DOI: 10.1002/adma.200502420 Protein-Coated Porous-Silicon Photonic Crystals for Amplified Optical Detection of Protease Activity** By Manuel M. Orosco, Claudia Pacholski, Gordon M. Miskelly , and Michael J. Sailor* There is a need for quick assays of protease activity because of their pivotal role in intra- [1–3] and intercellular [4,5] processes. Schemes to quantify protease activity involve either fluores- cent [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 gener- ally 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 em- ploy substrates that have been modified from their native forms in order to incorporate the relevant indicator chemis- tries. For example, a standard method to detect protease activ- ity 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 work- up procedure. [9] The unique optical properties of photonic crystals made from porous Si have been harnessed to generate very sensitive chemical or biochemical detectors. [10–12] Precise control of the current used to etch porous Si provides peaks in the reflectiv- ity spectrum that are much sharper than those that can be ob- tained with molecular dyes or quantum dots, [13] and allows the use of such materials in high-throughput, multiplexed as- says. [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 spectropho- tometer, 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 dif- fraction 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 metha- nol (10 mg mL –1 ) is spin-coated onto the methylated porous Si film, creating an even protein coating. Zein tends to form multilayered films on Si surfaces through molecular and electrostatic interactions. [18] In the present case, spectral mea- surements indicate that a similar layer is produced on the porous Si film; the pores are small enough (ca. 10 nm) that only a small amount of the intact protein (24 kDa (1 Da = 1.660 540 × 10 –27 kg), 16 nm × 4.6 nm × 1.2 nm) [18] in- COMMUNICATIONS Adv. Mater. 2006, 18, 1393–1396 © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1393 – [*] Prof. M. J. Sailor, M. M. Orosco, Dr. C. Pacholski Department of Chemistry and Biochemistry The University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92039-0358 (USA) E-mail: msailor@ucsd.edu Dr. G. M. Miskelly Department of Chemistry, The University of Auckland Private Bag 92019, Auckland (New Zealand) [**] This project has been funded in part with Federal funds from the Air Force Office of Scientific Research (Grant# F49620-02-1-0288) and the National Science Foundation (Grant# DMR-0503006). M. J. S. is a member of the Moores UCSD Cancer Center and the UCSD NanoTUMOR Center under which this research was conducted and partially supported by NIH grant U54 CA 119335. C. P. thanks the Deutsche Forschungsgemeinschaft (PA 925/1-1) for a postdoctoral fellowship. The authors thank Anne Ruminski for experimental as- sistance and Prof. Farooq Azam for helpful discussions. Supporting Information is available online from Wiley InterScience or from the author. spin-coat zein silicon porous Si photonic crystal etch 1μl protease one-hour digest methylation color change Figure 1. Enzymatic assay using a protein-coated porous Si photonic crystal. A hydrophobic, 1D photonic crystal is prepared by electrochemi- cal etching of Si, followed by electrochemical grafting of methyl species to the inner pore walls. A thin layer of the protein zein is then added. The assay is carried out by addition of a small drop of solution containing active protease (pepsin or pronase E in the present work), which digests the protein layer. Infiltration of proteolytic cleavage products to the pho- tonic crystal leads to a color change that is readily observable to the naked eye.