Frauke Rininsland

Biosensors from conjugated polyelectrolyte complexes

A charge neutral complex (CNC) was formed in aqueous solution by combining an orange light emitting anionic conjugated polyelectrolyte and a saturated cationic polyelectrolyte at a 1:1 ratio (per repeat unit). Photoluminescence (PL) from the CNC can be quenched by both the negatively charged dinitrophenol (DNP) derivative, (DNP-BS−), and positively charged methyl viologen (MV2+). Use of the CNC minimizes nonspecific interactions (which modify the PL) between conjugated polyelectrolytes and biopolymers. Quenching of the PL from the CNC by the DNP derivative and specific unquenching on addition of anti-DNP antibody (anti-DNP IgG) were observed. Thus, biosensing of the anti-DNP IgG was demonstrated.

Applications of fluorescent polymer superquenching to high throughput screening assays for protein kinases.

Protein kinases are involved in the regulation of cellular metabolism, growth, differentiation, and proliferation. Aberrations in their function can lead to diseases such as cancer and inflammation. Protein kinases are therefore possible targets for drug therapies. To address the need for high throughput screening of potential inhibitors, QTL has developed a homogeneous and robust kinase assay for use in multiwell plate format. The QTL Lightspeed fluorescence superquenching-based kinase assays do not require specialized equipment, nor do they involve the use of radioactive hazardous materials or antibodies. QTL Lightspeed kinase assays directly measure the enzymatic activity of the target and do not involve secondary (detector) enzyme. In this article, we compare QTL Lightspeed protein kinase assays using Protein Kinase A, Protein Kinase Balpha/Akt1, and ribosomal S6 kinase-2 as examples with other commercially available kinase kits. Our data show that QTL Lightspeed kinase assays offer significant advantages over the current commercial kits in terms of both sensitivity and performance. The QTL Lightspeed kinase assay also offers a kinetic assay mode where the substrate phosphorylation can be monitored in real-time.

A Robust Screen for Inhibitors and Enhancers of Phosphoinositide-3 Kinase (PI3K) Activities by Ratiometric Fluorescence Superquenching

Aberrant regulation of phosphoinositide 3-kinase (PI3K) activity is implicated in various diseases such as cancer and diabetes. Thus, high-throughput screening (HTS) of small-molecule inhibitors for PI3 kinases is an appealing strategy for drug development. Despite the attractiveness of lipid kinases as drug targets, screening for inhibitors for PI3K activities has been hampered by limited assay formats adaptable for HTS. The authors describe a homogeneous, direct, and nonradioactive assay for highly sensitive detection of PI3Kα, β, δ, and γ activities, which is suitable for HTS. The assay is based on fluorescence superquenching of a conjugated polymer upon metal-ion-mediated association of phosphorylated and dye-labeled substrates. As a result of phosphorylation, quencher and polymer are brought into proximity, and fluorescent energy transfer occurs. This event can be monitored as either fluorescence quench of the polymer or as enhanced emission from the quencher. Ratiometric analysis of the wavelengths eliminates interferences from autofluorescing compounds, which are present in HTS libraries. The platform has been adapted for the 384-well microplate format and delivers Z factors of > 0.6 at substrate conversions as low as 7%. Using this assay platform, several unreported inhibitors and activators of PI3Ks were identified in an 84- compound screen.