Baixe Enzyme-Responsive Nanoparticle Systems e outras Notas de estudo em PDF para Engenharia Elétrica, somente na Docsity! R E S E A R CDOI: 10.1002/adma.200703158H N E W S Enzyme-Responsive Nanoparticle Systems**By James E. Ghadiali and Molly M. Stevens*Inorganic nanoparticles and their accompanying diverse physical properties are now virtually in routine use as imaging tools in cell-biology. In addition to serving as excellent contrast agents, their size- and environment-dependent optical andmagnetic properties can be harnessed to create enzyme biosensor devices of extremely high sensitivity, whilst circumventing the numerous technical limitations associated with traditional enzyme assays. In this Research News article we discuss recent advances in field of enzyme-responsive nanoparticle systems, where the activity of an enzyme elicits a specific response in the nanoparticle assembly to produce a signal relating to enzyme activity, focusing on three important systems: DNA-structured nanoparticles, protein kinases and proteases.1. Introduction Whilst inorganic nanoparticles can be utilized in electronic and optical devices and chemical catalysis, their integration into biological systems provides the possibility of creating new high-sensitivity biological labels and drug-delivery systems.[1] Another key application of biofunctional metallic nanoparti- cles is the development of biosensor devices for the rapid detection of disease-related proteins, with a view to creating new diagnostic and high-throughput screening techniques. Such approaches often rely on utilizing the size-dependent phenomena associated with metallic nanoparticles, namely localized surface plasmon resonance, superparamagnetism, and quantum confinement, to devise high-sensitivity readout platforms. Furthermore, prior to integration into any biosensor device, inorganic nanoparticles need to be rendered water- soluble and appended with various biofunctional surface ligands.[2,3] For an up-to-date discussion of the numerous available bioconjugation strategies involved in interfacing inorganic nanoparticles with biological systems, Rotello et al. provide a comprehensive and detailed Review in this issue.[4][*] Dr. M. M. Stevens, J. E. Ghadiali Department of Materials, Imperial College London London, SW7 2AZ (UK) E-mail: m.stevens@imperial.ac.uk Dr. M. M. Stevens, J. E. Ghadiali Institute of Biomedical Engineering, Imperial College London London, SW7 2AZ (UK) [**] We gratefully acknowledge the support of the EPSRC (EP/E007627/1) and The Leverhulme Trust. Adv. Mater. 2008, 20, 4359–4363 2008 WILEY-VCH Verlag GHigh-sensitivity detection of enzymes and determination of their activity and kinetic parameters is of great importance in the development of novel pharmaceuticals and medical diagnostic devices. Protein detection methods have largely been dominated by enzyme-linked immunological assays (i.e., ELISA), where specific antibody binding to a target analyte is amplified by a secondary colorimetric enzyme reaction. Although this is a well-established procedure found in many clinical settings, the technique, in its traditional sense, is limited by the requirement of producing high-quality antibodies, multiple washing steps, and low capacity for multiplexed analyte detection. Many of these limitations are now being addressed by the development of novel detection strategies that incorporate the magnetic, optical, and electronic proper- ties associated with metallic nanoparticles into high-sensitivity sensing devices for a broad range of target analytes. For instance, the amenability of antibody-conjugated magnetic particles to physical separation, coupled with the enormous amplification potential of enzymatic DNA replication (poly- merase chain reaction) can be used to detect ultralow concentrations of disease-related proteins in serum.[5,6] Furthermore, protein detection using optical methods can be carried out by homogeneous nanoparticle assays, including whole blood, without the need for additional separation and purification.[7] For example, antibody-functionalized gold nanoshells have been tailored to specifically detect target analytes in a protein-rich background using the principles of a sandwich-immunoassay, where analyte-driven aggregation of the nanoshells results in a red-shift in their absorption spectra.[6]mbH & Co. KGaA, Weinheim 4359 R E S E A R C H N E W S J. E. Ghadiali and M. M. Stevens / Enzyme-Responsive Nanoparticle Systems Table 1. Key points for consideration during the development of ‘‘traditional’’ and nanoparticle-based enzyme assays. Radioisotope and fluorometric assays Nanoparticle-based assays Labelled substrate analogues can be costly. Nanoparticles are synthetically accessible using routine methods. Substrates are often commercially available. Many nanoparticle derivatives are commercially available at reasonable cost. Substrate can be added to enzyme reaction medium without any additional preparative steps. Nanoparticle-substrate conjugates must be prepared. Decay of substrate and photochemical stability of substrates can limit shelf-life and necessitates careful handling. Nanoparticles are often chemically robust. Colloidal stability is important factor with respect to shelf-life and compatibility with enzyme reaction media. Requires scintillation counting, autoradiography, or fluorometric detection equipment. Fluorometric, SERS, relaxivity, absorbance, or visual detection. Multistep procedures are often required. Possibility for homogeneous, one-step reactions. 4360Despite the sensitivity and versatility of immunological detection methods for enzymes, it is often useful to directly measure their activity. Nanoparticle-based materials are attractive alternatives to the fluorescence- or radiolabeled enzyme substrates of traditional enzymatic assays, owing to their photostability, ease of synthesis, and ability to conjugate to biological molecules (Table 1). In the vast majority of circumstances, excess of a thiolated derivative of a biological molecule of interest (e.g. DNA/peptide) coordinates directly to the nanoparticle’s surface, namely gold or silver, whilst any excess of themolecule is readily removed by centrifugation and washing steps. Nanoparticle enzyme sensors, in the most basic sense, consist of a biological substrate molecule immobilized onto the nanoparticle surface. The activity of an enzyme results in some form of modification of the substrate and a subsequent change in the nanoparticles’ local environment, leading to the generation of a user-readable signal, for example, optical or electronic. Being able to probe the activity of the enzyme directly, as opposed to simply detecting its presence as in immunological assays, yields several key benefits, as the enzyme’s activity and continuous turnover serves as an inherent means of signal amplification and also provides a route to screening potential inhibitors. Here, we provide an overview of recent examples of enzymes that have been examined using a range of nanoparticle-based strategies, focusing on enzymes involved in DNA metabolism, protein kinases, and proteases owing to the extent of their characterization and important implications in numerous disease states.Figure 1. Enzyme-driven modification of DNA nanostructures by a) endo- nuclease/ligation mechanisms, and b) DNA methyltransferase enzymes.2. Enzyme-Responsive Nanoparticle Systems 2.1. Enzymatic Control of DNA Nanostructures The site-specific addressability of DNA base-pairing is now well-known as a tool to drive the assembly of many diverse higher-order structures and controlled aggregation of inor- ganic nanoparticles.[8,9] There are now several examples demonstrating that such DNA–nanoparticle conjugates can serve as substrates for a range of enzymes involved in DNA processing, with the resultant changes in particle dispersionwww.advmat.de 2008 WILEY-VCH Verlag GmbH &used to drive precise structural rearrangements as a signal- output for enzyme activity.[10] Disassembly of gold nanoparticles crosslinked by comple- mentary duplex strands of DNA can be achieved by incubation with an endonuclease that cleaves double-stranded DNA in a site-specific manner.[11] The resultant dispersions can be subsequently characterized by absorption spectroscopy, trans- mission electron microscopy (TEM), and polyacrylamide gel electrophoresis. Furthermore, employing DNA ligases, in conjunction with similar specific endonucleases to generate cohesive DNA ends, provides a means to selectively link DNA-functionalized gold nanoparticles and form higher-order assemblies (Fig. 1).[12] These principles of enzyme-driven assembly/disassembly have also been extended to real-time colorimetric screening of endonuclease inhibitors and to discriminate chemically modified DNA from an unmodified form.[13,14] In the first example, a DNA-cleaving enzyme was added to a DNA– gold-nanoparticle crosslinked network, in the presence or absence of various DNA binding agents known to inhibit the enzyme. Monitoring the intensity of the plasmon absorbance band as a function of time provided rapid, semiquantitative information on relative inhibitor potency and kinetics. For a more in-depth profiling of reaction kinetics and mechanistic details of the cleavage reaction, surface-immobilization and single-molecule imaging of DNA–nanoparticle conjugates isCo. KGaA, Weinheim Adv. Mater. 2008, 20, 4359–4363