Annex-V
PK- 1/ 1
SVKM’S NMIMS
SHOBHABEN PRATAPBHAI PATEL SCHOOL OF PHARMACY & TECHNOLOGY MANAGEMENT, MUMBAI
SYNOPSIS
MINOR PROJECT - M.Pharm. +MBA
Academic Year- (2017-2018)
Name of the Student: Ms. Madhushree Phalak
Programme and Specialization: M.Pharm. (Quality Assurance)+MBA
Name of the Guide: Dr. Prashant S. Kharkar
Title: Applications of Fluorescent Probes for Esterase detection.
Introduction:
Esterases are hydrolase enzyme present in all the living organisms which can be used to provide information on the metabolic state of the bacterial cells. [1]
Enzyme-activated, fluorescence probes are powerful tools for studying bacterial pathogens, including Mycobacterium tuberculosis. These small molecule probes are versatile tools as they target specific enzyme classes in complex mixtures of living organisms. [2]
Detecting and characterizing these esterases has been difficult because most of them form inclusion bodies in heterologous hosts , fluorogenic enzyme probes can directly detect enzyme activities in intact organisms or in protein gel-resolved lysates without the need for isolated enzyme. [3]
Since the probe excites above 600nm, cellular autofluorescence and light scattering is minimal in this region of the spectrum, giving the probe superior fluorescent properties for imaging in living systems. [4]
A variety of enzyme-targeted probes have been used to better understand the bacterial strains and substrate analogues have been employed to annotate the genome, identify therapeutic targets, and characterize enzymatic activity. [4]
Objectives:
- To carry out enzyme kinetic evaluation of the prepared Fluorogenic Probes, using various sources of esterases.
- With the prepared fluorogenic probes, we look forward to developing assays that can stratify bacterial strains, annotate enzyme function, and track specific esterase activities during different stages of bacterial growth.
Plan of Work:
- Synthesis of the Fluorescence Probes from the acquired materials.
- Characterization of Fluorescence Probes
- Spectral Characteristics will be determined (Absorbance and Emission spectra’s , Extinction co-efficients ,Quantum yields )
- Assessment of the hydrolytic stability of the prepared fluorescence probes.
- Examination of the sensitivity of the probes by determining the PLE detection limits for the prepared probes.
- Evaluation of the probes for enzyme kinetics.
- Conducting gel based assay for the probes.
Time Line of the Project:
Sr. | Nature of work | Duration in weeks |
No. | ||
1. | Literature and patent search and Review Article. | 1 |
2. | Listing and procurement of materials | 2 |
3. | Synthesis of the Fluorescence Probes from the acquired materials | |
4. | Characterization of Fluorescence Probes | |
5. | Assessment of the hydrolytic stability of the prepared fluorescence probes | |
6. | Examination of the sensitivity of the probes by determining the PLE detection limits for the prepared probes | |
7. | Evaluation of the probes for enzyme kinetics. | |
8. | Conducting gel based assay for the probes. | |
9. | Patent/Research Publication | 1 |
10. | Thesis submission | 1 |
Total | 24 Weeks |