[PubMed] [Google Scholar] 2. this allows label-free quantification of enzyme kinetics in real-time within a volume of approximately 5 nanoliters. A common method to quantify protease activity is based on Fluorescence Resonance Energy Transfer5 (FRET) measurements using fluorescently tagged protein substrates. A major drawback to Setrobuvir (ANA-598) this approach is that a chemically modified substrate may not exhibit the same reactivity as the native substrate. While fluorescent labels can provide high sensitivity, the attachment of a probe requires an extra step that increases analysis time and complexity. In addition, fluorescent probes can be subject to photochemical degradation and pH-dependent responses. Porous Si-based interferometers operate by measuring a change in refractive index in a volume of solution contained within the porous nanostructure, and they have been shown to provide a simple, label-free method for detection of proteins that minimizes the quantity of reagents used6-9. Recently, the advantage of using more complicated porous Si optical structures to correct for zero-point drift or non-specific binding has been exhibited10,11. Additionally, recent work has shown how catalytic reactions can be harnessed to improve sensitivity of these nanosensory systems12,13. The two-layer porous Si nanostructure was prepared by electrochemical etching of silicon in an HF-containing electrolyte. The double layer was Setrobuvir (ANA-598) then thermally oxidized (600 C for 1.5 h, in air) to generate a stable silicon oxide (SiO2) surface. The size, shape, and population of the pores in a film are determined by the current, allowing one to dial in a specific porosity pattern14. Thus a double layer containing large pores on top Setrobuvir (ANA-598) of small pores is usually prepared by decreasing the applied current Rabbit Polyclonal to 5-HT-2C density midway through the etch, resulting in the abrupt decrease in pore size shown in the cross-sectional scanning electron microscope (SEM) image of Fig. 1. The tunability of the Setrobuvir (ANA-598) pore etching process provides a convenient means to build such nanostructured matrices that can act as reservoirs15 or size exclusion membranes4,16. The programmability can also be used to build optical structures with useful sensing properties. Open in a separate window Open in a separate window Physique 1 Scanning electron micrographs(secondary electron image, 5 kV) of the porous Si double-layer structure used in this study. (a) Image showing both porous layers, the top interface, and the Si substrate. (b) Close-up view of the interface between the two porous layers. The 1st (top) layer contains pore sizes ranging from 80-120 nm and acts as a host layer for the protease pepsin in the present work. The 2nd (bottom) layer has pores sizes ranging from 2-10 nm. This lower layer excludes the protease and large proteins such as -casein (25 kDa) but admits the degradation fragments resulting from action of the enzyme on casein. Top layer is Setrobuvir (ANA-598) usually 3.2 micron thick, 75% porous; bottom layer is usually 4.4 micron thick, 37% porous. The average diameter of the pores in the present structure is usually 100 nm in the top layer and 6 nm in the bottom. Being smaller than the wavelength of visible light, the features in this nanostructure do not scatter light, and the sample displays well-resolved Fabry-Prot interference fringes in the optical reflectivity spectrum (Fig. 2). The reflectivity spectrum displays an interference pattern that arises from a combination of Fabry-Prot.
