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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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.

In this study, we report immobilization of cysteine-labeled oligopeptides on UV activated surfaces decorated with N,N-dimethyl-n-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP). Our result shows that cysteine group, regardless of its position in the oligopeptide, is essential for successful immobilization of oligopeptide on the UV-activated surface. A possible reaction mechanism is nucleophilic addition of thiolates to surface aldehyde groups generated during UV activation. By using this technique, we are able to incorporate anchoring points into oligopeptides through cysteine residues. Furthermore, immobilized oligopeptides on the UV-activated surface is very stable even under harsh washing conditions. Finally, we show that an HPQ-containing oligopeptide can be immobilized on the UV-activated surface, but the final surface density and its ability to bind streptavidin are affected by the position of cysteine and HPQ. An oligopeptide with a cysteine at the N-terminus and a HPQ motif at the C-terminus gives the highest binding signal in the streptavidin-binding assay. This result is potentially useful for the development of functional oligopeptide microarrays for detecting target protein molecules., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We report a surface plasmon resonance (SPR) biosensor for online detection of glyphosate. The surface of the sensing element is decorated with an oligopeptide, TPFDLRPSSDTR, which is identified by using phage display library. This oligopeptide shows high binding specificity for glyphosate (KD = 8.6 μM), probably because of the presence of R and D in the oligopeptide. To detect glyphosate in buffer solution, an SPR gold sensor chip is modified by using the oligopeptide with a surface density of 0.6 1/nm(2). The sensitivity of this oligopeptide-functionalized SPR biosensor is 1.02 RU/μM whereas the limit of detection (LOD) is 0.58 μM. This oligopeptide functionalized SPR biosensor also shows good specificity against other analytes such as glycine, thiacloprid, and imidacloprid.

By using phage display library, we identified two highly specific oligopeptide sequences RKRIRRMMPRPS and RNRHTHLRTRPR for binding neonicotinoids such as thiacloprid and imidacloprid. The former shows high affinity for thiacloprid whereas the latter shows high affinity for imidacloprid. Surprisingly, cross binding is minimal despite the similarity of the two molecules. To develop a neonicotinoid biosensor, these two oligopeptides are synthesized and immobilized on the surface of a surface plasmon resonance (SPR) chip with a bare-gold surface. This oligopeptide functionalized SPR biosensor can rapidly detect thiacloprid and imidacloprid in buffer solutions in a real-time manner. The limit of detection (LOD) for thiacloprid and imidacloprid is 1.2 μM and 0.9 μM, respectively., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Semiconducting nanowires have the potential to function as highly sensitive and selective sensors for label-free detection of biological and chemical species. In this paper, we describe the preparation and applications of oligopeptide-modified single crystal silicon nanowire (SiNW) arrays as multichannel metal ion sensors. Because these SiNW arrays were fabricated by using a "top-down" process, a very high density of individually addressable SiNW (180 SiNWs grouped into 36 clusters) can be manufactured on a single chip. Furthermore, after two different SiNW clusters are modified with Pb(2+)-selective and Cu(2+)-selective oligopeptide, respectively, concentrations of Pb(2+) and Cu(2+) in aqueous solutions can be detected simultaneously and selectively in two different channels. By using the SiNW arrays, we successfully achieved a detection limit of 1 nM for Pb(2+) and 10 nM for Cu(2+), respectively. These results demonstrate the potential utility of oligopeptide-modified SiNW arrays for highly sensitive and selective chemical and biological sensing applications.

We describe a highly sensitive, substrate-specific, label-free, and multiplexed protease assay which reports proteases activities as an optical bar chart, allowing test results to be easily assessed by laymen with the naked eye. First, an oligopeptide microarray having six rows of immobilized oligopeptides, with well-controlled orientations and concentration gradients, is immersed in a buffer solution containing proteases. Then, a thin layer liquid crystal is supported on the microarray to transduce the oligopeptide cleavage event into an optical bar chart of different colors and lengths. This type of optical bar chart provides very rich information such as protease concentration, incubation time, surface densities of oligopeptides, etc. Both trypsin and chymotrypsin can be detected by using this assay within 3 h. The capability of the multiplexed protease assay opens up possibilities for detecting toxins such as botulinum neurotoxins which are known to cleave proteins and affect the docking and fusing synaptic vesicles.

This letter reports a strategy of using N-terminal cysteine labels for controlling the immobilization of oligopeptides on aldehyde-terminated surfaces through the formation of stable thiazolidine rings. We also study the effect of cysteine position (either N-terminal or C-terminal) and lysine residue on the immobilization of oligopeptides. On the basis of our ellipsometry and quartz crystal microbalance (QCM) results, we conclude that the proposed immobilization strategy is highly site-specific. It works only when cysteine is in the N-terminal position, and the formation of thiazolidine is much faster than the formation of imines between lysine residues and aldehydes, even in the presence of a reducing agent such as NaBH(3)CN. By labeling an oligopeptide CSNKTRIDEANNKATKML with an N-terminal cysteine, we immobilize this oligopeptide on an aldehyde-terminated surface and investigate the enzymatic activity of trypsin acting on the oligopeptide. It is found that trypsin is able to cleave the immobilized oligopeptide having a single anchoring point at the N-terminal cysteine. No cleavage is observed when the oligopeptide is immobilized through multiple anchoring points at lysine residues.

Short oligopeptides that complex with metal ions with high affinity and high specificity are of interest to the design of chemical sensors. In this study, we compare the complexation properties of two copper-selective tripeptides, Gly-Gly-His and His-Gly-Gly, either in aqueous solutions or immobilized on solid surfaces. Our results show that the copper complex formed by Gly-Gly-His is more stable than the complex formed by His-Gly-Gly in aqueous solutions, because the position of histidine (His) in the Gly-Gly-His permits the formation of a tetragonal copper complex with a high stability. However, when the tripeptides are immobilized on aldehyde-decorated silicon wafer surfaces under a reaction condition that gives rise to near maximum surface densities of tripeptides, both immobilized Gly-Gly-His and His-Gly-Gly experience strong steric hindrance on the over-crowded surfaces. The surface crowding effect causes less complexation with copper ions than that in aqueous solutions. To ensure a proper surface density on the surface for complexation with copper ions, a so-called two-dimensional (2D) metal-ion imprinting technique is employed to avoid the surface crowdedness. By immobilizing Gly-Gly-His in the presence of copper ions, we create a tripeptide-functionalized surface that exhibits high complexation capability for copper ions. We attribute the higher copper complexation capability to the proper intermolecular distances obtained from the ion-imprinting procedure that gives the copper-tripeptide complex a preferential tetragonal geometry. Our results show that the amounts of copper complexed to a copper-imprinted surface functionalized with Gly-Gly-His are 62% higher than those of a nonimprinted surface.

A protease assay is reported by using enzymatic deposition of silver particles to report protease activity as optical readouts. In this assay, a biotinylated oligopeptide, which is a protease substrate, is first immobilized on a glass surface. In the absence of trypsin, streptavidin−alkaline phosphatase (SA−ALP) complex can bind to biotin and catalyze enzymatic silver deposition (ESD), which leads to a layer of highly reflective silver particles deposited on the surface. In the presence of trypsin, the immobilized oligopeptide is cleaved and biotin is removed. As a result, SA−ALP does not bind to the surface, and ESD does not happen. The limit of detection (LOD) of this assay is closely related to the surface density of oligopeptide substrate. When the surface density of oligopeptide is 0.12 molecule nm−2, the LOD for trypsin can reach 10 ng mL−1 (400 × 10−12 m). It is also demonstrated that this ESD-based protease assay can be applied to screening of protease inhibitors, such as benzamidine hydrochloride. [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]

Abstract: Immunobiosensors are emerging as powerful tools for diagnostic applications. In this paper, we exploit optical properties of liquid crystal (LC) and the specific epitope–antibody interaction to develop an optical immunobiosensor which can be used to detect monoclonal anti-FLAG M2, a model antibody. The sensitive layer of this immunobiosensor is a monolayer of immobilized oligopeptide CDYKDDDDK (FLAG) at an LC/aqueous interface. The linear oligopeptide can function as an epitope which can be recognized by anti-FLAG M2. When the LC is modified with 50μM of FLAG and then exposed to an aqueous solution containing anti-FLAG, anti-FLAG binds to the immobilized FLAG epitope and triggers orientational transition of LC. Because of the optical birefringence of the LC, the orientational transition is accompanied by changes in the optical images of LC from dark to bright under crossed polarizers. The detection limit for the system is 27ng/mL of anti-FLAG, and the response time is <1h. The binding of anti-FLAG to its motif FLAG oligopeptide is also very specific. When a variant epitope CDAKDDDDK is used, no apparent optical response can be observed after the addition of anti-FLAG. [Copyright &y& Elsevier]

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]