Shibsekhar Roy

Novel Multiparametric Approach to Elucidate the Surface Amine-Silanization Reaction Profile on Fluorescent Silica Nanoparticles

This Article addresses the important issue of the characterization of surface functional groups for optical bioassay applications. We use a model system consisting of spherical dye-doped silica nanoparticles (NPs) that have been functionalized with amine groups whereby the encapsulated cyanine-based near-infrared dye fluorescence acts as a probe of the NP surface environment. This facilitates the identification of the optimum deposition parameters for the formation of a stable ordered amine monolayer and also elucidates the functionalization profile of the amine-silanization process. Specifically, we use a novel approach where the techniques of fluorescence correlation spectroscopy (FCS) and fluorescence lifetime measurement (FL) are used in conjunction with the more conventional analytical techniques of zeta potential measurement and Fourier transfer infrared spectroscopy (FTIR). The dynamics of the ordering of the amine layer in different stages of the reaction have been characterized by FTIR, FL, and FCS. The results indicate an optimum reaction time for the formation of a stable amine layer, which is optimized for further biomolecular conjugation, whereas extended reaction times lead to a disordered cross-linked layer. The results have been validated using an immunoglobulin (IgG) plate-based direct binding assay where the maximum number of IgG-conjugated aminated NPs were captured by immobilized anti-IgG antibodies for the NP sample corresponding to the optimized amine-silanization condition. Importantly, these results point to the potential of FCS and FL as useful analytical tools in diverse fields such as characterization of surface functionalization.

Fluorescence Lifetime Analysis and Fluorescence Correlation Spectroscopy Elucidate the Internal Architecture of Fluorescent Silica Nanoparticles

Fluorescence lifetime (FL) analysis and fluorescence correlation spectroscopy (FCS) have been successfully employed to reveal detailed information about the internal architecture of fluorescent silica nanoparticles (NPs). The dual-component lifetime behavior shows a two-domain dye distribution in the NP as a function of solvent accessibility. The introduction of an undoped silica shell serves to stabilize the outer dye fraction that is manifest as an increase in lifetime. The FCS not only shows a size-dependent increase in the cross-correlation decay constant but also demonstrates a significant relaxation in the FCS signal with the introduction of an undoped silica shell.

Protein synthesis in plant mitochondria II. Glutamate and glutamine incorporation and a study of initial steps and streptomycin effect

Abstract 1. 1. l -Glutamic acid and l -glutamine were incorporated nearly to the same extent into the protein of mitochondria isolated from 48-h seedlings of Vigna sinensis (Linn.) Savi. This, however, was not due to mutual interconversion. 2. 2. d -Glutamic acid was not incorporated into the mitochondrial protein, and inhibited the incorporation of l -glutamic acid. 3. 3. Adenosine 5′-triphosphate-pyrophosphate exchange, dependent on amino acids, was demonstrated in the mitochondrial preparation. The exchange values are however not high. Glutamic acid and its amide catalysed this exchange to nearly the same extent. 4. 4. Ribonuclease (EC 2.7.7.16) was without any effect on the incorporation, unless the mitochondria were treated with digitonin. 5. 5. Incorporation of l -glutamic acid into mitochondrial ribonucleic acid was demonstrated. 6. 6. Chloramphenicol inhibited the incorporation of l -glutamic acid into both mitochondrial protein and ribonucleic acid. 7. 7. Incorporation of l -glutamic acid into the protein and ribonucleic acid of mitochondria was inhibited by streptomycin. 8. 8. Oxidative processes and concomitant phosphorylation were unaffected in presence of streptomycin. 9. 9. Streptomycin had no appreciable effect on the normal release of nucleotides, ribonucleic acid and proteins from mitochondria. 10. 10. A complex of streptomycin with mitochondria and the ribonucleic acid therein was detected.

Deoxyribonucleic acid and the synthesis of protein in plant mitochondria. IV.

Abstract In the 5-time washed mitochondria from 48-h seedlings of Vigna sinensis (Linn) Savi, 9–11 μg of DNA per mg of protein is present. Actinomycin D and mitomycin C inhibit the incorporation of amino acids into the mitochondrial protein, implicating mitochondrial DNA in the process. The DNA has been isolated from the mitochondria and rendered free of protein, but a portion of the RNA (about 5 %) could not be removed. The ultraviolet absorption spectrum, the sedimentation velocity pattern of this DNA fraction, and the action of deoxyribonuclease upon it, indicate that the sample is DNA and probably consists of one major component.

From particle to platelet: Optimization of a stable, high brightness fluorescent nanoparticle based cell detection platform

UNLABELLED Nanoparticles are increasingly used as diagnostic tools due to the ease with which their surface chemistry, optical and physical properties can be controlled. Molecules, drugs, enzymes and fluorophores can be protected within the particle core or conjugated externally conferring nanoparticle biocompatibility, target specificity or environmental sensitivity. This study details the development and characterisation of stable, bright, dye-doped silica nanoparticles which are surface functionalised with PAMAM dendrimers to enable efficient conjugation to platelet activation-specific antibodies. We present the physical and optical properties and demonstrate colloidal stability. We also provide the first evidence of how NPs can be employed to specifically label human platelets immobilised on a lab-on-a-chip platform. Using a single step protocol, we demonstrate highly specific platelet labelling with the distribution of antibody-conjugated NPs matching that expected for the platelet GPIIb/IIIa receptor. The work highlights the potential of functionalized fluorescent NPs as diagnostic tools for cardiovascular disease. FROM THE CLINICAL EDITOR This study details the development and characterization of PAMAM dendrimer functionalized, stable, and bright dye-doped silica nanoparticles that enable efficient conjugation to platelet activation-specific antibodies. These fluorescent NPs may specifically label human platelets that can be used as diagnostic tools for cardiovascular disease.

Controllable self-assembly from fibrinogen–gold (fibrinogen–Au) and thrombin–silver (thrombin–Ag) nanoparticle interaction

Fibrinogen conjugated gold nanoparticles (fibrinogen–Au) and thrombin conjugated silver nanoparticles (thrombin–Ag) were synthesized by heating (90 °C) the proteins (50 μg protein/ml) with 1 mM AgNO3 or AuCl3. The resultant particles were harvested and examined by flow cytometry, scanning electron microscopy (SEM), transmission emission microscopy (TEM), optical microscopy and dynamic light scattering. SEM and TEM images revealed that the fibrinogen–Au and thrombin–Ag particles interacted. The emergent bio‐nanoconjugate population could be controlled by addition of thrombin–Ag. The method may be exploited in parametrizing coagulation factors and other clinically important protein–protein interactions.

Dendrimer driven self-assembly of SPR active silver-gold nanohybrids.

A fourth generation PAMAM dendrimer has been successfully employed for the development of a single step synthesis strategy for self-assembled Ag-Au nanohybrid structures. The surface plasmon resonance properties and the degree of self-assembly of the nanohybrid are strongly correlated with the stoichiometry of the metals which gives rise to enhanced plasmonic properties. The enhanced plasmonic response of the nanohybrids is modeled and is validated experimentally in a model HRP (horseradish peroxidise) bioassay carried out on an SPR-based biochip platform.

Thermal hysteresis of some important physical properties of nanoparticles.

Gold nanoparticles show thermal hysteresis with properties such as surface plasmon absorption, conductivity, and zeta potential. The direction of the incremental change in plasmon peak position and its extinction depend on the nature of surface conjugation. The thermal profile of a surface plasmon resonance spectrum for nanoparticles may serve as a signature for the associated small molecule or macromolecule on which it is seeded. The thermal responses of zeta potential and conductivity profile are found to be independent of the surface conjugation with the later being subjected to a phase transition phenomenon as revealed by a temperature criticality.

Synthesis and characterization of model silica?gold core?shell nanohybrid systems to demonstrate plasmonic enhancement of fluorescence

In this work, gold-silica plasmonic nanohybrids have been synthesized as model systems which enable tuning of dye fluorescence enhancement/quenching interactions. For each system, a dye-doped silica core is surrounded by a 15 nm spacer region, which in turn is surrounded by gold nanoparticles (GNPs). The GNPs are either covalently conjugated via mercapto silanization to the spacer or encapsulated in a separate external silica shell. The intermediate spacer region can be either dye doped or left undoped to enable quenching and plasmonic enhancement effects respectively. The study indicates that there is a larger enhancement effect when GNPs are encapsulated in the outer shell compared to the system of external conjugation. This is due to the environmental shielding provided by shell encapsulation compared to the exposure of the GNPs to the solvent environment for the externally conjugated system. The fluorescence signal enhancement of the nanohybrid systems was evaluated using a standard HRP-anti-HRP fluorescence based assay platform.

Label-Free Optical Characterization Methods for Detecting Amine Silanization-Driven Gold Nanoparticle Self-Assembly

Fluorescence lifetime correlation spectroscopy (FLCS) is presented as a single-step label-free detection method for probing the amine silanization-driven spontaneous 3D self-assembly of freestanding gold nanoparticles (GNPs) in solution. Unlike the conventional methods of studying self-assembly, for example, UV-vis spectroscopy and electron microscopy, FLCS utilizes the intrinsic gold fluorescence. The significance of this approach is to amalgamate the measurement of optical and hydrodynamic size properties simultaneously to achieve a more coherent description of the self-assembly pathway. GNP self-assembly has two-stage kinetics. Electrostatic interaction drives the initial amine silanization, and this is followed by siloxane bond formation between hydrolyzed ethoxy groups of GNP-attached APTES, resulting in the formation of micrometer-sized superstructures. The self-assembly has resulted in a 5-fold increase in the fluorescence lifetime (FL), and the FLCS study has shown an 8- to 10-fold increase in the diffusion coefficient using the pure diffusion model. This result is consistent with the transmission electron microscopy (TEM) observation, which shows a few hundred fold increase in the diameter due to assembly formation by the GNPs.