FLUOstar Omega

Metallic nanoparticles became subject of intensive research because of their potential antibiotic properties. Nanoparticles such as silver, gold or zinc oxide particles are easily and cost-effectively synthesized by blending metal salts with plant extracts that reduce the metal. Different extracts, varying in the plant or the part of the plant used for the extract, are currently investigated in regard to their capacity to form nanoparticles and their antimicrobial efficacy. The formation of nanoparticles can be verified by UV-Vis spectroscopy due to surface plasmon resonance of the particles that lead to a characteristic spectrum defined by the underlying metal and particle size. Subsequent analysis of nanoparticles on microbial growth is typically tested by methods based on absorbance changes.

Here, we present how the spectrometer-based BMG LABTECH instruments are used to quickly confirm Ag and ZnO nanoparticle formation and their inhibitory effect on the diarrhea-causing bacteria Vibrio cholerae and enterotoxic Escherichia coli (ETEC).

The cell membrane is a bilayer of phospholipids with embedded proteins. It contains cholesterol that determines the membrane's fluidity, permeability and activity of membrane proteins. Changes in membrane cholesterol are implicated in diseases such as Alzheimer's and cancer, demanding its investigation.

NR12S is a fluorophore that exhibits emission maxima at 560 and 630 nm dependent on the liquid order of the membrane. Incorporation of the dye in a liquid ordered phase (increased cholesterol) shifts the emission to 560 nm, whereas in a liquid disordered phase (lower cholesterol) it emits at 630 nm. Hence, the 560/630 ratio correlates with the membrane cholesterol content.

Inhibition of cholesterol synthesis by lovastatin or incubation with methyl-§-cyclodextrin decreased membrane cholesterol in hematological cells and was reported by decreases in the 560/630 ratio. Both emissions were conveniently measured on the filter-based FLUOstar^® Omega microplate reader and could be analyzed with one-click using the MARS analysis software.

SPARCL refers to an immunoassay that does not require washing steps. An analyte in a solution is detected by binding two antibodies: one is coupled to horse radish peroxidase (HRP), the other is coupled to the HRP substrate Acridan. In the presence of the analyte, binding of both antibodies brings the enzyme and substrate into proximity. The enzymatic reaction is started by the addition of H₂O₂, which immediately produces a light proportional to the amount of analyte. Thus, SPARCL assays require to be read at the same time as the injection of the trigger solution.

The CLARIOstar^® microplate reader, which is equipped with the dedicated H3 injection needle for simultaneous injection and reading, fulfils this task. The analyte IgG was detected down to a concentration of 45 ng/ml in 96 wells and down to 15.6 ng/ml in 384 well format. BMG LABTECH's MARS analysis software facilitates subsequent calculation of analyte concentration.

Measuring the protein concentration of liquid samples is a routine analysis in many life science laboratories. Accurate quantification is often a critical step for subsequent analyses such as protein characterization or western blots. Absorbance-based methods are well-established, easy to handle and cheap. They can be performed in microplates, allowing for high sample numbers processed at a time and low reagent volumes.

Spectrometer-based BMG LABTECH readers capture absorbance spectra or absorbance at discrete wavelengths from 220-1000 nm in less than a second per well and therefore can easily read these assays. The most common methods absorbance at 280 nm, Bradford, Bicin-Choninic Acid (BCA) and Lowry assay are presented here.

G-protein coupled receptors (GPCRs) are predominant drug targets. Therefore, monitoring GPCR activity upon compound addition in a cellular context is required. To this end, Montana Molecular developed biosensors that detect G-protein-driven cAMP generation. The sensor consists of a specific cAMP binding domain and a fluorescent label. It changes its fluorescence intensity if the second messenger cAMP is bound.

HEK293 cells that expressed the biosensor were treated with Isoproterenol, a well-known activator of the G protein subunit Gs. The resulting increase in cAMP was mirrored by changes in fluorescence intensity. Similarly, upon addition of Quinpirole that acts on the inhibitory G-protein subunit Gi, a drop of fluorescence intensity was detected which represented the expected decrease in cAMP.

Detection of Montanta Molecular's biosensors with the CLARIOstar^® microplate reader resulted in high Z' factors which prove the robustness of the assay.