Luminol-Based Turn-On Fluorescent Sensor for Selective and Sensitive Detection of Sulfur Mustard at Ambient Temperature.

We have explored a novel turn-on fluorescence detection of sulfur mustard (SM) at "room temperature". The innovative protocol that uses the combination of luminol and an ionic liquid in water exhibits fluorescence detection of SM within seconds. In this simple, fast, and low-cost chemosensing method, luminol acts as the receptor as well as a signaling element, and the ionic liquid (1-ethyl-3-methylimidazolium dicyanamide) provides the requisite and polarizing medium to realize the detection at "room temperature". Interestingly, with a higher concentration of a probe (0.56 mM), SM sensing can be visualized with the naked eye, leading to the formation of a fluorescent green color within a minute, thus expanding the application of the developed sensing technique for chromo-fluorogenic detection of SM. Excellent selectivity, sensitivity (LOD: 6 ppm), and chemosensing at ambient temperature make this methodology completely field-deployable.

Surface plasmon resonance sensing of Ebola virus: a biological threat.

Here, different monoclonal antibodies (mAb1, mAb2 and mAb3) of Ebola virus were screened in a real-time and label-free manner using surface plasmon resonance (SPR) to select an appropriate antibody for biosensor applications against a biological warfare agent. For this purpose, a gold SPR chip was modified with 4-mercaptobenzoic acid (4-MBA), and modification was confirmed by FTIR-ATR and EIS. The 4-MBA-modified gold SPR chip was used for immobilization of the recombinant nucleoprotein of Ebola (EBOV-rNP), and the interactions of mAb1, mAb2 and mAb3 were then investigated to determine the best mAb based on the affinity constant (KD), expressed as equilibrium dissociation constant. KD values of 809 nM, 350 pM and 52 pM were found for the interaction of mAb1, mAb2 and mAb3 of Ebola with the immobilized EBOV-rNP, respectively, thus reflecting the high affinity of mAb3. This was confirmed by ELISA results. The thermodynamic parameters (ΔG, ΔH and ΔS) for the interaction between mAb3 and EBOV-rNP were also determined, which revealed that the interaction was spontaneous, endothermic and driven by entropy. The SPR limit of detection of EBOV-rNP with mAb3 was 0.5 pg ml-1, showing mAb3 to be the best high-affinity antibody in our study. This study has opened up new possibilities for SPR screening of different monoclonal antibodies of BWA through the convergence of materials science and optical techniques.

Development and validation of immunoassay for whole cell detection of Brucella abortus and Brucella melitensis.

Brucella is alpha-2 Proteobacteria mainly responsible for multi-factorial bacterial zoonotic disease brucellosis with low concentration (10-100 CFU) required to establish the infection. In this study, we developed sandwich ELISA with detection range of 102 to 108 cells mL-1 and limit of detection at 103 cells mL-1 by employing polyclonal rabbit IgG (capture antibody, 10 µg mL-1) and mice IgG (detection antibody, 50 µg mL-1) antibody for its detection. Surface Plasmon Resonance evaluated the interaction of detection antibody with whole cell spiked serum samples at LOD of 102 cells mL-1 along with non co-operative interaction of protein albumin. Further, kinetic evaluation study using detection antibody against cell envelope antigen was performed whereby, Equilibrium Dissociation Constant (KD) and Maximum Binding Capacity (Bmax) were found to be 16.48 pM and 81.67 m° for Brucella abortus S99 and 0.42 pM and 54.50 m° for Brucella melitensis 16 M, respectively. During interference study, sandwich ELISA assay cross-reacted with either of the polyclonal antibody of above Brucella species. Upon validation, no cross-reactivity observed with bacteria-closely related to Brucella. In conclusion, developed semi-quantitative sandwich immunoassay is sensitively rapid in whole cell detection of Brucella and will be useful in development of detection assays from environmental and clinical matrices.

Photoelectrocatalytic degradation of vesicant agent using Eu/ZnO/pPy nanocomposite.

Herein, we demonstrate a nanocomposite material Eu/ZnO/pPy for enhanced performance in photoelectrocatalytic degradation of chemical warfare agent sulphur mustard (SM) at ambient conditions which is growing concern of the Scientific Community amidst the current climate of terrorism. Eu/ZnO/pPy was electrochemically prepared on Au electrode at ambient conditions and was used for electrocatalytic reductive elimination of chloride from SM and results indicated one electron involvement process for the cleavage of the carbon-chloride bond. Surface morphology of Eu/pPy, ZnO/pPy and Eu/ZnO/pPy composites were characterized by SEM and confirmed the formation of the nanoparticles and nanorods on the modified electrode which leads to provide more surface area for the reductive elimination reaction. The elemental composition, functional groups and phase of materials on the modified electrode were deduced using EDX, Raman spectroscopy and XRD, respectively. Eu/ZnO/pPy/Au electrode was utilized for the photoelectrocatalytic degradation of SM as it exhibit excellent electrocatalytic activity and degradation products were analyzed by GC-MS. In the reductive elimination of SM, the following parameters were deduced (i) heterogeneous rate constant (0.127 s-1), (ii) transfer coefficient (0.32) and (iii) number of electron involved (1.0). The enhanced photoelectrocatalytic capability of this nanocomposite could serve as a novel and promising catalyst in defence and environmental applications.

DNA-probe-target interaction based detection of Brucella melitensis by using surface plasmon resonance.

Surface plasmon resonance (SPR) immunosensor using 4-mercaptobenzoic acid (4-MBA) modified gold (4-MBA/Au) SPR chip was developed first time for the detection of Brucella melitensis (B. melitensis) based on the screening of its complementary DNA target by using two different newly designed DNA probes of IS711 gene. Herein, interaction between DNA probes and target molecule are also investigated and result revealed that the interaction is spontaneous. The kinetics and thermodynamic results derived from the experimental data showed that the interaction between complementary DNA targets and probe 1 is more effective than that of probe 2. Equilibrium dissociation constant (KD) and maximum binding capacity of analyte (Bmax) values for the interaction of complementary DNA target with the immobilized DNA probes were calculated by using kinetic evaluation software, and found to be 15.3 pM (KD) and 81.02m° (Bmax) with probe 1 and 54.9pM and 55.29m° (Bmax), respectively. Moreover, real serum samples analysis were also carried out using immobilized probe 1 and probe 2 with SPR which showed the applicability of this methodology and provides an alternative way for the detection of B. melitensis in less than 10min. This remarkable sensing response of present methodology offer a real time and label free detection of biological warfare agent and provide an opportunity to make miniaturized sensor, indicating considerable promise for diverse environmental, bio-defence, clinical diagnostics, food safety, water and security applications.

Direct electrochemistry of horseradish peroxidase bonded on a conducting polymer modified glassy carbon electrode.

Direct electron transfer process of immobilized horseradish peroxidase (HRP) on a conducting polymer film, and its application as a biosensor for H2O2, were investigated by using electrochemical methods. The HRP was immobilized by covalent bonding between amino group of the HRP and carboxylic acid group of 5,2':5',2"-terthiophene-3'-carboxylic acid polymer (TCAP) which is present on a glassy carbon (GC). A pair of redox peaks attributed to the direct redox process of HRP immobilized on the biosensor electrode were observed at the HRPmid R:TCAPmid R:GC electrode in a 10 mM phosphate buffer solution (pH 7.4). The surface coverage of the HRP immobilized on TCAPmid R:GC was about 1.2 x 10(-12) mol cm(-2) and the electron transfer rate (ks) was determined to be 1.03 s(-1). The HRPmid R:TCAPmid R:GC electrode acted as a sensor and displayed an excellent specific electrocatalytic response to the reduction of H2O2 without the aid of an electron transfer mediator. The calibration range of H2O2 was determined from 0.3-1.5 mM with a good linear relation.

Surface plasmon resonance characterization of monoclonal and polyclonal antibodies of malaria for biosensor applications.

Surface plasmon resonance (SPR) screening of monoclonal and polyclonal antibodies of Plasmodium falciparum (MoabPf and PoabPf) for recombinant Histidine rich protein-II antigen (Ag) of Pf (rHRP-II Ag) was conducted in a real-time and label-free manner to select an appropriate antibody (Ab) for biosensor applications. In this study 4-mercaptobenzoic acid (4-MBA) modified gold SPR chip was used for immobilizing the Ag and then Ab was interacted. SEM image showed modification of SPR chip with 4-MBA and EDAX confirmed the presence of 4-MBA on the SPR chip. Equilibrium constant (KD) and maximum binding capacity of analyte (Bmax) values for the interaction of MoabPf or PoabPf with the immobilized rHRP-II Ag were calculated and found to be 0.517 nM and 48.61 m° for MoabPf and 2.288 nM and 46.80 m° for PoabPf, respectively. In addition, thermodynamic parameters such as ΔG, ΔH and ΔS were determined for the interaction between rHRP-II Ag and MoabPf or PoabPf and the values revealed that the interaction is spontaneous, exothermic and driven by entropy. The kinetics and thermodymanic results of this study revealed that the interaction between MoabPf and rHRP-II Ag is more effective than that of PoabPf due to the fact that MoabPf was derived from a single epitope (single clone) whereas the PoabPf was from the mixture of a number of epitopes (polyclones). Finally, SPR methodology was developed for the sensing of malarial antibodies. The limit of detection was found to be 5.6 pg with MoabPf which was found to be the best in our study.

Detection of protective antigen, an anthrax specific toxin in human serum by using surface plasmon resonance.

In this study, surface plasmon resonance (SPR) technology was used for the sensitive detection of protective antigen (PA), an anthrax specific toxin in spiked human serum samples. A monoclonal antibody raised against Bacillus anthracis PA was immobilized on carboxymethyldextran-modified gold chip, and its interaction with PA was characterized in situ by SPR. By using kinetic evaluation software, KD (equilibrium constant) and Bmax (maximum binding capacity of analyte) were found to be 20 fM and 18.74 m°, respectively. The change in Gibb's free energy (∆G= -78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 1 pg/mL purified PA. In PA-spiked human serum samples, 10 pg/mL of PA could be detected. Presence of PA in blood samples serves as an important early diagnostic marker for B. anthracis infections. Thus, SPR test can be a sensitive assay for detection of anthrax at early stages of infection.

Deletion mutant comprising 198 residues of BoNT/A toxin receptor binding domain retained gt1b binding property but failed to induce protective antibody response in a mouse model.

The most effective protection against toxin is inducing protective immune response through vaccination that will produce neutralizing antibodies. Antibodies will bind to and clear toxin from the circulation before it can enter nerve cells and block neurotransmission and can also be used for development of detection system. In the present study we constructed a deletion mutant of the binding domain (1098-1296) to produce smallest toxin fragment as vaccine candidate against BoNT/A. The BoNT/A-HCC protein was highly expressed in Escherichia coli SG13009 and found to form inclusion bodies. The purified inclusion bodies were solubilized in 6 M guanidine-HCl containing 10 mM ß-mercaptoethanol and 20 mM imidazole and the rBoNT/A-HCC was purified and refolded in a single step on Ni2+ affinity column. The purified protein was ∼98 % pure as assessed by SDS-polyacrylamide gel with the yield of 8 mg/L and showed binding to polysialoganglioside (GT1b). The rBoNT/A-HCC at dose of 40 µg/mouse generated high IgG antibody titre with predominance of IgG1 subtype, but failed to protect animals against BoNT/A challenge. Antibody titre in serum was determined by enzyme linked immunosorbent assay and specific binding to rBoNT/A-HCC was demonstrated by surface plasmon resonance (SPR), with a dissociation constant of 0.8 pM.