Protein Sizing on a Microchip

• Published in: Analytical chemistry (Washington) 2001-03, Vol.73 (6), p.1207-1212
• Main Authors: Bousse, Luc, Mouradian, Stephane, Minalla, Abdel, Yee, Herman, Williams, Kathi, Dubrow, Robert
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbonic Anhydrases - chemistry; Chemistry; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Instruments; Micelles; Microchemistry - instrumentation; Protein Denaturation; Proteins; Proteins - chemistry; Sodium Dodecyl Sulfate
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac0012492

We have developed a microfabricated analytical device on a glass chip that performs a protein sizing assay, by integrating the required separation, staining, virtual destaining, and detection steps. To obtain a universal noncovalent fluorescent labeling method, we have combined on-chip dye staining with a novel electrophoretic dilution step. Denatured protein−sodium dodecyl sulfate (SDS) complexes are loaded on a chip and bind a fluorescent dye as the separation begins. At the end of the separation channel, an intersection is used to dilute the SDS below its critical micelle concentration before the detection point. This strongly reduces the background due to dye molecules bound to SDS micelles and also increases the peak amplitude by 1 order of magnitude. Both the on-chip staining and SDS dilution steps occur in the 100-ms time scale and are ∼104 times faster than their conventional counterparts in SDS−PAGE. This represents a much greater speed increase due to microfabrication than has been obtained in other assay steps such as electrophoretic separations. We have designed and tested a microchip capable of sequentially analyzing 11 different samples, with sizing accuracy better than 5% and high sensitivity (30 nM for carbonic anhydrase).