A Rhodiola rosea root extract protects skeletal muscle cells against chemically induced oxidative stress by modulating heat shock protein 70 (HSP70) expression.

Rhodiola rosea is a perennial plant in the Crassulaceae family, recently postulated to exert its adaptogenic functions partially by modulating the expression of molecular factors such as heat shock proteins (HSP). The aim of this study was to analyze the efficacy of a Rhodiola rosea extract (Rhodiolife) in protecting murine skeletal muscle cells (C2 C12 myotubes) from chemically induced oxidative stress and to establish whether modulation of HSP70 expression is observed. C2 C12 cells treated with Rhodiolife did not experience any loss of viability (p > 0.05) at concentrations of 1-100 µg/mL for up to 24 h. In control cultures, viability decreased 25% following exposure to 2 mM H2 O2 (1 h). However, no significant decrease in viability in cells pre-treated with extract at concentrations as low as 1 µg/mL was observed. HSP70 mRNA levels were up-regulated two-fold in cell cultures treated with Rhodiolife (10 µg/mL), and expression was further enhanced by exposure to H2 O2 (six-fold, p < 0.05). HSP70 protein levels were maintained in pre-treated cell cultures compared to controls but was significantly lower (-50%) in cells lacking treatment exposed to H2 O2 . The present results indicate that Rhodiolife protects C2 C12 myotubes against peroxide-induced oxidative stress through the modulation of the molecular chaperone HSP70.

Immunoassay arrays fabricated by dip-pen nanolithography with resonance Raman detection.

Here, we report the first use of resonance Raman scattering for the detection of miniaturized microscale arrays fabricated by dip-pen nanolithography. Antibody arrays for prostate-specific antigen (PSA) were printed, and a sandwich immunoassay was carried out. An enzyme-linked detection antibody was used to provide an insoluble and stable colored microdot in the recommended size range for microarray readers, which could be read with resonance Raman scattering. This gives quantitative detection as well as an improved detection limit and a larger dynamic range than that previously achieved by direct fluorescent detection methods. By Raman mapping across the arrayed area, the microdots were easily detected with very little background signal from surrounding areas. Levels of PSA as low as 25 pg/mL were detected using this method, which could be extended to a large number of useful biomarkers.

Tracking bisphosphonates through a 20 mm thick porcine tissue by using surface-enhanced spatially offset Raman spectroscopy.

Track it down: A recognized surface-enhanced Raman scattering (SERS) nanotag signal was monitored from a thin, dispersed layer of bisphosphonate-functionalized nanotags on a bone sample, through a 20 mm thick specimen of porcine muscle tissue by surface-enhanced spatial offset Raman spectroscopy (SESORS; see picture). The result demonstrates the great potential for non-invasive in vivo bisphosphonate drug tracking.

Quantitative detection of human tumor necrosis factor α by a resonance raman enzyme-linked immunosorbent assay.

Tumor necrosis factor α is an inflammatory cytokine which has been linked with many infectious and inflammatory diseases. Detection and quantification of this key biomarker is commonly achieved by use of an enzyme-linked immunosorbent assay (ELISA). This fundamental technique uses the spectroscopic detection of a chromogen such as 3,3',5,5'-tetramethylbenzidine (TMB). Horseradish peroxidase (HRP), bound to the detection antibody, catalyzes the oxidation of TMB by hydrogen peroxide to generate colored products which may be measured spectrophotometrically. In this study we have used a conventional ELISA kit and shown that, by replacing the traditional colorimetric detection with resonance Raman spectroscopy, we can achieve 50 times lower detection limits and the potential for multiplexed analysis is increased. In this approach, the laser wavelength was tuned to be in resonance with an electronic transition of the oxidized TMB. The relative intensity of the enhanced Raman bands is proportional to the amount of TMB, thus providing a means of improved quantification. Furthermore, TMB is one of the most widely used chromogenic substrates for HRP-based detection and commercial ELISA test kits, indicating that this detection technique is applicable to a large number of target analytes.

Fabricating protein immunoassay arrays on nitrocellulose using dip-pen lithography techniques.

Advancements in lithography methods for printing biomolecules on surfaces are proving to be potentially beneficial for disease screening and biological research. Dip-pen nanolithography (DPN) is a versatile micro and nanofabrication technique that has the ability to produce functional biomolecule arrays. The greatest advantage, with respect to the printing mechanism, is that DPN adheres to the sensitive mild conditions required for biomolecules such as proteins. We have developed an optimised, high-throughput printing technique for fabricating protein arrays using DPN. This study highlights the fabrication of a prostate specific antigen (PSA) immunoassay detectable by fluorescence. Spot sizes are typically no larger than 8 µm in diameter and limits of detection for PSA are comparable with a commercially available ELISA kit. Furthermore, atomic force microscopy (AFM) analysis of the array surface gives great insight into how the nitrocellulose substrate functions to retain protein integrity. This is the first report of protein arrays being printed on nitrocellulose using the DPN technique and the smallest feature size yet to be achieved on this type of surface. This method offers a significant advance in the ability to produce dense protein arrays on nitrocellulose which are suitable for disease screening using standard fluorescence detection.

Isolation of calcium phosphate crystals from complex biological fluids using bisphosphonate-modified superparamagnetic beads.

The surface of superparamagnetic magnetic beads was modified with bisphosphonates to selectively capture calcium phosphate crystals from complex biological fluids (i.e. synovial fluid).

Multidentate macromolecules for functionalisation, passivation and labelling of metal nanoparticles.

A new class of SERRS-active macromolecule designed to protect silver nanoparticle surfaces against salt corrosion whilst retaining colloidal stability of the particles is reported.

New approaches in the detection of calcium-containing microcrystals in synovial fluid.

BACKGROUND: The presence of calcium phosphate crystals such as basic calcium phosphate and calcium pyrophosphate dihydrate in intra-articular fluid is linked to a number of destructive arthropathies and detection of these deposits is often pivotal for early diagnosis and appropriate management of such disease. RESULTS: We describe the use of a calcium-sensitive dye, Fluo-4, to selectively label calcium-containing mineral deposits in synovial fluid, which can then be easily visualized using a standard fluorescence microscope. Furthermore, we have combined the fluorescent properties of the tagged crystals with flow cytometry as a fast and semi-quantitative method of detection. CONCLUSION: Dot-plots were used to quantify differences between various types of arthropathies and confirmed by visual observation of the crystals under a fluorescence microscope.

Determination of calcium in synovial fluid samples as an aid to diagnosing osteoarthritis.

BACKGROUND: Microscopic inorganic crystals are commonly observed in the synovial fluid of patients suffering from arthritic diseases. Basic calcium phosphate (BCP) crystals are known to occur quite commonly in the joint fluid of osteoarthritis (OA) patients and are insoluble at physiological pH. Current analysis of patient synovial fluid depends on light microscopy and staining with Alizarin Red-S. Both methods cannot identify crystals < 1µm in size and are highly subjective. This article investigates the use of o-cresolphthalein complexone (OCP), a colorimetric reagent, to quantify calcium from crystals isolated from synovial fluid samples as a means of identifying the presence of BCP and, hence, improving the diagnosis of OA. RESULTS: Inorganic crystals were isolated following degradation of the biological sample matrix with hyaluronidase. 1-M HNO(3) was used for crystal dissociation into ions and the colorimetric response of OCP to calcium was measured in a basic environment of 2-amino-2-methyl-1-propanol. The average calcium content in OA patient samples was up to 40% higher than in rheumatoid arthritis (RA) patient samples. RA samples were used as a comparison, because they are generally accepted to be crystal free. Within the OA group, higher levels of calcium were detected in three out of 12 synovial fluid samples, which correlated with a significantly greater number of BCP crystals detected during microscopic examination. CONCLUSIONS: A simple method based on colorimetry for measurement of calcium content and semiquantification of BCP crystals in synovial fluid samples has been described. Sample pretreatment following addition of hyaluronidase proved to be effective in reducing viscosity and aiding the dissociation of BCP crystals in synovial fluid samples.

Detection of calcium phosphate crystals in the joint fluid of patients with osteoarthritis - analytical approaches and challenges.

Clinically, osteoarthritis (OA) is characterised by joint pain, stiffness after immobility, limitation of movement and, in many cases, the presence of basic calcium phosphate (BCP) crystals in the joint fluid. The detection of BCP crystals in the synovial fluid of patients with OA is fraught with challenges due to the submicroscopic size of BCP, the complex nature of the matrix in which they are found and the fact that other crystals can co-exist with them in cases of mixed pathology. Routine analysis of joint crystals still relies almost exclusively on the use of optical microscopy, which has limited applicability for BCP crystal identification due to limited resolution and the inherent subjectivity of the technique. The purpose of this Critical Review is to present an overview of some of the main analytical tools employed in the detection of BCP to date and the potential of emerging technologies such as atomic force microscopy (AFM) and Raman microspectroscopy for this purpose.

SERRS labelled beads for multiplex detection.

Beads labelled using surface enhanced resonance Raman scattering (SERRS) are highly sensitive and specific tags, with potential applications in biological assays, including molecular diagnostics. The beads consist of a nucleus containing dye labelled silver-nanoparticle aggregates surrounded by a polymer core. The nuclei generate strong SERRS signals. To illustrate the coding advantage created by the sharp, molecularly specific SERRS signals, four specially designed SERRS dyes have been used as labels and three of these have been combined in a multiplex analysis. These dyes use specific groups such as benzotriazole and 8-hydroxyquinoline to improve binding to the surface of the silver particles. The aggregation state of the particles is held constant by the polymer core, this nucleus also contains many dye labels, yielding a very high Raman scattering intensity for each bead. To functionalise these beads for use in biological assays an outer polymer shell can be added, which allows the attachment of oligonucleotide probes. Oligonucleotide modified beads can then be used for detection of specific oligonucleotide targets. The specificity of SERRS will allow for the detection of multiple targets within a single assay.