Bifunctionalized nanobioprobe based rapid color-shift assay for typhoid targeting Vi capsular polysaccharide.

Typhoid fever is an acute illness caused by Salmonella Typhi and the current diagnostic gap leads to inaccurate, over-diagnosis of typhoid leading to excessive use of antibiotics. Herein, to address the challenges we describe a new rapid color-shift assay based on a novel bifunctional nanobioprobe (Vi-AgNP probe) that is functionalized with specific biomarker Vi polysaccharide and also has the co-presence of Ag as urease inhibitor. The immunoreactions between the Vi with specific antibodies (Abs) present in typhoid patient sample forms a shielding barrier over Vi-AgNP probe rendering the urease to be active, generating colored output. Vi polysaccharide coating on the AgNP was visualized using HRTEM. TEM was performed to get insight into shielding barrier formation by the Abs. MST (microscale thermophoresis) data showed less binding Kd of 7.43 µM in presence of Abs whereas probe with urease showed efficient binding with Kd 437 nM. The assay was validated using 53 human sera samples and proven effective with 100% sensitivity. The assay showed relative standard deviation (RSD) of 4.3% estimated using rabbit anti-Vi Abs. The entire procedure could be completed within 15 min. Unlike lateral flow based assays, our assay does not require multiple combination of Abs for detection. The assay format was also found compatible in paper strip test that provides promising opportunities to develop low-cost on-spot assay for clinical diagnostics.

UV-FIA: UV-induced fluoro-immunochemical assay for ultra-trace detection of PETN, RDX, and TNT.

Fluorescence quenching based immunoassay format for the detection of a trace amount of some nitro-explosives with a high degree of selectivity is reported in this study. The immunoassay comprises anti-explosive antibodies functionalized microtitre strips specific to the targeted explosives, pentaerythritol tetranitrate (PETN), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and 2,4,6-trinitrotoluene (TNT). UV induced photolysis of nitro-explosive bound to targeted antibodies generates primarily nitrite ions which after the quick reaction with the detector molecule, 2,3-diaminonaphthalene (DAN), a fluorophore, quenches its fluorescence intensity, however, proportionately undergo cyclization to produce a highly fluorescent product, 2,3-naphthotriazole (NAT). The synthesized product, NAT, was verified using various chromatographic and spectrophotometric techniques. This newly developed antibody-based detection method, utilizing DAN dye, demonstrated a high selectivity towards PETN, RDX, and TNT. This method can be used as an economical testing kit for direct quantification of explosives, implying the great potential for quick, low-cost trace detection of explosives.

Trace detection of some nitro-explosives using thermal mediated immunochemical defragmented method.

A new immunoassay format using thermally induced defragmentation of some nitro-explosives with a high degree of selectivity is reported. Specific antibodies against three widely used explosives, 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and pentaerythritol tetranitrate (PETN) were generated by designing suitable haptens using geometry optimization modules. These in-house generated antibodies were used in a newly developed thermal mediated immunochemical biosensing technique which involves the binding of specific antibodies to respective nitro-explosives on a microtiter strip, resulting in the formation of specific immunocomplex. Heating the specific immuno-complex formed on microtiter wells resulted in thermal lysis of nitro-explosives to generate nitrite ions. These ions react with Griess reagent to form a colored chromophore which correlates the concentration of individual explosive in the sample. The present work fulfills the need for an improved explosive detecting system that is highly specific and capable of quickly determining the presence of nitrate containing explosives from a mixture pool.

Aptamer functionalized MoS2-rGO nanocomposite based biosensor for the detection of Vi antigen.

We report a novel aptamer functionalized MoS2-rGO based electrochemical method for Vi polysaccharide antigen mediated detection of enteric fever. Herein, highly selective anti-Vi aptamers were screened from a pool of oligonucleotides using a microtitre based SELEX approach and characterized for its specificity and stability. The MoS2-rGO nanocomposite was synthesized using a liquid assisted exfoliation by taking optimum ratio of MoS2 and rGO. The nanocomposite presented synergistic effect owing to easy biomolecular functionalization and enhanced conductivity. The screened anti-Vi aptamers were embedded on the MoS2-rGO nanocomposite via thiol linkage to give a stable biointerface. The developed aptasensor was characterized and further evaluated for its performance with different concentrations of Vi antigen using ferrocene labeled boronic acid as an electroactive probe. The aptasensor responded linearly in the range between 0.1 ng mL-1 to 1000 ng mL-1with a detection limit of 100 pg mL-1, and did not show any cross-reactivity with other bacterial polysaccharides indicating high specificity. The applicability of the developed aptasensor was further validated in urine and sera specimens spiked with Vi antigen.

An immunochromatographic dipstick as an alternate for monitoring of heroin metabolites in urine samples.

Heroin use and addiction pose serious risks and side effects due to overdose. Quantification of heroin in biological samples is challenging due to rapid deacetylation of heroin to its active metabolites. In this study, we report the quantification of metabolic degradation of heroin by-products in biological urine samples. The presence of the drug was monitored after oral administration of heroin at different time intervals. Various biophysical techniques, such as high performance liquid chromatography (HPLC) and mass spectrometry (MS) were used to evaluate the presence of the drug. A competitive fluorescence based immunoassay was developed with a limit of detection (LOD) up to 0.01 ng mL-1 and the IC50 value was 0.1 ng mL-1, while the dipstick assay shows a LOD up to 5 ng mL-1. Rapid detection of narcotic drugs was carried out for biological urine samples collected at various time points. Validation of the developed dipstick was carried out for the standard as well as the spiked urine samples by fluorescence based immunoassay (FIA), using anti-morphine antibodies. A strong correlation (R = 0.94) was obtained between the developed dipstick and FIA assay for biological urine samples collected at various time points. The developed immunochromatographic dipstick is highly sensitive, field applicable and cost effective, and can serve as a first choice for the monitoring of narcotic drugs in blood, urine and saliva in drug addicts and athletes.

A label-free electrochemical immunosensor for the detection of cardiac marker using graphene quantum dots (GQDs).

A label-free immunosensor based on electrochemical impedance spectroscopy has been developed for the sensitive detection of a cardiac biomarker myoglobin (cMyo). Hydrothermally synthesized graphene quantum dots (GQDs) have been used as an immobilized template on screen printed electrodes for the construction of an impedimetric sensor platform. The GQDs-modified electrode was conjugated with highly specific anti-myoglobin antibodies to develop the desired immunosensor. The values of charge transfer resistance (Rct) were monitored as a function of varying antigen concentration. The Rct value of the immunosensor showed a linear increase (from 0.20 to 0.31kΩ) in the range of 0.01-100ng/mL cMyo. The specific detection of cMyo was also made in the presence of other competing proteins. The limit of detection for the proposed immunosensor was estimated as 0.01ng/mL which is comparable to the standard ELISA techniques.

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

A specific surface antigen, OmpD has been reported first time as a surface biomarker in the development of selective and sensitive immunosensor for detecting Salmonella typhimurium species. The OmpD surface antigen extraction was done from Salmonella typhimurium serovars, under the optimized growth conditions for its expression. Anti-OmpD antibodies were generated and used as detector probe in immunoassay format on graphene-graphene oxide (G-GO) modified screen printed carbon electrodes. The water samples were spiked with standard Salmonella typhimurium cells, and detection was done by measuring the change in impedimetric response of developed immunosensor to know the concentration of serovar Salmonella typhimurium. The developed immunosensor was able to specifically detect S. typhimurium in spiked water and juice samples with a sensitivity upto 10(1)CFUmL(-1), with high selectivity and very low cross-reactivity with other strains. This is the first report on the detection of Salmonella typhimurum species using a specific biomarker, OmpD. The developed technique could be very useful for the detection of nontyphoidal Salmonellosis and is also important from an epidemiological point of view.

Combined molecular dynamics and continuum solvent approaches (MM-PBSA/GBSA) to predict noscapinoid binding to γ-tubulin dimer.

γ-tubulin plays crucial role in the nucleation and organization of microtubules during cell division. Recent studies have also indicated its role in the regulation of microtubule dynamics at the plus end of the microtubules. Moreover, γ-tubulin has been found to be over-expressed in many cancer types, such as carcinomas of the breast and glioblastoma multiforme. These studies have led to immense interest in the identification of chemical leads that might interact with γ-tubulin and disrupt its function in order to explore γ-tubulin as potential chemotherapeutic target. Recently a colchicine-interacting cavity was identified at the interface of γ-tubulin dimer that might also interact with other similar compounds. In the same direction we theoretically investigated binding of a class of compounds, noscapinoids (noscapine and its derivatives) at the interface of the γ-tubulin dimer. Molecular interaction of noscapine and two of its derivatives, amino-noscapine and bromo-noscapine, was investigated by molecular docking, molecular dynamics simulation and binding free energy calculation. All noscapinoids displayed stable interaction throughout simulation of 25 ns. The predictive binding free energy (ΔGbind) indicates that noscapinoids bind strongly with the γ-tubulin dimer. However, bromo-noscapine showed the best binding affinity (ΔGbind = -37.6 kcal/mol) followed by noscapine (ΔGbind = -29.85 kcal/mol) and amino-noscapine (ΔGbind = -23.99 kcal/mol) using the MM-PBSA method. Similarly using the MM-GBSA method, bromo-noscapine showed highest binding affinity (ΔGbind = -43.64 kcal/mol) followed by amino-noscapine (ΔGbind = -37.56 kcal/mol) and noscapine (ΔGbind = -34.57 kcal/mol). The results thus generate compelling evidence that these noscapinoids may hold great potential for preclinical and clinical evaluation.

Molecular mechanism of poly(vinyl alcohol) mediated prevention of aggregation and stabilization of insulin in nanoparticles.

It is a challenge to formulate polymer based nanoparticles of therapeutic proteins as excipients and process conditions affect stability and structural integrity of the protein. Hence, understanding the protein stability and complex aggregation phenomena is an important area of research in therapeutic protein delivery. Herein we investigated the comparative role of three kinds of surfactant systems (Tween 20:Tween 80), small molecular weight poly(vinyl alcohol) (SMW-PVA), and high molecular weight PVA (HMW-PVA) in prevention of aggregation and stabilization of hexameric insulin in poly(lactide-co-glycolide) (PLGA) based nanoparticle formulation. The nanoparticles were prepared using solid-in-oil-in-water (S/O/W) emulsification method with one of the said surfactant system in inner aqueous phase. The thermal unfolding analysis of released insulin using circular dichroism (CD) indicated thermal stability of the hexameric form. Insulin aggregation monitored by differential scanning calorimetry (DSC) suggested the importance of nuclei formation for aggregation and its prevention by HMW-PVA. Additional guanidinium hydrochloride based equilibrium unfolding and in silico (molecular docking) studies suggested maximum stability of released insulin from formulation containing HMW-PVA (F3). Furthermore, in vivo studies of insulin loaded nanoparticle formulation (F3) in diabetic rats showed its bioactivity. In conclusion, our studies highlight the importance of C-terminal residues of insulin in structural integrity and suggest that the released insulin from formulation containing HMW-PVA in inner aqueous phase was conformationally and thermodynamically stable and bioactive in vivo.

Recent advances in immunosensor for narcotic drug detection.

INTRODUCTION: Immunosensor for illicit drugs have gained immense interest and have found several applications for drug abuse monitoring. This technology has offered a low cost detection of narcotics; thereby, providing a confirmatory platform to compliment the existing analytical methods. METHODS: In this minireview, we define the basic concept of transducer for immunosensor development that utilizes antibodies and low molecular mass hapten (opiate) molecules. RESULTS: This article emphasizes on recent advances in immunoanalytical techniques for monitoring of opiate drugs. Our results demonstrate that high quality antibodies can be used for immunosensor development against target analyte with greater sensitivity, specificity and precision than other available analytical methods. CONCLUSION: In this review we highlight the fundamentals of different transducer technologies and its applications for immunosensor development currently being developed in our laboratory using rapid screening via immunochromatographic kit, label free optical detection via enzyme, fluorescence, gold nanoparticles and carbon nanotubes based immunosensing for sensitive and specific monitoring of opiates.

One step in-situ synthesis of amine functionalized graphene for immunosensing of cardiac marker cTnI.

2-Aminobenzyl amine (2-ABA) functionalized graphene is proposed for the ultrasensitive immunosensing of Cardiac Troponin I (cTnI). 2-ABA was electrochemically polymerized on the graphene decorated interdigitated electrode to obtain the amine functionalized graphene (f-GN). The f-GN electrode was then modified with monoclonal anti-cTnI antibodies via Schiff reaction based chemistry. Detailed characteristics of the processes involved and the finally developed antibody conjugated f-GN interdigitated electrode have been studied. The above micro-device was used in a drain source configuration for the sensing of cTnI. A wide dynamic linear range of antigen detection (0.01-1ng/mL) is achieved with the limit of detection of 0.01ng/mL. The utility of the proposed sensing technique is demonstrated by successfully testing the antigen concentration in spiked serum samples.

Immuno-fluorescence based Vi capsular polysaccharide detection for specific recognition of Salmonella enterica serovar Typhi in clinical samples.

Typhoid fever is a life threatening bacterial infection that remains a major global health concern. This continued high burden associated with significant morbidity and mortality rate demands specific and rapid detection technique. This work reports a new sandwich type fluorescence immunoassay format using polymyxin B, a cationic receptor molecule, as a binder agent while anti-Vi antibody served as the capturing agent for specifically detecting Salmonella enterica serovar Typhi. Anti-Vi IgG antibody raised against Vi-BSA conjugate revealed affinity of 7.779nM(-1) signifying immunodominancy of O-acetyls groups in Vi polysaccharide. The detection limit of the developed assay was around 10(1) cellsmL(-1) of Vi expressing Salmonella enterica serovar Typhi with a correlation coefficient (R(2)) equal to 0.97. Positive response obtained for all the tested serovar Typhi clinical isolates as well as the pathogen spiked blood samples recommended specificity and accuracy of Vi antigen as a biomarker during typhoid fever. The intra- and inter-assay precision with Vi spiked samples were satisfactory revealing coefficient of variance (CV%) with a mean of 4.05% and 5.97% respectively. This may be the novel attempt and constructive report on the fluorescence based detection of Vi antigen of serovar Typhi in the epidemic as well as pandemic outbreaks.

Biofunctionalized rebar graphene (f-RG) for label-free detection of cardiac marker troponin I.

One-step microwave-assisted unscrolling of carbon nanotubes to form functionalized rebar graphene (f-RG) is reported. The well-characterized f-RG on an interdigitated electrode biochip in a FET configuration showed enhanced electronic properties, as demonstrated with I-V characteristics. The developed device was biofunctionalized with specific anti-cTnI antibodies exhibiting a shift of threshold voltage from -2.15 V to -0.5 V and decrease in electron mobility from 3.609 × 10(4) to 8.877 × 10(3) cm(2) V(-1) s(-1). The new sensing strategy holds great promise for its applicability in diagnostics exhibiting high sensitivity (∼ 1 pg/mL) and specificity toward cardiac marker (cTnI).

Hybrid aptamer-antibody linked fluorescence resonance energy transfer based detection of trinitrotoluene.

Combining synthetic macromolecules and biomolecular recognition units are promising in developing novel diagnostic and analysis techniques for detecting environmental and/or clinically important substances. Fluorescence resonance energy transfer (FRET) apta-immunosensor for explosive detection is reported using 2,4,6-trinitrotoluene (TNT) specific aptamer and antibodies tagged with respective FRET pair dyes in a sandwich immunoassay format. FITC-labeled aptamer was used as a binder molecule in the newly developed apta-immunoassay format where the recognition element was specific anti-TNT antibody labeled with rhodamine isothiocyanate. The newly developed sensing platform showed excellent sensitivity with a detection limit of the order of 0.4 nM presenting a promising candidate for routine screening of TNT in samples.

Graphene-gated biochip for the detection of cardiac marker Troponin I.

We report lithium ion intercalation mediated efficient exfoliation of graphite to form monolithic graphene sheets which have subsequently been investigated for the development of highly sensitive label-free electrochemical detection platform for cardiac biomarker, Troponin I (cTnI). The spectroscopic and morphological analysis demonstrated the formation of defect free graphene sheets which were successfully employed to fabricate an inter-digited microdevice in a drain-source configuration on a silicon biochip. The graphene gated biochip functionalized with anti-cTnI antibodies used in label free detection of cTnI which exhibited an excellent sensitivity in the picogram range (~1 pg mL(-1)) for cTnI without the use of any enzymatic amplification that promises its potential applicability for bio-molecular detection in clinical diagnosis.

Nanobioprobe mediated DNA aptamers for explosive detection.

Specific nucleic acid aptamers using the microtiter plate based modified SELEX method against explosive trinitrotoluene are reported. Efficient partitioning of dsDNA was carried out using streptavidin labeled gold nanoprobes for the selection of specific aptamers. The selected binders having an affinity of ~10(-7) M were used in the newly developed electrochemical aptasensor, exhibiting a detection limit of around 1 ppb for trinitrotoluene.

Plasmon enhanced fluoro-immunoassay using egg yolk antibodies for ultra-sensitive detection of herbicide diuron.

Plasmon enhanced fluorescence immunoassay (PEFI) format has been reported in developing a sensitive heterogeneous fluoroimmunoassay for monitoring the phenylurea herbicide diuron. Computer-assisted molecular modeling was carried out to study the conformational and electrostatic effects of synthesized hapten for producing highly specific egg yolk antibody against a phenyl urea herbicide diuron. The generated antibodies were labeled with fluorescein isothiocyanate at different molar ratios and used as tracer in the developed fluorescence based immunoassay. The sensitivity of the assay format was enhanced by using silver nanoparticles tagged with bovine serum albumin as a new blocking reagent in the developed PEFI format. Enhancer treatment on the developed immunoassay showed a significant improvement of fluorescence signal intensity with approximately 10 fold increase in assay sensitivity. The immunoassay has a detection limit of 0.01 ng mL(-1) with good signal precision (~2%) in the optimum working concentration range between 1 pg mL(-1) to 10 µg mL(-1) of diuron. These findings facilitate high throughput fluorescence-based processes that could be useful in biology, drug discovery and compound screening applications.

Characterization of Hapten-Protein Conjugates: Antibody Generation and Immunoassay Development for Pesticides Monitoring.

The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In the present study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density of two different class of pesticides (atrazine and 2,4-dichlorophenoxyacetic acid in this study) coupled to carrier protein. The study proved useful for monitoring the course of hapten-protein conjugation for the production of specific antibodies against small molecules. Well-characterized hapten-protein conjugates enabled obtaining highly sensitive anti-atrazine and anti-2,4-D antibodies with IC50 values equal to 12 and 70 ng mL-1 for atrazine and 2,4-D respectively. These antibodies were used for developing a fluorescence-based immunoassays format demonstrating a detection limit of atrazine and 2,4-D in standard water samples 2 and 7 ng mL-1, respectively. The developed immunoassay format could be used as convenient quantitative tools for sensitive and specific screening of pesticides in samples.

CdTe nanobioprobe based optoelectrochemical immunodetection of diabetic marker HbA1c.

Highly luminescent water soluble CdTe quantum dots (QDs) were synthesized and conjugated with anti-HbA1c antibody to generate specific nanobioprobe. A sandwich immunoassay model was employed using capture HbA1c antibody as a specific receptor molecule and the QD-labeled secondary antibody as a dual (fluorescence cum electrochemical) tracer to quantify the concentration of HbA1c. A linear increase in current was observed for HbA1c analytical standards with a R(2) value of 0.990 and coefficient of variance ~5%. A comparison between HPLC and dual immunoassay for clinical samples showed a correlation coefficient of 89% and 96% for fluorescence and electrochemical detection methods respectively. The QD-based immunoassay shows great promise for rapid reproducible and cost effective analysis of HbA1c in clinical samples.

Amine functionalized graphene oxide/CNT nanocomposite for ultrasensitive electrochemical detection of trinitrotoluene.

Binding of electron-deficient trinitrotoluene (TNT) to the electron rich amine groups on a substrate form specific charge-transfer Jackson-Meisenheimer (JM) complex. In the present work, we report formation of specific JM complex on amine functionalized reduced graphene oxide/carbon nanotubes- (a-rGO/CNT) nanocomposite leading to sensitive detection of TNT. The CNT were dispersed using graphene oxide that provides excellent dispersion by attaching to CNT through its hydrophobic domains and solubilizes through the available OH and COOH groups on screen printed electrode (SPE). The GO was reduced electrochemically to form reduced graphene that remarkably increases electrochemical properties owing to the intercalation of high aspect CNT on graphene flakes as shown by TEM micrograph. The surface amine functionalization of dropcasted and rGO/CNT was carried out using a bi-functional cross linker ethylenediamine. The extent of amine functionalization on modified electrodes was confirmed using energy dispersive X-ray (EDX), X-ray photoelectron spectroscopy (XPS) and confocal microscopy. The FTIR and Raman spectra further suggested the formation of JM complex between amine functionalized electrodes and TNT leading to a shift in peak intensity together with peak broadening. The a-rGO/CNT nanocomposite prepared electrode surface leads to ultra-trace detection of TNT upto 0.01 ppb with good reproducibility (n=3). The a-rGO/CNT sensing platform could be an alternate for sensitive detection of TNT explosive for various security and environmental applications.