Spectroscopic investigations of lactose oxidase and its CDs conjugates

Abstract

To study Langmuir monolayer properties of lactose oxidase and CDs-Conjugate and to further investigate the interactions between the ligand on the subphase and the monolayers Background Surface chemistry is an approach in biophysical chemistry that can be used to understand biological functions such as the nature of the cell membrane interfacial, properties of various peptides and proteins. The best way to understand the interactions of surface proteins with their membrane and subphase environment is to study their air-subphase behavior by using the Langmuir film technique. Basic information like protein packing and conformation studies can be obtained from measurement of the surface pressure, average molecular area and surface potential, however, detailed information can be obtained by employing additional spectroscopic and microscopic studies at different surface pressures. In Langmuir monolayer, amphiphilic molecules are spread at the air-water interface. In doing so, the molecular packing is controlled by compression using movable barriers. The study of Langmuir film is basically used to measure the surface pressure as a function of surface area available to surfactant molecules at constant temperature which is so called as surface pressure-area (À-A) isotherm. This measurement gives information about the existence of different phase transitions and the stability of the film at specific temperature and pH .The isotherm behavior of the Langmuir film is usually estimated by the physical and chemical properties of amphiphilic molecules, temperature and composition of the subphase. Lactose oxidase (LO) oxidizes lactose to lactobionic acid, producing H2O2 that could actas an antimicrobial and activate the lactoperoxidase system (LPS), an antimicrobial system present in milk. As per our knowledge, the Langmuir monolayer property of this important enzyme is unknown till this date. In this project we aim to study the interfacial behavior of the LO. Further, we aim to conjugate the LO with carbon dots (CDs) and study the surface property of the conjugate. As we were interested to study the LO Langmuir monolayer at air/subphase interface, we will utilize the Langmuir monolayer film technique to learn basic surface chemistry properties and to investigate the behavior of LO at the air-subphase interface at various surface pressures that correspond to different states, ranging from the gaseous phase to the liquid compressed phase. Further, we will investigate the LO Langmuir monolayer in absence and presence of CDs of varying concentration on the water subphase. Moreover, the conjugation of enzyme with carbon dots provides various nanohybrids with new possibilities in biosensor applications. Also, the activity and stability of enzyme can be enhanced due to the larger surface area of nanomaterials. Methodology: To achieve our goal, we will follow four basic aims as shown below: Aim 1: Langmuir monolayer and Langmuir Blodgett (LB) studies of Lactose oxidase at different pH and temperatures. Aim 2: Langmuir monolayer and LB studies of the CDs-Lactose oxidase conjugates. Aim 3: Interactions with the ligand (lactose) in the subphase, both with Lactose oxidase andconjugates. Aim 4: Use of the conjugate to develop a biosensing platform. (AU)