C. Raman Suri

Synthesis and capping of water-dispersed gold nanoparticles by an amino acid: bioconjugation and binding studies.

We report a novel strategy for the synthesis of aqueous stable, carboxylated gold nanoparticles (GNPs) by using glutamic acid as the reducing agent. The ratio of chloroaurate ions, AuCl(-)(4) to glutamic acid was optimized in the reaction medium to obtain monodispersed GNPs. Glutamic acid reduced gold nanoparticles were characterized by UV-visible, FTIR, dynamic light scattering and transmission electron microscopy, which demonstrated high stability in aqueous solution over a period of time indicating stabilization via surface-bound amino acid. Functionalized nanoparticles were conjugated with protein molecules through electrostatic attraction between the surface-terminated negatively charged carboxylate groups (COO(-)) of glutamic acid and the positively charged amino groups (NH(+)(3)) of the protein. The conjugation efficiency of the GNP:protein conjugates was confirmed qualitatively and quantitatively through gel electrophoresis and critical flocculation concentration analysis. The interaction between functionalized GNPs with protein molecules was investigated using fluorescence spectroscopy showing the fluorescence quenching of the tryptophan residues of protein molecules after conjugation. Circular dichroism (CD) studies of the conjugates confirmed that the protein undergoes a more flexible conformational state on the boundary surface of GNPs after conjugation. There was substantial conformational transition from alpha-helix to beta-sheet structure after conjugation of protein to GNPs.

Interaction of gold nanoparticles with protein: A spectroscopic study to monitor protein conformational changes

Gold nanoparticles (GNPs) conjugated with biomolecules are promising building blocks for assembly into nanostructured functional materials for developing biomarker platforms because of their size dependent optical and electrical properties. Biocompatible GNPs were synthesized using glutamic acid as a reducing agent and the interaction between bovine serum albumin (BSA) and GNPs was investigated using fluorescence and circular dichroism (CD) spectroscopies. The binding constant (Kb) of protein (BSA) to GNPs was determined by measuring the quenching of the fluorescence intensity of tryptophan residues of the protein molecules after conjugation. The conformational change in BSA at its native form after conjugation with GNPs confirmed that protein undergoes a more flexible conformational state on the boundary surface of GNPs after bioconjugation. The CD studies further showed a decrease in the α-helical content after conjugation. The results confirmed that the change in conformation was larger at higher conce...

Bio-functionalized graphene-graphene oxide nanocomposite based electrochemical immunosensing

We report a novel in-situ electrochemical synthesis approach for the formation of functionalized graphene-graphene oxide (fG-GO) nanocomposite on screen-printed electrodes (SPE). Electrochemically controlled nanocomposite film formation was studied by transmission electron microscopy (TEM) and Raman spectroscopy. Further insight into the nanocomposite has been accomplished by the Fourier transformed infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) spectroscopy. Configured as a highly responsive screen-printed immunosensor, the fG-GO nanocomposite on SPE exhibits electrical and chemical synergies of the nano-hybrid functional construct by combining good electronic properties of functionalized graphene (fG) and the facile chemical functionality of graphene oxide (GO) for compatible bio-interface development using specific anti-diuron antibody. The enhanced electrical properties of nanocomposite biofilm demonstrated a significant increase in electrochemical signal response in a competitive inhibition immunoassay format for diuron detection, promising its potential applicability for ultra-sensitive detection of range of target analytes.

A novel bacterial isolate Stenotrophomonas maltophilia as living factory for synthesis of gold nanoparticles.

BackgroundThe synthesis of gold nanoparticles (GNPs) has received considerable attention with their potential applications in various life sciences related applications. Recently, there has been tremendous excitement in the study of nanoparticles synthesis by using some natural biological system, which has led to the development of various biomimetic approaches for the growth of advanced nanomaterials. In the present study, we have demonstrated the synthesis of gold nanoparticles by a novel bacterial strain isolated from a site near the famous gold mines in India. A promising mechanism for the biosynthesis of GNPs by this strain and their stabilization via charge capping was investigated.ResultsA bacterial isolate capable of gold nanoparticle synthesis was isolated and identified as a novel strain of Stenotrophomonas malophilia (AuRed02) based on its morphology and an analysis of its 16S rDNA gene sequence. After 8 hrs of incubation, monodisperse preparation of gold nanoparticles was obtained. Gold nanoparticles were characterized and found to be of ~40 nm size. Electrophoresis, Zeta potential and FTIR measurements confirmed that the particles are capped with negatively charged phosphate groups from NADP rendering them stable in aqueous medium.ConclusionThe process of synthesis of well-dispersed nanoparticles using a novel microorganism isolated from the gold enriched soil sample has been reported in this study, leading to the development of an easy bioprocess for synthesis of GNPs. This is the first study in which an extensive characterization of the indigenous bacterium isolated from the actual gold enriched soil was conducted. Promising mechanism for the biosynthesis of GNPs by the strain and their stabilization via charge capping is suggested, which involves an NADPH-dependent reductase enzyme that reduces Au3+ to Au0 through electron shuttle enzymatic metal reduction process.

Immunosensors for pesticide analysis: antibody production and sensor development.

ABSTRACT:  Immunosensors, a type of affinity biosensor, are based on the binding interactions between an immobilized biomolecule (antibody/antigen) on the electronic transducer surface with the analyte of interest (antigen/antibody), resulting in a detectable signal. The sensor system takes advantage of the high selectivity provided by the molecular recognition characteristic of an antibody, which binds reversibly with a specific antigen. This review article presents the current status of immunosensors, highlighting their potential benefits and limitations for pesticide analysis. The basic criteria for generating specific antibodies against low-molecular-mass pesticides, which are usually nonimmunogenic in nature, are briefly discussed. The article also describes the fundamentals of important transducer technologies and their use in immunosensor development.

Strip-based immunochromatographic assay using specific egg yolk antibodies for rapid detection of morphine in urine samples.

Using specific egg yolk antibodies (IgY), a strip-based immunochromatographic assay was developed for rapid detection of morphine in urine samples. IgY type antibody against morphine was generated by immunizing chickens with well-characterized monoacetyl morphine-protein conjugate. The antibody was labeled with gold nanoparticles and used as an immunoprobe in the dipstick format for the visual detection of morphine in urine samples. The dipstick was developed using three membranes: an application pad made of glass fiber membrane to hold the tracer, a signal generation test line on nitrocellulose membrane (detection zone) and a cellulose membrane used as an absorption pad. Analytes of interest (morphine and its analogues) added to the sample well, dissolved the labeled antibody (tracer), and the antigen-antibody complex formed was transported by the flow caused by capillary action to the test line. The color signal of test line in proportion to the morphine concentration in urine samples was measured using a detector. The developed dipstick assay format was optimized, showing the average IC(50) values of morphine as low as 9.45 ng/mL, the detection range of 1-1000 ng/mL and the lowest detection limit 2.5 ng/mL under optimal conditions of analysis. The correlation between the developed dipstick and ELISA was 0.948 in the analysis of urine samples spiked with morphine. The developed dipstick could be a highly sensitive and convenient tool for rapid detection of opiate drugs in samples with high degree of stability.

Molecular mechanism of polyethylene glycol mediated stabilization of protein.

The effect of different molar ratios of polyethylene glycol (PEG) on the conformational stability of protein, bovine serum albumin (BSA), was studied. The binding of PEG with BSA was observed by fluorescence spectroscopy by measuring the fluorescence intensity after displacement of PEG with chromophore ANS and had further been confirmed by measuring the intrinsic fluorescence of tryptophan residues of BSA. Co-lyophilization of BSA with PEG at optimum BSA:PEG molar ratio led to the formation of the stable protein particles. Circular dichroism (CD) spectroscopy study suggested that a conformational change had occurred in the protein after PEG interaction and demonstrated the highest stability of protein at the optimum BSA:PEG molar ratio of 1:0.75. Additional differential scanning calorimetry (DSC) study suggested strong binding of PEG to protein leading to thermal stability at optimum molar ratio. Molecular mechanism operating behind the polyethylene glycol (PEG) mediated stabilization of the protein suggested that strong physical adsorption of PEG on the hydrophobic core of the protein (BSA) along with surface adsorption led to the stability of protein.

A novel disposable electrochemical immunosensor for phenyl urea herbicide diuron.

A disposable electrochemical immunosensor has been developed for the determination of phenyl urea herbicide-diuron using a low cost laser ablated gold electrodes (LC-LAGE) fabricated on polystyrene substrate. The electrodes were electrochemically deposited with prussian blue-gold nanoparticle (PB-GNP) film, and a competitive inhibition immunoassay was performed on LC-LAGE by using a specific hapten-protein conjugate. The binding of available diuron specific antibody on conjugate coated electrode was detected using alkaline phosphatase rabbit anti-IgG antibody. The addition of 1-naphthyl phosphate substrate resulted in the production of electrochemically active product, 1-naphthol, which was monitored using square wave voltammetry technique. The assay exhibited an excellent sensitivity and specificity showing the dynamic response range between 1 ppt and 10 ppm for diuron with detection limit around 1 ppt. This study provides insight into development of a rapid and high-throughput screening of pesticides in environmental samples at a very low cost.

Activating piezoelectric crystal surface by silanization for microgravimetric immunobiosensor application

The development of a microgravimetric immunobiosensor using a piezoelectric quartz crystal as a detector requires a stable and reproducible immobilization method for ligand binding. The method of silanization using 3-aminopropyltriethoxysilane (APTES) has been widely used for activating the carrier surface. In the present study, APTES deposition on a piezoelectric crystal surface was studied under various solvent conditions. A fluorescence method, using fluorescence isothiocyanate as a dye, was demonstrated for the quantification of amino groups on the silanized piezoelectric crystal surface. The optimum binding conditions of APTES deposition on a piezoelectric crystal surface were incorporated for the covalent immobilization of protein on the crystal surface in developing a stable and sensitive microgravimetric immunobiosensor. Determination of immunoglobulin G (IgG) concentration was performed using APTES modified piezoelectric crystals coated with protein G. The resonant frequency shift, resulting from the formation of protein G-IgG complex on the crystal surface, correlated with the concentration of IgG in the range 10 ng/ml to 0.1 mg/ml. The APTES modified, protein G coated crystal were found to be quite stable and did not show a significant loss of sensitivity even after 12 weeks of storage at 4 degrees C in a desiccator.

Facile synthesis and functionalization of water-soluble gold nanoparticles for a bioprobe

In this work, we report the size tunable synthesis of water-dispersed gold nanoparticles by using octadecylamine (ODA) as the reducing agent, that electrostatically complexes with the chloroaurate ions, reduces them, and subsequently caps the gold nanoparticles. Amine-capped gold nanoparticles, thus formed, were subsequently coordinated with a secondary monolayer of an anionic surfactant, sodium bis(2-ethylhexyl)-sulfosuccinate (AOT) which helps in providing sufficient hydrophilicity to the gold nanoparticles. Functionalized gold nanoparticles were characterized by UV-vis, IR spectrophotometric, dynamic light scattering, zeta-potential and transmission electron microscopic techniques, which demonstrated high stability of gold nanoparticles in aqueous media, indicating stabilization via bilayers of ODA and AOT. The gold nanoparticles were further conjugated with a protein (bovine serum albumin) and the interaction was investigated by circular dichroism studies as well as by measuring the fluorescence quenching of the tryptophan residues of protein molecules after the binding of nanoparticles to specific sites of the protein. The binding constant and the stoichiometry values indicated that the particles with larger core size are less site-specific but show higher binding affinity with protein molecules. The use of a bio-compatible synthetic process and the stabilization of the gold nanoparticles by ODA and AOT are interesting from the point of view of making bioprobes for life science applications.