Investigation into the Applicability of the Centrifugal Microfluidics Platform for the Development of Protein−Ligand Binding Assays Incorporating Enhanced Green Fluorescent Protein as a Fluorescent Reporter

• Published in: Analytical chemistry (Washington) 2004-12, Vol.76 (24), p.7263-7268
• Main Authors: Puckett, Libby G, Dikici, Emre, Lai, Siyi, Madou, Marc, Bachas, Leonidas G, Daunert, Sylvia
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Antidepressive Agents - analysis; Binding sites; Calmodulin - chemistry; Chemistry; Exact sciences and technology; Fluorescence; General, instrumentation; Green Fluorescent Proteins - chemistry; Ligands; Microfluidics - methods; Pharmaceuticals; Propranolol - analysis; Protein Binding; Proteins; Proteins - chemistry; Spectrometric and optical methods; Technology, Pharmaceutical - methods; Trifluoperazine - analysis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac049758h

The incorporation of a protein−ligand binding assay into a centrifugal microfluidics platform is described. The platform itself is a disc-shaped polymer substrate, upon which a series of microfluidic channels and reservoirs have been machined. Centrifugal microfluidics platforms require no internal moving parts, and fluid propulsion is achieved solely through rotation of the disc. Fluid flow is controlled by passive valves, the opening of which is dependent on the angular frequency of the rotating plat-form, the channel dimensions, and the physical properties of the fluid. To evaluate the effectiveness of incorporating a protein-based assay onto the centrifugal microfluidics analytical platform, a class-selective, homogeneous assay for the detection of phenothiazine antidepressants was employed. This class of drugs is known to bind to calmodulin, a calcium binding protein. Specifically, a fusion protein between calmodulin and enhanced green fluorescent protein was utilized. Calmodulin undergoes a conformational change upon binding to phenothiazines that alters the fluorescence properties of the attached fluorescent protein, which can be correlated to the concentration of the drug present. Another important aspect of this work was to study the efficacy of the platform to perform reconstitution assays. To do this, the biological reagent was dried on the platform and rehydrated to carry out the assay. The ability to prealiquot reagents on the platform should enhance its versatility and portability. The integration of protein-based assays in this platform should be useful in the design of analytical systems for high-throughput screening of pharmaceuticals and clinical diagnostics.