Plasmon Resonance Energy Transfer Nanoruler for Pinpointing Molecular Distance and Interaction on the Living Cell Membrane.

Developing novel strategies to measure nanoscale distance and molecular interaction on a living cell membrane is of great significance but challenging. Here we develop a model of a linker-free plasmon resonance energy transfer, termed "PRET nanoruler", which is composed of a single-sized nanogold-antibody conjugates donor (G26@antiCD71) and a fluorophore-labeled XQ-2d aptamer receptor (XQ-2d-Cy3), that produces a separation distance (r) dependent energy transfer (ηPRET). Both the theoretical finite element simulation and experiments evidence the observable PRET between single G26NPs and XQ-2d-Cy3. Regardless of the size of ηPRET, we could confirm r is less than 5 nm, the separation of two binding sites is in the range of 13.0-18.0 nm. There is a competitive binding of Tf and XQ-2d-Cy3 on CD71 receptors. PRET nanoruler realizes the estimation of the nanoscale separation distance, and determines the molecular interaction and competitive binding. It is an alternative tool for observing nanoscale single molecular events in the future.

Gold nanoparticles modified reduced graphene oxide nanosheets based dual-quencher for highly sensitive detection of carcinoembryonic antigen.

In the current scenario, the dominance of cancer is becoming a disastrous threat to mankind. Therefore, an advanced analytical approach is desired as the need of the hour for early diagnosis to curb the menace of cancer. In this context, the present work reports the development of nano surface energy transfer (NSET) based fluorescent immunosensor for carcinoembryonic antigen (CEA) detection utilizing protein functionalized graphene quantum dots (anti-CEA/amine-GQDs) and a nanocomposite of nanostructured gold and reduced graphene oxide (AuNPs@rGO) as energy donor-acceptor pair, respectively. The obtained AuNPs@rGO nanocomposite has been characterized by different advanced analytical techniques. The functionality of the biosensor depends on quenching the fluorescence of anti-CEA/amine-GQDs donor species by AuNPs@rGO acceptor species, followed by the gradual recovery of GQDs' fluorescence after CEA addition. The efficient energy transfer kinetics have been envisaged by utilizing the AuNPs@rGO nanocomposite as a dual-quencher nanoprobe that revealed improved energy transfer and quenching efficiency (∼62 %, 88 %) compared to AuNPs (∼43 %, 81 %) as a single quencher. Further, the developed biosensing platform successfully detected CEA biomarker with notable biosensing parameters, including a wider linear detection range (0.001-500 ng mL-1), fast response time (24 min), and a significantly low detection limit (0.35 pg mL-1).

Rat Embryonic Stem Cell Transgenesis.

The availability of reliable germline competent rat embryonic stem cell (ESC) lines that can be genetically manipulated provides an important tool for generating new rat models. Here we describe the process for culturing rat ESCs, microinjecting the ESCs into rat blastocysts, and transferring the embryos to surrogate dams by either surgical or non-surgical embryo transfer techniques to produce chimeric animals with the potential to pass on the genetic modification to their offspring.

Binding interactions of cationic gemini surfactants with gold nanoparticles-conjugated bovine serum albumin: A FRET/NSET, spectroscopic, and docking study.

This work demonstrates binding interactions of two cationic gemini surfactants, 12-4-12,2Br- and 12-8-12,2Br- with gold nanoparticles (AuNPs)-conjugated bovine serum albumin (BSA) presenting binding isotherms from specific binding to saturation binding regions of surfactants. The binding isotherm has been successfully constructed using Förster's resonance energy transfer (FRET) and nanometal surface energy transfer (NSET) parameters calculated based on fluorescence quenching of donor, tryptophan (Trp) residue by acceptor, AuNP. Energy transfer efficiency (ET) changes due to alteration in the donor-acceptor distance when surfactants interact with bioconjugates. A solid reverse relationship between α-helix and ß-turn contents of BSA-AuNPs-conjugates is noted while interacting with surfactants. 12-8-12,2Br- shows stronger binding interactions with BSA-bioconjugates than 12-4-12,2Br-. The effect of bioconjugation on secondary/tertiary structures of BSA in the absence and presence of a surfactant is studied through circular dichroism, fluorescence, and Fourier transform infrared spectroscopic measurements. Motional restrictions imposed by AuNPs on Trp residues of folded and unfolded BSA have been investigated using red edge emission shift (REES) measurements. Finally, the molecular docking results present the modes of interactions of 12-4-12,2Br- and 12-8-12,2Br-, and Au-nanoclusters (Au92) with BSA. An approach to describe the binding isotherms of surfactants using AuNPs-bioconjugates as optical-based molecular ruler and possible effects of AuNPs on microenvironment and conformations of the protein is presented.

Nonsurgical Embryo Transfer Protocol for Use with the NSET™ Device.

Genetically modified embryos must be transferred to a suitable female recipient for development to pups. Nonsurgical embryo transfer is a fast and efficient method used to deliver blastocyst stage embryos to the uterine horn of recipient females. The efficiency of recovery of pups after nonsurgical embryo transfer is similar to the efficiency after surgical transfer. However, nonsurgical transfer eliminates the pain and distress caused by the surgical procedure and provides a refinement in accordance with Russel and Burch's "3Rs" (The principles of humane experimental technique. Methuen & Co., London, 1959), an ethical framework for animal research. This method is also useful for rederivation of mouse strains. Rederivation is important for either removal of potential pathogens from an incoming mouse strain after shipping, or within a facility to obtain a clean mouse colony.

Systematic Analyses and Prediction of Human Drug Side Effect Associated Proteins from the Perspective of Protein Evolution.

Identification of various factors involved in adverse drug reactions in target proteins to develop therapeutic drugs with minimal/no side effect is very important. In this context, we have performed a comparative evolutionary rate analyses between the genes exhibiting drug side-effect(s) (SET) and genes showing no side effect (NSET) with an aim to increase the prediction accuracy of SET/NSET proteins using evolutionary rate determinants. We found that SET proteins are more conserved than the NSET proteins. The rates of evolution between SET and NSET protein primarily depend upon their noncomplex (protein complex association number = 0) forming nature, phylogenetic age, multifunctionality, membrane localization, and transmembrane helix content irrespective of their essentiality, total druggability (total number of drugs/target), m-RNA expression level, and tissue expression breadth. We also introduced two novel terms-killer druggability (number of drugs with killing side effect(s)/target), essential druggability (number of drugs targeting essential proteins/target) to explain the evolutionary rate variation between SET and NSET proteins. Interestingly, we noticed that SET proteins are younger than NSET proteins and multifunctional younger SET proteins are candidates of acquiring killing side effects. We provide evidence that higher killer druggability, multifunctionality, and transmembrane helices support the conservation of SET proteins over NSET proteins in spite of their recent origin. By employing all these entities, our Support Vector Machine model predicts human SET/NSET proteins to a high degree of accuracy (∼86%).

Facile synthesis of reduced graphene oxide-gold nanohybrid for potential use in industrial waste-water treatment.

Here, we report a facile approach, by the photochemical reduction technique, for in situ synthesis of Au-reduced graphene oxide (Au-RGO) nanohybrids, which demonstrate excellent adsorption capacities and recyclability for a broad range of dyes. High-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) data confirm the successful synthesis of Au-RGO nanohybrids. The effect of several experimental parameters (temperature and pH) variation can effectively control the dye adsorption capability. Furthermore, kinetic adsorption data reveal that the adsorption process follows a pseudo second-order model. The negative value of Gibbs free energy (ΔG0) confirms spontaneity while the positive enthalpy (ΔH0) indicates the endothermic nature of the adsorption process. Picosecond resolved fluorescence technique unravels the excited state dynamical processes of dye molecules adsorbed on the Au-RGO surface. Time resolved fluorescence quenching of Rh123 after adsorption on Au-RGO nanohybrids indicates efficient energy transfer from Rh123 to Au nanoparticles. A prototype device has been fabricated using Au-RGO nanohybrids on a syringe filter (pore size: 0.220 µm) and the experimental data indicate efficient removal of dyes from waste water with high recyclability. The application of this nanohybrid may lead to the development of an efficient reusable adsorbent in portable water purification.

Development of a Triplet-Triplet Absorption Ruler: DNA- and Chromatin-Mediated Drug Molecule Release from a Nanosurface.

Triplet-triplet (T-T) absorption spectroscopy has been used successfully as a molecular ruler to understand the actual release process of sanguinarine as a drug molecule from a gold nanoparticle surface in the presence of cell components, that is, DNA and chromatin. The obtained results have been verified by fluorescence and surface-enhanced Raman spectroscopy (SERS), and a plausible explanation has been put forward to describe the underestimation and overestimation of the percentage (%) of the release of drug molecules measured by fluorescence- and SERS-based techniques, respectively, over the highlighted T-T absorption spectroscopy. Because of the intrinsic nature of absorption, the reported T-T absorption spectroscopic assay overpowers fluorescence- and SERS-based assays, which are limited by the long-range interaction and nonlinear dependence of the concentration of analytes, respectively.

Fluorescence quenching of quantum dots by gold nanoparticles: a potential long range spectroscopic ruler.

The dependence of quantum dot (QD) fluorescence emission on the proximity of 30 nm gold nanoparticles (AuNPs) was studied with controlled interparticle distances ranging from 15 to 70 nm. This was achieved by coassembling DNA-conjugated QDs and AuNPs in a 1:1 ratio at precise positions on a triangular-shaped DNA origami platform. A profound, long-range quenching of the photoluminescence intensity of the QDs was observed. A combination of static and time-resolved fluorescence measurements suggests that the quenching is due to an increase in the nonradiative decay rate of QD emission. Unlike FRET, the energy transfer is inversely proportional to the 2.7th power of the distance between nanoparticles with half quenching at ∼28 nm. This long-range quenching phenomena may be useful for developing extended spectroscopic rulers in the future.

Molecular recognition of proteolytic activity in metastatic cancer cells using fluorogenic gold nanoprobes.

We describe the development of biomarker-sensitive nanoprobes based on nanoparticle surface energy transfer (NSET) effect that enabling recognition of the expression of membrane type-1 matrix metalloproteinase (MT1-MMP) anchored on invasive cancer cells and its proteolytic activity simultaneously. First of all, we confirmed invasiveness of cancer cell lines (HT1080 and MCF7) via migration and invasion assay. We also prepared gold nanoparticle (GNP) acts as a quencher for fluorescein isothiocyanate (FITC). This FITC is conjugated in end-terminal of activatable fluorogenic peptide (ActFP). The ActFP attach to surface of GNP (GNP-ActFP) for a targeting moiety and proteolytic activity ligand toward MT1-MMP. The GNP-ActFP can generate fluorescence signal when ActFP is cleaved by proteolytic activity after targeting toward MT1-MMP. In order to study specificity for MT1-MMP, GNP-ActFP is treated to HT1080 and MCF7 cells, and then, we determine the in vitro targeting potential and fluorogenic activity of GNP-ActFP for MT1-MMP via fluorescence multi-reader. We also confirmed fluorogenic activity of GNP-ActFP via confocal microscopic imaging, and finally, endocytosis of GNP-ActFP is observed via cellular transmission electron microscopic imaging.