Biosensor for on-line fluorescent detection of trifluoroperazine based on genetically modified calmodulin

Abstract

This paper describes the development of a novel on-line biosensor based on a fluorescently labeled human calmodulin (CaM), hCaM M124C-mBBr, immobilized on controlled-pore glass (CPG), for the analysis of trifluoroperazine (TFP); a phenothiazine drug in human urine samples. The device was automated by packing hCaM M124C-mBBr-CPG in a continuous-flow microcell connected to a monitoring system, composed of a bifurcated optical fiber coupled to a spectrofluorometer. Operating parameters of the on-line biosensor (flow rate, sample injection volume, and carrier solution and buffer pH) were studied and optimized. Under the optimal conditions, the biosensor provides a detection and a quantification limit of 0.24 and 0.52 μg mL(-1), respectively, and a dynamic range from 0.52 to 61.05 μg mL(-1) TFP (n = 5, correlation coefficient 0.998). The response time (t(100)) was shorter than 42 s (recovery time <4.5 min) and reproducibility and repeatability of the TFP measurements, within the linear response range, were lower than 1.4 and 2.7%, respectively. The device was successfully applied to the analysis of TFP in spiked human urine samples with recoveries ranging between 97 and 101% and with RSDs lower than 5.9%.

Insights in Fungal Bioprospecting in Mexico

“…The last fungus selected for the search of α-glucosidase inhibitors was M. flavorosea. From the active extract prepared from the grain-based culture, two new polyketides, namely, 8-chloroxylarinol A (27) and flavoroseoside (28), along with the known compounds xylarinol A (29), xylarinol B (30), massarigenins B and C (31 and 32), and clavatol (33), were isolated [25] (▶ Fig. 5).…”

“…For the discovery of CaM inhibitors, several methods have been used, which were described in a recent review [26]. In our investigations, we have employed functional enzymatic assays with bovine brain CaM-sensitive cAMP phosphodiesterase (PDE1) and/or peas CaM-dependent nicotinamide adenine dinucleotide kinase (NADK) as reporter enzymes, SDS-PAGE, and/or fluorescent biological sensors (FBS) built with human CaM (hCaM) developed in recent years [27][28][29][30][31][32][33]. In some cases, in silico analyses were used to explore the potential binding site of the inhibitors.…”

New Insights on Optical Biosensors: Techniques, Construction and Application

“…Nevertheless, amazing development ondetection methods in biosensors, their construction, even at nanoscale, and applications have been reported in the last decade. [16][17][18][19][20][21][22]…”

Bisindolylmaleimides New Ligands of CaM Protein

“…In many cases, slight modifications in chemical structure can significantly alter a compound’s ability to bind to CaM and inhibit its activity. A direct detection tool developed by our group is the calmodulin biosensor ( h CaM-M124C- mBBr ), which we have used to detect the binding of various ligands [ 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. On the other hand, theoretical and computational studies, such as cheminformatics, docking, and molecular dynamics simulations (MD), are considered tools of great value to complement the experimental data.…”