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Abnormal protease activities are associated with cancers, vascular diseases and Alzheimer diseases. Therefore, detection of protease activities has become increasingly important in recent years. Herein, we report a semi-quantitative, liquid crystal (LC)-based protease assay for naked-eye detection of protease activity. In this assay, casein molecules are cleaved by proteases into small peptide fragments, which can be quantified either by using Lowry’s method or using LC. In the latter, peptide fragments adsorb on a solid surface and disrupt LC to produce a bright spot for naked-eye detection. The bright spot is observed only when the surface-adsorbed peptide density exceeds a critical value. In the assay, a major challenge is how to separate undigested casein and remaining protease from peptide fragments to prevent their interferences with LC. To overcome this issue, trichloroacetic acid (TCA) is added to precipitate casein and proteases. By using the assay, we are able to detect 10 ng/mL of protease (activity 7.23 U/mg protease, R 2  = 0.991) or 6.5 μg/mL of peptide fragments. Finally, a continuous protease assay is developed to minimize manual sampling and reduce errors in kinetic studies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Polyethylene glycol (PEG) hydrogel is permeable to biomolecules, but its permeability depends on the molecular weight of monomers and the concentration of monomer solutions. In this study, we show that PEG hydrogel made from 20% to 30% of PEG700 monomer is permeable to amino acids yet impermeable to oligopeptides. Because of its unique permeability, the gel can be used to detect protease by separating amino acids from oligopeptides when proteases cleave the oligopeptides and release amino acids. Based on this principle, an UV crosslinked gel array is fabricated on a chip for simultaneous detection of protease in up to 40 samples with only 1 µl of volume required for each sample. As a proof of concept, the on-chip protease assays are used to detect trypsin in buffer and serum. The detection limits are 1.2 nM in buffer and 17.7 nM in serum, which are comparable to conventional protease assays. Moreover, because only 1 µl of liquid is required, as little as 1.2 fmol of trypsin can be detected by using the on-chip assay. The protease assay also shows good specificity for trypsin and chymotrypsin. The gel array chip could be a useful miniaturized platform for high-throughput detection of different proteases and screening of their inhibitors. [ABSTRACT FROM AUTHOR]

We reported an oligopeptide immobilization strategy for improving stability and sensitivity of liquid-crystal (LC) based protease assay. In this strategy, oligopeptides are immobilized by using N -terminal cysteine or free amine groups on lysine residues to react with surface aldehyde groups and form multiple anchoring points. Our results show that the number of anchoring points is an important factor for improving stability of the immobilized oligopeptides. Oligopeptides with multiple anchoring points are very stable on the surface. They can only be cleaved from the surface by using proteases such as trypsin or α-chymotrypsin. This phenomenon provides a principle for developing an assay for the detection of proteases. On the other hand, oligopeptides with a single anchoring point cannot form stable linkage on the surface, and they can be easily washed off during rinsing steps. By immobilizing oligopeptides with different surface densities, concentrations of trypsin and α-chymotrypsin can be estimated simply by counting the number of bright LC spots. The higher the concentrations of trypsin and α-chymotrypsin, the less LC spots can be observed. In addition, the limit of detection (LOD) of the assay can be lowered by allowing a very large oligopeptide fragment to desorb from the surface after the cleavage. This task can be accomplished by immobilizing an oligopeptide with all anchoring points located at both ends while the cleavage sites are located near the anchoring points. Based on the principle, optimized LODs for trypsin and α-chymotrypsin can reach 1 and 0.1 ng/mL, respectively. [ABSTRACT FROM AUTHOR]

Abstract: We report a functional protease assay in which liquid crystals (LCs) are used as signal reporters to transduce the test results into optical signals. In this assay, an oligopeptide substrate (CLSELDDRADALQAGASQFESSAAKLKRKYWWKNLK) is used as a probe. This oligopeptide can be cleaved by α-chymotrypsin at multiple locations and become smaller fragments after the cleavage. When the original oligopeptide is immobilized on a solid surface, its long flexible oligopeptide chain is able to influence the orientation of a thin layer of LC supported on the surface, as is evident as a bright spot on the surface. In contrast, when the shorter oligopeptide fragments are immobilized on the same surface, their shorter, less flexible chains cannot disrupt the orientation of LC, and a dark spot is observed. On the basis of the dark or bright signal from LC, α-chymotrypsin in buffer solution or complex media such as chicken broth can be detected by using the naked eye. However, when the incubation time is 3h, the limit of detection (LOD) for α-chymotrypsin in buffer solution is 50ng/mL, whereas that in chicken broth is only 500ng/mL. Unlike traditional antibody-based assays which show little difference between active and inactive α-chymotrypsin, only active protease can be detected in this assay. This study shows the potential utility of LCs for detecting functional proteases with good specificity and sensitivity. [Copyright &y& Elsevier]

• A liquid crystal-based protease inhibition assay is developed. • This assay is used to assess inhibition efficiency of a protease inhibitor pefabloc. • IC 50 value of pefabloc can be detected by using the color of liquid crystal. • The assay provides detailed information about the transition period from reversible to irreversible inhibition. • Pefabloc inhibits protease activity irreversibly if the incubation time is more than 60 min. Protease inhibitors are essential drug molecules which can be used for treatments of protease-related diseases. Herein, we report an LC-based protease inhibition assay built inside a millifluidic device. This assay can be used to study the inhibition efficiency and reversibility of pefabloc (a serine protease inhibitor) against proteases. LC is employed for the detection of peptide fragments produced from protease activity inside the millifluidic device. LC gives a bright spot when the amount of peptide fragment exceeds a minimum value. By using this assay, we find that IC 50 value of pefabloc for the immobilized protease is 0.45 mg/mL, which is lower than the IC 50 value of pefabloc (0.90 mg/mL) obtained in a homogeneous assay. Moreover, proteases can be immobilized on the millifluidic device to build a heterogeneous protease assay. In this assay, pefabloc blocks the immobilized protease irreversibly after 60 min, which is longer than that in a homogenous protease assay. This reversibility study provides useful information about the transition period of pefabloc from reversible to irreversible inhibition. This protease inhibition assay is potentially useful for high throughput screening of unknown proteases and their inhibitor in a small sample volume. Moreover, this method can be used for dosage test of novel drug molecules which are protease inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: A colorimetric protease assay based on the cleavage of gold nanoparticles (AuNPs) aggregates cross-linked with oligopeptides is developed. In this assay, when oligopeptide YHPQMNPYTKAGGGC is added to a solution containing citrate-stabilized AuNPs, cysteine and lysine on the oligopeptide causes rapid aggregation of AuNPs, resulting in color change from red to blue. However, in the presence of a model protease trypsin, the oligopeptide is cleaved by trypsin into two fragments. As a result, AuNPs do not aggregate and no color change can be observed. By using this principle, trypsin can be detected with the naked eye at a concentration as low as 0.5nM. This assay can also be applied to detect trypsin inhibitors such as benzamidine hydrochloride. [Copyright &y& Elsevier]