Anodic stripping voltammetry of anti-Vi antibody functionalized CdTe quantum dots for the specific monitoring of Salmonella enterica serovar Typhi

Abstract

Recent trends in electrochemical immunoassays have paved the way for metal nanoparticles bursting as a very promising technique for sensitive evaluation. In this work, we report the competence of bioconjugated CdTe quantum dot (QD) and gold nanoparticle (GNP) for the detection of Vi capsular polysaccharide antigen of Salmonella Typhi. The QD and GNP were bioconjugated with anti-Vi antibody and characterized to quantify the loading of respective nanoparticles. Cadmium chloride and gold chloride reference standards were used for the evaluation of the respective metal atoms in the nanobioprobes. The bursting of QD and GNP released 6.91 nmol of cadmium and 83.21 nmol of gold ions in 1 μL each, whereas, the anti-Vi nanobioprobe bursting resulted in the release of 17.29 nmol of cadmium and ∼34.75 nmol of gold atoms per 1 μg of antibody. The results are indicative of conjugation of multiple QDs per antibody molecule in marked contrast to the GNPs which can interact and bind with many antibodies owing to its larger size. CdTe–IgG nanobioprobe was, therefore, made use for developing a new sandwich type stripping voltammetry immunoassay in the presence of polymyxin B, a cationic receptor molecule, as a capture molecule. The stripping response observed was much convincing in the range 1 ng to 625 ng of Vi antigen indicating feasibility and reliability of the QD based stripping assay. The results provided an insight into the governing factors of immunostripping inferring the potency of biofunctionalized semiconductor/inorganic nanodots for electroanalytical applications.

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1. Introduction

The unique physicochemical properties of QDs and GNPs have proved to be useful for their application in developing highly sensitive sensing platforms.^1–4 QDs are luminescent semiconductor molecules which are, in general, made up of few hundred atoms constituting II–VI, III–V and IV–VI group elements of the periodic table.^5,6 On the other hand, GNPs are suspension of submicrometre-size gold particles distributed in colloidal manner in a fluid. Bioconjugation of these nanoparticles with receptors provides hybrid nanobioprobes that combine the unique physicochemical properties of nanoparticles with the specific and selective binding behaviour of antibodies.^7–9 These nanobioprobes have become very important from a commercial point of view and have great capability to replace enzymes in immunoassays. Couple of biosensing devices available now a days are based on metalloimmunoassay principle (http://www.agplusdiagnostics.com). Keeping in view the high importance of metal nanoparticles in electrochemical immunoassays, an attempt was made in this study to understand the competence of two differently biofunctionalized nanoparticle bioprobes in an immunoassay for monitoring Vi capsular polysaccharide of Salmonella enterica serovar Typhi, whose expression is host restricted during systemic infection.

Typhoid, being an enteric fever, is a life threatening bacterial infection caused by invasive Salmonella serovar Typhi.^10–12 The global burden of typhoid fever has been estimated to be approximately 17–22 million cases leading to 0.2–0.6 million deaths each year as per WHO records.^13,14 Although there were considerable advancements in hygienic and public sanitation systems, typhoid continues to be endemic in most of the developing countries due to inadequate sewage treatment, chaotic conditions and lack of affordable diagnostic techniques.^15,16 Detection strategies based on the conventional culturing followed by biochemical metabolite identification may not be high-throughput screening technique particularly in endemic areas as they often lag behind to diagnose within time schedule. Eventually, these inconsistent extensive laborious procedures necessitated developing a novel diagnostic strategy for clinical samples with higher sensitivity and specificity. Current identification techniques of serovar Typhi is dominated by biochemical and agglutination tests using blood isolates of patients for the identification of antibody against O (polysaccharide) and H (flagellar protein) antigen. However, O antigen is prone to cross reactivity with bacterial species other than Salmonella¹⁷ that could be a limiting factor for accurate diagnosis. From clinical point of view, the high prevalence of Vi antigen in endemic areas could be interesting as an alternate specific surface marker for identification of serovar Typhi as ∼99% of blood isolates results positive for Vi agglutination tests.¹⁸ Therefore, Vi antigen has been considered as a potent biomarker in the present study for the specific monitoring of Salmonella enterica serovar Typhi.

Metalloimmunoassays have increasingly attracted great attention in recent years.¹⁹ The large amount of metal ions quickly liberated upon bursting of a metal nanoparticles attached to an antibody probe has been widely publicized as a substitute to enzyme labelling.²⁰ The liberated metal ions are quantitated by anodic stripping voltammetry (ASV) that has proved to be exceedingly very effective and widely adopted for high sensitivity metal analysis. It has been reported that ASV is routinely capable of analyzing and quantifying trace components as low as nM level with excellent sensitivity and selectivity.²¹ ASV based detection platforms based on nanoparticles such as QDs and GNPs has inspired scientific community due to their controlled size monodispersity, very high surface area and suitability for conjugations with bioreceptors. ASV employs the initial electrodeposition of analyte of concern on the operational electrode followed by its oxidation in the stripping step. QDs and GNPs are promising for the development of sensing platforms, however, the inherent properties of these nanoparticles, which could empower the assay sensitivity in ASV is a much needed study. Hence, in the present study, the competence of these nanomaterials has been compared for their applications to immunoassay before and after their conjugation with anti-Vi IgG antibody. The strategy can be made use for other toxins/pathogens and environmental pollutants aimed at high sensitive detection platform development.

2. Experimental section

2.1. Materials and instruments

Tellurium powder (Te), cadmium chloride (CdCl₂·2.5H₂O), sodium borohydride (NaBH₄), 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), tetrachloroauric acid (HAuCl₄), 3-mercaptopropionic acid (MPA) and tri-sodium citrate were procured from Sigma-Aldrich India Pvt. Ltd (New Delhi). Maxisorp microtitre plates (flat bottom) were a product of Nunc (Roskilde, Denmark). Antibiotic polymyxin B was procured from Himedia laboratories Pvt. Ltd. (Mumbai, India). All reagents used were of analytical grade and acquired from standard suppliers. The Zensor carbon screen printed electrodes (SPEs, model TE100) were a product of CH Instruments, USA. Morphological characterization of nanoparticles was done with TEM microscope (Jeol) operating at 100 kV using carbon coated copper grids. Electrophoresis of nanoparticles and their respective nanobioprobes were carried out using Bio-Rad electrophoresis apparatus. Particle size distributions of samples were carried out using Zetasizer Nano series operating at laser 632.2 nm (Model Nano-ZS from Malvern Instruments, UK). Gel Permeation Chromatography (GPC) was carried out with Viscotek GPC system equipped with a VF-1122 solvent delivery system, P-4000 protein SEC column and VE-3580 RI detector (Malvern Instruments Ltd, Enigma Business Park, United Kingdom). All reagents were prepared in Milli-Q water with a resistivity of 18.2 MΩ cm.

2.2. Synthesis and characterization of nanoparticles

CdTe QD of ∼2.5 nm having a band gap of ∼2.4–2.6 eV and citrate capped colloidal GNP of ∼15 nm were synthesized by aqueous route as reported previously.^22,23 In brief, CdTe QD was synthesized by refluxing 25 mL of aqueous solution (pH to 9.2) of 0.02 M cadmium chloride with sodium hydrogen telluride (NaHTe) in the presence 0.05 M of MPA for 120 min under continuous argon flow. Sodium hydrogen telluride (NaHTe) was synthesized by reacting 0.03 M of NaBH₄ and 0.01 M of Te purged with argon gas till the complete dissolution of the metal leaving a faint pinkish coloured solution. Similarly, GNP was synthesized by refluxing 0.25 mM HAuCl₄ with 0.5 mM tri-sodium citrate in 18.2 MΩ water till the solution turns into intense rich red colour. Both NPs were characterized by spectral photo-absorption, zeta potential charge distribution (NPs were dissolved in 10 mM phosphate buffer, pH 7.4, at a concentration of 0.5 nM for GNP and 10 nM for CdTe QD) and transmission electron microscopic studies. The average particle size of CdTe QD and GNPs were determined by using a transmission electron microscopy (TEM, Jeol JEM-2100) operated at 120 kV. The TEM samples were prepared by placing a drop of diluted CdTe QD and GNPs separately on a carbon coated copper grid. The films on the TEM grids were allowed to dry for 5 min at room temperature before analysis.

The metal components of NPs were quantified by ASV over a SPE electrode. The SPE is made up of a carbon working electrode, carbon counter electrode, and Ag/AgCl reference electrode.^24–26 Cadmium chloride and gold chloride were used as respective standards in the range 0.3125 to 80 nmol 100 μL⁻¹. For the measurement of oxidation current, the droplet configuration of the biosensor was used employing 50 μL of acetate buffer (0.1 M, pH 4.8) and saline spread over the measurement area and covering the three screen printed electrodes. Initially, electrode was preconditioned with a potential of 0.6 V for 60 s followed by electrodeposition at −1.2 V for 5 min, and stripping from −1.2 to 0 V for cadmium and −1 V to 1.8 V for gold with 4 mV potential steps, 25 mV amplitude at a frequency of 15 Hz. The assay was realised in a microtiter plate. For the dissolution of ions, CdTe QD was treated with 0.1 N HCl and GNP with 100 μL of diluted aqua regia (1 : 10 dilution of 15.7 M HNO₃ and 11.3 M HCl in the ratio 3 : 1) and incubated for 20 minutes to oxidize the respective metal ions. Furthermore, 50 μL of 0.1 M sodium acetate buffer was added to cadmium solution and 50 μL of saline were added to gold solution for the electrochemical reduction of metal ions on the screen printed electrode (SPE) before subjecting them to square wave voltammetry (SWV) using CH-660D electrochemical workstation.

2.3. Bioconjugation and characterization of nanobioprobes

Both nanoparticles were bioconjugated with anti-Vi antibody to develop nanobioprobes in separate experiments. Anti-Vi antibody was generated in house by immunizing white New Zealand rabbit with Vi polysaccharide antigen of S. Typhi.¹² Initially, Vi antigen was purified from an over expressing culture of S. Typhi and further confirmed for its purity by gel permeation chromatography (GPC). Immunodominancy of purified Vi antigen was assessed by estimating of O-acetyl content according to WHO standard. Further, 200 μg of column purified Vi capsular polysaccharide antigen was immunized subcutaneously as per standard immunization protocol with Freund's adjuvant for three booster doses at an intervals of 21 days. Sera were drawn 7 days after each booster immunization separately and processed to isolate IgG (please see ESI† for details). CdTe QD was conjugated to anti-Vi IgG antibody (∼3 : 1) by conventional carbodiimide protocol as reported previously.^27,28 In brief, 50 μL of CdTe QD (3 μM) was activated separately in presence of 0.05 M EDC and 5 mM NHS in PBS (50 mM, pH 7.4) at 32 °C for 15 minutes at 110 RPM. Anti-Vi IgG antibody (∼0.18 mg mL⁻¹, 1 μM) in PBS was added to the activated CdTe QD and further incubated at 32 °C for 150 minutes at 110 RPM. The solution was then kept at 4 °C overnight to deactivate the remaining EDC–NHS. The conjugate solutions were purified by dialyzing against PBS using Spectra/Por dialysis membranes of 6–8 kDa cut off.

GNPs synthesized as above were used for preparing the antibody–gold conjugate (Ab–GNPs). The molar ratio of antibody and synthesized optimum GNP for conjugation was determined by critical flocculation assay principle as discussed in ESI.† During optimization, antibody concentration was varied in the range 66 pmol to 666 pmol keeping colloidal GNP at ∼250 nmol concentration. Anti-Vi IgG antibody prepared in phosphate buffer (1 mM, pH 7.4) was added drop-wise to GNPs solution under mild stirring conditions. The pH of the GNPs solution was maintained at 7.4 by addition of 10 mM K₂CO₃ before adding the antibody. The conjugation samples were treated with 50 μL of 10% NaCl and further absorption spectral change was recorded. Finally, the bioconjugate with optimized concentration was prepared and centrifuged at 10 500 g for 30 min to remove unconjugated antibody from the solution. The pellet was resuspended in phosphate buffer (10 mM, pH 7.4) and stored at 4 °C for further use.

Both nanobioprobes were characterized by spectral photo-absorption (400–700 nm) and ASV techniques as described in previous section. Variation in size as well as charge distribution was recorded by particle size analyzer.

2.4. Stripping voltammetric immunosensing of Vi capsular polysaccharide

A microtiter plate was coated with 100 μL of 0.5 mg mL⁻¹ polymyxin B in phosphate buffer saline (PBS, 100 mM, pH 7.4) and incubated overnight at 4 °C.^12,28 The plate was washed three times with PBS and was blocked by 5% sucrose. 100 μL of Vi capsular polysaccharide antigen (1 ng to 1250 ng) was added to each well in PBS and incubated for 1 h at 37 °C followed by washing three times with PBS containing 0.05% Tween-20 (PBST). The anti-Vi antibody conjugates of GNP and CdTe QD (1 : 1000) prepared in PB (100 mM, pH 7.4) was added to each well in separate experiments. The plate was then incubated for 1 h at 37 °C. After washing three times with PBS-T, assay was realised by subjecting to ASV as discussed in previous section. The principle of ASV based immunoassay has been illustrated in Scheme 1.

Scheme 1 Schematic presentation of anodic stripping voltammetry (ASV) based electro-immunoassay. The immunoassay was realised in a microtitre plate followed by bursting of the nanoparticles. The bursted solution was analyzed by ASV in a screen printed electrode. (1) Microtiter plate, (2) polymyxin B, (3) Vi capsular polysaccharide, (4) QD–anti-Vi IgG conjugate, (5) GNP–anti-Vi IgG conjugate, (6) Screen Printed Electrode (SPE).

3. Results and discussion

3.1. Characterization of QDs and GNPs

QD and GNPs were synthesized by aqueous route to obtain colloidal particles with homogenous distribution. Both NPs were characterized by spectral absorption, zeta potential charge distribution and transmission electron microscopic studies. Initial absorption spectral analysis revealed characteristic broad absorption spectrum for CdTe QD with first excitonic peak at 493 nm and characteristic plasmon absorption maxima at 519 nm for GNPs confirming the successful NPs synthesis (Fig. 1a). TEM studies revealed the uniform shape and homogenous distribution of respective nanoparticles (Fig. 1b and c). Zeta potential distribution of thus synthesized NPs was −48.2 mV and −39.1 mV respectively for CdTe QD and GNP (Fig. 2a and b). Further, both NPs were electrochemically characterized and their respective metal components were quantified using cadmium chloride and gold chloride salts as reference standards. The response vs. concentration of ions were fitted with double logarithmic power fit equations and was convincing with a regression coefficient R² = 0.987 for cadmium and R² = 0.99 for gold ions in the range 0.3125 to 80 nmol (Fig. 3a and b). From the standard graphs it was calculated that the CdTe QD and GNP consist of 6.91 nmol of cadmium and 83.21 nmol of gold atoms respectively in 1 μL solution (please see ESI† for details). The relative response change obtained against the concentration gradient was better with cadmium stripping in comparison to gold stripping. Eventually, this trend with cadmium ion could be a better choice for electro-analytical technique development as the limit of saturation of response in terms of current against concentration may be far away from working range. This difference could be attributed to the basic electrochemical behaviour of respective metal ions.

Fig. 1 Characterization of nanoparticles (a) absorption spectra of CdTe QD, GNP and GNP–IgG, (b) transmission electron micrographs of GNPs, (c) transmission electron micrographs of CdTe QD.

Fig. 2 Zeta potential distribution of CdTe QD, GNP, IgG and their conjugates.

Fig. 3 Standard graphs of (a) cadmium and (b) gold atoms generated by SWV. Voltammetry analysis was done at an initial current of −1.2 to 0 V for cadmium and −1 V to 1.8 V for gold with 4 mV potential steps, 25 mV amplitude at a frequency of 15 Hz. Inset picture shows the response graph of respective metals in SWV.

3.2. Stripping voltammetry behaviour of gold and cadmium

Cadmium being a transition metal shows oxidation at −0.8 V and for gold it is around +0.7 to +1 V (inset of Fig. 3a and b) consistent with previous reports.^24,26 It was reported that gold tends to form mixed chlorohydroxy complexes at pH > 6 resulting from the exchange of Cl⁻ by OH⁻.²⁴ This tendency of gold to form mixed chlorohydroxy complexes other than most stable [AuCl₄]⁻ state from reduced Au⁰ might be the reason behind the gradual shifting of oxidation peak towards higher potential over an increasing concentration with a shoulder peak at +1.1 V. The oxidation range observed (0.75 V to 0.95 V) infers the presence of mixed chlorohydroxy complexes such as [AuCl_x(OH)_4−x]⁻, where x = 0–4. This fundamental difference in turn might have influenced the potential oxidation of respective metal ions alongside other factors such as electronic configuration and ionization energy thereby reducing the potency of gold ions to get oxidised when compared to easy oxidation of cadmium. Generally, it is difficult to pullout valence electrons from gold ion due to its higher ionization energy in comparison to that of cadmium whose ionization energy is lower.^29,30 Therefore, over a wide concentration range, gold ions could not effectively get oxidised as it was in the case of cadmium ions stripping. The positive reduction potential of gold (Au³⁺ + 3e⁻ → Au⁰ at ∼+1.692 V) shifts its redox equilibrium preferably towards reduced state, as reported^31,32 consequently contributed for declined oxidation trend. On the other hand, cadmium with net negative reduction potential in the electrochemical series prefers to get oxidised and hence might have not reached saturation even at higher concentration (Fig. 3a and b). The results of stripping analysis showed higher analytical performance for bare QDs in comparison with the GNPs.

3.3. ASV based immunosensing of Vi capsular polysaccharide

3.3.1. Biofunctionalization of nanoparticles for immunosensing. Both CdTe QD and GNP were biofunctionalized by conjugating them with anti-Vi antibodies generated in house. The antibodies were characterized for their selectivity and specificity, as has been reported in our previous work.¹² The anti-Vi antibody was covalently conjugated with CdTe QD and confirmed based on the difference in size and net molecular charge on the conjugate as reported by Vinayaka and Thakur.²⁷ On the other hand the bioconjugation of GNP with anti-Vi antibody was performed using charge-coupled electrostatic interactions and confirmed by shift in its plasmon absorption. The characteristic peak shift in the plasmon resonance absorption towards lower energies from 519 nm to 527 nm indicated change in local dielectric constant as a result of successful bioconjugation (Fig. 1a).^33,34 Both nanobioprobes were subjected to dynamic light scattering (DLS) studies to comprehend the effect of anti-Vi antibody on respective nanoparticles upon bioconjugation. There was a significant shift in the zeta potential distribution of the anti-Vi antibody after conjugation with nanoparticles. The negative potential increased from −13.1 mV for anti-Vi antibody to −31.8 mV for CdTe–IgG conjugate after covalent conjugation and −25.1 mV for GNP–IgG conjugate after electrostatic interaction (Fig. 2a and b). Hence, a decrease in the net zeta potential distribution of anti-Vi antibody is an inference of the utilization of positively charged primary amine functional moieties by both nanoparticles although conjugation procedures are different. Consequently, an increase in the net negative potential distribution stabilized these nanobioprobes rendering more inter-particle repulsive force. In addition, these observations were also endorsed by the increased molecular size of anti-Vi antibody after conjugation. The average hydrodynamic size (in diameter) of anti-Vi antibody increased from 8.72 nm to 15.69 nm for CdTe bioprobe and 18.17 nm for GNP bioprobe wherein, GNP was having a hydrodynamic size of 13.54 nm (Fig. 4a and b). This shift in size is an indicative of lesser Brownian motion and diffusion coefficient rate as a consequence of increased particle size and density. There was also an increase in the size of nanobioprobe, which was revealed by agarose gel electrophoresis, as the mobility of nanobioprobe got decreased under electric field (Fig. S1†). Thus, spectral and DLS analysis as well as gel electrophoresis confirmed the successful bioconjugation of both the nanoparticles.

Fig. 4 Size distributions of (a) IgG, GNP, GNP–IgG conjugates and (b) CdTe and CdTe–IgG conjugate.

The difference in biomolecule loading capacity of both the nanoparticles may significantly affect their performance in the immunoassay. The quantification of the nanoparticles loading of biomolecules in the nanobioprobes was performed by electrochemical evaluation method employing the standard plot generated with respective metal ions (Fig. 3a and b). After respective nanoparticle bursting followed by metal ion stripping, it was quantified that 1 μg of anti-Vi antibody (6.67 pmol) was loaded with 17.29 nmol of cadmium that corresponds to ∼7–8 QDs per IgG molecule considering ∼330 Cd ions per QD molecule. In the contrary, it was estimated that 1 μg of anti-Vi antibody was loaded with ∼34.75 nmol of gold ions that corresponds to ∼11 IgG molecules per GNP (please see ESI† for details).

In general, bioconjugation potency of nanoparticles and proteins relies upon various factors that determine the efficacy of a bioanalytical technique.²⁶ In the present study, charge-coupled electrostatic interactions were basically adopted for biofunctionalization of citrate capped GNPs with anti-Vi antibody and CdTe QD were conjugated by covalent coupling chemistry. ASV quantified values indicated ∼8.2 times more loading of CdTe QD on anti-Vi antibody in marked contrast to the GNP's that was ambiguous to the findings on hydrodynamic studies of the respective conjugates with DLS. There was a minor difference in the hydrodynamic size (in diameter) of both nanobioprobes (Fig. 4a and b) though molecular size ratio between them is significantly larger. These results infer possibility of vital role of molecular size in their conjugation efficacy. CdTe QD used in this study was about 2.5 nm in diameter, whereas GNP was about 13.5 nm. Thus, anti-Vi antibody has a molecular size ratio (in nm) of 3.488 with CdTe QD (IgG : CdTe) whereas it was only 0.644 with GNP (IgG : GNP) (Fig. 4a and b). Consequently, probability of steric hindrance affecting the conjugation was higher in the case of GNP due to its bulkiness and direct interactions with antibody. On the other hand, QDs were smaller in size having an organic capping layer that acts as a linker molecule facilitated covalent conjugation keeping QDs sufficiently away from the proximity of the antibody to overcome the steric hindrance effects. These aspects in turn might have allowed anti-Vi antibody to have higher load of CdTe QDs. The outcome, although not surprising considering the differences in the electrochemical response of respective nanoparticles, supports our argument that bursting of antibody conjugated GNP was not efficient enough probably due to the insulation of GNPs with antibodies. Considering these factors, immunostripping assay for the detection of S. Typhi was developed with IgG–CdTe QD nanobioprobe in a sandwich immunoassay format.

3.3.2. Application of CdTe nanobioprobes in sandwich immunoassay format. The well characterized CdTe nanobioprobe was used as electrochemical reporter in a sandwich immunoassay format for monitoring Vi antigen of S. Typhi. Previously we have reported the potency of Vi polysaccharide as a specific biomarker for bioassay development.¹² Vi being a capsular polysaccharide is expressed during systemic infection by Salmonella enterica serovar Typhi and hence has been purified by culture grown under simulated physiological conditions. GPC confirmed the purity of the extracted Vi polysaccharide sample with a single major peak observed at a retention volume of 12.85 mL. The O-acetyl content in the purified Vi antigen was ∼13 micromoles per mL that inferred the immunodominancy of antigen. O-Acetyls groups that protrude outside from the planar center of the Vi polysaccharide molecule provide the structural complexity necessary for high affinity antibody generation. Thus, anti-Vi IgG antibody raised against Vi antigen exhibited an affinity of 6 × 10⁹ M⁻¹ signifying immunodominancy of O-acetyls groups.¹²

Polymyxin B was used as a primary capturing reagent in this sandwich assay. Polymyxin B is a cationic polypeptide possessing bactericidal activity, produced by Bacillus polymyxa. The interaction between polymyxin B and Vi capsular polysaccharide antigen are mainly electrostatic charged based interactions.^12,35 The binding ability of polymyxin B towards anionic lipopolysaccharides renders a unique advantage for the development of a unique sandwich immunoassay employing single antibody. The response observed with CdTe–IgG as reporter probe was convincing in the range 1 ng to 625 ng of Vi antigen concentration tested with a regression coefficient R² = 0.979 (Fig. 5). The response in terms of current was convincing even at higher concentrations indicating feasibility and reliability of the QD based stripping assay without saturation of the sensor surface (Fig. S2†).

Fig. 5 Standard graph for Vi antigen detection (1 ng to 625 ng) generated by ASV using IgG–CdTe QD nanobioprobe.

4. Conclusion

In summary, present study was focussed to provide an insight into the influence of unique physicochemical properties of QDs and GNPs in ASV technique. Perspectives on the fundamental physicochemical properties of these nanomaterials and their bioconjugates were a much needed study as they could empower the assay sensitivity. In this regard, an interdisciplinary approach has been adopted by integrating them with immunosensing principle for developing an electroanalytical sensing platform. Anodic stripping response observed for cadmium chloride was better than gold chloride suggesting its ability to cover broad working concentration range advocating its suitability as a prime choice for electro-analytical technique development. ASV also quantified bioconjugated nanoparticles and indicated ∼8 times more loading of CdTe QD on anti-Vi antibody than GNP. The response observed with CdTe–IgG as reporter probe in ASV based sandwich immunoassay was much convincing and satisfactory. Thus, the response observed with CdTe–IgG as reporter probe was convincing in the range 1 ng to 625 ng of Vi antigen concentration tested. The combination of Vi antigen as a specific surface marker and the binding ability of polymyxin B towards anionic lipopolysaccharides demonstrated high specificity and accuracy of the developed immunoassay for serovar Typhi. This study provides a way to quantify the exact ionic concentration of QDs and GNPs and further to characterize the efficiency of their respective immunoconjugates. The developed immuno-stripping technique has great potential to be used for regular monitoring of typhoid fever in diagnostic laboratories and can also be adoptable for various other food and environmental contaminants.

Acknowledgements

The authors thank Department of Science and Technology (DST), India for providing the required financial support for this project. S. K. P. and A. C. V. are thankful to CSIR, India, for providing research fellowships. Authors also thank Dr V. K. Bhalla, Scientist, CSIR-IMTECH for his kind help.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra13465j

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