Research

An overview                                                                                  

♠ Development of a novel method for counting water molecules in cross-linked polymer materials using FLIM super-resolution microscopy.

♠ Study of Nile red and polymer dynamics in NIPAM polymer using FCS and TCSPC.

♠ Ca2+ sensing in polymer fiber using FLIM imaging.

♠ Development of Twin-FRET probe and their application as biological nano ruler.

♠ Development  of  novel  cryogenic  microscope for  measurement of single-molecule emission, excitation  spectra  and corresponding images, simultaneously with different excitation wavelength at the same time.

♠ Study of  single molecule spectroscopy of Photosystem I (PSI) at 77K  temperature   to establish the fluorescence blinking mechanism.

♠ Study  of retinal protein  Gloeobacter  rhodopsin and  its binding kinetics with the  synthetic retinal chromo-    phore analoge and carotene and origin of the CD spectra.

♠ Study  of various  photo-induced  processes  like excited state Intramolecular charge transfer  (ICT), excited      state Intramolecular  proton transfer  (ESIPT)  etc.  in newly designed  molecular  systems containing Donor-     Chromophore-Acceptor or proton donor-acceptor groups by steady state and time resolved spectroscopy.

♠  Combined and competitive ICT and ESIPT processes in single aromatic and Schiff base molecules.

♠  Interaction of various ICT and  ESIPT probes with biological, biomimicking and supramolecular  assemblies to extract novel insights into the fundamental mechanism of interaction.

♠  Sensing of biologically important and Environmentally pollutant ions such as Zn2+, Cd2+, F-, OAc- etc.

♠  Molecular  Modelling  for correlation between the  experimental and  computational results by DFT and ab-initio calculations , Molecular docking and Molecular dynamics simulations.

In details

1. Development of a novel method for counting water molecules in cross-linked polymer materials using FLIM super-resolution microscopy

Water molecules play an important role in the structure, function, and dynamics of (bio-)materials, and an estimation of the number of water molecules inside different compartments is thus important. In presence of H2O, the fluorescence of red emissive dyes like Atto 655 is quenched by transferring the excited energy to the surrounding H2O molecules. Exchanging H2O with D2O increases the fluorescence quantum yield and concomitantly also increases the fluorescence lifetime. Here I used Fluorescence lifetime imaging (FLIM) to count water molecules around the reporter dye Atto 655, which is covalently embedded into microgels, an interesting class of nanoparticles with high potential in drug delivery, medicinal chemistry, and biochemistry. I recorded FLIM images in different H2O:D2O ratios in the swollen state of the microgels at 22 ºC and in their collapsed state at 40 ºC. Stern-Volmer analysis allowed us to calculate the number of water molecules in the direct surroundings of the dye molecule inside the microgels at different temperatures. Additionally, the combination of FLIM with localization-based super-resolution microscopy allows for an estimation of the spatial distribution of water inside microgels. [Angew. Chem. Int. Ed., 2024, e202318421, DOI: 10.1002/anie.202318421 ].

2.  Study of Nile red and polymer dynamics in NIPAM polymer using FCS and TCSPC

Study of Nile red and polymer dynamics in NIPAM polymer using FCS and TCSPC The biocompatible homopolymer, poly(N-isopropylacrylamide) (pNIPAM) has been extensively studied microscopically and spectroscopically due to its applications in the fields of drug delivery, bioimaging, and polymer physics. For further progress in these applications, a detailed understanding of the properties of pNIPAM is essential. Studies with the solvatochromic dye Nile Red open new possibilities to understand the structure of pNIPAM gels and the solvent conditions in them. Also, Nile Red has been found to be an ideal dye for PAINT studies in pNIPAM microgels. We studied the interaction between Nile Red and two linear pNIPAM polymers with different chain lengths of molecular mass 2.5 and 1600 kg/mol, respectively, and different concentrations in aqueous solution. The diffusion coefficient of Nile Red was determined by FCS and the Nile Red fluorescence lifetime measured with TCSPC revealed its surroundings. Surprisingly, the diffusion coefficient of Nile Red does not reflect one of the polymer chains which means that it does not permanently bind to pNIPAM. That binding occurs, however, can be shown with the fluorescence lifetimes which can be fitted with two main components, one for Nile Red in the surrounding of (dense) pNIPAM and the other one rather resembling Nile Red in aqueous solution. [Conf. Proc., Single Mol. Workshop 22, PicoQuant, Berlin, P16, 62]

3. Ca2+ sensing in polymer fiber using FLIM imaging.

Casein is a milk protein with a spherical quaternary structure (size ~50 to 100 nm) which is called casein micelles (CMs). In CMs, colloidal calcium phosphates are distributed throughout the micelle in the form of calcium phosphate nanoclusters. The casein fibre regenerated from rennet-treated casein micelles in a calcium chloride coagulation bath contains a lot of free Ca2+. I estimated the free Ca2+ in casein fibre by quantitative FLIM imaging using a calcium sensing dye Oregon Green 488 BAPTA-6F. I measured FLIM images by a z-stack scanning through the fiber within a range of 50 μm with 10 μm step size. In each FLIM image, a region of 257 × 257 μm2 was scanned with a spatial resolution of 0.519 μm pixel−1. The fluorescence intensity decays (figure on the right) were fitted using a biexponential decay model for the selected pixels (figure on the left) using SymPhoTime 64 software. Reference measurements were performed for calibration with different Ca2+ concentrations for a fixed dye concentration. Comparing with the reference measurement I easily determined the free Ca2+ in casein fibre and out of the casein fibre in solution. This method can also be applied to live cells to examine calcium regulation.

S. Jana et al, Fine Structure and Swelling Properties of Fibers from Regenerated Rennet Treated Casein Micelles, Macromol. Mater. Eng., 307(2022) 2200272,

4. Development of Twin-FRET probe and their application as biological nano ruler

Conformational changes in biomolecules underpin all biological processes and being able to quantify these structural changes in solution is crucial to understand cellular function. By carefully choosing and positioning two fluorophores within the biomolecule, it is possible to use a property, so-called fluorescence resonance energy transfer (FRET) that is highly dependent on the relative separation of the two labels (FRET pair), to act as a molecular ruler and measure the desired distance. However, the current techniques still require to incorporate a FRET pair involving two different labels. For proteins in particular, labelling with two different fluorophores remains a substantial challenge. Currently, this can be done stochastically by covalently attaching the markers to two cysteine residues, which leads to a difficult-to-purify mixture of species, or by using orthogonal chemistries for each fluorophore that require far from trivial protein-specific modifications. To overcome this limitation, I am developing a barrier-breaking user-friendly strategy that removes entirely the need to incorporate two different fluorescent molecules to measure a distance. We are developing the novel concept of ‘TWIN-FRET’ where two identical fluorescent molecules act as the FRET pair. The TWIN-FRET molecule has been chosen so that it exhibits an acid-base equilibrium, at physiologically relevant pH values, between a neutral (FRET donor) and an anion (FRET acceptor) species emitting at different wavelengths. Crucially, each molecule at each position has the FRET pair encoded in its equilibrium chemical structure and therefore the ability to act as a donor or as an acceptor in the presence of an identical molecule placed nearby. We are going to simplify enormously the way biomolecular distances are measured in solution and we will deliver fluorophores and bioassays ready for commercialization and use in any academic or biotechnological environment.

Biophysical Journal 116 (2019) 565A, Supplement 1

5.

Development of novel cryogenic microscope to measure single molecule emission, excitation spectra and imaging in photosynthetic research. Study of single molecule PSI at 77K temperature to establish fluorescence blinking mechanism

Solar energy is the most abundant source of renewable energy, and this energy is harvested and stored via photosynthesis in plants, algae, and cyanobacteria etc. Therefore, our interest is to completely understand the fundamental processes of photosynthesis and chemical conversion of different types of energy. Till date, photosynthetic research has achieved to its molecular level for understanding the actual mechanism, but, there are some mechanisms not fully understood due to lack of proper instruments by which we can study optical spectra of single photosynthetic proteins.

Recently, our group, reported the blinking of fluorescence intensity of PSI at liquid N2 temperature. Till now, its exact  mechanism is not established. We proposed a model to explain this phenomenon. To established this proposed model and to explain the reason of blinking of fluorescence intensity we have to measure the excitation spectra and images of single PSI molecule with different excitation wavelength at the same time. For this purpose we have designed and developed a new microscope to measure the emission and excitation spectra and their corresponding images of single molecule PSI at cryogenic temperature.

6.

Study of retinal protein Gloeobacter rhodopsin and  its binding kinetics with the  synthetic retinal analogue in presence of carotene and origin of the CD spectra

Retinal proteins like bacteriorhodopsin, proteorhodopsin, xanthorhodopsin, halorhodopsin, sensory rhodopsin etc. play important roles in transporting of proton through the cell membrane following light absorption and transformation of energy into an electrochemical gradient. In addition, retinal proteins act as well as ion pumps and channels and as photo sensors. The gR protein shows very fast photocycle, and acts as a light driven proton pump, and transfers the proton from the cytoplasmic region to the extracellular region of a cell following light absorption.

The main goal of this research is to explore the mechanism of retinal binding kinetics and role of salinixanthin (sal) for pigment formation. We have tried to establish that how does the sal interact with the retinal and affect the synthetic retinal binding rates to the apo protein of gR (apo-gR). We want to know that, first retinal goes into the binding pocket and then formation of protonated Schiff base followed by fixation of the carotene or vice versa. Which part of the retinal is important for formation of protonated Schiff base and also the rate? Is pH has any effect on the retinal binding? For this purpose, we have examined the retinal binding process to the apo-gR in different pH and determined binding rates in presence and absence of sal and also evaluated pH effect on the binding process. To establish the role of retinal ring on the binding kinetics to apo-gR, we have used a series of synthetic retinal analogues by modifying in their retinal ring and side chain.

7.

Photo-induced ICT process in a series of newly designed flexible molecular systems containing Donor-Chromophore-Acceptor groups and ESIPT process in some drug derivatives

Photo-induced charge transfer has been found to produce polarity sensitive emission properties in an impressively massive number of ‘donor-acceptor’ systems: molecules having a charge donor and an acceptor group attached to a central aromatic chromophore. These molecules have been widely studied throughout past few decades due to their vast applications such as pH and ion detectors, thin film transistors, electro-optical switches, solar cells, creation of new opto-electronic devices such as electroluminescence devices and chemical sensors for free volume measurement in polymers, probes for the study of micro-heterogeneous environments, degree of water penetration into the surfactant aggregates and sensing the local polarity of the micro-environment at binding sites on proteins and so on. keeping these utilities of ICT molecules in mind, we have designed some flexible ICT molecules by introducing extra C=C double bond between the chromophore and acceptor groups which control the ICT properties. Our main objective is to design, synthesize and study the photophysical properties of molecules which show tunable ICT property.

Proton transfer in hydrogen bonded systems is a central mechanistic and kinetic step in many important reactions and plays a crucial role in many elementary reactions of living systems. Various types of proton transfer reactions, depending on acidity and basicity, adiabatic and non-adiabatic interactions, strong versus weak hydrogen bonding interactions and whether the reaction is taking place in the ground or excited state, have been categorized. Among them, excited state intra-molecular proton transfer (ESIPT) reaction is particularly of immense importance and has received considerable attention due to their vast applications in the fields of biochemistry, analytical chemistry, electrochromic modulation, laser dyes, molecular memory storage devices, fluorescent probes, polymer protectors, metabolic process of living systems and so on. In this area we focus mainly on unique systems with proton donor and acceptor sites within the same molecule where cyclic intra-molecular hydrogen bonded (IMHB) ring in the ground state facilitates proton transfer in the excited state of the molecule.

8.

Combined and competitive ICT and ESIPT processes in single aromatic and Schiff base molecules

Literature reports suggest that the ICT and ESIPT molecules have wide range of  applications in  various basic and  applied  fields. But  when  both  the  ICT  and  ESIPT  properties  are  combined in  a single  molecule, it is expected  that their  utility and applications  will be  magnified manifold.  For  this purpose we  have designed, synthesized and established  the photophysical properties of some aromatic and simple Schiff base molecules containing both the ICT  and  ESIPT moieties. At the same time, we are interested in exploring whether both ICT and ESIPT processes occur independently or are there some interactions  like  proton transfer assisted charge  transfer or suppression of ICT process by ESIPT process etc.

9.

Interaction of various ICT and ESIPT probes with biological, biomimicking and supramolecular assemblies to extract novel insights into the fundamental mechanism of interaction

Fuorescent  probe  spectroscopy  is  rapidly  gaining  importance  as  a  non-invasive  efficient  technique  for studying  proteins  and  related  biological  systems as  well  as  chemical  unfolding  of  proteins  induced  by denaturing agents like guanidine hydrochloride,  urea and surfactants  etc.  and also to extract  novel insights into  the  fundamental  mechanism  of  interactions,  thermodynamics  and  kinetics of  interaction  processes between the probe  and  protein molecules.  For  this  purpose,  our designed  and photophysically established  long  chain  ICT  and  ESIPT  molecules  have   been   utilized   for   studying  the  biological  and   biomimiking environments  like  model   proteins   human  and  bovine   serum   albumins,  miceller  and  reverse   miceller  systems  and supramolecular macrocyclic host like cyclodextrins etc.

10.

Sensing of biologically important and Environmentally pollutant ions

Minute quantity of transition  metal ions  such as  Cu2+,  Fe2/3+,  Zn2+ etc,  and  biologically  important  anions namely F-,  OAc-, H2PO4- etc are  essential  for human body.  At the  same time,  these are  dangerous to health or to  the  environment when it exceeds their natural  accumulation limit. On  the other  hand, Hg2+, Cd2+, As3+, Sb3+ etc.  are  always  toxic  even  they  present  in  minute  quantity.  Therefore,  the development  of  artificial chromogenic/ fluorogenic sensors (host) for environmentally and biologically important  and pollutant  ions is the  focus  of our current  research  interest. Here,  we  are  interested  with  the  flexibility  dependent  sensory  activity of the sensor  and showed  the change of  binding  mode of the  sensor after  introduction of flexibility within  the sensor.  Flexible  sensor  also  shows ratiometric  displacement  of  an  heavy toxic metal  ion by the essential transition metal ion.

11.

Quantum chemical calculations and molecular modelling

Advanced quantum chemical techniques are used to get the better insight about the photophysical properties (ICT & ESIPT) of molecules  like  ground  and excited  electronic states calculation, potential energy curve and surface generation,  calculation of  oscillator  strength  for  electronic  transition,  absorption  and  emission spectral band maxima, HOMO-LUMO diagram generation using Gaussian 03/09 suit. Protein-probe molecular docking and molecular dynamics simulations are also performed for the study the various weak  interactions (particularly hydrogen bonding,  electrostatic,  Van  der  Walls  interactions  etc. ),  calculation of Gibbs'  free energy changes, association/ binding constants, RMSD, RMSF and radii of  gyration etc. after the  binding  of our ICT and ESIPT probes with the protein or other macro-molecules.

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