Research Interests

In the general areas of Fluorescence Spectroscopy and Physical Photochemistry, Dr Mishra’s research group has been working on a variety of diverse systems and problems, ranging from basic photophysical studies to newer applications of fluorescence. Two major directions in recent years have been (i) Developing novel paradigms for understanding the fluorescence behavior of complex multi-fluorophoric systems, and (ii) Developing fluorescent molecular probes for organized media based on various photophysical concepts.

(i) Developing novel paradigms for understanding the fluorescence behavior of complex multi-fluorophoric systems

  • Excitation-Emission-Matrix Fluorescence (EEMF), Synchronous Fluorescence Spectroscopy (SFS) and Total Synchronous Fluorescence Spectroscopy (TSFS)
  • Integration of Chemometry with fluorescence based analytical methods
  • Design and development of Fiber optic based fluorimetric techniques
  • Use of White light excitation fluorescence (WLEF) as a low cost, fast response alternative in fluorimetric instrumentation

(ii) Developing fluorescent molecular probes for organized media based on various photophysical concepts

  • Excited state prototropism and its applications
  • Fluorescent molecular probing of Biological and Biomimetic System
  • Understanding the organization and dynamics of polymeric hydrogel
  • Fluorescent drugs and their interactions with various substrates
  • Fluorescence based sensors
Figure 1. Recent works in our laboratory

Analytical fluorimetry- Chemometrics

Several new analytical methods have been introduced by employing multidimensional fluorescence techniques such as Excitation-Emission-Matrix Fluorescence (EEMF), Synchronous Fluorescence Spectroscopy (SFS), Total Synchronous Fluorescence Spectroscopy (TSFS) along with conventional fluorescence. Using the above developed methods, fluorescence based characterization of otherwise difficult multifluorophoric samples such as petroleum fuels, mixture of polycyclic aromatic hydrocarbons (PAHS), humic acid, transformer oil etc.

By employing Chemometrics, the multivariate calibration methods such as Principal Component analysis (PCA), Principal Component Regression (PCR), Partial Least-Squares Regression (PLSR), Cluster Analysis, Multivariate curve resolution alternating Least square (MCR-ALS) analysis, Parallel factor analysis (PARAFAC), N-way Partial Least-Squares (NPLS) etc. on SFS and EEMF data appreciable reduction in processing time has been achieved. The analytical potential of fluorescence spectroscopy on multifluorophoric samples could be enhanced by using multivariate methods. Currently, the above method has been applied for classification of Ayurvedic drugs

Biological and Biomimetic Systems

  • Bile Salts
The physiological importance of bile salts lies in their ability to solubilise and emulsify cholesterol, dietary lipids and fat-soluble vitamins. Due to their extraordinary solubilising capacity, these have got much recognition as delivery systems for medicines, cosmetics and several other chemicals. Being amphiphilic they tend to form micellar aggregates in aqueous medium. The micellisation behavior of bile salts has been investigated by using fluorescent molecular probes. The effect of bile salts on phospholipid bilayers and thermosensitive polymers (PNIPAm) has also been evaluated. Taking advantage of the intrinsic fluorescence of drugs such as Amphotericin B, Fisetin, Carvedilol, Camptothesin etc., interaction of these drugs with bile salt aggregates has been explored.
  • Dendrimers
Dendrimers have long been the interest of researchers because of their possible applications in different fields, from drug-delivery to catalysis. The nanoenvironment created in the core of the dendrimer resembles that of biological systems such as proteins, primarily because of the branching architecture.The potential application of dendrimers in various fields makes it important that their properties be understood. We have studied the photophysical behavior of newkome and polyglycerol type dendrimers by attaching to the core fluorescent molecules such as pyrene and dansyl chloride, which act as fluorescent sensors.
  • Liposomes
Phospholipids when dispersed in aqueous medium rearrange themselves into spherical entities known as liposome or vesicles, which are used as model for cell membrane. Several conceptually different fluorescent molecules have been introduced as molecular probes to study properties of lipid bilayer membranes. Fluorescence of such probes have shown sensitive response to (i) acyl chain melting phase transition in membranes constituted of synthetic and natural (lipids of human erythrocyte membrane) lipids, (ii) membrane property modifications induced by cholesterol, surfactants, charged lipids, electrolytes, peptide, bile salts and (iii) ethanol induced bilayer interdigitation. Owing to their importance in drug delivery the interaction of various drugs with lipid bilayer membrane has been evaluated.
  • Proteins and Peptides
7-Aminocoumarins show remarkable changes in fluorescence intensity, solvatochromism fluorescence anisotropy, lifetime on binding to bovine and human serum albumins. The probable location and structure of binding in the domain II of HSA have been obtained by modeling studies. The results are encouraging regarding the application of aminocoumarins as fluorescent molecular probes for serum albumins.

Fibre Optic Fluorimetry

A novel fiber optic based fluorescence technique, White Light Excitation Fluorescence (WLEF) has been introduced as a method of choice for the analysis of multifluorophoric systems. It provides a sensitive low-cost option in analytical fluorimetry. In WLEF, the entire white light is used as the excitation source. The technique is cost saving as there is no need for the use of band pass/cut off filters and/or a monochromator. WLEF spectra retain the spectral integrity of fluorescence spectra. The difference spectrum, in which the lamp scatter profile is subtracted from the overall WLEF spectrum, is fairly adequate to get back the true spectrum of a fluorophore, thereby enabling simple in situ fluorescence analysis, even when the solutions are non-transparent. One of the major advantages of this novel WLEF analytical method would be that one need not know the excitation wavelength of an analyte! WLEF would easily capture multiple fluorescing species in such cases. The ability of WLEF to see the entire fluorescence spectral signature of a sample can used in the analysis of complex multifluorophoric systems. The intrinsic ability of WLEF to measure the fluorescence spectral signature of every fluorophore in complex multifluorophoric systems is a strong case for carrying out a detailed study of the applicability of WLEF in the analysis of such systems. This technique could be useful for the analysis of (i) poly aromatic compounds (PAC) in aqueous media, (ii) PAC in petroleum products like petrol, diesel, kerosene and their mixtures, (iii) PAC in transformer oil, with reference to its aging, (iv) PAC in industrial solvents, (v) interaction of drug with bio-fluids blood plasma and blood serum, (vi) pigment content in ripen fruit (vii) humic substances etc.

Excited state prototropism and its applications

Certain molecules (like naphthols, carbazoles, hydroxy flavones) show remarkable enhancement in their acidity in the excited singlet state. The excited state prototropic equilibrium is highly sensitive to the microenvironment around the fluorophore. Since emission maxima of the neutral and anionic forms of the excited state acids are usually widely separated, such fluorophores as two-state fluorescent probes enable convenient single or dual wavelength monitoring of fluorescence intensity and lifetime. This concept has been successfully introduced as a probe concept to study properties of lipid bilayer membranes.

Fluorescent Drugs

Intrinsically fluorescent drugs have been used to evaluate the interaction of drugs with bile salts. The use of bile salts and other bio-colloids such as liposomes, cholesterol and proteins offer better drug delivery systems. Interactions of certain fluorescent drugs such as Amphoterecin B, Fisetin etc with sodium deoxycholate, sodium cholate, sodium taurodeoxycholate and sodium taurocholate have been evaluated.

Bromadiolone is an anti-coagulant rodenticide, widely used to control the rodent population. Residues of Bromadiolone in bodies of dead rodents can be toxic to scavengers, predators and indirectly to man. Toxicity of Bromadiolone is directly linked to its binding with serum albumin. Interaction of Bromadiolone, having a fluorescent hydroxycoumarin moiety with CTAB micelles was characterized. The interaction of Bromadiolone with human and bovine serum albumin was explored using fluorescence resonance energy transfer experiments.

Introducing new fluorescent probes

The success of fluorescent molecular probes for the study of organised assemblies is because of the distinct advantages offered in terms of high sensitivity, selectivity, fast response time, non-invasive nature and minimal perturbation to the microenvironment to be probed. There is always a constant demand for new chromophores that can serve as fluorescent probes for specific applications. Our research has been active in developing new fluorescent molecular probes and probe concepts for various applications.

Molecular modeling

Amphotericin B (AmB) is a widely used antifungal drug. Colloidal suspensions of sodium deoxycholate (NaDC) have been commercially used as a medium for AmB delivery. The feasibility of stable Ambile salt association through hydrophobic interactions has been explored by geometry optimization of AmB bile salt systems using Hyperchem software with inbuilt molecular mechanics calculations. An AMBER force field and Polak-Ribiere conjugate gradient optimizer are used. The heptaene face of a single molecule of AmB is allowed to interact with steroidal face of a single bile salt molecule under periodic boundary conditions of di mensions121023 A  (z), containing 63 water molecules. In this manner,AmB and bile salt molecules are allowed to interact through the hydrophobic faces and hydrophilic faces associate with the surrounding water molecules.

 

Polymers

Polymer gels are three dimensionally cross-linked polymer networks containing fluid. In their ability to retain a significant amount of water, hydrogels are quite similar to natural living tissues, rendering them useful for a wide variety of biomedical applications. Polymeric systems that are used for different biochemical and biomedical applications have been of interest to us and are studied using fluorescence spectroscopy. Gels are characterized by a heterogeneous distribution of microstructures or inhomogeneties. The heterogeneity and microstructures of hydrogels such as Poly-acrylamide, and Poly-vinylalcohol has been explored using solvent polarity probe, viscosity sensitive fluorescent probes, excimer forming fluorophores and excited state proton transfer probes. The growth of the polymer, as experienced by the fluorescent probe due to increased local rigidity and enhancement in local hydrophobic surface is reflected in the emission parameters of the fluorophores.

Hydrogels that undergo sol-gel transition in response to change in temperature are known as thermosensitive gels. Poly-N-isopropyacrylamide (PNIPAAm) is a thermoreversibile gel, which attracts much attention, because it can be readily dissolved in water and its phase transition temperature is close to body temperature, and results in phase transition. Due to their temperature-sensitive properties, at suitable temperatures, PNIPAAm gel and modified PNIPAAm are important as vehicles for drug delivery. Physicochemical properties of Poly-N-iso-propylacrylamide were characterized. Subsequently, the effect of bile salts on the thermoreversible gelation of PNIPAAm and its aggregation to nano and macrogels has been characterized.

Other research interests

In addition to the above, some other lines of research has been: mechanism of fluorescence quenching, solubilization by l3-cyclodextrin, thermal relaxation in nylon copolymers, estimation of total lipid in fungal systems, monitoring failure of XPLE cables, luminescence determination of europium etc.