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 and magnetic 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.
Bioconjugates of quantum dot nanocrystals possess unique optical properties that allow them to serve as exceptional biological imaging and sensing reagents. Protein kinases are an important family of enzymes that phosphorylate serine, threonine, or tyrosine side chains and are critical in cell signaling and cancer biology, but despite their biomedical and pharmaceutical significance, their activity has been little explored using quantum dot technology. We demonstrate that self-assembled peptide-quantum dot conjugates can serve as surrogate substrates in a simple homogeneous assay for protein kinase activity. Enzymatic phosphorylation of the peptide-conjugates is detected by means of a complementary FRET-acceptor labeled antiphosphotyrosine antibody, with formation of the immunocomplex resulting in energy transfer between the quantum dot and FRET acceptor molecules. This approach should facilitate the development of new assays for protein kinases and other enzymes based on quantum dot FRET donors.
Kinases are one of the largest families of homologous enzymes encoded by the human genome and are essential regulators of cell communication, immune regulation, stem cell differentiation and many other important metabolic pathways in the body. The identification of kinases, their substrates, and prospective inhibitors is thus critical for understanding various signal transduction cascades and for potential diagnostic and drug discovery applications. [1] Conventional methods for kinase detection typically involve radiolabelling of the substrate using g-32P-ATP or use of a variety of fluorescence-based approaches. [2,3] In this paper, we describe a simple, homogeneous and generic one-step approach for colorimetric kinase detection using functionalized gold nanoparticles (NPs) in solution. The use of inorganic NPs as biosensing platforms is favorable due to their remarkable broad range of optical, chemical, electronic and structural properties which can be used to address some of the limitations of isotopic and fluorescence based assays. [4] The dispersion-dependent absorbance exhibited by suspensions of gold NPs due to interparticle plasmon coupling can be used to provide a convenient optical signal for the detection of protein kinase activity. This general concept has been exploited in several enzyme-responsive gold NP studies. [5,6] For example, kinase-catalyzed biotinylation of peptide-coated gold NPs by means of a g-biotin ATP derivative has been detected using streptavidin-coated gold NPs. The resultant particle aggregates arising from the specific high-affinity streptavidin-biotin interaction leads to a marked color change of the colloid suspension, which can be readily monitored spectroscopically or by eye. [7] The detection format of the assay, however, has the drawback of being a two-stage process. Moreover, there exists no standard method for peptide assembly which can be applied across different colloidal systems without considerably compromising the NP stability. This cumbersome need for case-bycase optimization of the NP assembly thus limits the facile implementation of the technology.The simple and effective assay format we propose comprises a single step and eliminates the need for any labeled ATP derivatives. The system consists of two populations of gold NPs; namely one population coated with a protein kinase substrate peptide and the other coated with complementary antiphosphotyrosine antibodies. Simultaneous addition of enzyme and ATP to the two particle types results in enzymatic phosphorylation of the NP-immobilized peptide substrate and interparticle cross-linking due to specific recognition by the antibody-functionalized particles (Figure 1). This is marked by changes in absorbance intensity at the plasmon resonance peak at 529 nm (typical of stable gold dispersions), and in the visible deposition of NP clusters. Aggregation does not occur in the absence of enzyme or ATP, or in the presence of an inhibitor of either the kinase or the antibody, indicating that this is a specific enzyme-driv...
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