Biosensors based on the two-dimensional layered nanomaterials transition metal dichalcogenides such as WS2 and MoS2 have shown broad applications, while they largely rely on the utilization of single stranded DNA as probe biomolecules. Herein we have constructed novel WS2- and MoS2- based biosensing platforms using peptides as probe biomolecules. We have revealed for the first time that the WS2 and MoS2 nanosheets display a distinct adsorption for Arg amino acid and particularly, Arg-rich peptdies. We have demonstrated that the WS2 and MoS2 dramatically quench the fluorescence of our constructed Arg-rich probe peptide, while the hybridization of the probe peptide with its target collagen sequence leads to the fluorescence recovery. The WS2-based platform provides a sensitive fluorescence-enhanced assay that is highly specific to the target collagen peptide with little interferences from other proteins. This assay can be applied for quantitative detection of collagen biomarkers in complex biological fluids. The successful development of WS2- and MoS2- based biosensors using non-ssDNA probes opens great opportunities for the construction of novel multifunctional biosensing platforms, which may have great potential in a wide range of biomedical field.

The mechanism by which enzymes recognize the "uniform" collagen triple helix is not well understood. Matrix metalloproteinases (MMPs) cleave collagen after the Gly residue of the triplet sequence Glyϳ[Ile/Leu]-[Ala/Leu] at a single, unique, position along the peptide chain. Sequence analysis of types I-III collagen has revealed a 5-triplet sequence pattern in which the natural cleavage triplets are always flanked by a specific distribution of imino acids. NMR and MMP kinetic studies of a series of homotrimer peptides that model type III collagen have been performed to correlate conformation and dynamics at, and near, the cleavage site to collagenolytic activity. A peptide that models the natural cleavage site is significantly more active than a peptide that models a potential but non-cleavable site just 2-triplets away and NMR studies show clearly that the Ile in the leading chain of the cleavage peptide is more exposed to solvent and less locally stable than the Ile in the middle and lagging chains. We propose that the unique local instability of Ile at the cleavage site in part arises from the placement of the conserved Pro at the P 3 subsite. NMR studies of peptides with Pro substitutions indicate that the local dynamics of the three chains are directly modulated by their proximity to Pro. Correlation of peptide activity to NMR data shows that a single locally unstable chain at the cleavage site, rather than two or three labile chains, is more favorable for cleavage by MMP-1 and may be the determining factor for collagen recognition.The degradation of collagen, the major structural component of connective tissues in skin, bone, tendon, and ligament, is an integral part in many biological processes such as wound healing, cell migration, tissue remodeling, and organ morphogenesis (1-4). Accelerated breakdown of collagen may result in many diseases such as arthritis, tumor cell invasion, glomerulonephritis, and metastasis (5-7). Types I, II, and III collagens, also called interstitial collagens, are the most abundant (8 -10), and contain a characteristic triple-helical conformation, which consists of three polyproline II-like helices supercoiled around a common axis (11,12). The close packing of the three chains can only accommodate Gly as every third residue, generating the repetitive (Gly-Xaa-Yaa) n sequence pattern. The Gly residues are all buried in the center, and the structure is stabilized by interchain N-H (Gly) . . . CϭO (Xaa) hydrogen bonds. The residues at the X and Y positions can be almost any amino acid, but they are frequently Pro and 4-hydroxyproline (Hyp 2 or O), respectively.The triple helical structure allows collagen to be degraded by only a few proteinases including a group of matrix metalloproteinases (MMPs) (5, 13). These MMPs (MMP-1, MMP-2, MMP-8, MMP-13, MMP-14, MMP-18) can bind and cleave interstitial collagens at a unique locus approximately threefourths away from the N terminus of the collagens (14). The cleavage site is after the Gly residue in the sequence of Glyϳ[Ile/Leu]-[Ala/Leu]...

NMR spectroscopy is used to investigate the heterotrimeric nature of a collagen model peptide. Two distinct peptide chains (A and B) were synthesized to model a site in heterotrimeric basement membrane type IV collagen. For NMR studies, four amino acids in the B chain were labeled with 15 N/ 13 C. CD spectroscopy and DSC thermal stability results on a solution with both A and B peptides (molar ratio 2A:1B) are consistent with the presence of one heterotrimeric triple-helical molecular species. HSQC experiments on homotrimers of the B peptide show trimer peaks which disappear at temperatures higher than 10°C, while the 2A:1B mixture has trimer peaks with increased stability and altered chemical shifts. The reduction in the number of Leu trimer peaks from three to one and the increased stability of trimer resonances confirm the participation of B chains in an AAB heterotrimer molecule.Triple-helical peptides serve as valuable models for collagen, a critical protein in normal tissue structure and in disease. Close packing of 3 polyproline-like chains in the collagen triple-helix structure generates the requirement for glycine as every third residue, (Gly-X-Y) n . 1 Peptides with Gly as every third residue and a high imino acid content will spontaneously self-assemble into homotrimers with a triple-helical structure. Such stable homotrimer peptides have been well characterized in terms of stability, folding, and dynamics, and molecular structures have It has proved more difficult to obtain and characterize hybrid triple-helical peptides composed of chains with different amino acid sequences (heterotrimers), which can serve as models for heterotrimeric collagens such as Type I collagen in bone and type IV collagen in basement membranes.Heterotrimer peptide design strategies have included covalent linkage to force the selection of three chains and their alignment, 3 while recent studies used electrostatic interactions within the (Gly-X-Y) n sequences to direct desired self-assembly. 4 Thus far, techniques for biophysical characterization of heterotrimers have probed average properties of the triple-helix. For instance, Gauba and Hartgerink used circular dichroism (CD) spectroscopy to follow the thermal stabilities of single peptide vs. mixed peptide solutions, and differences in stability are interpreted in terms of homotrimer and heterotrimer molecules. 4 In contrast, NMR has the capacity to follow the properties of individual residues and individual chains within trimers. 2a Complexes where one subunit is labeled and others are not have been studied by NMR to define the features of the labeled complex and its interaction. 5 Here, a similar strategy is applied to the triple-helix, using NMR to monitor labeled residues within one peptide chain to follow specific structural and chemical properties within a heterotrimer versus a homotrimer context.The design strategy presented here is based on mixing an 15 N/ 13 C labeled peptide chain which has a low propensity to self-associate into a triple-helix together w...