A range of microgels with two different functionalities were synthesized, and their utility in novel bioapplications was examined. Cationic microgels with varying properties were developed by tuning synthesis conditions. Their size and primary amine content was analyzed, and one microgel system was selected as a model construct. Its primary amine groups were conjugated to two dyes with properties favorable for utilization as contrast agents in photoacoustic imaging. The concentration of contrast agent in single particles was determined. The implications of a high local dye concentration in the generation of high intensity photoacoustic signals, are discussed. The second bioapplication involved the targeted delivery of fibrinolytics to fibrin clots, in order to bring about dissolution of abnormal thrombi. For this purpose, core/shell microgels with carboxylic acid groups in their shells were synthesized in three size ranges. Following this, their dimension based differential localization in and around porous fibrin clots was examined. Fibrin-specific peptides were then conjugated onto the shells of these particles and the conjugates were shown to demonstrate strong interactions with the fibrin clots. The microgels conjugated to the peptide with the highest binding affinity to fibrin, were observed to bring about disruption of fibrin clots, merely through interference in the dynamic interactions among clot fibers, due to the equilibrium nature of the fibrin polymer. The implications of these novel results and future studies required to facilitate a better understanding of the phenomena involved, are discussed.

Polyketides are natural products with complex chemical structures and immense pharmaceutical potential that are synthesized via secondary metabolic pathways. The in-vitro synthesis of these molecules requires high supply of building blocks such as acetyl Co-enzyme A, and cofactors (adenosine triphosphate (ATP). These precursor and cofactor are synthesized from respective soluble enzymes. Owing to the expensive nature of the enzymes, it is important to immobilize enzymes to improve the process economics by enabling multiple uses of catalyst and improving overall productivity and robustness. The polymer-based particles of nano and submicron size have become attractive material for their role in the life sciences. With the advances in synthetic protocols of the microgels and commercial availability of many of the monomers, it is feasible to tune the properties of the particles as per the process requirement. The core shell microgel with functional shell allows high loading of ligands onto the microgel particles due to increased availability of functional group on the outer surface. The aim of the thesis thus was to study biochemical reactions on the smart microgels support using single (acetyl CoA synthetase (Acs)) and dual (pyruvate kinase (Pk) and L-lactic dehydrogenase (Ldh)) enzyme/s systems. The study indicated that the enzyme immobilization significantly depends on the enzyme, conjugation strategy and the support. The covalent immobilization provides rigidity to the enzyme structure as in case of Acs immobilized on PNIPAm-AEMA microgels but at the same time leads to loss in enzyme activity. Whereas, in the case of covalent immobilization of Ldh on microgel showed improved in enzyme activity. On the other hand adsorption of the enzyme via ionic interaction provide better kinetic profile of enzymes hence the membrane reactor was prepared using PNIPAm-PEI conjugates for acetyl CoA synthesis. The better outcome of work with PNIPAm-PEI resulted in its further evaluation for dual enzyme system. This work is unique in the view that the immobilization strategies were well adapted to immobilize single and dual enzymes to achieve stable bioconjugates for their respective applications in precursor biosynthesis (Acetyl Co enzyme A) and co-factor dependent processes (ACoA and ATP). The positive end results of microgels as the support (particles in solution and as the thin film (membrane)) opens further prospective to explore these systems for other precursor biomolecule production.

Polyketides are natural products with complex chemical structures and immense pharmaceutical potential that are synthesized via secondary metabolic pathways. The in-vitro synthesis of these molecules requires high supply of building blocks such as acetyl Co-enzyme A, and cofactors (adenosine triphosphate (ATP). These precursor and cofactor are synthesized from respective soluble enzymes. Owing to the expensive nature of the enzymes, it is important to immobilize enzymes to improve the process economics by enabling multiple uses of catalyst and improving overall productivity and robustness. The polymer-based particles of nano and submicron size have become attractive material for their role in the life sciences. With the advances in synthetic protocols of the microgels and commercial availability of many of the monomers, it is feasible to tune the properties of the particles as per the process requirement. The core shell microgel with functional shell allows high loading of ligands onto the microgel particles due to increased availability of functional group on the outer surface. The aim of the thesis thus was to study biochemical reactions on the smart microgels support using single (acetyl CoA synthetase (Acs)) and dual (pyruvate kinase (Pk) and L-lactic dehydrogenase (Ldh)) enzyme/s systems. The study indicated that the enzyme immobilization significantly depends on the enzyme, conjugation strategy and the support. The covalent immobilization provides rigidity to the enzyme structure as in case of Acs immobilized on PNIPAm-AEMA microgels but at the same time leads to loss in enzyme activity. Whereas, in the case of covalent immobilization of Ldh on microgel showed improved in enzyme activity. On the other hand adsorption of the enzyme via ionic interaction provide better kinetic profile of enzymes hence the membrane reactor was prepared using PNIPAm-PEI conjugates for acetyl CoA synthesis. The better outcome of work with PNIPAm-PEI resulted in its further evaluation for dual enzyme system. This work is unique in the view that the immobilization strategies were well adapted to immobilize single and dual enzymes to achieve stable bioconjugates for their respective applications in precursor biosynthesis (Acetyl Co enzyme A) and co-factor dependent processes (ACoA and ATP). The positive end results of microgels as the support (particles in solution and as the thin film (membrane)) opens further prospective to explore these systems for other precursor biomolecule production.