A sensitive and rapid method for detecting fluorescent dyes at low concentrations in homogenous solution is experimentally demonstrated. Fluorescent-labeled DNA probes are detected by attaching magnetic beads and applying alternating magnetic field gradient. This condenses the fluorescent probes into a small detection volume and eliminates the scattering noise from solution by synchronous detection. For DNA probes concentration of 1 x 10(-13) M the detection signal was 3.3 times higher than the noise, thereby implying detection sensitivity of 3 x 10(-14) M.
This paper reviews the development of a magnetic modulation biosensing (MMB) system for rapid, simple and sensitive detection of biological targets in homogeneous solution at low concentrations. It relies on condensation and modulation of the fluorescent-labeled probes attached to magnetic beads using an alternating magnetic field gradient. Condensation of the beads from the entire volume increases the signal while modulation separates the signal from the background noise of the non-magnetized solution. We first discuss the motivation and challenges in specific DNA sequences detection as well as current approaches to overcome some of these challenges. We then present the MMB system, DNA detection schemes and magnetic beads manipulation in solution. Rapid detection at sub-picomolar concentrations of fluorescent-labeled probes as well as of coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) without any washing or separation step is also reviewed. Finally, we show preliminary results of protein detection using a 'sandwich'-based assay.
Glutathione synthesis and growth properties were studied in the gamma-glutamyl transpeptidase(GGT)-negative, non-tumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. gamma-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with gamma-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a gamma-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources.
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