Simultaneous fingerprinting of multiplex collagen biomarkers in connective tissues by multicolor quantum dots-based peptide probes.

The accurate detection of multiplex collagen biomarkers is vital for diagnosing and treating various critical diseases such as tumors and fibrosis. Despite the attractive optical properties of quantum dots (QDs), it remains technically challenging to create stable and specific QDs-based probes for multiplex biological imaging. We report for the first time the construction of multi-color QDs-based peptide probes for the simultaneous fingerprinting of multiplex collagen biomarkers in connective tissues. A bipeptide system composed of a glutathione (GSH) host peptide and a collagen-targeting guest peptide (CTP) has been developed, yielding CTP-QDs probes that exhibit exceptional luminescence stability when exposed to ultraviolet irradiation and mildly acidic conditions. The versatile bipeptide system allows for facile one-pot synthesis of high-quality multicolor CTP-QDs probes, exhibiting superior selectivity in targeting critical collagen biomarkers including denatured collagen, type I collagen, type II collagen, and type IV collagen. The multicolor CTP-QDs probes have demonstrated remarkable efficacy in simultaneously fingerprinting multiple collagen types in diverse connective tissues, irrespective of their status, whether affected by injury, diseases, or undergoing remodeling processes. The innovative multicolor CTP-QDs probes offer a robust toolkit for the multiplex fingerprinting of the collagen suprafamily, demonstrating significant potential in the diagnosis and treatment of collagen-related diseases.

A robust and versatile host-guest peptide toolbox for developing highly stable and specific quantum dot-based peptide probes for imaging extracellular matrices and cells.

Multiplex fluorescence imaging plays a vital role in precision medicine for targeting complex diseases with diverse biomolecular signatures. Quantum dot (QD) probes with vibrant colors are promising candidates for multiplex imaging, but their stability and specificity are frequently compromised by the current tedious post-modification process. We have herein developed a robust and versatile host-guest peptide (HGP) toolbox for creating highly stable and specific QD-based peptide probes for imaging extracellular matrices and cells. The HGP system comprises a host peptide and a guest peptide with a shared sequence pattern of cysteine and negatively charged amino acids, allowing for QD stabilization and specificity towards targeted biomarkers. HGP has been demonstrated as a convenient one-step approach to construct hydrophilic QD-based peptide probes with superior stability under various conditions. Six multicolor HGP-modified QDs have been developed to specifically target extracellular matrix proteins such as collagen, laminin, and nidogen, as well as major cellular elements like the membrane, nucleus, and cytoplasm, providing an efficient tool for real-time monitoring of high-resolution interactions between cancer cells and the extracellular matrix. The HGP system represents a next-generation approach to developing QDs with unprecedented stability and specificity, holding great potential in multiplex imaging and precision medicine.

A robust collagen-targeting MRI peptide contrast agent for in vivo imaging of hepatic fibrosis.

We herein report the construction of a robust MRI peptide contrast agent Gd-ICTP with superior selectivity for type I collagen, enabling the accurate and non-invasive detection of hepatic fibrosis in vivo.

Highly specific recognition of denatured collagen by fluorescent peptide probes with the repetitive Gly-Pro-Pro and Gly-Hyp-Hyp sequences.

Denatured collagen is a key biomarker for various critical diseases such as cancer. Peptide probes with the repetitive (Gly-Pro-Hyp)n sequences have recently been found to selectively target denatured collagen; however, thermal or UV pretreatment is required to drive the peptides into the monomer conformation, which poses a substantial challenge for clinical applications. We herein construct two peptide probes, FAM-GOO and FAM-GPP, consisting of the repetitive (Gly-Hyp-Hyp)8 and (Gly-Pro-Pro)8 sequences, respectively. The CD, fluorescence and colorimetric studies have consistently revealed that FAM-GOO showed strong capability of forming the triple helical structure, while FAM-GPP pronouncedly displayed the single stranded conformation at temperatures as low as 4 °C. The binding experiments have indicated that both peptide probes could recognize denatured collagen with high specificity, and FAM-GPP remarkably did not need the preheating treatment. The tissue staining results have shown that preheated FAM-GOO and unheated FAM-GPP could target denatured collagen in a wide variety of rat frozen and human FFPE tissue sections. Compared with antibodies specific for a certain type of collagen, both FAM-GOO and FAM-GPP act as broad-spectrum probes for the selective detection of denatured collagen of different types and from different species. Importantly, FAM-GPP possessed the unique capability of maintaining the monomer conformation by itself, thus avoiding the potential risks of the thermal or UV pretreatment. This novel peptide probe provides a handy and versatile biosensor for specifically targeting denatured collagen, which has attractive potential in the diagnosis and therapeutics of collagen-involved diseases.

In Situ Imaging of Pathological Collagen by Electrostatic Repulsion-Destabilized Peptide Probes.

The development of robust collagen assays is crucial in the diagnosis and treatment of various pathological conditions. Peptide probes composed of the (Gly-Pro-Hyp)n sequences have received extensive attention for their remarkable collagen-targeting capability, which unfortunately has been severely impaired by their high triple helical stability. Herein, we report an efficient strategy to reduce the triple helical propensity of the (Gly-Pro-Hyp)n sequences by electrostatic repulsion. A series of peptides consisting of the (Gly-Pro-Hyp)7 sequence and a number of charged amino acid Asp have been investigated, indicating that the presence of six additional Asp pronouncedly weakened the triple helical stability of peptide probe FAM-PCTP-D6 under physiological conditions (pH 7.4). FAM-PCTP-D6 could be directly applied without any pretreatment to recognize denatured collagen with high selectivity, whereas another dye-labeled peptide probe ROX-PCTP-D6 specifically targeted pathological collagen in various connective tissues of animal disease models and human patients. The inclusion of extra charged natural amino acids has been demonstrated as a convenient approach to create biocompatible collagen-targeting peptide probes with much weaker triple helical stability. Without the need for preheating treatment, these electrostatic repulsion-driven peptide probes provide a handy tool for histopathology staining, showing promising applications in collagen-involved diseases.

Peptide Probes with Aromatic Residues Tyr and Phe at the X Position Show High Specificity for Targeting Denatured Collagen in Tissues.

The construction of potent peptide probes for selectively detecting denatured collagen is crucial for a variety of widespread diseases. However, all of the denatured collagen-targeting peptide probes found till date primarily utilized the repetitive (Gly-X-Y) n sequences with exclusively imino acids Pro and Hyp in the X and Y positions, which stabilized the triple helical conformation of the peptide probes, resulting in severe obstacles for their clinical applications. A novel series of peptide probes have been constructed by incorporating nonimino acids at the X position of the (GPO)3GXO(GPO)4 sequence, while the X-site residue is varied as Tyr, Phe, Asp, and Ala, respectively. Peptide probes FAM-GYO and FAM-GFO containing aromatic residues Tyr and Phe at the X position showed similarly high binding affinity and tissue-staining efficacy as the well-established peptide probe FAM-GPO, while peptide probes FAM-GDO and FAM-GAO with the corresponding charged residue Asp and the hydrophobic residue Ala indicated much weaker binding affinity and tissue-staining capability. Furthermore, FAM-GYO and FAM-GFO could specifically detect denatured collagen in different types of mouse connective tissues and efficiently stain various human pathological tissues. We have revealed for the first time that the incorporation of nonimino acids, particularly aromatic residues at the X and Y positions of the repetitive (Gly-X-Y) n sequences, may provide a convenient strategy to create novel robust collagen-targeting peptide probes, which have promising diagnostic applications in collagen-involved diseases.

A single stranded fluorescent peptide probe for targeting collagen in connective tissues.

We herein report the construction of a novel single stranded fluorescent collagen mimetic peptide by introducing a bulky FAM dye in the central region rather than the N terminus. Without the need for any prior thermal or ultraviolet treatment, the peptide probe can be conveniently applied to specifically target collagen in connective tissues for fluorescence imaging.

Paper-based FRET for the direct detection of collagen triple helix.

In order to develop improved diagnosis and therapies for the highly prevalent chronic fibroproliferative diseases, it is of utmost importance to construct efficient assays to detect collagen biomarkers. We have successfully created a paper-based FRET assay for the fast, inexpensive and direct detection of collagen triple helix. We have demonstrated that the adsorption of the dye-labeled probe peptide onto the GO-immobilized paper quenches the fluorescence of the dye, while the hybridization of the probe peptide with the target collagen peptide results in the desorption of the probe peptide from GO, thus restoring the fluorescence. We have also shown that this novel assay is highly specific to the target collagen peptide sequence with little interference from other proteins, and it can be applied for quantitative detection in complex biological fluids. Our integration of the GO-based FRET assay with a patterned paper provides a powerful new tool for the detection of collagen molecules with many superior features: tiny volumes of samples, multichannel detection mode, easy operation and low-cost equipment. This assay may have promising applications to fast screen multiple target collagen sequences for the discovery of new collagen biomarkers.

Immobilized trypsin on hydrophobic cellulose decorated nanoparticles shows good stability and reusability for protein digestion.

The preparation of biocatalysts based on immobilized trypsin is of great importance for both proteomic research and industrial applications. Here, we have developed a facile method to immobilize trypsin on hydrophobic cellulose-coated silica nanoparticles by surface adsorption. The immobilization conditions for the trypsin enzyme were optimized. The as-prepared biocatalyst was characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and elemental analysis. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and denaturants. In addition, the immobilized trypsin showed good durability for multiple recycling. The general applicability of the immobilized trypsin for proteomic studies was confirmed by enzymatic digestion of two widely used protein substrates: bovine serum albumin (BSA) and cytochrome c. The surface adsorption protocols for trypsin immobilization may provide a promising strategy for enzyme immobilization in general, with great potential for a range of applications in proteomic studies.