Recent developments in protease activity assays and sensors

Abstract

Proteases play a pivotal role in regulating important physiological processes from food digestion to blood clotting. They are also important biomarkers for many diseases such as cancers. The importance of proteases has led to extensive efforts in the screening of proteases and their inhibitors as potential drug molecules. For example, human immunodeficiency virus (HIV) patients have been treated with HIV-1 protease inhibitors to prolong the life expectancy of patients. Such a close relationship between diseases and proteases provides a strong motivation for developing sensitive, selective, and robust protease assays and sensors, which can be exploited to discover new proteases and inhibitors. In this aspect, protease assays based on levels of proteolytic activities are more relevant than protease affinity assays such as immunoassays. In this review, recent developments of protease activity assays based on different detection principles are discussed and compared. For homogenous assays, fluorescence-based techniques are the most popular due to their high sensitivity and quantitative results. However, homogeneous assays have limited multiplex sensing capabilities. In contrast, heterogeneous assays can be employed to detect multiple proteases simultaneously, given the microarray technology that is already available. Among them, electrochemical methods, surface spectroscopy techniques, and enzyme-linked peptide protease assays are commonly used. Finally, recent developments in liquid crystal (LC)-based protease assays and their applications for detecting proteases and their inhibitors are discussed.

AL-PHA beads: bioplastic-based protease biosensors for global health applications

“…In response, biotechnological innovations have led to the development of an array of technologies that help address the evolving needs of protease research applications. Classical protease activity assays typically incorporate fluorogenic small molecule, peptide or nanoparticle substrates, Fluorescence Resonance Energy Transfer (FRET)-probes, electrochemical components or zymography methods [106][107][108][109]. Whereas, recent synthetic biology approaches have led to the development of more sophisticated modelling-led design strategies, the embedding of protease biosensors within smart materials and also increasingly complex whole-cell bioreporters [3,[11][12][13]37].…”

Miniaturisation of a peptide-based electrochemical protease activity sensor using platinum microelectrodes

“…1 As such, they are attracting widespread attention as key biomarkers of disorders including cardiovascular diseases, HIV, Alzheimer's disease, thrombosis, diabetes and cancer. [2][3][4][5][6][7][8][9] Therefore, the development of both sensitive and selective assays and sensors that monitor protease activity has generated considerable interest. According to Ong et al, these can be classified into two main groups: homogeneous assays (that include those based on colorimetry, mass spectrometry and fluorescence resonance energy transfer) and heterogeneous systems (that include electrochemical assays, surfaceenhanced Raman scattering and surface plasmon resonance).…”

Transducing Protease Activity into DNA Output for Developing Smart Bionanosensors

“…Many types of assays have been studied as protease monitors, with the gold standard typically being based on Förster resonance energy transfer (FRET) or colorimetric substrate‐based biosensors . The interested reader is referred to several informative review articles on this subject . In this paper, we instead examine a new class of sensors where the transduction is from protease to DNA oligomer for subsequent potential downstream information processing.…”