There is a need for quick assays of protease activity because of their pivotal role in intra- [1][2][3] and intercellular [4,5] processes.Schemes to quantify protease activity involve either fluorescent [6,7] or colorimetric assays [8,9] that are time consuming and require relatively large quantities of enzyme. For example, the limits of detection for colorimetric protease assays are generally in the low nanogram range and require a few hundred microliters of analyte reagent. Incubation times up to 24 h are necessary to obtain the lowest detection limits. Conventional fluorescent and colorimetric assays must employ substrates that have been modified from their native forms in order to incorporate the relevant indicator chemistries. For example, a standard method to detect protease activity uses a fluorescent molecule conjugated to casein or bovine serum albumin substrates. [7] Some concerns with the use of fluorescent conjugated substrates are that the presence of the dye may affect the proteolytic cleavage rate, quantification requires a sensitive fluorimeter, and the reagents are costly. The well-known colorimetric assay using the Folin-Ciocalteu reagent operates on native substrates, but it is less sensitive than fluorescence methods and it requires an extensive workup procedure.[9]The unique optical properties of photonic crystals made from porous Si have been harnessed to generate very sensitive chemical or biochemical detectors. [10][11][12] Precise control of the current used to etch porous Si provides peaks in the reflectivity spectrum that are much sharper than those that can be obtained with molecular dyes or quantum dots, [13] and allows the use of such materials in high-throughput, multiplexed assays. [14] The present work takes advantage of these features, using color changes induced in a porous Si photonic crystal as a sensitive probe of protease activity. Amounts of protease as small as a few picomoles in a 1 lL aliquot can be detected as a color change that is visible to the naked eye. By contrast, current colorimetric assays require the use of a spectrophotometer, and they must use 100-500 lL aliquots to obtain comparable detection limits (7 pmol or 240 ng for pepsin). Fabrication of the photonic crystal involves anodic etching of a p-type, boron-doped Si wafer polished on the (100) face ( Fig. 1).[15] A sinusoidal etch waveform produces a film with an alternating porosity gradient in the <100> direction that acts as a 1D photonic crystal, displaying a distinct optical diffraction peak in the white-light reflectivity spectrum. [13,16] The wavelength of the peak is determined by the period of the waveform used in the preparation. The porous Si layer is then methylated using an electrochemical grafting procedure [17] to impart stability and hydrophobicity. A protease sensor is prepared from the porous Si photonic crystal following the procedure shown in Figure 1. A solution of the hydrophobic, naturally occurring protein zein in methanol (10 mg mL -1 ) is spin-coated onto the methylated porous Si f...