Picking up the Pieces: A Generic Porous Si Biosensor for Probing the Proteolytic Products of Enzymes

• Published in: Analytical chemistry (Washington) 2013-02, Vol.85 (3), p.1951-1956
• Main Authors: Shtenberg, Giorgi, Massad-Ivanir, Naama, Moscovitz, Oren, Engin, Sinem, Sharon, Michal, Fruk, Ljiljana, Segal, Ester
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Biosensors; Cattle; Enzymes; Enzymes, Immobilized - analysis; Enzymes, Immobilized - genetics; Hybridization; Mass spectrometry; Molecular Sequence Data; Porosity; Protein Structure, Secondary; Proteolysis; Proteomics; Silicon Dioxide - chemistry; Surface Properties
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac303597w

A multifunctional porous Si biosensor that can both monitor the enzymatic activity of minute samples and allow subsequent retrieval of the entrapped proteolytic products for mass spectrometry analysis is described. The biosensor is constructed by DNA-directed/reversible immobilization of enzymes onto a Fabry–Pérot thin film. We demonstrate high enzymatic activity levels of the immobilized enzymes (more than 80%), while maintaining their specificity. Mild dehybridization conditions allow enzyme recycling and facile surface regeneration for consecutive biosensing analysis. The catalytic activity of the immobilized enzymes is monitored in real time by reflective interferometric Fourier transform spectroscopy. The real-time analysis of minute quantities of enzymes (concentrations at least 1 order of magnitude lower, 0.1 mg mL–1, in comparison to previous reports, 1 mg mL–1), in particular proteases, paves the way for substrate profiling and the identification of cleavage sites. The biosensor configuration is compatible with common proteomic methods and allows for a successful downstream mass spectrometry analysis of the reaction products.