Kamla Rawat

Single electron transfer-driven multi-dimensional signal read-out function of TCNQ as an "off-the-shelf" detector for cyanide.

Herein we report the first applications of TCNQ as a rapid and highly sensitive off-the-shelf cyanide detector. As a proof-of-concept, we have applied a kinetically selective single-electron transfer (SET) from cyanide to deep-lying LUMO orbitals of TCNQ to generate a persistently stable radical anion (TCNQ(•-)), under ambient condition. In contrast to the known cyanide sensors that operate with limited signal outputs, TCNQ(•-) offers a unique multiple signaling platform. The signal readability is facilitated through multichannel absorption in the UV-vis-NIR region and scattering-based spectroscopic methods like Raman spectroscopy and hyper Rayleigh scattering techniques. Particularly notable is the application of the intense 840 nm NIR absorption band to detect cyanide. This can be useful for avoiding background interference in the UV-vis region predominant in biological samples. We also demonstrate the fabrication of a practical electronic device with TCNQ as a detector. The device generates multiorder enhancement in current with cyanide because of the formation of the conductive TCNQ(•-).

Interactions in globular proteins with polyampholyte: coacervation route for protein separation

In this work, we report exclusive separation of Bovine Serum Albumin (BSA) from a solution where this protein was present with β-lactoglobulin (β-Lg) in 1 : 0.75 (w/v) ratio at their common isoelectric pH (5 ± 0.02). A polyampholytic polypeptide Gelatin B (GB) also having the same pI was used to extract protein (BSA or β-Lg) molecules selectively from this solution through a process called complex coacervation. In our study, the protein-rich condensate, called coacervate, comprised of GB–BSA complexes while the supernatant mostly contained β-Lg molecules. For the separation of BSA from BSA–GB coacervate, we used ethyl alcohol, which removed the BSA to the supernatant. The differential binding affinity of BSA versus β-Lg to GB chains was established through fluorescence quenching and fluorescence resonance energy transfer (FRET) studies. The BSA–GB binding protocol followed a surface selective patch binding mechanism and these results were obtained from an array of experimental methods such as UV-vis and fluorescence spectroscopy, small angle neutron scattering (SANS), FTIR and circular dichroism spectroscopy. Herein, it is clearly established that selective coacervation at pI can be used as a method for protein separation.

Surface patch binding and mesophase separation in biopolymeric polyelectrolyte-polyampholyte solutions.

Surface patch binding (SPB) induced mesophase separation causing complex coacervation between biopolymers: gelatin A-gelatin B, chitosan-gelatin A, chitosan-gelatin B, and, agar-gelatin B was investigated with and without salt (I=0-0.3 M NaCl). SPB was induced by pH change and three characteristic pHs identified transitions in a turbidity plot: intermolecular interactions ensued at pHc, coacervation transition occurred at pHΦ and phase separation was noticed at pHprep. Associative interactions lead to formation of soluble complexes at pHc exclusively through SPB whereas the coacervation transition was driven by electrostatic binding (EB). Neither pHc nor pHΦ displayed discernible ionic strength (till 50 mM) or temperature dependence, but coacervate yield reduced with increase in ionic strength. Coacervation was completely suppressed beyond 50 mM NaCl. Linear combination of attractive and repulsive parts operating between a polyelectrolyte (charged rod) with a polyampholyte (dipole or point charge) was used to model the interaction potential as function of ionic strength. Relative strength of SPB vis a vis EB was used as SPB index to establish a linear relationship with zeta potential ratio of binding partners. Different phase diagrams could be constructed which clearly identified distinct interaction regimes encountered in solutions undergoing coacervation transition.

Size-dependent CdSe quantum dot–lysozyme interaction and effect on enzymatic activity

Herein, protocols for modifying hydrophobic CdSe quantum dots with 3-mercaptopropionic acid (MPA) to generate hydrophilic moieties and their size-dependent (2.5 and 6.3 nm) interaction with lysozyme are reported comprehensively. The interaction of MPA-capped water-soluble quantum dots with lysozyme (Ly) was investigated, and a range of techniques such as static fluorescence spectroscopy and synchronous fluorescence spectroscopy were used to quantify QD–lysozyme binding isotherms, exchange rates, critical flocculation concentrations, and the compositions of mixed QD–lysozyme complexes. The results demonstrated that the binding of QDs with lysozyme induced conformational changes in lysozyme. QDs were able to enhance the enzymatic activity of lysozyme in a highly efficient dose-dependent manner. It was concluded that smaller-sized QDs were found to bind poorly to lysozyme, but produced a greater enhancement in enzymatic activity compared with larger QDs. In summary, a comprehensive characterization of the stability of lysozyme-bound QDs is a necessary step for their potential use as intracellular delivery vectors and imaging agents.

Biocompatible capped iron oxide nanoparticles for Vibrio cholerae detection

We report the studies relating to fabrication of an efficient immunosensor for Vibrio cholerae detection. Magnetite (iron oxide (Fe(3)O(4))) nanoparticles (NPs) have been synthesized by the co-precipitation method and capped by citric acid (CA). These NPs were electrophoretically deposited onto indium-tin-oxide (ITO)-coated glass substrate and used for immobilization of monoclonal antibodies against Vibrio cholerae (Ab) and bovine serum albumin (BSA) for Vibrio cholerae detection using an electrochemical technique. The structural and morphological studies of Fe(3)O(4) and CA-Fe(3)O(4)/ITO were characterized by x-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, and dynamic light scattering (DLS) techniques. The average crystalline size of Fe(3)O(4), CA-Fe(3)O(4) nanoparticles obtained were about 29 ± 1 nm and 37 ± 1 nm, respectively. The hydrodynamic radius of the nanoparticles was found to be 77.35 nm (Fe(3)O(4)) and 189.51 nm (CA-Fe(3)O(4)) by DLS measurement. The results of electrochemical response studies of the fabricated BSA/Ab/CA-Fe(2)O(3)/ITO immunosensor exhibits a good detection range of 12.5-500 ng mL(-1) with a low detection limit of 0.32 ng mL(-1), sensitivity 0.03 Ω/ng ml(-1) cm(-2), and reproducibility more than 11 times.

Cadmium-free aqueous synthesis of ZnSe and ZnSe@ZnS core–shell quantum dots and their differential bioanalyte sensing potential

Herein we report a facile and cadmium-free approach to prepare water-soluble fluorescent ZnSe@ZnS core–shell quantum dots (QDs), using thioglycolic acid (TGA) ligand as a stabilizer and thiourea as a sulfur source. The optical properties and morphology of the obtained core–shell QDs were characterized by UV–vis and fluorescence spectroscopy, transmission electron microscopy (TEM), energy-dispersive x-ray analysis (EDX), x-ray diffraction (XRD), electrophoresis and dynamic light scattering (DLS) techniques. TEM analysis, and electrophoresis data showed that ZnSe core had an average size of 3.60 ± 0.12 nm and zeta potential of −38 mV; and for ZnSe@ZnS QDs, the mean size was 4.80 ± 0.20 nm and zeta potential was −45 mV. Compared to the core ZnSe QDs, the quantum yield of these core–shell structures was higher (13% versus 32%). These were interacted with five common bioanalytes such as, ascorbic acid, citric acid, oxalic acid, glucose and cholesterol which revealed fluorescence quenching due to concentration dependent binding of analytes to the core only, and core–shell QDs. The binding pattern followed the sequence: cholesterol < glucose < ascorbic acid < oxalic acid < citric acid for ZnSe, and cholesterol < glucose < oxalic acid < ascorbic acid < citric acid for core–shell QDs. Thus, enhanced binding was noticed for the analyte citric acid which may facilitate development of a fluorescence-based sensor based on the ZnSe core-only quantum dot platform. Further, the hydrophilic core–shell structure may find use in cell imaging applications.

Hierarchical Surface Charge Dependent Phase States of Gelatin–Bovine Serum Albumin Dispersions Close to Their Common pI

We report interaction between bovine serum albumin ([BSA] = 1% (w/v)) and gelatin B ([GB] = 0.25-3.5% (w/v)) occurring close to their common isoelectric pH (pI). This interaction generated distinguishable multiple soft matter phases like opaque coacervates (phase I) and transparent gels (phase II), where the former are composed of partially charge neutralized intermolecular complexes (zeta potential, ζ ≤ 0) and the latter of overcharged complexes (ζ ≥ 0) that organized into a network pervading the entire sample volume. These phase states were completely governed by the protein mixing ratio r = [GB]:[BSA]. Coacervates, when heated above 32 °C, produced thermoirreversible turbid gels (phase III), stable in the region 32 ≥ T ≤ 50 °C. When the transparent gels were heated to T ≥ 34 °C, these turned into turbid solutions that did form a turbid fragile gel (phase IV) upon cooling. Mechanical and thermal behaviors of aforesaid coacervates (phase I) and gels (phase II) were examined; coacervates had lower storage modulus and melting temperature compared to gels. Cole-Cole plots attributed considerable heterogeneity to coacervate phase, but gels were relatively homogeneous. Raman spectroscopy data suggested differential microenvironment for these phases. Coacervates were mostly hydrated by partially structured water with degree of hydration dependent on gelatin concentration whereas for gels hydration was invariant of [GB]. Small-angle neutron scattering (SANS) data gave static structure factor profiles, I(q), versus wavevector q, that were remarkably different. For transparent gels, data could be split into two distinct regions: (i) 0.01 < q < 0.1 Å(-1), I(q) = IOZ(0)/(1 + q(2)ζgel(2))(2) (Debye-Bueche function) with ζgel = 9-13 nm, and (ii) 0.1 < q < 0.35 Å(-1), I(q) = IOZ(0)/(1 + q(2)ξgel(2)) (Ornstein-Zernike function) with ξgel = 3.1 ± 0.6 nm. Similarly, for coacervate, the aforesaid two q-regions were described by (i) I(q) = IPL(0)q(-α) with α = 1.7 ± 0.1 and (ii) I(q) = IOZ(0)/(1 + q(2)ξcoac(2)) with ξcoac = 1.6 ± 0.2 nm, a value close to the persistence length of gelatin chain (lp ≈ 2 nm). Phase transition from one equilibrium state to another, i.e., phase I to II, was hierarchical in the charge state of the protein-protein complex. Within the same charge state, transition from phase I to III and from phase II to IV was thermally activated. The aforesaid mechanisms are captured in a unique ζ-T phase diagram.

Au@carbon dot nanoconjugates as a dual mode enzyme-free sensing platform for cholesterol

In this report, we present a novel application of gold-carbon dot nanoconjugates (Au@CDs) of an average size of around 12.6 nm as a sensor for the detection of cholesterol. The Au particles perform the dual function of displaying colorimetric sensing, and fluorescence quenching in response to cholesterol in the concentration range of 10-100 ppm (0.208-2.08 mM), wherein the carbon dots act as the fluorescent entity. Interestingly, the nanoconjugates were observed to show a high specificity to cholesterol resulting in their precipitation. A visible change in colour of the assay mixture along with fluorescence quenching was seen in the reaction mixture on treatment with cholesterol. The synthesized nanoconjugates had high selectivity towards cholesterol, even in the presence of interfering analytes, and a minimum detection limit of 0.12 ppm (0.0025 mM) in the linear range of 50-300 ppm (1-6.25 mM). We anticipate that these Au@CDs can be employed for the fabrication of enzyme-free strip-based biosensors for the detection of cholesterol.

Charge heterogeneity induced binding and phase stability in β-lacto-globulin–gelatin B gels and coacervates at their common pI

An understanding of the interactions between gelatin B (GB) and β-lacto-globulin (β-Lg) mainly arising from surface selective patch binding occurring at their common pI (≈5.0 ± 0.5) in the absence of added salt. Heterogeneous surface charge distribution on β-Lg facilitated such interaction at different mixing ratio ([GB]: [β-Lg] = r) and the GB–β-Lg complexes carried distinctive surface charge (seen through their zeta potential, ζ). For r 1:1 (overcharged regime, ζ > 0) the dispersion remained transparent and homogeneous which gives no phase separation, but the dispersion formed a gel on waiting. The overcharged gels were homogeneous, more rigid and higher melting temperature in comparison to coacervate. In the coacervate phase, the intensity of the scattered light Is, and its time-correlation function [g2(t) − 1] did not evolve with time. In contrast, the gel phase displayed considerable change with aging time tw. For gels, as tw → ∞ the system moved from an ergodic to non-ergodic state. At tw = 0, the correlation function exhibited one relaxation mode due to the system residing deeply inside the ergodic phase and purely mirroring Brownian dynamics. After a characteristic waiting time, tw an additional mode (slow relaxation) appeared which was attributed to inter-chain interaction induced reorganization of entanglements. This characteristic time was the time required for the system to get dynamically arrested, similar observation was made from rheology measurements too. A comprehensive phase diagram depicting the stability of the dispersion in various charged soft matter states of the complex under various temperature conditions was established.

Electrochemical response of agar ionogels towards glucose detection

We have reported a sensing platform comprising of agar ionogels (IGs) made in ionic liquid solutions (1-octyl-3-methyl imidazolium chloride [C8mim][Cl] and 1-ethyl-3-methylimidazolium chloride [C2mim][Cl]) and used it for glucose oxidase (GOx) immobilization for glucose detection. The ionogels were deposited onto an indium tin oxide (ITO) coated glass plate using the drop-casting technique. These agar–[C8mim][Cl]/ITO (Ag–C8/ITO) and agar–[C2mim][Cl]/ITO (Ag–C2/ITO) substrates were used for immobilization of GOx, which was selected as a model enzyme to investigate its interaction with these electrodes using electrochemical techniques. Structural and morphological studies of these GOx/Ag–C8/ITO and GOx/Ag–C2/ITO electrodes were performed by using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and electrochemical techniques (CV). These GOx/Ag–C8/ITO and GOx/Ag–C2/ITO bioelectrodes exhibited improved glucose detection capability in the concentration range of 0.27–16.7 mM and 0.28–5.6 mM with sensitivity ≈4.1 μA mM−1 cm−2 and 14.6 μA mM−1 cm−2, respectively. The values of apparent Michaelis–Menten constant (Km) were 0.023 mM and 0.0007 mM for the aforesaid two cases respectively. It is shown that customization of agar hydrogels in green solvent medium widens the scope of their potential applications in glucose sensing in real samples.