Amrita Srivastava

Highly sensitive and selective hyphenated technique (molecularly imprinted polymer solid-phase microextraction–molecularly imprinted polymer sensor) for ultra trace analysis of aspartic acid enantiomers

The present work is related to combination of molecularly imprinted solid-phase microextraction and complementary molecularly imprinted polymer-sensor. The molecularly imprinted polymer grafted on titanium dioxide modified silica fiber was used for microextraction, while the same polymer immobilized on multiwalled carbon nanotubes/titanium dioxide modified pencil graphite electrode served as a detection tool. In both cases, the surface initiated polymerization was found to be advantageous to obtain a nanometer thin imprinted film. The modified silica fiber exhibited high adsorption capacity and enantioselective diffusion of aspartic acid isomers into respective molecular cavities. This combination enabled double preconcentrations of d- and l-aspartic acid that helped sensing both isomers in real samples, without any cross-selectivity and matrix complications. Taking into account 6×10(4)-fold dilution of serum and 2×10(3)-fold dilution of cerebrospinal fluid required by the proposed method, the limit of detection for l-aspartic acid is 0.031ngmL(-1). Also, taking into account 50-fold dilution required by the proposed method, the limit of detection for d-aspartic acid is 0.031ngmL(-1) in cerebrospinal fluid.

Molecularly imprinted micro solid-phase extraction technique coupled with complementary molecularly imprinted polymer-sensor for ultra trace analysis of epinephrine in real samples.

A simple hyphenation approach was adopted to obtain a new molecularly imprinted micro solid-phase extraction fiber (as a selective extraction tool) and complementary molecularly imprinted polymer coated pencil graphite electrode (as a detection tool) for the selective and sensitive analysis of epinephrine, which is a disease biomarker prevalent at ultra trace level in biological fluids. In both extraction and detection processes, the functionalized multiwalled carbon nanotubes (CNT-mers) were preferred to multiwalled carbon nanotubes (unmodified) in order to obtain a stable homogeneously dispersed imprinted polymer matrix of better electroconductivity and adsorptive characteristics. The hyphenation of both tools helped dual pre-concentration of epinephrine so as to achieve the stringent limit [limit of detection: 0.002 ng mL(-1), S/N=3] of clinical detection, without any problems of non-specific contributions and cross-reactivity.

Electrochemically grown imprinted polybenzidine nanofilm on multiwalled carbon nanotubes anchored pencil graphite fibers for enantioselective micro-solid phase extraction coupled with ultratrace sensing of d- and l-methionine

An alternative method is presented for the modification of pencil graphite fibers using surface imprinting technology. In this new approach, we have adopted surface initiated electropolymerization of benzidine monomer, with simultaneous imprinting of template (d- and l-methionine), on carboxylated multiwalled carbon nanotubes anchored pencil graphite fiber. This yielded a nanostructured ultrathin imprinted film (58.3nm) uniformly coated all along the perimeter and length of pencil graphite fiber, for micro-solid phase extraction with substantial adsorption capability. The same film is coated over the exposed tip of the pencil graphite fiber to serve as a complementary molecularly imprinted polymer-sensor. Both extraction and sensing devices are not capable to measure the stringent limit (0.016ngmL(-1)) of clinical detection of methylenetetrahydrofolate reductase (MTHFR) gene mutation caused by acute methionine depletion, when used alone. However, on combination of both techniques, a successful enantioselective analysis of d- and l-methionine with excellent analytical figures of merit [limit of quantitation range: 0.03-30.00ngmL(-1), limit of detection: 0.0098ngmL(-1) (RSD=2.04, S/N=3)] could be achieved without any problem of non-specific false-positive contribution and cross-reactivity, in real samples.

Molecularly imprinted polymer-matrix nanocomposite for enantioselective electrochemical sensing of D- and L-aspartic acid.

A new molecularly imprinted polymer-matrix (titanium dioxide nanoparticle/multiwalled carbon nanotubes) nanocomposite was developed for the modification of pencil graphite electrode as an enantioselective sensing probe for aspartic acid isomers, prevalent at ultra trace level in aqueous and real samples. The nanocomposite having many shape complementary cavities was synthesized adopting surface initiated-activators regenerated by electron transfer for atom transfer radical polymerization. The proposed sensor has high stability, nanocomposite uniformity, good reproducibility, and enhanced electrocatalytic activity to respond oxidative peak current of L-aspartic acid quantitatively by differential pulse anodic stripping voltammetry, without any cross-reactivity in real samples. Under the optimized operating conditions, the L-aspartic acid imprinted modified electrode showed a wide linear response for L-aspartic acid within the concentration range 9.98-532.72 ng mL(-1), with the minimum detection limit of 1.73-1.79 ng mL(-1) (S/N=3) in aqueous and real samples. Almost similar stringent limit (1.79 ng mL(-1)) was obtained with cerebrospinal fluid which is typical for the primitive diagnosis of neurological disorders, caused by an acute depletion of L-aspartic acid biomarker, in clinical settings.

Sodium transport and mechanism(s) of sodium tolerance in Frankia strains

The mechanism(s) underlying differential salt sensitivity/tolerance were investigated in the terms of altered morphological and physiological responses against salinity such as growth, electrolyte leakage, Na+ uptake, efflux, accumulation and intracellular concentrations of macronutrients among the Frankia strains newly isolated from Hippöphae salicifolia D. Don. Growth was minimally reduced at 500 and 250 mM NaCl respectively in HsIi10 and rest of the strains (HsIi2, HsIi8, HsIi9) which proved that 500 and 250 mM NaCl are the critical concentrations for the respective strains. The differences in the sodium influx/efflux rate was responsible for the differential amount of remaining sodium among the frankial strains and might be one of the primary determinants for the reestablishment of macronutrients (Mg2+, Ca2+ and K+) during salinity. Secondly, the interactive effect of sodium influx/efflux rate, remaining sodium and intracellular macronutrients (Mg2+, Ca2+ and K+) concentration has been responsible for the extent of membrane damage and growth sustenance of the tolerant/sensitive frankial strains during salinity. HsIi10 showed better co‐regulation of various factors and managed to tolerate salt stress up to considerable extent. Therefore, HsIi10 can serve as a potential biofertilizer in the saline soil.

Salt stress–induced changes in antioxidative defense system and proteome profiles of salt-tolerant and sensitive Frankia strains

ABSTRACT An appreciation of comparative microbial survival is most easily done while evaluating their adaptive strategies during stress. In the present experiment, antioxidative and whole cell proteome variations based on spectrophotometric analysis and SDS-PAGE and 2-dimensional gel electrophoresis have been analysed among salt-tolerant and salt-sensitive Frankia strains. This is the first report of proteomic basis underlying salt tolerance in these newly isolated Frankia strains from Hippophae salicifolia D. Don. Salt-tolerant strain HsIi10 shows higher increment in the contents of superoxide dismutase, catalase and ascorbate peroxidase as compared to salt-sensitive strain HsIi8. Differential 2-DGE profile has revealed differential profiles for salt-tolerant and salt-sensitive strains. Proteomic confirmation of salt tolerance in the strains with inbuilt efficiency of thriving in nitrogen-deficient locales is a definite advantage for these microbes. This would be equally beneficial for improvement of soil nitrogen status. Efficient protein regulation in HsIi10 suggests further exploration for its potential use as biofertilizer in saline soils.

Deciphering the evolutionary affiliations among bacterial strains (Pseudomonas and Frankia sp.) inhabiting same ecological niche using virtual RFLP and simulation-based approaches

To decipher an evolutionary lineage between two different but important bacterial groups, i.e., Pseudomonas strain (γ-Proteobacteria) and Frankia strain (actinobacteria) growing in the same ecological niche in and around of an actinorhizal plant Hippophae salicifolia D. Don, genetic diversity and comparative molecular phylogeny have been investigated using 16S rRNA gene sequences and computer-simulated and virtually directed restriction fragment length polymorphism (RFLP) through 10 restriction enzymes. Bayesian and coalescent analyses on the basis of 16S rRNA gene sequences suggested three major groups with close proximity between Pseudomonas and Frankia isolates. This result has been further validated based on the data observed through similarity coefficient value and computational RFLP. Principal component analysis and Mandel h and k statistical analysis also confirmed and strengthen the findings. Approximately 458 aligned sequence of all the taxa were used to decipher nucleotide diversity, polymorphism and gene flow between these taxa. Thus, our results suggest for a possible co-evolution or a heterologous gene transfer of distantly related microbial forms. Further, our study also advocate for the use of computer aided, virtual RFLP analysis as a cost effective and rapid identification tool.

Phylogeny and evolutionary genetics of Frankia strains based on 16S rRNA and nifD-K gene sequences.

16S rRNA and nifD–nifK sequences were used to study the molecular phylogeny and evolutionary genetics of Frankia strains isolated from Hippöphae salicifolia D. Don growing at different altitudes (ecologically classified as riverside and hillside isolates) of the Eastern Himalayan region of North Sikkim, India. Genetic information for the small subunit rRNA (16S rRNA) revealed that the riverside Frankia isolates markedly differed from the hillside isolates suggesting that the riverside isolates are genetically compact. Further, for enhanced resolutions, the partial sequence of nifD (3′ end), nifK (5′ end) and nifD–K IGS region have been investigated. The sequences obtained, failed to separate riverside isolates and hillside isolates, thus suggesting a possible role of genetic transfer events either from hillside to riverside or vice versa. The evolutionary genetic analyses using evogenomic extrapolations of gene sequence data obtained from 16S rRNA and nifD–K provided differing equations with the pace of evolution being more appropriately, intermediate. Values of recombination frequency (R), nucleotide diversity per site (Pi), and DNA divergence estimates supported the existence of an intermixed zone where spatial isolations occurred in sync with the temporal estimates. J. Basic Microbiol. 2015, 54, 1–9