlled sizes, composition, and surface properties [41, 42]. These problems may perhaps be resolved by chemically coating these magnetic nanoparticles with peptides [43], polymers [44, 45], silanes [46, 47] metal oxides [48] and metals [49]. In this context, gold coated magnetic nanoparticles have been predicted to be promising [50, 51]. The core shell of AuNPs along with Fe3O4 NPs may be used for ultrasensitive detection of analytes [52] to obtain enhanced electron transfer, across the interface [53, 54]. Furthermore, the core–shell of AuFe3O4 NPs can be used to improve kinetics of the immuno-reactions and increase binding [55].
In our previous study, we have observed that QCM measurement becomes more efficient when combined with electrochemistry. We have received wider linear range, low detection limit and higher sensitivity with EQCM technique rather than QCM [56]. Here we report results of the studies relating to development of a reusable, label free, competitive sensitive sandwich-type electrochemical piezoelectric quartz crystal (EQCM) immunosensor based on 4-amino thiophenol self assembled monolayers to detect AFB1. The secondary rIgG- Au-Fe3O4 NPs conjugate structure has been synthesized to utilize it as a signal enhancer and sensor regeneration through external magnet. The constructed biosensor yields about a tenfold lower detection limit than the previously reported competitive sandwich immunosensor in real samples and can be regenerated with negligible loss of activity using a strong magnet. 2. Experimental Methods 2.1. Chemicals and reagents Monoclonal anti-aflatoxin B1 (aAFB1) antibodies, aflatoxin B1 (AFB1), bovin serum albumin (BSA), polyclonal IgG antibodies from rabbit (r-IgG), 4 amino thio phenol (4-ATP), Nethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), ferrous chloride hexahydrate (FeCl2.6H2O), ferric chloride tetrahydrate (FeCl3.4H2O), sodium hydroxide (NaOH) and chloroauric acid (HAuCl4.H2O), were procured from Sigma-Aldrich. All reagents were of analytical grade and used without further purification. De-ionized water (resistance ~ 18MΩ cm) from Millipore direct Q3 purification system, was used for the preparation of desired aqueous solutions. The gold coated (diameter: 6.7 mm) quartz resonator (AT cut quartz crystal, 13.7 mm dia, 6 MHz) was procured from Autolab, the Netherlands. A nylon membrane used during the extraction of aflatoxins extract from the corn flakes, was obtained from Whatman 2.2. Solution preparation
Anti-AFB1antibody (1 mgmL-1) solution was prepared in 50 mM phosphate buffer (PBS), 50 mM, pH 7.4 and 0.15 M NaN3 was used as a preservative. r-IgG antibody (2 mgmL-1) solution was prepared in 50 mM PBS (50 mM, pH 7.4). The stock solution of AFB1 was prepared in PBS (50 mM, pH 7.4) with 10% (v/v) methanol and dispensed in different working concentrations and stored at -20 °C. A solution of bovine serum albumin (BSA, 1 mgmL-1) was prepared in PBS (50 mM, pH 7.0) and used for blocking for the nonspecific binding sites. 2.3. Pretreatment of quartz crystals The quartz crystals were immersed in 1M NaOH for 5 min and 1M HCl for 2 min in sequence. Later, freshly prepared piranha solution {1:3 (30% v/v) H2O2–H2SO4} was dropped on the gold surface for 2 min, taking special care to avoid contamination of the electrode leads. The quartz crystals were rinsed twice with deionized water followed by ethanol, and dried in a stream of nitrogen after each pretreatment and then the initial resonance frequency (F0) was recorded. After cleaning, the quartz crystal was ready for surface modification and antibody immobilization. 2.4. Synthesis of Fe3O4 and Au-coated Fe3O4 Fe3O4 NPs were synthesized simply by the co-precipitation method reported earlier [57] with some modification. Solutions of 0.07 M FeCl3·6H2O and 0.04 M FeCl2.4H2O (2:1, w/w ratio) were dissolved in 25 mL deionized water and then this mixture was added drop wise to 100 mL solution of 0.15 mM NaOH with stirring under a N2 atmosphere at room temperature. A black precipitate of Fe3O4 NPs was obtained. This precipitate was dissolved in 20 mL citrate buffer (1.6 gm citric acid and 0.8 gm tri-sodium citrate) to stabilize ferrofluid in solution at pH of 6.3.
The Au-Fe3O4 core shell NPs were prepared using 3 mL of synthesized colloidal Fe3O4 nano-suspension (0.1 M), boiled with 25 mL of ultra pure water under vigorous stirring conditions. Then 3 mL of 0.2 mM HAuCl4 was added, followed by the addition of 3mL of 10 mM tri-sodium citrate and the reaction mixture was kept boiling and stirred for 15 min till the color of the solution turned from black to red. The gold coated Fe3O4 NPs (Au- Fe3O4 NPs) solution was allowed to cool and was stored in a dark glass bottle at 4 ̊ C before use. 2.5. Synthesis of r-IgG-Au- Fe3O4 The synthesized Au-Fe3O4 nano- suspension was treated with 10-3 M aqueous solution of cysteamine hydrochloride in a 1:15 volume ratio for 12 h at 25 ̊C. The cysteamine functionalized Au-Fe3O4 NPs (Au-Fe3O4) were separated and purified by centrifugation at 10,000 rpm for 10 min. The purification and centrifugation processes were repeated 4-5 times for removing nonbonded cysteamine. Then cysteamine functionalized Au-Fe3O4 core shell NPs were re-dispersed in PBS (50 mM, pH 7.0) solution. Cysteamine forms a self assembled layer on Au-Fe3O4 NPs which provides NH2 groups to bind with COOH functional groups of the polyclonal rabbitimmunoglobulin antibodies (r-IgG) during immobilization. The r-IgG antibodies were mixed with Au-Fe3O4 solution in 1:3 (v/v ratio) [58], followed by addition of 0.2M EDC and 0.05M NHS for the activation of –COOH groups present in the antibody. Furthermore, to block the nonspecific sites on the r-IgG-Au-Fe3O4 conjugates, 100µL BSA (1mgmL-1) was added and incubated for 2h at 25 ̊C. The mixture was centrifuged at 10,000 rpm for 10 min and washed 4-5 times with deionized water. Finally, the r-IgG-Au-Fe3O4 conjugate was re- suspended in PBS of (50 mM, pH 7.4) and stored at 4 ̊C until use. Scheme S1 represents the formation of r-IgG-AuFe3O4 conjugate. [See Supplementary Scheme S1] 2.6. Fabrication of AFB1/BSA/aAFB1/4-ATP/Au immuno sensor
Pretreated quartz crystal was immersed in 2 mM solution of 4-ATP in ethanol for 24h at 25 ̊C for SAM formation. However, a uniform and steady 4-ATP film was obtained [56]. The crystal was subsequently washed with ethanol followed by rinsing with water to remove any unbound ATP molecules. 10µL of 40 µgmL-1 of monoclonal anti aflatoxin B1 (aAFB1), activated with 0.2 M EDC and 0.05M NHS for about 2h, was spread over the electrode and incubated overnight at 4 °C for the amide bond formation between aAFB1 and 4-ATP. In this study, an optimized concentration of 40 µgmL-1 of aAFB1 was used. The non-specific sites of fabricated immuno electrodes were blocked with BSA in PBS of (50 mM, pH 7.0) (1mgmL-1). These fabricated BSA/aAFB1/4-ATP/Au immuno electrodes were exposed to saturated concentration of AFB1 (5ngmL-1) for 35 min at 25 °C. 2.7. Detection of AFB1 The fabricated AFB1/BSA/aAFB1/4-ATP/Au immuno electrode was allowed to compete with free AFB1 with a fixed concentration of secondary antibody conjugate. The immuno electrode was
exposed to 3 mL solution
of
5 mM [Fe(CN)6]3−/4− mediator in
phosphate buffer saline (50 mM, pH 7.4, 0.9% (w/v) NaCl) containing 10 µL of saturated concentration (30 µgmL-1) of r-IgG-Au- Fe3O4 conjugate and 10 µL of AFB1 standards in PBS buffer (0–5 ngmL-1). To determine the optimum concentration of r-IgG antibody conjugate, AFB1/BSA/aAFB1/4-ATP/Au immuno electrode was allowed to interact with different concentrations of r-IgG-Au- Fe3O4 conjugate i.e. 10-50 µg/mL and the corresponding response current was observed with EQCM-CV. During the competition process, secondary antibodies easily access to free AFB1, while rest of the r-IgG-Au- Fe3O4 conjugates form a sandwiched structure with the coated AFB1. Therefore, with increased concentrations of free AFB1, the availability of r-IgG-Au-Fe3O4 conjugate on coated AFB1 gradually decreases resulting in a
decrease in response current. After competition, the crystal was rinsed with PB (containing 0.1% (w/v) Tween-20) dried under a N2 stream. All experiments were performed in triplicate and the experimental temperature was controlled at 25 ̊C. 2.8. Pretreatment and analysis of cereal samples Cereal samples (corn-flakes) were spiked with AFB1 and then were crushed to powder using a hand-held blender. 2g of powdered cereals were added to methanol: water (7:3, v/v) solution on a sonication bath for 45 min. The extract was centrifuged for 7 min at 5000 rpm to remove the solids. The supernatants were collected and allowed to evaporate to dryness under nitrogen at 25°C. Evaporation was necessary to avoid inhibition of the antibody-antigen binding caused by methanol. The residues were re-suspended in 5 mL PBS and filtered through 0.45 µm Whatman nylon membranes [59]. Finally, the extract was spiked with the concentrations of 0.05, 2 and 5 ng mL-1 of aAFB1. 2.9. Instrumentation The resonant frequency of quartz crystal and electrochemical studies was monitored using
a
Autolab
Potentiostat/Glavanostat
Model
AUT83945
(PGSTAT302N).
The
electrochemical quartz crystal cyclic voltammetric (CV) studies were carried out in a three electrode cell using a modified gold coated quartz crystal as the working electrode, gold wire as the auxiliary electrode, and saturated Ag/AgCl as the reference electrode in PBS, (50 mM, pH 7.4, 0.9% (w/v) NaCl) containing 5 mM [Fe(CN)6]3−/4− as a redox species. The Au-Fe3O4 core shell magnetic nano particles were characterized by scanning electron microscopy (ZEISS EVO18), vibrating sample magnetometry (VSM) (Microsense, ADE-Model EV9), transmission electron microscopy (JEOL JEM (Model 1200F) and X-ray diffractometry ( Bruker AXS, XRD). The structural and surface morphological characterizations of 4-ATP/Au, aAFB1/4-
ATP/Au, BSA/aAFB1/4-ATP/Au, and the AFB1/BSA/aAFB1/4-ATP/Au electrodes were carried out using Fourier transform Infrared spectroscopy (FT-IR, Perkin-Elmer, and Model 2000),
scanning
electron
microscopy (ZEISS
EVO-18)
and
potentiometry Autolab
Potentiostat/Glavanostat Model AUT83945 (PGSTAT302N). 3. Results and discussion 3.1. Characterization of Fe3O4 and Au-Fe3O4 core shell structure Fig. 1A shows the UV-Visible absorption spectrum of pure magnetic Fe3O4 NPs, Au NPs and Au-Fe3O4 core shell NPs. A typical absorption spectra of pure Fe3O4 NPs (curve a) exhibits
an absorption edge at ∼ 340 nm [60,61] that is due to iron oxide. A sharp peak at 527 nm (curve
b) exhibits strong absorption for pure AuNPs. The absorption band maxima for AuNPs between 520-532 nm is assigned to presence of spherical AuNPs nanoparticles [62, 63]. The UV-Visible absorption spectrum of the Au-Fe3O4 core shell (curve c) structure shows a broad peak at 532 nm. The shifting of the peak position towards longer wavelength (red shift) and disappearance of the peak edge arise due to presence of Fe3O4, indicating the formation of a bimetallic core shell structure [64]. Au covers most of the Fe3O4 NPs surface and the observed shift can be ascribed to the inherent surface plasmon resonance property of Au NPs. The magnetic properties of Fe3O4 NPs and Au-Fe3O4 core shell structure were analyzed by a vibrating sample magnetometer (VSM) at 17 K. Fig.1B shows the hysteresis loop measured for the Fe3O4 NPs (curve a), Fe3O4 NPs in citrate buffer (curve b) and Au- Fe3O4 core shell
structure (curve c). The values of saturated magnetization from the hysteresis curve of the pure Fe3O4 NPs and Fe3O4 NPs in buffer were found to be 0.0028 and 0.0085 emu g-1, respectively, at 17 K. The saturated magnetization of the Fe3O4 NPs dispersed in citrate buffer increases by ~ 4 times compared to that of the precipitated Fe3O4 NPs indicating uniform dispersion of Fe3O4 particles in citrate buffer. In the dispersed form, each nanoparticle acts like a tiny magnet, resulting in higher magnetic moment density than that of precipitated Fe3O4 NPs. The saturated specific magnetization of Au-Fe3O4 core shell structure decreases to 0.0022 emu g-1. This decrease may perhaps be due to the diamagnetic contribution of the Au nanoparticles in the core shell particles [65] indicating that the gold is successfully coated on Fe3O4 NPs to form AuFe3O4 core shell. The EQCM-CV (Fig. 1C) of Fe3O4 NPs dispersion and Au-Fe3O4 NPs was studied in PBS buffer 50 mM, pH 7.4, 0.9% (w/v) NaCl containing 5 mM [Fe(CN)6]3−/4−. 100 µL of NP dispersion was added in buffer to conduct CV at a scan rate of 100 mV/s in the potential range −0.2 to 0.8 V {Figs.1C (a, b and c)}. The curve a represents the EQCM-CV of [Fe (CN)6]3−/4− redox system in PBS buffer. The magnitude of the peak current increases after adding Fe3O4 NPs (curve b) which further increases on adding the Au-Fe3O4 core shell (curve c) showing an enhanced electron transform rate through the medium to the surface of electrode confirming that Au is successfully coated onto Fe3O4 NPs. To confirm the formation of Au-Fe3O4 NPs, EDX analysis was performed for elemental composition in Fe3O4 and Au-Fe3O4 NPs. Fig.2A, image (a) and (b) for Fe3O4 and Au-Fe3O4 NPs, shows the presence of a Fe peak at 6.8keV the and absence of an Au peak in image (a), while image (b) shows peaks for both Au at 2.4 keV and 9.5 keV and Fe at 0.58 keV, 6.5 keV
and 7.1 keV, respectively. The weight percentage of these elements, shown as inset of respective images, indicates the presence of the corresponding elements. Fig 2B shows the TEM images of Fe3O4 NPs, Fe3O4 NPs in citrate buffer and Au- Fe3O4 NPs. The average particle sizes of Fe3O4 NPs, Fe3O4 NPs in citrate buffer and Au- Fe3O4 NPs were ̴ 8 nm, ̴ 13 nm and ̴19 nm, respectively. The image (i) indicates that Fe3O4 NPs overlaps with each other, while image (ii) shows uniform distribution of the Fe3O4 NP in buffer as an ionic citrate layer surroundings the Fe3O4 NPs. Image (iii) shows the Au-Fe3O4 core shell NPs structure with a dark center of Fe3O4 NPs surrounded by a lighter layer of Au NPs. The molecular d spacing is 0.48 nm for the darker part and 0.23nm (from image J software) for lighter part of shell. The lattice distances measured for the shell correspond to the known Au lattice parameters for the (111) plane and those measured for the core match well with the Fe3O4 lattice parameters for the (311) plane. The presence of these two phases is also confirmed by Xray diffraction (XRD) analysis. The XRD pattern of the Fe3O4 NPs and Au-Fe3O4 core shell NPs, is demonstrated in Fig. 2C. Fig. 2C (spectra a) show diffraction peaks at 2θ 30.150, 35.760,43.20, 53.60, 57.60 and 62.960 which exhibit indexed (220), (311), (400), (422), (511) and (440) for lattice plane of Fe3O4 (JCPDS 79-0418). While spectra b shows some additional diffraction peaks at 2θ (38.280, 44.430, 59.10, 64.700 and 77.810), marked by their indices (111), (200), (220), (311) and (222) are observed for Au (JCPDS 04-0784) [66, 67]. Spectra b exhibits the diffraction peaks for both Fe3O4 and Au NPs, the peak intensity of Fe3O4 Nps decreases and some lattice plane peaks merge with Au when compared to the X-RD pattern of Fe3O4 alone (spectra a), which may perhaps due to the heavy atom effect from Au NPs surrounds the Fe3O4 NP. This revealed that the core shell like structure of Au-Fe3O4 NPs was successfully synthesized.
3.2. Characterization of r-IgG-Au-Fe3O4 The conjugate formation of r-IgG-Au-Fe3O4 was confirmed by UV-absorption spectroscopy (Fig.3A). The absorbance maxima for the pure r-IgG antibody solution appears at 250 nm (curve a) whereas two absorption bands are observed at 258 nm and 529 nm (curve b) for the r-IgG-AuFe3O4 conjugate. Broadening in peaks and the slight red shift is also observed in both the peaks due to interaction of r-IgG antibody with functionalized AuNPs. The peak at 250 nm for pure rIgG antibody and 258 nm in the conjugate system arises due to p-p* transition from the tryptophan and tyrosine residues in the antibody [68]. The EQCM-CV (Fig.3B) of the cysteamine functionalized Au-Fe3O4 NPs and r-IgG- AuFe3O4 conjugate corroborate the fabrication of secondary antibody conjugate with Au-Fe3O4 NPs. EQCM-CV studied in PBS buffer of 50 mM, pH 7.4, 0.9% (w/v) NaCl containing 5 mM [Fe(CN)6]3−/4−. 100 µL of the conjugate dispersion is added to the buffer to conduct CV at a scan rate of 100 mV/s in the potential range of −0.2 to 0.8 V {Fig.3B (a, b and c)}. Curve a represents the EQCM-CV of Au-Fe3O4 nanoparticles in the [Fe (CN)6]3−/4− redox system in PBS buffer. The magnitude of the peak current decreases after functionalization of the Au-Fe3O4 nanoparticles with cysteamine (curve b), decrease in current occuring due to the insulating nature of cysteamine. The EQCM- CV of r-IgG- Au-Fe3O4 conjugate (curve c) in Fe(CN)6]3−/4− redox system results in increase in current, due to the presence of carboxyl and amine groups throughout the IgG antibodies indicating the formation of r-IgG-Au- Fe3O4 conjugates. 3.3. Characterization of the immuno sensor 3.3.1. Electrochemical characterization of the immuno electrode
EQCM-CV (Fig.4A) was conducted in PBS, 50 mM, pH 7.4, 0.9% (w/v) NaCl containing 5 mM [Fe(CN)6]3−/4− as a redox species at a scan rate of 100 mV/s in the potential range of −0.2 to 0.8 V. Fig. 4A shows CV of [Fe (CN)6]3−/4− on a bare Au QCM electrode (curve a), 4-ATP/Au electrode (curve b), aAFB1/4-ATP/Au (curve c) and BSA/aAFB1/4-ATP/Au electrode (curve d). After the SAM deposition, the magnitude of anodic peak current (2.49x10-4 A) decreases (curve b), due to insulating properties of the thin layer of thiol which hinders the electron movement through the gold surface of the electrode. The presence of aAFB1 over the surface enhances the peak current up to 6.48 x 10-4 A (curve c) due to the presence of polar groups on the amino acids of the antibody such as carboxyl and amine moieties. There is a slight decrease in the magnitude of current response (6.17 x 10-4 A) after immobilization of BSA on to the surface of aAFB1/4-ATP/Au electrode, indicating enhanced barrier to electrons. After the competition, the magnitude of current increases (8.31 x 10-4) due to the presence of r-IgG-AuFe3O4 conjugate over the surface of AFB1/BSA/aAFB1/4-ATP/Au immuno electrode. It reveals that the IgG antibodies interact with AFB1 coated over the surface. 3.3.2. SEM studies The surface morphological studies of aAFB1/4-ATP/Au, AFB1/BSA/aAFB1/4-ATP/Au and r-IgG-Au-Fe3O4/AFB1/BSA/aAFB1/4-ATP/Au electrodes and immuno electrodes were examined by scanning electron microscopy (SEM). Fig.4B represents the surface morphology of aAFB1/4-ATP/Au electrode (image a), showing highly dense globular morphology with bright streaks confirming the immobilization of aAFB1 onto 4-ATP/Au surface. Fig.4B image (b) shows the surface morphology of AFB1/BSA/aAFB1/4-ATP/Au electrode surface. The observed morphological changes seen in the SEM image after incubation of AFB1 indicate binding of AFB1 to the aAFB1/4-ATP/Au electrode surface. Image c shows surface morphology of the r-
IgG-Au-Fe3O4/AFB1/BSA/aAFB1/4-ATP/Au immuno electrode and the surface is saturated with r-IgG-Au-Fe3O4 conjugate. Inset (image c), shows morphology of the r-IgG-AuFe3O4/AFB1/BSA/aAFB1/4-ATP/Au at high magnification. It reveals that the secondary antibody interacts with coated antigen and forms a ‘sandwich-like’ structure. Further, this fact is confirmed by EDX of this surface (Fig.4B, image d) containing Fe and Au along with other elements and the weight % shown in the inset of image d. The presence of these elements confirms the interaction of secondary antibody r-IgG-Au-Fe3O4 conjugate over the AFB1/BSA/aAFB1/4-ATP/Au immuno electrode. 3.3.3. FT-IR studies Figure 4C demonstrates FT-IR spectrum of the thiol monolayer between 500 and 3000 cm-1. The band in the range of 624-640 cm-1 assigned to Au-S stretching mode (spectrum a) reveals the formation of 4-ATP SAM over the gold coated electrode. The observed bands at 804 cm-1, 1461 cm-1 and broad band at 3340 cm-1 due to =C–H deformation of the benzene ring, aromatic –C=C– in-plane vibrations and N–H vibration of NH2, further confirm the presence of 4-ATP on Au surfaces. The appearance of two intense amide bands in spectrum b is a signature of aAFB1 adsorption on the 4-ATP/Au electrode surface; amide I at 1687 cm−1 corresponding to carbonyl C=O stretching vibration, amide II band at 1594 cm−1 is due to the coupled C-N stretching and -N-H bending mode, these bands indicate successful immobilization of monoclonal antibodies [69]. Fig.4C {spectrum (c)} represents FT-IR spectrum of AFB1/BSA/aAFB1/4-ATP/Au i.e. after recognition of AFB1 by BSA/aAFB1/4-ATP/Au immuno sensor. The presence in Fig. 4C (spectrum (c)) of a band at 1474 cm−1 is indicative of a methyl adjacent to an epoxy ring, 1308 cm−1 for in-plane –CH bending of phenyl clearly indicates the presence of AFB1 on the surface of aAFB1/4-ATP/Au surface [70]. The band at
1098 cm−1 for symmetric stretching of =C–O–C or symmetric bending of phenyl and 938 cm−1 for possibly isolated H further confirms interaction between coated AFB1– aAFB1 on the immuno sensor surface. The band at 3414 cm-1 due to –N–H stretching of NH2 group of the antibody almost disappears in the spectrum of AFB1/BSA/aAFB1/4-ATP/Au (Fig. 4C (c) indicating strong interaction between the antigen epitope with paratope of the antibody. 3.3.4. Response studies of the immuno electrode The sensitivity and detection limit of an immuno sensor depends on antibody loading. Prior to sensing studies, we have optimized all the parameters such as the concentration of aAFB1 (40µgmL-1), incubation time (30-35 min) and pH (7.4) in our previous study [58] with the frequency measurements (Supplementary Fig. S1, S2, and S3). Further the concentration of rabbit-immunoglobulin antibodies (r-IgG) was optimized from 10-50 µgmL-1 (Supplementary Fig.S4). Fig. 5A represents the EQCM-CV response studies of AFB1/BSA/aAFB1/4-ATP/Au immuno sensor from 0–5 ngmL-1. It has been observed from EQCM-CV, that the peak current intensity of the redox mediator is inversely proportional to the amount of AFB1 in the sample. At zero concentration of AFB1, immuno electrode surface is fully covered with r-IgG-Au-Fe3O4 conjugate to obtain the maximum magnitude current (8.3x10-4). The peak current decreases with increase in concentration of AFB1 and this result is also supported by change in frequency (Supplementary S5) measured along with CV via EQCM, as mass increase on the electrode surface cause in change in vibration frequency of electrode. In this experiment, free sample AFB1 and coated antigen compete for the fixed amount of r-IgG-Au-Fe3O4 conjugate. The free antigens have a maximum probability to interact with r-IgG-Au-Fe3O4 conjugate secondary
antibodies. Scheme 1 represents the formation of this competitive sandwich type immuno electrode. Fig. 5B shows the calibration curve as a function of AFB1 concentration, the linearity is from 0.05 to 5 ngmL-1 after which it decreases revealing that at 5 ngmL-1 concentration becomes saturated. The calibration plot between anodic peak current and AFB1 concentration was determined. Each value was measured in triplicate and the regression equation obtained had a regression coefficient of ca. 0.98: I (A) = (6.93x10-4 A) – 9.40 x 10-5 A ng-1mL) × [AFB1] ngmL-1
Eq1
This corresponds to the sensitivity of ca. 335.7 µAng-1mLcm-2 for AFB1 with a calculated low detection limit (LOD) of 0.07 ngmL-1. The LOD has been calculated by using 3σ/m criteria, where σ is the standard deviation and m is the slope of the calibration curve [69]. Interestingly, under the same optimized conditions, we observed that the linear range using a competitive mode is higher than by the direct non-competitive method (shown in Supplementary file (Fig. S6)). It is, however, not linear and has a regression coefficient is 0.933 thus revealing that the competitive mode is highly sensitive in comparison to the non competitive mode. Table 1 shows characteristics of the AFB1/BSA/aAFB1/4-ATP/Au immuno sensor along with some of those reported in the literature (71-73, 19, 21, 23, 37, 62). Jin et.al have enhance the sensitivity by an insoluble product produced by labeled enzyme. From the literature survey, it has been found out the majority of the reported papers are based on labeled immunosensor. 3.3.5. Real Sample testing and selectivity of immuno electrode To evaluate the applicability of the developed immunosensor to real sample analysis, corn flakes samples were spiked with various concentrations of AFB1. For this corn flakes
sample were extracted with a methanolic solution of potassium bicarbonate. Prior evaporation to dryness and final reconstitution in PBS buffer was necessary to avoid the inhibition of the antibody-antigen binding caused by methanol. The extract sample was spiked with three different concentrations of AFB1 (0.05, 2 and 5ngmL-1) to examine the applicability of the proposed probe. During these experiments, the AFB1/BSA/aAFB1/4-ATP/Au immunosensor was dipped in the cell containing a mixture of different concentrations of AFB1 in spiked extracted samples and the optimized amount of r-IgG-Cyst/Au-Fe3O4 in PBS and incubated for 35 min. The EQCM-CV of AFB1/BSA/aAFB1/4-ATP/Au immuno sensor was examined with the cornflakes extract in PBS, and indicates the minimum interference. Variation of peak current in blank and corn flake extracts [Fig.6 (bar 1, 2)] is within 5%. It indicates that the AFB1/BSA/aAFB1/4-ATP/Au immuno sensor shows specificity towards AFB1, and the levels are not inflated by other constituents present in the corn flake sample extract. However, the response of the AFB1/BSA/aAFB1/4-ATP/Au immuno sensor changes when the corn flakes sample contains AFB1 and magnitude of the anodic peak further decreases as the AFB1 concentration increases in the corn flakes sample. The results obtained with this extracted solution in PBS and result from standard PBS within 3-5% variations, showing that the developed immuno sensor is highly specific to AFB1 and avoids interference of other materials present in corn flake extracts. 3.3.6. Reproducibility, shelf life and regeneration of immuno- electrode The reproducibility of the proposed immuno electrode was estimated by repetitive measurement of the current response using 2 ngmL-1 standard AFB1 solutions in PBS (50 mM, pH 7.4, 0.9% (w/v) NaCl, containing 5mM [Fe(CN)6]3-/4-). The results obtained in 5 repeated measurements show a relative standard deviation (RSD) of 2–3%, indicating that the obtained
data is reproducible. These results demonstrate the acceptable reproducibility and precision of the proposed immuno sensor. In addition, the immunosensor was stored at 4 ̊C for shelf life (stability) study. The stability of the BSA/aAFB1/4-ATP/Au immuno electrode was evaluated by EQCM- CV and the current response in the presence of 2 ngmL-1 standard AFB1 solutions in PBS (50 mM, pH 7.4, 0.9% (w/v) NaCl) was monitored at a regular interval of 7 days (Fig.7). The immuno electrode retains its activity up to 28 day with 5-7 % decrease in activity. The response of the immmunoelectrode decreased to 95% of the initial value after 1 week and it decreased to 90% after 1 month. The immuno sensor can be regenerated (Scheme 1) using an external strong magnet to remove the immuno r-IgG-Au-Fe3O4 conjugate. It was observed that the immunosensor could be used 15-16 times with 2-3% loss in activity using two different concentrations of AFB1 (1 and 2 ng/mL). Table 2 shows the response of AFB1/BSA/aAFB1/4-ATP/Au immunoelectrode, after the regeneration. 4. Conclusions A competitive, sandwiched immunoelectrode based on an Au-Fe3O4 core shell NPs was fabricated for the detection of AFB1 using EQCM. The prepared immuno electrode can be used to detect AFB1 in the concentration range of 0.05–5 ngmL-1 with the limit of detection as 0.07 ngmL-1. This immuno sensor is found to be highly promising for detection of AFB1 in corn flakes samples. This immuno sensor can be regenerated about 15-16 times with 2-3% loss of activity. Therefore, on comparing with direct mode, the competitive mode along with electrochemical piezoelectric tool offers a highly sensitive reusable immuno sensing platform. Efforts should be made to utilize this novel magneto immunosensor for detection of other food toxins such as OTA, OTB, fumonisins and zearalenone.
Acknowledgements We thank Dr. Ashok Kumar Chauhan (Founder President, Amity University Uttar Pradesh) for providing the facilities. We also thank to Dr. (Mrs) Balvinder Shukla, Vice Chancellor Amity University Uttar pradesh and Prof. L.M. Bharadwaj, Director, AINT. We also express our deep gratitude to Ms. Shuvra Singha, Department of Chemistry, University of Hyderabad, India for her help in conducting the XRD studies.
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Table 1 : Characteristics of the AFB1/BSA/aAFB1/4-ATP/Au immunoelectrode along with those reported in the literature for aflatoxin B1 detection, with their important parameters.
Bioelectrode
Detection Method
Sensitivity
Limit of Detection (ngmL−1)
Detection range (ngmL−1)
Stability (days)
Reference
BSA-anti-AFB1/Au NPs
Electrochemical
1.4 µS /ngmL−1
0.1
0.5-10
12
71
BSA/anti-AFB1/MWCNTs/ITO
Electrochemical
95.2 µA ng/mL cm−2
0.08
0.25–1.375
45
62
aAFB1/DSP /Au
QCM
-
0.5
0.5-10
-
19
Fe3O4/SiO2/aAFB1/BSA
Quartz crystal microbalance
20.39Hz/ngmL−1
0.3
0.3– 7.0
13
21
aAFB1/BSA-AFB1/MPA/Au
Quartz crystal microbalance
33.13 Hz/ngmL−1
0.01
0.01–10.0
-
23
Nano-size gold hollowballs (NGB)
Quartz crystal microbalance
-
0.05
0.6-12.5
-
72
GCE/chitosan/AuNP/aAFB1
Electrochemical
-
0.2
0.6-110
-
37
PTH/AuNP/GCE
Electrochemical
-
0.07
0.6-2.4
-
73
BSA/aAFB1/ATP/Au
EQCM-CV
335.7 µAng1 mLcm-2
0.07
0.05-5.0
28
Present work
Tabel 2: Regeneration of r-IgG –Au-Fe3O4/AFB1/ BSA/aAFB1/4-ATP/Au immunoelectrode.
S.No.
No. of cycles
response of immunoelectrode with 1ng/mL
response of immunoelectro de with 2ng/mL
1
1st
5.72 x 10-4
4.78 x 10-4
2
5th
5.81 x 10-4
4.62 x 10-4
3
12th
5.49 x 10-4
4.66 x 10-4
4
15th
5.53 x 10-4
4.52 x 10-4
S1369-703X(15)30010-3 http://dx.doi.org/doi:10.1016/j.bej.2015.07.002 BEJ 6243
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
4-3-2015 3-6-2015 4-7-2015
Please cite this article as: Ruchika Chauhan, Jay Singh, Pratima R.Solanki, T.Basu, Richard O’Kennedy, B.D.Malhotra, Electrochemical piezoelectric reusable immunosensor for aflatoxin B1 detection, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2015.07.002 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 proof before it is published in its final 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.
Electrochemical piezoelectric reusable immunosensor for AflatoxinB1 detection Ruchika Chauhan,1a Jay Singh,2b Pratima R. Solanki,3c T. Basu,4a* Richard O’Kennedy,d and B.D. Malhotra6e a
b
Amity Institute of Nanotechnology, Amity University Uttar Pradesh, NOIDA India
Department of Applied Chemistry & Polymer Technology, Delhi Technological University, Delhi-110042, India
c
Special Centre for Nano Sciences , Jawaharlal Nehru University, New Delhi-110067,India d
Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin 9,Ireland
e
Department of Biotechnology, Delhi Technological University, Delhi-110042 , India Email address: [email protected] , [email protected], 3
[email protected], [email protected], [email protected] 6
[email protected]
_________________________________________________________________________ *Corresponding author Email: [email protected] Highlights
•
Synthesis of gold coated iron oxide core shell nanoparticles (Au-Fe3O4 NPs).
•
Synthesis of antibody conjugates with nanoparticles (r-IgG-Cys-Au-Fe3O4 conjugate).
•
Iron oxide core shell are utilize for detection of aflatoxin B1
•
Fabricated reusable EQCM immunosensor.
•
Regeneration upto 15-16 times with negligible loss in activity.
Abstract A competitive, reusable, reliable and sensitive immunosensor based on electrochemical quartz crystal microbalance (EQCM) was developed for the detection of aflatoxin B1 (AFB1). The sensing platform was developed using a primary monoclonal anti-aflatoxin antibody (anti AFB1) covalently immobilized on a monolayer of 4- amino thiophenol self assembled on a gold coated quartz crystal electrode (anti AFB1/4-ATP/Au). The reusability was achieved by a sandwiched system using secondary rabbit-immunoglobulin antibodies (r-IgGs) tagged with core shell of gold coated iron oxide nanoparticles (r-IgG-Au-Fe3O4), which can be used to regenerate the bioelectrode. A competitive mode was employed, between free and coated AFB1 for fixed concentration of nanoparticle conjugate (r-IgG-Au-Fe3O4). After the competitive interaction, immunoelectrode was washed with phosphate saline buffer (PBS) at pH 7.4 and examined by electrochemical quartz crystal microbalance-cyclic voltammetry (EQCM-CV). Under the optimized conditions, the fabricated immunosensor can be used to detect AFB1 quantitatively with a linear range of 0.05 to 5 ngmL-1. For reliability, the fabricated immuno sensor was tested using cereal samples spiked with different concentrations of AFB1. In addition, this immuno electrode displays good reproducibility, and storage stability. The above immunosensor was regenerated with negligible loss in activity through removal of the r-IgG-Au-Fe3O4 conjugate using a strong magnet. It is shown that the r-IgG-Au-Fe3O4/AFB1/BSA/aAFB1/4-ATP/Au
immuno sensor is a new approach for developing sensitive reliable and reusable electrochemical piezoelectric immunosensors. Key words: Aflatoxin B1, immunosensor, EQCM, Fe3O4 NPs, Au-Fe3O4 NPs 1. Introduction Aflatoxin B1 (AFB1) is one of the most important mycotoxin, produced by strains of Aspergillus flavus and Aspergillus parasiticus and is a carcinogen. It is commonly present in various foods (e.g., corn, sorghum, peanuts, fruits, dried fruits, cocoa and spices) and causes human health disorders such as hepatocellular carcinoma, Reye’s syndrome and chronic hepatitis. The European Commission has set strict limit for the maximum level (2 µgkg−1) of AFB1 that can be allowed in food products [1-4]. Compared to conventional techniques [5-7] biosensors are a valuable alternative that can be used to quantify and detect the desired toxin molecules [8-11]. Some of the biosensing techniques that have been used for AFB1 detection include electrochemical biosensors [12-14], surface plasmon resonance based platforms [15,16], fluorescence based biosensors [17,18], and quartz crystal (piezoelectric) microbalance based sensors [19,20]. Among the signal detection techniques, quartz crystal microbalance (QCM) based detection systems are considered to be the most promising due to their affordable cost, real-time detection capability, label free format, compatibility with miniaturization, portability and high sensitivity [21]. The piezoelectric immunosensors are known to rely on interaction of an antibody with the antigen on the probe surface [22, 23]. However, self assembled monolayers (SAMs) have been predicted to play a potentially valuable role in the development of immunosensing surfaces in biosensing devices [24, 25].
Since AFB1 is a small molecule of low molecular weight its direct detection with a satisfactory detection limit and sensitivity is very difficult using QCM [26]. It was reported that small molecules such as TNT, ochratoxin A and aflatoxins could be detected with high sensitivity using a competitive immunoassay format with a signal enhancing label [27, 28]. This label may comprise of an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP) [29-30], a nano label (e.g. gold NPs) [31] or nanogold-attached carbon nanotubes [32]. It was found that some of these bionano labels are difficult to isolate and hence may cause interference during experiments. Several researchers are currently working on the reusability or regeneration of sensing elements functionalized with various biomolecules [33, 34]. To overcome these problem magnetic nanoparticles (MNPs) may be used for the signal amplification, rapid separation and purification of bioconjugates using an external magnet [35-37]. Iron oxide nanoparticles are being used in biomedical application due to their biocompatibility and magnetic properties [38,39]. Magnetic separation and concentration using nanoparticles was studied by Wu et. al. using antigen-modified magnetic nanoparticles as immunosensing probes and fluorescent bio labels [40]. Some of the current challenges include the synthesis of MNPs with well controlled sizes, composition, and surface properties [41, 42]. These problems may perhaps be resolved by chemically coating these magnetic nanoparticles with peptides [43], polymers [44, 45], silanes [46, 47] metal oxides [48] and metals [49]. In this context, gold coated magnetic nanoparticles have been predicted to be promising [50, 51]. The core shell of AuNPs along with Fe3O4 NPs may be used for ultrasensitive detection of analytes [52] to obtain enhanced electron transfer, across the interface [53, 54]. Furthermore, the core–shell of AuFe3O4 NPs can be used to improve kinetics of the immuno-reactions and increase binding [55].
In our previous study, we have observed that QCM measurement becomes more efficient when combined with electrochemistry. We have received wider linear range, low detection limit and higher sensitivity with EQCM technique rather than QCM [56]. Here we report results of the studies relating to development of a reusable, label free, competitive sensitive sandwich-type electrochemical piezoelectric quartz crystal (EQCM) immunosensor based on 4-amino thiophenol self assembled monolayers to detect AFB1. The secondary rIgG- Au-Fe3O4 NPs conjugate structure has been synthesized to utilize it as a signal enhancer and sensor regeneration through external magnet. The constructed biosensor yields about a tenfold lower detection limit than the previously reported competitive sandwich immunosensor in real samples and can be regenerated with negligible loss of activity using a strong magnet. 2. Experimental Methods 2.1. Chemicals and reagents Monoclonal anti-aflatoxin B1 (aAFB1) antibodies, aflatoxin B1 (AFB1), bovin serum albumin (BSA), polyclonal IgG antibodies from rabbit (r-IgG), 4 amino thio phenol (4-ATP), Nethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), ferrous chloride hexahydrate (FeCl2.6H2O), ferric chloride tetrahydrate (FeCl3.4H2O), sodium hydroxide (NaOH) and chloroauric acid (HAuCl4.H2O), were procured from Sigma-Aldrich. All reagents were of analytical grade and used without further purification. De-ionized water (resistance ~ 18MΩ cm) from Millipore direct Q3 purification system, was used for the preparation of desired aqueous solutions. The gold coated (diameter: 6.7 mm) quartz resonator (AT cut quartz crystal, 13.7 mm dia, 6 MHz) was procured from Autolab, the Netherlands. A nylon membrane used during the extraction of aflatoxins extract from the corn flakes, was obtained from Whatman 2.2. Solution preparation
Anti-AFB1antibody (1 mgmL-1) solution was prepared in 50 mM phosphate buffer (PBS), 50 mM, pH 7.4 and 0.15 M NaN3 was used as a preservative. r-IgG antibody (2 mgmL-1) solution was prepared in 50 mM PBS (50 mM, pH 7.4). The stock solution of AFB1 was prepared in PBS (50 mM, pH 7.4) with 10% (v/v) methanol and dispensed in different working concentrations and stored at -20 °C. A solution of bovine serum albumin (BSA, 1 mgmL-1) was prepared in PBS (50 mM, pH 7.0) and used for blocking for the nonspecific binding sites. 2.3. Pretreatment of quartz crystals The quartz crystals were immersed in 1M NaOH for 5 min and 1M HCl for 2 min in sequence. Later, freshly prepared piranha solution {1:3 (30% v/v) H2O2–H2SO4} was dropped on the gold surface for 2 min, taking special care to avoid contamination of the electrode leads. The quartz crystals were rinsed twice with deionized water followed by ethanol, and dried in a stream of nitrogen after each pretreatment and then the initial resonance frequency (F0) was recorded. After cleaning, the quartz crystal was ready for surface modification and antibody immobilization. 2.4. Synthesis of Fe3O4 and Au-coated Fe3O4 Fe3O4 NPs were synthesized simply by the co-precipitation method reported earlier [57] with some modification. Solutions of 0.07 M FeCl3·6H2O and 0.04 M FeCl2.4H2O (2:1, w/w ratio) were dissolved in 25 mL deionized water and then this mixture was added drop wise to 100 mL solution of 0.15 mM NaOH with stirring under a N2 atmosphere at room temperature. A black precipitate of Fe3O4 NPs was obtained. This precipitate was dissolved in 20 mL citrate buffer (1.6 gm citric acid and 0.8 gm tri-sodium citrate) to stabilize ferrofluid in solution at pH of 6.3.
The Au-Fe3O4 core shell NPs were prepared using 3 mL of synthesized colloidal Fe3O4 nano-suspension (0.1 M), boiled with 25 mL of ultra pure water under vigorous stirring conditions. Then 3 mL of 0.2 mM HAuCl4 was added, followed by the addition of 3mL of 10 mM tri-sodium citrate and the reaction mixture was kept boiling and stirred for 15 min till the color of the solution turned from black to red. The gold coated Fe3O4 NPs (Au- Fe3O4 NPs) solution was allowed to cool and was stored in a dark glass bottle at 4 ̊ C before use. 2.5. Synthesis of r-IgG-Au- Fe3O4 The synthesized Au-Fe3O4 nano- suspension was treated with 10-3 M aqueous solution of cysteamine hydrochloride in a 1:15 volume ratio for 12 h at 25 ̊C. The cysteamine functionalized Au-Fe3O4 NPs (Au-Fe3O4) were separated and purified by centrifugation at 10,000 rpm for 10 min. The purification and centrifugation processes were repeated 4-5 times for removing nonbonded cysteamine. Then cysteamine functionalized Au-Fe3O4 core shell NPs were re-dispersed in PBS (50 mM, pH 7.0) solution. Cysteamine forms a self assembled layer on Au-Fe3O4 NPs which provides NH2 groups to bind with COOH functional groups of the polyclonal rabbitimmunoglobulin antibodies (r-IgG) during immobilization. The r-IgG antibodies were mixed with Au-Fe3O4 solution in 1:3 (v/v ratio) [58], followed by addition of 0.2M EDC and 0.05M NHS for the activation of –COOH groups present in the antibody. Furthermore, to block the nonspecific sites on the r-IgG-Au-Fe3O4 conjugates, 100µL BSA (1mgmL-1) was added and incubated for 2h at 25 ̊C. The mixture was centrifuged at 10,000 rpm for 10 min and washed 4-5 times with deionized water. Finally, the r-IgG-Au-Fe3O4 conjugate was re- suspended in PBS of (50 mM, pH 7.4) and stored at 4 ̊C until use. Scheme S1 represents the formation of r-IgG-AuFe3O4 conjugate. [See Supplementary Scheme S1] 2.6. Fabrication of AFB1/BSA/aAFB1/4-ATP/Au immuno sensor
Pretreated quartz crystal was immersed in 2 mM solution of 4-ATP in ethanol for 24h at 25 ̊C for SAM formation. However, a uniform and steady 4-ATP film was obtained [56]. The crystal was subsequently washed with ethanol followed by rinsing with water to remove any unbound ATP molecules. 10µL of 40 µgmL-1 of monoclonal anti aflatoxin B1 (aAFB1), activated with 0.2 M EDC and 0.05M NHS for about 2h, was spread over the electrode and incubated overnight at 4 °C for the amide bond formation between aAFB1 and 4-ATP. In this study, an optimized concentration of 40 µgmL-1 of aAFB1 was used. The non-specific sites of fabricated immuno electrodes were blocked with BSA in PBS of (50 mM, pH 7.0) (1mgmL-1). These fabricated BSA/aAFB1/4-ATP/Au immuno electrodes were exposed to saturated concentration of AFB1 (5ngmL-1) for 35 min at 25 °C. 2.7. Detection of AFB1 The fabricated AFB1/BSA/aAFB1/4-ATP/Au immuno electrode was allowed to compete with free AFB1 with a fixed concentration of secondary antibody conjugate. The immuno electrode was
exposed to 3 mL solution
of
5 mM [Fe(CN)6]3−/4− mediator in
phosphate buffer saline (50 mM, pH 7.4, 0.9% (w/v) NaCl) containing 10 µL of saturated concentration (30 µgmL-1) of r-IgG-Au- Fe3O4 conjugate and 10 µL of AFB1 standards in PBS buffer (0–5 ngmL-1). To determine the optimum concentration of r-IgG antibody conjugate, AFB1/BSA/aAFB1/4-ATP/Au immuno electrode was allowed to interact with different concentrations of r-IgG-Au- Fe3O4 conjugate i.e. 10-50 µg/mL and the corresponding response current was observed with EQCM-CV. During the competition process, secondary antibodies easily access to free AFB1, while rest of the r-IgG-Au- Fe3O4 conjugates form a sandwiched structure with the coated AFB1. Therefore, with increased concentrations of free AFB1, the availability of r-IgG-Au-Fe3O4 conjugate on coated AFB1 gradually decreases resulting in a
decrease in response current. After competition, the crystal was rinsed with PB (containing 0.1% (w/v) Tween-20) dried under a N2 stream. All experiments were performed in triplicate and the experimental temperature was controlled at 25 ̊C. 2.8. Pretreatment and analysis of cereal samples Cereal samples (corn-flakes) were spiked with AFB1 and then were crushed to powder using a hand-held blender. 2g of powdered cereals were added to methanol: water (7:3, v/v) solution on a sonication bath for 45 min. The extract was centrifuged for 7 min at 5000 rpm to remove the solids. The supernatants were collected and allowed to evaporate to dryness under nitrogen at 25°C. Evaporation was necessary to avoid inhibition of the antibody-antigen binding caused by methanol. The residues were re-suspended in 5 mL PBS and filtered through 0.45 µm Whatman nylon membranes [59]. Finally, the extract was spiked with the concentrations of 0.05, 2 and 5 ng mL-1 of aAFB1. 2.9. Instrumentation The resonant frequency of quartz crystal and electrochemical studies was monitored using
a
Autolab
Potentiostat/Glavanostat
Model
AUT83945
(PGSTAT302N).
The
electrochemical quartz crystal cyclic voltammetric (CV) studies were carried out in a three electrode cell using a modified gold coated quartz crystal as the working electrode, gold wire as the auxiliary electrode, and saturated Ag/AgCl as the reference electrode in PBS, (50 mM, pH 7.4, 0.9% (w/v) NaCl) containing 5 mM [Fe(CN)6]3−/4− as a redox species. The Au-Fe3O4 core shell magnetic nano particles were characterized by scanning electron microscopy (ZEISS EVO18), vibrating sample magnetometry (VSM) (Microsense, ADE-Model EV9), transmission electron microscopy (JEOL JEM (Model 1200F) and X-ray diffractometry ( Bruker AXS, XRD). The structural and surface morphological characterizations of 4-ATP/Au, aAFB1/4-
ATP/Au, BSA/aAFB1/4-ATP/Au, and the AFB1/BSA/aAFB1/4-ATP/Au electrodes were carried out using Fourier transform Infrared spectroscopy (FT-IR, Perkin-Elmer, and Model 2000),
scanning
electron
microscopy (ZEISS
EVO-18)
and
potentiometry Autolab
Potentiostat/Glavanostat Model AUT83945 (PGSTAT302N). 3. Results and discussion 3.1. Characterization of Fe3O4 and Au-Fe3O4 core shell structure Fig. 1A shows the UV-Visible absorption spectrum of pure magnetic Fe3O4 NPs, Au NPs and Au-Fe3O4 core shell NPs. A typical absorption spectra of pure Fe3O4 NPs (curve a) exhibits
an absorption edge at ∼ 340 nm [60,61] that is due to iron oxide. A sharp peak at 527 nm (curve
b) exhibits strong absorption for pure AuNPs. The absorption band maxima for AuNPs between 520-532 nm is assigned to presence of spherical AuNPs nanoparticles [62, 63]. The UV-Visible absorption spectrum of the Au-Fe3O4 core shell (curve c) structure shows a broad peak at 532 nm. The shifting of the peak position towards longer wavelength (red shift) and disappearance of the peak edge arise due to presence of Fe3O4, indicating the formation of a bimetallic core shell structure [64]. Au covers most of the Fe3O4 NPs surface and the observed shift can be ascribed to the inherent surface plasmon resonance property of Au NPs. The magnetic properties of Fe3O4 NPs and Au-Fe3O4 core shell structure were analyzed by a vibrating sample magnetometer (VSM) at 17 K. Fig.1B shows the hysteresis loop measured for the Fe3O4 NPs (curve a), Fe3O4 NPs in citrate buffer (curve b) and Au- Fe3O4 core shell
structure (curve c). The values of saturated magnetization from the hysteresis curve of the pure Fe3O4 NPs and Fe3O4 NPs in buffer were found to be 0.0028 and 0.0085 emu g-1, respectively, at 17 K. The saturated magnetization of the Fe3O4 NPs dispersed in citrate buffer increases by ~ 4 times compared to that of the precipitated Fe3O4 NPs indicating uniform dispersion of Fe3O4 particles in citrate buffer. In the dispersed form, each nanoparticle acts like a tiny magnet, resulting in higher magnetic moment density than that of precipitated Fe3O4 NPs. The saturated specific magnetization of Au-Fe3O4 core shell structure decreases to 0.0022 emu g-1. This decrease may perhaps be due to the diamagnetic contribution of the Au nanoparticles in the core shell particles [65] indicating that the gold is successfully coated on Fe3O4 NPs to form AuFe3O4 core shell. The EQCM-CV (Fig. 1C) of Fe3O4 NPs dispersion and Au-Fe3O4 NPs was studied in PBS buffer 50 mM, pH 7.4, 0.9% (w/v) NaCl containing 5 mM [Fe(CN)6]3−/4−. 100 µL of NP dispersion was added in buffer to conduct CV at a scan rate of 100 mV/s in the potential range −0.2 to 0.8 V {Figs.1C (a, b and c)}. The curve a represents the EQCM-CV of [Fe (CN)6]3−/4− redox system in PBS buffer. The magnitude of the peak current increases after adding Fe3O4 NPs (curve b) which further increases on adding the Au-Fe3O4 core shell (curve c) showing an enhanced electron transform rate through the medium to the surface of electrode confirming that Au is successfully coated onto Fe3O4 NPs. To confirm the formation of Au-Fe3O4 NPs, EDX analysis was performed for elemental composition in Fe3O4 and Au-Fe3O4 NPs. Fig.2A, image (a) and (b) for Fe3O4 and Au-Fe3O4 NPs, shows the presence of a Fe peak at 6.8keV the and absence of an Au peak in image (a), while image (b) shows peaks for both Au at 2.4 keV and 9.5 keV and Fe at 0.58 keV, 6.5 keV
and 7.1 keV, respectively. The weight percentage of these elements, shown as inset of respective images, indicates the presence of the corresponding elements. Fig 2B shows the TEM images of Fe3O4 NPs, Fe3O4 NPs in citrate buffer and Au- Fe3O4 NPs. The average particle sizes of Fe3O4 NPs, Fe3O4 NPs in citrate buffer and Au- Fe3O4 NPs were ̴ 8 nm, ̴ 13 nm and ̴19 nm, respectively. The image (i) indicates that Fe3O4 NPs overlaps with each other, while image (ii) shows uniform distribution of the Fe3O4 NP in buffer as an ionic citrate layer surroundings the Fe3O4 NPs. Image (iii) shows the Au-Fe3O4 core shell NPs structure with a dark center of Fe3O4 NPs surrounded by a lighter layer of Au NPs. The molecular d spacing is 0.48 nm for the darker part and 0.23nm (from image J software) for lighter part of shell. The lattice distances measured for the shell correspond to the known Au lattice parameters for the (111) plane and those measured for the core match well with the Fe3O4 lattice parameters for the (311) plane. The presence of these two phases is also confirmed by Xray diffraction (XRD) analysis. The XRD pattern of the Fe3O4 NPs and Au-Fe3O4 core shell NPs, is demonstrated in Fig. 2C. Fig. 2C (spectra a) show diffraction peaks at 2θ 30.150, 35.760,43.20, 53.60, 57.60 and 62.960 which exhibit indexed (220), (311), (400), (422), (511) and (440) for lattice plane of Fe3O4 (JCPDS 79-0418). While spectra b shows some additional diffraction peaks at 2θ (38.280, 44.430, 59.10, 64.700 and 77.810), marked by their indices (111), (200), (220), (311) and (222) are observed for Au (JCPDS 04-0784) [66, 67]. Spectra b exhibits the diffraction peaks for both Fe3O4 and Au NPs, the peak intensity of Fe3O4 Nps decreases and some lattice plane peaks merge with Au when compared to the X-RD pattern of Fe3O4 alone (spectra a), which may perhaps due to the heavy atom effect from Au NPs surrounds the Fe3O4 NP. This revealed that the core shell like structure of Au-Fe3O4 NPs was successfully synthesized.
3.2. Characterization of r-IgG-Au-Fe3O4 The conjugate formation of r-IgG-Au-Fe3O4 was confirmed by UV-absorption spectroscopy (Fig.3A). The absorbance maxima for the pure r-IgG antibody solution appears at 250 nm (curve a) whereas two absorption bands are observed at 258 nm and 529 nm (curve b) for the r-IgG-AuFe3O4 conjugate. Broadening in peaks and the slight red shift is also observed in both the peaks due to interaction of r-IgG antibody with functionalized AuNPs. The peak at 250 nm for pure rIgG antibody and 258 nm in the conjugate system arises due to p-p* transition from the tryptophan and tyrosine residues in the antibody [68]. The EQCM-CV (Fig.3B) of the cysteamine functionalized Au-Fe3O4 NPs and r-IgG- AuFe3O4 conjugate corroborate the fabrication of secondary antibody conjugate with Au-Fe3O4 NPs. EQCM-CV studied in PBS buffer of 50 mM, pH 7.4, 0.9% (w/v) NaCl containing 5 mM [Fe(CN)6]3−/4−. 100 µL of the conjugate dispersion is added to the buffer to conduct CV at a scan rate of 100 mV/s in the potential range of −0.2 to 0.8 V {Fig.3B (a, b and c)}. Curve a represents the EQCM-CV of Au-Fe3O4 nanoparticles in the [Fe (CN)6]3−/4− redox system in PBS buffer. The magnitude of the peak current decreases after functionalization of the Au-Fe3O4 nanoparticles with cysteamine (curve b), decrease in current occuring due to the insulating nature of cysteamine. The EQCM- CV of r-IgG- Au-Fe3O4 conjugate (curve c) in Fe(CN)6]3−/4− redox system results in increase in current, due to the presence of carboxyl and amine groups throughout the IgG antibodies indicating the formation of r-IgG-Au- Fe3O4 conjugates. 3.3. Characterization of the immuno sensor 3.3.1. Electrochemical characterization of the immuno electrode
EQCM-CV (Fig.4A) was conducted in PBS, 50 mM, pH 7.4, 0.9% (w/v) NaCl containing 5 mM [Fe(CN)6]3−/4− as a redox species at a scan rate of 100 mV/s in the potential range of −0.2 to 0.8 V. Fig. 4A shows CV of [Fe (CN)6]3−/4− on a bare Au QCM electrode (curve a), 4-ATP/Au electrode (curve b), aAFB1/4-ATP/Au (curve c) and BSA/aAFB1/4-ATP/Au electrode (curve d). After the SAM deposition, the magnitude of anodic peak current (2.49x10-4 A) decreases (curve b), due to insulating properties of the thin layer of thiol which hinders the electron movement through the gold surface of the electrode. The presence of aAFB1 over the surface enhances the peak current up to 6.48 x 10-4 A (curve c) due to the presence of polar groups on the amino acids of the antibody such as carboxyl and amine moieties. There is a slight decrease in the magnitude of current response (6.17 x 10-4 A) after immobilization of BSA on to the surface of aAFB1/4-ATP/Au electrode, indicating enhanced barrier to electrons. After the competition, the magnitude of current increases (8.31 x 10-4) due to the presence of r-IgG-AuFe3O4 conjugate over the surface of AFB1/BSA/aAFB1/4-ATP/Au immuno electrode. It reveals that the IgG antibodies interact with AFB1 coated over the surface. 3.3.2. SEM studies The surface morphological studies of aAFB1/4-ATP/Au, AFB1/BSA/aAFB1/4-ATP/Au and r-IgG-Au-Fe3O4/AFB1/BSA/aAFB1/4-ATP/Au electrodes and immuno electrodes were examined by scanning electron microscopy (SEM). Fig.4B represents the surface morphology of aAFB1/4-ATP/Au electrode (image a), showing highly dense globular morphology with bright streaks confirming the immobilization of aAFB1 onto 4-ATP/Au surface. Fig.4B image (b) shows the surface morphology of AFB1/BSA/aAFB1/4-ATP/Au electrode surface. The observed morphological changes seen in the SEM image after incubation of AFB1 indicate binding of AFB1 to the aAFB1/4-ATP/Au electrode surface. Image c shows surface morphology of the r-
IgG-Au-Fe3O4/AFB1/BSA/aAFB1/4-ATP/Au immuno electrode and the surface is saturated with r-IgG-Au-Fe3O4 conjugate. Inset (image c), shows morphology of the r-IgG-AuFe3O4/AFB1/BSA/aAFB1/4-ATP/Au at high magnification. It reveals that the secondary antibody interacts with coated antigen and forms a ‘sandwich-like’ structure. Further, this fact is confirmed by EDX of this surface (Fig.4B, image d) containing Fe and Au along with other elements and the weight % shown in the inset of image d. The presence of these elements confirms the interaction of secondary antibody r-IgG-Au-Fe3O4 conjugate over the AFB1/BSA/aAFB1/4-ATP/Au immuno electrode. 3.3.3. FT-IR studies Figure 4C demonstrates FT-IR spectrum of the thiol monolayer between 500 and 3000 cm-1. The band in the range of 624-640 cm-1 assigned to Au-S stretching mode (spectrum a) reveals the formation of 4-ATP SAM over the gold coated electrode. The observed bands at 804 cm-1, 1461 cm-1 and broad band at 3340 cm-1 due to =C–H deformation of the benzene ring, aromatic –C=C– in-plane vibrations and N–H vibration of NH2, further confirm the presence of 4-ATP on Au surfaces. The appearance of two intense amide bands in spectrum b is a signature of aAFB1 adsorption on the 4-ATP/Au electrode surface; amide I at 1687 cm−1 corresponding to carbonyl C=O stretching vibration, amide II band at 1594 cm−1 is due to the coupled C-N stretching and -N-H bending mode, these bands indicate successful immobilization of monoclonal antibodies [69]. Fig.4C {spectrum (c)} represents FT-IR spectrum of AFB1/BSA/aAFB1/4-ATP/Au i.e. after recognition of AFB1 by BSA/aAFB1/4-ATP/Au immuno sensor. The presence in Fig. 4C (spectrum (c)) of a band at 1474 cm−1 is indicative of a methyl adjacent to an epoxy ring, 1308 cm−1 for in-plane –CH bending of phenyl clearly indicates the presence of AFB1 on the surface of aAFB1/4-ATP/Au surface [70]. The band at
1098 cm−1 for symmetric stretching of =C–O–C or symmetric bending of phenyl and 938 cm−1 for possibly isolated H further confirms interaction between coated AFB1– aAFB1 on the immuno sensor surface. The band at 3414 cm-1 due to –N–H stretching of NH2 group of the antibody almost disappears in the spectrum of AFB1/BSA/aAFB1/4-ATP/Au (Fig. 4C (c) indicating strong interaction between the antigen epitope with paratope of the antibody. 3.3.4. Response studies of the immuno electrode The sensitivity and detection limit of an immuno sensor depends on antibody loading. Prior to sensing studies, we have optimized all the parameters such as the concentration of aAFB1 (40µgmL-1), incubation time (30-35 min) and pH (7.4) in our previous study [58] with the frequency measurements (Supplementary Fig. S1, S2, and S3). Further the concentration of rabbit-immunoglobulin antibodies (r-IgG) was optimized from 10-50 µgmL-1 (Supplementary Fig.S4). Fig. 5A represents the EQCM-CV response studies of AFB1/BSA/aAFB1/4-ATP/Au immuno sensor from 0–5 ngmL-1. It has been observed from EQCM-CV, that the peak current intensity of the redox mediator is inversely proportional to the amount of AFB1 in the sample. At zero concentration of AFB1, immuno electrode surface is fully covered with r-IgG-Au-Fe3O4 conjugate to obtain the maximum magnitude current (8.3x10-4). The peak current decreases with increase in concentration of AFB1 and this result is also supported by change in frequency (Supplementary S5) measured along with CV via EQCM, as mass increase on the electrode surface cause in change in vibration frequency of electrode. In this experiment, free sample AFB1 and coated antigen compete for the fixed amount of r-IgG-Au-Fe3O4 conjugate. The free antigens have a maximum probability to interact with r-IgG-Au-Fe3O4 conjugate secondary
antibodies. Scheme 1 represents the formation of this competitive sandwich type immuno electrode. Fig. 5B shows the calibration curve as a function of AFB1 concentration, the linearity is from 0.05 to 5 ngmL-1 after which it decreases revealing that at 5 ngmL-1 concentration becomes saturated. The calibration plot between anodic peak current and AFB1 concentration was determined. Each value was measured in triplicate and the regression equation obtained had a regression coefficient of ca. 0.98: I (A) = (6.93x10-4 A) – 9.40 x 10-5 A ng-1mL) × [AFB1] ngmL-1
Eq1
This corresponds to the sensitivity of ca. 335.7 µAng-1mLcm-2 for AFB1 with a calculated low detection limit (LOD) of 0.07 ngmL-1. The LOD has been calculated by using 3σ/m criteria, where σ is the standard deviation and m is the slope of the calibration curve [69]. Interestingly, under the same optimized conditions, we observed that the linear range using a competitive mode is higher than by the direct non-competitive method (shown in Supplementary file (Fig. S6)). It is, however, not linear and has a regression coefficient is 0.933 thus revealing that the competitive mode is highly sensitive in comparison to the non competitive mode. Table 1 shows characteristics of the AFB1/BSA/aAFB1/4-ATP/Au immuno sensor along with some of those reported in the literature (71-73, 19, 21, 23, 37, 62). Jin et.al have enhance the sensitivity by an insoluble product produced by labeled enzyme. From the literature survey, it has been found out the majority of the reported papers are based on labeled immunosensor. 3.3.5. Real Sample testing and selectivity of immuno electrode To evaluate the applicability of the developed immunosensor to real sample analysis, corn flakes samples were spiked with various concentrations of AFB1. For this corn flakes
sample were extracted with a methanolic solution of potassium bicarbonate. Prior evaporation to dryness and final reconstitution in PBS buffer was necessary to avoid the inhibition of the antibody-antigen binding caused by methanol. The extract sample was spiked with three different concentrations of AFB1 (0.05, 2 and 5ngmL-1) to examine the applicability of the proposed probe. During these experiments, the AFB1/BSA/aAFB1/4-ATP/Au immunosensor was dipped in the cell containing a mixture of different concentrations of AFB1 in spiked extracted samples and the optimized amount of r-IgG-Cyst/Au-Fe3O4 in PBS and incubated for 35 min. The EQCM-CV of AFB1/BSA/aAFB1/4-ATP/Au immuno sensor was examined with the cornflakes extract in PBS, and indicates the minimum interference. Variation of peak current in blank and corn flake extracts [Fig.6 (bar 1, 2)] is within 5%. It indicates that the AFB1/BSA/aAFB1/4-ATP/Au immuno sensor shows specificity towards AFB1, and the levels are not inflated by other constituents present in the corn flake sample extract. However, the response of the AFB1/BSA/aAFB1/4-ATP/Au immuno sensor changes when the corn flakes sample contains AFB1 and magnitude of the anodic peak further decreases as the AFB1 concentration increases in the corn flakes sample. The results obtained with this extracted solution in PBS and result from standard PBS within 3-5% variations, showing that the developed immuno sensor is highly specific to AFB1 and avoids interference of other materials present in corn flake extracts. 3.3.6. Reproducibility, shelf life and regeneration of immuno- electrode The reproducibility of the proposed immuno electrode was estimated by repetitive measurement of the current response using 2 ngmL-1 standard AFB1 solutions in PBS (50 mM, pH 7.4, 0.9% (w/v) NaCl, containing 5mM [Fe(CN)6]3-/4-). The results obtained in 5 repeated measurements show a relative standard deviation (RSD) of 2–3%, indicating that the obtained
data is reproducible. These results demonstrate the acceptable reproducibility and precision of the proposed immuno sensor. In addition, the immunosensor was stored at 4 ̊C for shelf life (stability) study. The stability of the BSA/aAFB1/4-ATP/Au immuno electrode was evaluated by EQCM- CV and the current response in the presence of 2 ngmL-1 standard AFB1 solutions in PBS (50 mM, pH 7.4, 0.9% (w/v) NaCl) was monitored at a regular interval of 7 days (Fig.7). The immuno electrode retains its activity up to 28 day with 5-7 % decrease in activity. The response of the immmunoelectrode decreased to 95% of the initial value after 1 week and it decreased to 90% after 1 month. The immuno sensor can be regenerated (Scheme 1) using an external strong magnet to remove the immuno r-IgG-Au-Fe3O4 conjugate. It was observed that the immunosensor could be used 15-16 times with 2-3% loss in activity using two different concentrations of AFB1 (1 and 2 ng/mL). Table 2 shows the response of AFB1/BSA/aAFB1/4-ATP/Au immunoelectrode, after the regeneration. 4. Conclusions A competitive, sandwiched immunoelectrode based on an Au-Fe3O4 core shell NPs was fabricated for the detection of AFB1 using EQCM. The prepared immuno electrode can be used to detect AFB1 in the concentration range of 0.05–5 ngmL-1 with the limit of detection as 0.07 ngmL-1. This immuno sensor is found to be highly promising for detection of AFB1 in corn flakes samples. This immuno sensor can be regenerated about 15-16 times with 2-3% loss of activity. Therefore, on comparing with direct mode, the competitive mode along with electrochemical piezoelectric tool offers a highly sensitive reusable immuno sensing platform. Efforts should be made to utilize this novel magneto immunosensor for detection of other food toxins such as OTA, OTB, fumonisins and zearalenone.
Acknowledgements We thank Dr. Ashok Kumar Chauhan (Founder President, Amity University Uttar Pradesh) for providing the facilities. We also thank to Dr. (Mrs) Balvinder Shukla, Vice Chancellor Amity University Uttar pradesh and Prof. L.M. Bharadwaj, Director, AINT. We also express our deep gratitude to Ms. Shuvra Singha, Department of Chemistry, University of Hyderabad, India for her help in conducting the XRD studies.
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Table 1 : Characteristics of the AFB1/BSA/aAFB1/4-ATP/Au immunoelectrode along with those reported in the literature for aflatoxin B1 detection, with their important parameters.
Bioelectrode
Detection Method
Sensitivity
Limit of Detection (ngmL−1)
Detection range (ngmL−1)
Stability (days)
Reference
BSA-anti-AFB1/Au NPs
Electrochemical
1.4 µS /ngmL−1
0.1
0.5-10
12
71
BSA/anti-AFB1/MWCNTs/ITO
Electrochemical
95.2 µA ng/mL cm−2
0.08
0.25–1.375
45
62
aAFB1/DSP /Au
QCM
-
0.5
0.5-10
-
19
Fe3O4/SiO2/aAFB1/BSA
Quartz crystal microbalance
20.39Hz/ngmL−1
0.3
0.3– 7.0
13
21
aAFB1/BSA-AFB1/MPA/Au
Quartz crystal microbalance
33.13 Hz/ngmL−1
0.01
0.01–10.0
-
23
Nano-size gold hollowballs (NGB)
Quartz crystal microbalance
-
0.05
0.6-12.5
-
72
GCE/chitosan/AuNP/aAFB1
Electrochemical
-
0.2
0.6-110
-
37
PTH/AuNP/GCE
Electrochemical
-
0.07
0.6-2.4
-
73
BSA/aAFB1/ATP/Au
EQCM-CV
335.7 µAng1 mLcm-2
0.07
0.05-5.0
28
Present work
Tabel 2: Regeneration of r-IgG –Au-Fe3O4/AFB1/ BSA/aAFB1/4-ATP/Au immunoelectrode.
S.No.
No. of cycles
response of immunoelectrode with 1ng/mL
response of immunoelectro de with 2ng/mL
1
1st
5.72 x 10-4
4.78 x 10-4
2
5th
5.81 x 10-4
4.62 x 10-4
3
12th
5.49 x 10-4
4.66 x 10-4
4
15th
5.53 x 10-4
4.52 x 10-4