I hereby declare that the subject matter of this thesis is the result of research conducted by me in the Department of Chemistry, Indian Institute of Technology Guwahati, India under the guidance of Dr. The lecture reflected the enormous opportunities within the well-known research field. as 'nanoscience and nanotechnology' today.
Nanotechnology in medicine
- Delivery Vehicle
- Drugs
- Diseases and detection
- Surface Plasmon Resonance
- Nanoparticles in biodetection
- What makes Nanoparticles so desirable in biodetection?
- Basic principles involved in molecule detection
An intelligent selection or design of the drug carrier would thus be a major step towards effective drug delivery. Changing the dielectric constant of the surrounding material affects the oscillation frequency due to the varying ability of the surface to accommodate electron charge density from the NPs.
Gold Nanoparticles (Au NPs)
Shape dependent advantages
Some of the incident light falling on the NPs is scattered, but the absorbed light causes heating of the particles resulting in a highly localized temperature increase, which can be exploited in the various proposed photo-thermal therapeutic uses of Au NPs. We have used this principle to establish the interaction between Au NPs and proteins by measuring the intrinsic fluorescence of the tryptophan group of the protein in the presence of Au NPs under reaction conditions.
Biocompatibility of Au NPs
The large field enhancement in the vicinity of Au nanocrystals is known to lead to the surface-enhanced Raman scattering effect (SERS), and developments in this area may potentially change the picture. Mirkin and colleagues79 have shown that it is possible to detect a wide range of biological macromolecules through binding events involving Au nanocrystals coated with specific molecules that provide a distinct Raman signature.
Au NPs in nanomedicine
Not only can changes in SPR absorbance be detected to detect adsorbed species in chemical, biochemical, sensory, and medical fields, but also the scattering signal can be used in imaging techniques to observe various interactions with functionalized NPs. Thus, from imaging to treatment, Au NP finds great application in the field of nanomedicine.
Proteins: The biological working horses
14 - differentiation of cancer cells from non-cancerous cells by dark-field light scattering imaging and absorption spectroscopy of solid ~40 nm Au nanospheres immunotargeted to EGFR overexpressed on cancer cells.89 Along with cancer imaging and diagnostic applications, the ability of Au NPs to effectively the conversion of absorbed light into localized heat can easily be used for therapy based on photothermal destruction of cancer cells.90-92 For example, Hirsch et al.90 used NIR-absorbing Au core-shell silica particles for photothermal destruction of cells human breast carcinoma in vitro as well as solid tumors in vivo. For example, the ability of antibody-conjugated magnetic particles for physical separation, together with their enormous potential to amplify enzymatic DNA replication (polymerase chain reaction), can be used to detect ultralow concentrations of disease-associated proteins in serum.93,94 In addition, protein detection using optical methods can be performed with homogeneous NP assays, including whole blood, without the need for additional separation and purification.95 For example, antibody-functionalized Au nanoshells have been adapted for specific detection of target analytes in a protein-rich background using sandwich-immunoassay principles where analyte-driven aggregation of nanoshells causes a red shift in their absorption spectra.94.
Motivation behind the thesis
16 - For example, any change caused in the structure of a biomolecule such as protein/enzyme due to the presence of NPs can lead to positive or negative effects on the activity/properties of the latter. The effect of the presence of NPs on enzyme activity has been an important concern and studied by different groups.
Layout of the thesis
It also led to the development of a method to follow starch digestion (in the form of a composite) by UV-vis spectroscopy, monitoring the change in the surface plasmon resonance (SPR) peak of the Au NPs in the composite. Our results suggested that the specificity of release of encapsulated NPs could be achieved with an appropriate combination of encapsulation materials and the choice of an appropriate enzyme that would cleave the encapsulation to release the NPs.
Introduction
In this work we have also tried to check for any changes in the biological activity of the enzyme after NP synthesis. The Au NP-α-amylase complex was purified, characterized, and tested for the functional activity of the NP-bound enzyme.
Materials and Methods 1 Enzymes and Reagents
- Spectroscopy and Microscopy Studies
- Modification of Free Thiol Groups of a-Amylase
The images were recorded immediately after the synthesis of the NPs, and also after studying the kinetics of starch degradation by Au NP-enzyme complex. The UV-vis spectrum of the sample was measured to determine the modification of the free cysteine thiol groups of α-amylase.
Results and Discussions
- Enzymatic Synthesis of Au Nanoparticles
We also observed that the synthesis of Au NP with α-amylase led to the decrease in the pH of the medium. However, in the present study, the enzymatic activity of the Au NP-α-amylase complex was maintained.
Summary
In the case of soluble (free) enzyme, the activity of the enzyme is based solely on the collision frequency between the enzyme and the substrate molecule and their steric orientations. This means that a significant number of enzyme molecules are preferentially oriented toward the medium and away from Au NPs, facilitating the reaction between starch and the enzyme molecules.
Introduction
An example of this would be to monitor the mechanism of enzymatic digestion of a starch-NP composite versus that of starch alone and the fate of the NPs after digestion. Herein, we report the results of the studies on the enzymatic release of Au NPs encapsulated in starch.
Materials and Methods
- Preparation of Starch-Au NP complex
- Estimation of starch in the composite using GOD-POD Method
- Enzymatic Starch Digestion Studies
- Experiment for SPR based kinetics study
Digestion of the starch–Au NP composite by α-amylase and subsequent recovery of the precipitate after digestion was performed as previously mentioned. The UV-vis spectra of the enzyme-treated starch-Au NP composite were recorded at different time intervals.
Results and Discussions
Interestingly, the presence of Au NPs in the starch-Au NP composite did not inhibit the formation of starch-iodine complex. Also, the presence of Au NPs in the iodine-treated composite was confirmed by X-ray diffraction (XRD; Figure 4). Essentially, the above results support the validity of the current method for estimating starch by iodine in the presence of Au NPs.
Summary
Dissolution kinetics followed by iodine test using the same reaction condition as in group (A) 1. However, the results clearly show that the enzymatic digestion of the compound can also be followed by SPR absorption of Au NPs. Interestingly, the studies found that the rate of digestion of free and compound starch followed the same kinetics.
Introduction
Here, we report the development of a new method for the rapid and efficient quantification of proteins in aqueous solution based on changes in the SPR absorption of citrate-capped Au NPs in the medium. However, there was no change in the spectral behavior above the critical concentration of the protein. To account for the changes in the absorption spectra, we proposed a model based on the agglomeration of Au NPs in the presence of proteins.
Materials and Methods
- Preparation of citrate stabilized Au NPs
- Preparation of denatured enzyme/protein solution
- Particle size analysis by DLS method
However, in the case of AMG, BSA and GFP, enzyme/protein solution was added in 10.0 µL increments. All the peak areas were then calculated (after performing proper volume correction) and plotted against the concentration of the protein in the solution. The ratio of enzyme:Au NPs was kept the same as in the other experiments.
Results and Discussion
The change in the spectral characteristics continued to occur upon further addition of the protein. Further investigations by TEM measurements indicated agglomeration of Au NPs in the presence of native form of the protein. Further, Lorentzian fitting of the absorption spectra of Au NPs in the presence of different.
No. Enzyme/Protein
Summary
The method relies on the interaction of the protein with citrate-stabilized Au NPs, leading to the changes in the absorption spectra due to the NPs. For example, the change in the area under the UV-vis extinction spectrum of citrate-stabilized Au NPs as a result of the addition of a protein solution varied linearly with the concentration of the protein. The results have been explained by Mie scattering theory based on the changes in the dielectric constant of the stabilizer as a result of interaction of citrate-stabilized Au NPs with protein.
Materials and Methods
- Preparation of citrate stabilized Au NPs
- Preparation of native/denatured protein solution
- Preparation of TGA-stabilized Au NPs
- UV-vis extinction spectra of citrate-stabilized Au NPs in the presence of proteins The citrate stabilized Au NPs solution as prepared was diluted (in phosphate buffer of
- Calculation of the area under the UV-vis spectrum
- Temperature dependent denaturation study of proteins
- Sample preparation for TEM analysis
- Fluorescence studies of proteins
It can be mentioned here that a new cuvette was used to record each spectrum in order to avoid sticking of the protein-stabilized Au NPs to the walls of the cuvette. The ratio of the peak area of the UV-visible spectra of Au NPs plus protein to that of Au NPs alone was calculated and plotted against the corresponding protein mole fraction. The ratio of the peak area for citrate-stabilized Au NPs with proteins to that of Au NPs alone was calculated using the same method as already described in the Experimental Section.
Results and Discussion
UV-vis spectra of citrate-stabilized Au NPs before and after addition of protein solution with increasing mole fraction of the native form of (A) α-amylase and (B) BSA. UV-vis extinction spectra of citrate-stabilized Au NPs before and after addition of protein solution with increasing mole fraction of the native form of AMG. Ratio of the area under the UV-vis spectrum of citrate-stabilized Au NPs in the presence of AMG to that of citrate-stabilized Au NPs (only) for different composition of native:denatured AMG (the enzyme is denatured at 80 0C).
Summary
- Preparation of Citrate Stabilized Au NPs
- Preparation of Dinitrosalicylic Acid (DNS) reagent The reagent consists of following two components
- Enzyme activity by DNS method
- To check Au NP interference with the DNS test
- Estimation of amount of free protein in solution
- Preparation of TEM samples
Herein, we report as high as 8-fold increase in the specific activity of the digestive enzyme α-amylase in the presence of citrate-stabilized Au NPs. Control experiments were performed for each of the maltose concentrations (by replacing Au NPs with buffer). From the concentrations of the proteins in the supernatants, the concentrations of protein bound to Au NPs were obtained.
Results and Discussion
Furthermore, experiments revealed that changing the concentration of Au NPs (other than those used herein) did not change the values of the highest activity of the enzyme, substrate affinity or rate of product formation (Figure 3 and Table 2 ). The fraction of enzyme attached to Au NPs and active (x), free enzyme (y) and the fraction attached to NPs but inactive (z). Also, TEM images of the enzyme-Au NP mixture in presence of additional Au NPs are shown in Figure 9 .
Summary
Overview of the work done
This indicated the preferential binding of α-amylase enzyme to Au NPs compared to AMG to Au NPs. The change in SPR of citrate-stabilized Au NPs due to the presence of a specific protein was a parameter to quantify the amount of protein. The degree of agglomeration of NPs, dependent on the enzyme:Au NP ratio, was found to be the main reason for the change in the specific activity of the enzyme.
Future prospects
Single exponential fits of data obtained in time-dependent heat denaturation studies for ΒSΑ. Single exponential fits of data obtained in time-dependent heat denaturation studies for ΑΜG. UV-vis spectra of Au NPs in the presence of different amount of supernatant (same solution) after separation from Au NP bound enzyme by centrifugation.
Deka, J
