{'fraction_non_alphanumeric': 0.0354251012145749, 'fraction_numerical': 0.004723346828609987, 'mean_word_length': 5.879350348027843, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Peptide‐Functionalized Spherical Polyelectrolyte Nanobrushes for Real‐Time Sensing of Protease Activity

Proteases play vital roles in physiological processes from simple protein catabolism to highly regulated cascades, such as blood coagulation, cell regulation and signaling and immune defense, as well as pathological processes, including inflammation and tumor growth.[1] Consequently, interest in the activity of proteases, as well as their inhibition mechanism, is increasingly directed toward biochemical research, diagnosis of protease-related diseases, and the development of potential therapeutic drugs. Over the past decade, many methods for detecting protease activity have been reported, such as high-performance liquid chromatography (HPLC), [2] polyacrylamide gel electrophoresis,[3] capillary electrophoresis,[4] and FRET-based methods (fluorescence resonance energy transfer),[5] but most of them are time-consuming and impractical for a real-time, multiplex or high-throughput format. Therefore, there is a high demand for a simple, rapid, sensitive, and high-throughput routine assay to detect protease activity. Recently, some inorganic nanomaterials, such as quantum dots and gold nanoparticles, have been widely explored for use in protease assays. [6] Herein, we demonstrate that peptide-functionalized spherical polyelectrolyte brushes (SPB), as a new type of biocompatible colloidal nanostructure with good water-solubility, can be exploited to generate a highly sensitive fluorescence polarization (FP) assay for protease activity. SPB consists of a solid polystyrene core with a diameter of 50-100 nm onto which linear polyelectrolyte poly(acrylic acid) chains (PAA) are grafted.[7] By its excellent dispersibility and good retention, PAA has been shown to be an attractive carrier for drug delivery systems, biosensors, immunoassays, and biocatalysts. Ballauff group have done a systematic work of nanobrushes (SPB) in the biological application,[8] especially in the protein immobilization onto the SPB. Scheme 1 illustrates the principle of the SPB-based FP assay proposed in this work. Briefly, fluorophore-labeled peptide substrates are rapidly assembled onto the thin polyelectrolyte brushes to form Peptide-SPB composite probes, which, in turn, are capable of displaying a high fluorescence polarization value. The FP value of a fluorophore is sensitive to changes in the rotational motion of fluorescently labeled molecule. Fluorophore-labeled peptides confined in SPB nanobrushes will have bigger anisotropy values due to their slow rotation. In the presence of protease, the Peptide-SPB composite probes are recognized and specifically cleaved to release the fluorophore-labeled peptide segments, resulting in depolarization of the system. This SPB-peptide hydrolysis event is then translated into measurable FP decrease by free cleaved dye-labeled peptide segments, which, in turn, results in the sensing of protease activity.