Biotechnology and Biomedical

Tryptophan phosphorescence within protein molecules is gaining attention as a probe of protein dynamics and structure. The tryptophan phosphorescence lifetime, τ, varies with the protein molecule’s local environment and conformation.

The sample described here were DNA 35 base fragments thiol-grafted deposited on a thick gold layer. Although this experiment can be performed in air it is important to study such samples in a biosensor configuration, where the desired interaction between the grafted DNA and different analyte solutions can be characterised. The performance of the UVISEL Sepctroscopic Phase Modulated Ellipsometer allows very sensitive and selective study of the kinetics and quantification of such interactions.

This application note shows that the HORIBA Scientific SPRi platform is suitable for the analysis of aptamer-based molecular interactions. For this purpose, the interaction between human IgE protein, an antibody involved in allergic reactions, and a human IgE specific aptamer4 is presented as proof-of-concept.

Description of the advantages of combining the use of peptide chips with direct label-free detection as achieved by SPR imaging (SPRi).

Whilst biochips dedicated to cell analysis have generated interest in developing new tools for diagnostic and research purposes, microarray technology is playing a crucial role in detecting interactions; Further, optical detection by Surface Plasmon Resonance imaging (SPRi) combines both thechniques and offers great interest for the real-time direct measurement of label-free cell-surface binding.

Surface-enhanced Raman scattering (SERS) is a powerful plasmonics-based analytical technique for biosensing. SERS effect relies on nanostructures that need to be designed to maximize enhancement factors and molecular specificity. In addition to numerical modeling, an analytical tool capable of imaging localized enhancement would be an added value.

The coupling approaches of SPR to LC-MS and ProteinChip - based mass spectrometry (SELDI) as a means of identifiying proteins captured on DNA surfaces has been compared.

G-protein-coupled receptors (GPCRs), the largest family of integral membrane proteins, participate in the regulation of many physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases (approximately 30% of currently marketed drugs). However, despite their pharmacologic potential, clinically useful ligands do not exist for the majority of GPCRs. GPCRs are extremely hydrophobic, characterized by seven transmembrane-spanning α-helices. These receptors require a lipid environment to maintain the native structure, conformation and activity. Typically, GPCRs are expressed with low levels in native tissues.

The use of low molecular weight molecules in the industries of drug discovery and agri-food has become increasingly important over the course of a century of drug research and science development in agriculture and in pharmaceutical field.

Among the possible fluorescence biosensors for medical and biochemical monitoring and imaging are the flavonoids, compounds that occur in many plants and their products, such as tea, chocolate, and red wine.

This application highlights the development of an oligosaccharide chip (surface functionalization, density of immobilization, spacer arm length) on the same SPRi platform using conductive polymers. These results are highlighted through the example of interactions between HS and SDF-1 α chemokine.

Although the fluorescence time-scale is well-suited to follow many biological processes, often there is a requirement for processes to be monitored over longer time-scales.

Genes can be mutated in several different ways. The simplest type of mutation involves a change in a single base along the sequence of a particular gene (much like a typographical error in a word that has been misspelled). In other instances, one or more bases may be added or deleted. Sometimes too, segments of a DNA molecule are accidentally repeated, deleted or moved.

Our SPRi technology (Surface Plasmon Resonance imaging), is able to measure multiplexed label-free biological interactions in a complex media such as serum. We will demonstrate the ability to monitor specific interactions between immobilized peptides and proteins issued from non purified immune sera, using a cystamine/glutaraldehyde SAM surface chemistry on the bio-chip surface.

The prevalence of allergies in the world is between 30 and 40%. Even though it exists medical treatments such as antihistaminic and desensitization this number is constantly increasing. Thus, in this application note, the identification and chemical characterization of aeroallergens by Raman microscopy will allow to prevent people who are affected by respiratory allergies of the presence of this type of allergens in indoor and outdoor air.

Over 20% of the European population suffers from respiratory allergies. Many factors can cause these immune dysfunctions. The purpose of this proof of concept is to show that Surface Plasmon Resonance imaging (SPRi) is a suitable technique to detect allergens.

Characterization of protein-based samples utilizing fluorescence spectroscopy is fundamental to advancing our understanding of biomolecular structure, interactions, and dynamics, which are crucial for drug discovery, biophysics, and cellular biology. Yet, researchers face persistent challenges.

HORIBA provides the pharmaceutical industry, biotech companies and academic research laboratories with high performance instrumentation based on SPRi technology (Surface Plasmon Resonance imaging). To demonstrate the power of this technology, we carried out an experiment based on an antibody-antigen interaction. Original surface chemistry combined with an electrochemical process allows the rapid coupling of biomolecules to the gold layer (polypyrrole co-polymerisation) on the top of a glass prism.

Samples can be valuable. Either you don’t want to waste them, or you may not have very much starting material. Biological proteins and enzymes, for example, are often obtained in small volumes and may be expensive.

Various substitutes for bone materials have been researched and developed in orthopedic, clinic and surgical use today. In recent years, research has been actively conducted on bone prosthetic materials having a composition similar to living bone, and the need for a method of measuring carbon content in the materials is increasing.

This application reports on TERS characterization of binary phospholipid bilayer systems deposited onto a gold coated coverslip. Such samples are used as model samples for directly and chemo-selectively visualizing the distribution of a lipid constituent at the nanometer scale.

A wide range of elements are present in plants. Some elements exist to play important roles as plant nutrients or enzymes, and some elements accumulate unexpectedly due to environmental pollution. While it is important to understand the bulk concentrations of individual elements from a nutritional and ecotoxicological perspective, the distribution of elements is also important for a better understanding of the mechanisms of metabolism and uptake in plants.

Elemental distribution is important to understand metabolism in biological samples. Biological samples contain water or gas and therefore cannot be measured in a full vacuum environment because they will be significantly affected or damaged. The unique partial vacuum mode or He-purge mode with the XGT-9000 Series enables analysis of biological samples without compromising sensitivity to the light elements.

Calcium micro-nodules are identified within mulberry leaves, and XRF mapped imaging provides a correlation between nodule concentration and leaf age. In a seperate study, the uptake of heavy element pollutants by plants is investigated, with high resolution images quickly acquired to show the distribution of lead through the leaf.

The effect of zinc on gastric ulcer healing has been investigated - mapped imaging of tissue samples shows evidence of zinc accumulation within the ulcerated tissue. In a separate study a fish otolith ('ear bone') has been analyzed to reveal its heterogeneous elemental composition and physical structure.

Raman and resonance Raman spectroscopy have proven to be important research tools to investigate structure-function relationships in enzymes.

An imaging platform that combines Raman microscopy with enhanced darkfield and hyperspectral imaging technology was used to study lung tissue sections containing carbon nanotubes. It enabled easy identification of the regions containing the carbon nanotubes followed by spectroscopic characterization of chemical composition.

Silica is currently one of the most important industrial materials, whose nanoparticles are formed via a sol-gel process.

The study of singlet oxygen (1O2) is of interest, principally, as it is a highly reactive species. It can be produced by photosensitisation, usually of a molecule such as a dye or porphyrin. Thus, by the appropriate selection of sensitiser, the presence of oxygen and light, 1O2 can be selectively generated. From a biological aspect it has the ability to damage and destroy cells, which has lead to interest in its use as an anticancer agent in photodynamic therapy (PDT).

Quick and quantitative analytical evaluation of tooth decay is required in any study of the environmental factors promoting such decay. This information is essential to any study of which dental products are most effective in slowing or arresting the decay.

Recent developments CMOS technology allow widefield lifetime imaging, with the ability to measure FLIM in real time, to be employed to investigate mobile samples and kinetics, as well as remote imaging.

This application note describes FLIM technology and its implementation using HORIBA's FLIMera and InverTau instruments. These instruments, based on time-correlated single-photon counting (TCSPC), enhance fluorescence microscopy by providing an additional contrast mechanism based on fluorescence lifetime.

Rapid mixing accessories to perform stopped flow measurements have found application in characterizing interactions and reactions occurring in solution.

This technical note describes kinetic fluorescence as an analytical technique to quantify non-fluorescent species. The technique is applied to the determination of thiamine (vitamin B1) in solution.

The choice of protecting group is of crucial importance in the success of many steps in organic synthesis and the manipulation of polyfunctional molecules, since they can prevent the formation of undesired side products and reactions.

This study demonstrates the application of Excitation Emission Matrix measurement to monitor cell culture medium conditions during the proliferation of mammalian cells.

When Dr. Charles Mazel of Physical Sciences (Andover, MA) decided to make fluorescence measurements of corals, he began searching for the right instrument. The instrument would have to be transportable and able to withstand the pitch and yaw aboard a ship.

Biological processes can be traced through study of gene-expression via a “molecular beacon” (single-stranded DNA, ssDNA), a hairpin-shaped oligonucleotide with a fluorophore (donor) and a quencher (acceptor).

Polarized light striking a fluorescent molecule results in polarized fluorescence. This polarized emission gradually returns to unpolarized fluorescence depending on rotational diffusion and other factors.

The fluorescence spectra of intrinsic protein fluorophores have been studied extensively and used in investigating biological events. Native fluorophores in human tissue include NADH, collagen, elastin, tryptophan, tyrosine, porphyrins and FAD.

The potential health benefits stemming from the antioxidant activity of curcumin, commonly found in turmeric (Curcuma longa L), has attracted the interest of several research groups.

This application note explores the use of Duetta for a common absorbance application, the Protein A280 application.

Through its user-friendly software, Duetta allows different measurements related to biomolecules like DNA, RNA and proteins including protein concentration, DNA/RNA purity, finding the concentration of an unknown sample based on a standard-based calibration curve, Pass/Fail testing and all standard fluorescence and absorbance related measurements.

A mouse model for human atherosclerosis has been developed in which the animals lack ApoE; these animals are called ApoE knock-out mice. These two measurements -the results of the aorta of an ApoE knock-out mouse and the results of the chemical study - indicate that the globule deposits contain cholesterol dissolved in fatty acid, and thus can be used as an early indicator of the atherosclerosis process in animal models during drug development.

Confocal Raman spectroscopy is beginning to be recognized as a high potential technique for the non invasive study of biological tissues and human skin under in vivo conditions. Raman spectroscopy can be applied to obtain information regarding the molecular composition of the skin down to several hundred micrometers below the skin surface.

By coupling Raman spectroscopy with microscopy, it is possible to obtain full spectral information with a spatial resolution in the order of 1 µm. However, Raman is a weak phenomenon, and its sensitivity is not always ideal, particularly when analysing what are often relatively low concentration of intracellular components.

Raman microscopy is a technique which has proven highly valuable in a wide range of applications. Within biomedical research, its relevance to highly selective identification of biomolecules including amino acids and proteins, DNA/RNA and chemotherapeutic drugs is fast being realised. However, the efficiency of Raman scattering is low, and collection times can be long, and detection limits high.

Raman Spectroscopy was evaluated as a non-invasive method of analysis of sperm DNA and the influence of UV irradiation on the sperm. The results show that Raman Spectroscopy, combined with multivariate analysis provide the reproducible and accurate information on DNA of sperm and the effect and location of damage.

Fast and non-invasive methods for clinical and non clinical investigations for biological tissue are more and more required. Raman imaging at micro scale can answer to crucial questions about the monkey brain tissue morphology and structural evolution.

Identification of contaminating microbes is an important public safety issue in many fields such as the control of food quality and the manufacture of pharmaceutical and cosmetic products. Previous research has shown the potential for Raman spectroscopy in combination with chemometric methods to investigate bulk samples as well as a single bacterium and yeast cell for classification on the species and strain level.

Traditionally, Raman has been a technique of the material scientist, physicist or chemist, but as instrumentation continues to evolve, the power of Raman in biological and medical applications is fast being realized, not least because of the high information content provided and an excellent tolerance for water.

Decreasing turnaround time is a paramount objective in clinical diagnosis. We evaluated the discrimination power of Raman spectroscopy when analyzing microcolonies from nine bacterial and one yeast species directly on solid culture medium after a shortened incubation time of 6 h (compared to 24 hour for macrocolonies). This approach, that minimizes sample preparation and culture time, would allow resuming culture after identification to perform downstream antibiotic susceptibility testing.

In this application note, SERS is used to study the behaviour of glycoprotein αIIbβ3 which plays a critical role in thrombosis (pathological blood clotting which can reduce or completely stop blood supply to tissues and organs, leading to myocardial infarction or stroke).

In this study, we demonstrate the ViewSizer 3000’s capabilities as a next generation NTA based analysis instrument in order to accurately and efficiently measure and characterize extracellular vesicles (EV) via particle size and concentration.

Nanoparticles used for drug delivery is an important research field combining the life sciences with nanotechnology. While the strict definition of a nanoparticle is between 1 – 100 nm, we can extend the upper size range in the context of this application up to approximately 500 nm. Laser diffraction provides the advantage of being able to measure larger particles.

Considerable research has been conducted exploring the use of nanoparticles composed of biopolymers as a drug delivery vehicle. Much of the particle size analysis in this field has been done using dynamic light scattering (DLS). However, certain materials will feature larger particles that are outside the upper size range of DLS, but within the capability of laser diffraction.

Liposomes are bilayer vesicles made of phospholipids derived from natural or man-made materials. They are mainly used in the pharmaceutical field for treatment of cancers as carriers of chemotherapeutic drugs to the tumor area. Accurate and rapid measurement of the size of liposomes is essential for novel and effective drug delivery systems.