Assay For Label-free Detection Research Articles

Label free detection of auramine O by G-quadruplex-based fluorescent turn-on strategy

Auramine o (AO) is a synthetic dye used in paper and textile industries. Although it has been an unauthorized food additive in many countries due to its toxic and carcinogenic possibility, its illegal uses have been detected in certain food products such as pasta, semolina and spices and also in pharmaceuticals. The presence of AO in food products should be monitored, therefore, to minimize the negative health effects on consumers. In this study, a simple, highly sensitive and selective label free detection method was investigated for AO by G-quadruplex-based fluorescent turn-on strategy. The optimum fluorescent detection assay was achieved with a specific G-quadruplex DNA sequence, c-myc, at 400 nM in Tris-HCl buffer at pH 7.4. The linearity of fluorescence intensity depending on AO concentration ranged from 0 to 0.07 µM and LOD and LOQ were 3 nM and 10 nM, respectively. The G-quadruplex-based detection assay was highly specific for AO as compared to other two synthetic food colorings and successfully applied to determine AO in pasta, bulgur and curry powder with recoveries in the range from 70.33% to 106.49%. This G-quadruplex-based label free detection assay has a significant potential to be used in the detection of AO in food products.

Protamine-Induced Supramolecular Self-Assembly of Red-Emissive Alkynylplatinum(II) 2,6-Bis(benzimidazol-2'-yl)pyridine Complex for Selective Label-Free Sensing of Heparin and Real-Time Monitoring of Trypsin Activity.

A label-free detection assay is developed based on the design and synthesis of a new anionic alkynylplatinum(II) 2,6-bis(benzimidazol-2'-yl)pyridine complex with water-soluble pendants. With the aid of electrostatic interaction and noncovalent metal-metal and π-π stacking interactions, protamine is shown to induce supramolecular self-assembly of platinum(II) complexes with drastic UV-vis absorption and red emission changes. On the basis of the strong binding affinity of protamine and heparin, the ensemble has been further employed to probe heparin by monitoring the spectroscopic changes. Other than heparin, this ensemble can also detect the activity of trypsin, which can hydrolyze protamine into fragments, leading to the deaggregation of platinum(II) complexes. By modulation of the self-assembly properties of platinum(II) complexes via real-time UV-vis absorption and emission studies, the reported assay has been demonstrated to be a sensitive and selective detection method for trypsin, as well as trypsin inhibitor screening, which is essential for drug discovery.

Advantages and application of label-free detection assays in drug screening

Background: Adoption is accelerating for a new family of label-free optical biosensors incorporated into standard format microplates owing to their ability to enable highly sensitive detection of small molecules, proteins and cells for high-throughput drug discovery applications. Objective: Label-free approaches are displacing other detection technologies owing to their ability to provide simple assay procedures for hit finding/validation, accessing difficult target classes, screening the interaction of cells with drugs and analyzing the affinity of small molecule inhibitors to target proteins. Methods: This review describes several new drug discovery applications that are under development for microplate-based photonic crystal optical biosensors and the key issues that will drive adoption of the technology. Results/conclusions: Microplate-based optical biosensors are enabling a variety of cell-based assays, inhibition assays, protein–protein binding assays and protein–small molecule binding assays to be performed with high-throughput and high sensitivity.