Aggregation-induced signal amplification strategy based on peptide self-assembly for ultrasensitive electrochemical detection of melanoma biomarker.

The detection of melanoma circulating biomarker in liquid biopsies is current under evaluation for being potentially utilized for earlier cancer diagnosis and its metastasis. Herein, we developed a non-invasive electrochemical approach for ultrasensitive detection of the S100B, serving as a potential promising blood circulating biomarker of melanoma, based on an aggregation-induced signal amplification (AISA) strategy via in-situ peptide self-assembly. The fundamental principle of this assay is that the designed amphiphilic peptides (C16-Pep-Fc), fulfilling multiple functions, feature both a recognition region for specific binding to S100B and an aggregation (self-assembly) region for the formation of peptide nanomicelles under mild conditions. The C16 tails were encapsulated within the hydrophobic core of the aggregates, while the relatively hydrophilic recognition fragment Pep and Fc tag were exposed on the outer surface for subsequent recognition of S100B and signal output. AISA provided remarkable accumulation of electroactive Fc moieties that enabled ultrasensitive S100B detection of as low as 0.02 nM, which was 10-fold lower than un-amplified approach and better than previously reported assays. As a proof-of-concept study, further experiments also highlighted the good reproducibility and stability of AISA and demonstrated its usability when applied to simulated serum samples. Hence, this work not only presented a valuable assay tool for ultrasensitive detecting protein biomarker, but also advocated for the utilization of aggregation-induced signal amplification in electrochemical biosensing system, given its considerable potential for future practical applications.

Identification of hybrid amyloid strains assembled from amyloid-ßand human islet amyloid polypeptide.

Cross-fibrillation of amyloid-ß(Aß) peptides and human islet amyloid polypeptides (hIAPP) has revealed a close correlation between Alzheimer's disease and type 2 diabetes (T2D). Importantly, different amyloid strains are likely to lead to the clinical pathological heterogeneity of degenerative diseases due to toxicity. However, given the complicated cross-interactions between different amyloid peptides, it is still challenging to identify the polymorphism of the hybrid amyloid strains and reveal mechanistic insights into aggregation, but highly anticipated due to their significance. In this study, we investigated the cross-fibrillation of Aßpeptides and different hIAPP species (monomers, oligomers, and fibrils) using combined experimental and simulation approaches. Cross-seeding and propagation of different amyloid peptides monitored by experimental techniques proved that the three species of hIAPP aggregates have successively enhanced Aßfibrillation, especially for hIAPP fibrils. Moreover, the polymorphism of these morphologically similar hybrid amyloid strains could be distinguished by testing their mechanical properties using quantitative nanomechanical mapping, where the assemblies of Aß-hIAPP fibrils exhibited the high Young's modulus. Furthermore, the enhanced internal molecular interactions andß-sheet structural transformation were proved by exploring the conformational ensembles of Aß-hIAPP heterodimer and Aß-hIAPP decamer using molecular dynamic simulations. Our findings pave the way for identifying different hybrid amyloid strains by quantitative nanomechanical mapping and molecular dynamic simulations, which is important not only for the precise classification of neurodegenerative disease subtypes but also for future molecular diagnosis and therapeutic treatment of multiple interrelated degenerative diseases.

Fluorescent probe for highly selective detection of cysteine in living cells.

Cys play an important physiological role in the human body. Abnormal Cys concentration can cause many diseases. Therefore, it is of great significance to detect Cys with high selectivity and sensitivity in vivo. Because homocysteine (Hcy) and glutathione (GSH) have similar reactivity and structure to cysteine, few fluorescent probes have been reported to be specific and efficient for cysteine. In this study, we designed and synthesized an organic small molecule fluorescent probe ZHJ-X based on cyanobiphenyl, which can be used to specifically recognize cysteine. The probe ZHJ-X exhibits specific selectivity for cysteine, high sensitivity, short reaction response time, good anti-interference ability, and has a low detection limit of 3.8 × 10-6 M. The probe ZHJ-X was successfully applied to the visualization of Cys in living cells and had great application prospects in cell imaging and detection.

Design and Synthesis of Novel Fluorescent Probe Based on Cyanobiphenyl and its Application in Detection of Hypochlorite.

Hypochlorite is an important biological reactive oxygen species, which plays a pivotal role in various life activities. Excessive presence in the human body or excessive intake in life causes a series of diseases. To monitor the hypochlorite level in living cells, organisms and environment water samples, we herein designed and synthesized three organic small molecule fluorescent probes with different recognition sites based on nitrile biphenyl. Through performance comparison, it was found that probe A-HM exhibited the best detection performance for hypochlorite with a low detection limit of 2.47 × 10-6 M. The introduction of hypochlorite will induce probe fluorescence A-HM to turn on, and the fluorescence colour will change from colourless to green. The application of A-HM in biological systems has been demonstrated by the imaging monitoring of hypochlorite in MCF-7, L929 cells and zebrafish. Furthermore, A-HM was also used for the accurate determination of the hypochlorite level in real water samples with high sensitivity and good recoveries.

Electric Field Effect on Inhibiting the Co-fibrillation of Amyloid Peptides by Modulating the Aggregation Pathway.

With the revelation of the close link between Alzheimer's disease (AD) and type II diabetes (T2D) and the possible assembly of multiple amyloid peptides therein, it is critical to understand and regulate the co-fibrillation pathway between related amyloid peptides. Here, we show experimentally and theoretically that electric field (EF) inhibited hybrid amyloid fibrillation of ß-amyloid peptide (Aß) and human islet amyloid peptide (hIAPP) by modulating the hetero-aggregation pathway. Experimental results confirm that the ß-sheet secondary structure of amyloid peptides would be disrupted under small static EF and accompanied by transforming fibril aggregates into amorphous particles in vitro. Molecular dynamics simulations further demonstrate that even with the transformation of the secondary structure from ß-sheet to random coil, the strong interaction between Aß and hIAPP peptides would remain largely unaffected under the small static EF, leading to the formation of amorphous nanoparticles observed in the experiments. This inhibitory effect of EF on the co-fibrillation of multiple amyloid peptides might contribute to reducing the mutual deterioration of different degenerative diseases and show great potential for the noninvasive treatment of amyloid-related diseases.

Identifying Heterozipper ß-Sheet in Twisted Amyloid Aggregation.

Amyloid peptide (AP) self-assembly is a hierarchical process. However, the mechanistic rule of guiding peptides to organize well-ordered nanostructure in a clear and precise manner remains poorly understood. Herein we explored the molecular insight of AP motif aggregates underlying hierarchical process with helical fibrillar structure by atomic force microscope, cryo-electron microscopy (cryo-EM), and molecular dynamics simulation. AP assembly encompasses well-ordered twisted fibrils with uniform morphology, size, and periodicity. More importantly, a heterozipper ß-sheet was identified in a protofilament of AP assembly determined by cryo-EM with a high resolution of 3.5 Å. Each peptide heterozipper was further composed of two antiparallel ß strands and arranged by an alternative manner in a protofilament. The hydrophobic core and hydrophilic area in each zipper played the significant role for peptide assembling. This work proposed and verified the rule facilitating the basic building unit to form twisted fibrils and gave the explanation of peptide hierarchical assembling.

Oriented Nanoarchitectonics of Bacteriorhodopsin for Enhancing ATP Generation in a Fo F1 -ATPase-Based Assembly System.

Energy conversion plays an important role in the metabolism of photosynthetic organisms. Improving energy transformation by promoting a proton gradient has been a great challenge for a long time. In the present study, we realize a directional proton migration through the construction of oriented bacteriorhodopsin (BR) microcapsules coated by Fo F1 -ATPase molecular motors through layer-by-layer (LBL) assembly. The changes in the conformation of BR under illumination lead to proton transfer in a radial direction, which generates a higher proton gradient to drive the synthesis of adenosine triphosphate (ATP) by Fo F1 -ATPase. Furthermore, to promote the photosynthetic activity, optically matched quantum dots were introduced into the artificial coassembly system of BR and Fo F1 -ATPase. Such a design creates a new path for the use of light energy.

Structural conversion of human islet amyloid polypeptide aggregates under an electric field.

Electric fields (EFs) in biological systems are well known, and their presence implies the activity of protein ion channels and pumps in various cells. The aggregation of islet amyloid polypeptides (IAPP) was recently found in human brain tissue, and this was related to the electrical activity of neurons and caused neuronal loss. However, the association between amyloid formation and the electric field is still unknown. Herein a direct method to stimulate the formation of the hIAPP peptide under an EF is reported.

Studying the Pyroelectric Effects of LiNbO3 Modified Composites.

LiNbO3 (LN) crystal has been widely used as a pyroelectric material due to its spontaneous electric polarization, which could be recharged easily and can directly convert heat energy into electricity. LN crystal's heat-resistant, low-cost, and low dielectric loss properties make it possible for its applications in room-temperature pyroelectric devices and thermal sensors. However, LN crystal suffers from fragility, inflexibility, and other mechanical properties, which limit its suitability for many applications in various fields. In this study, the LN modified flexible pyroelectric films, composed of LN micro-particles, polypropylene (PP) matrix, and multiwalled carbon nanotubes (MWCNTs), are successfully fabricated. The pyroelectric effects of LN crystal and LN/PP/MWCNT composite films are characterized by monitoring the patterned self-assembly of nanoparticles and the output pyroelectric currents. The excellent pyroelectric properties of the composites have potential applications in energy harvesters or sensors.

An instant, biocompatible and biodegradable high-performance graphitic carbon nitride.

Polymer graphitic carbon nitride (g-C3N4) materials have attracted growing interest owing to their impressive applicability in photocatalysis and optoelectronic devices. However, further applications of g-C3N4 materials are greatly restricted by their chemical inertness and insolubility in most solvents. Regarding the rising prospect of g-C3N4 nanosheets in the biomedicalfield, high solubility and biocompatibility are required for the further development of g-C3N4 materials. In this study, a simple one-step thermal polymerization method was designed to prepare fast-soluble mesoporous g-C3N4 nanosheets by using NH4HSO4 as the critical adjuvant. The products, especially the optimal g-C3N4 NSs-4, showed impressive solubility, biocompatibility and partial biodegradability. The enriched surface hydrophilic groups (-NH2 and -OH) may contribute to improving the solubility of g-C3N4 nanosheets, while the partial biodegradability can be ascribed to the presence of the disulfide bond in the g-C3N4 framework. In this system, the NH4HSO4 adjuvant acted not only as O and S sources, but also as a bubbling agent that endows the g-C3N4 a porous structure with greatly enlarged specific surface area and high separation efficiency of photogenerated electron-hole pairs. These integrative positive factors also greatly contributed to the photocatalytic activity of the g-C3N4 nanosheets. This facile, economic and general fabrication strategy for mesoporous, fast-soluble and biocompatible g-C3N4 with superior visible-light photocatalytic activity is promising in environmental, energy and biomedical fields.

Antifouling and pH-Responsive Poly(Carboxybetaine)-Based Nanoparticles for Tumor Cell Targeting.

Nanocarriers with responsibility and surface functionality of targeting molecules have been widely used to improve therapeutic efficiency. Hence, we report the assembly of pH-responsive and targeted polymer nanoparticles (NPs) composed of poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) as the core and poly(carboxybetaine methacrylate) (PCBMA) as the shell, functionalized with cyclic peptides containing Arginine-Glycine-Aspartic acid-D-Phenylalanine-Lysine (RGD). The resulting polymer NPs (PDPA@PCBMA-RGD NPs) can maintain the pH-responsivity of PDPA (pKa ~6.5) and low-fouling property of PCBMA that significantly resist non-specific interactions with RAW 264.7 and HeLa cells. Meanwhile, PDPA@PCBMA-RGD NPs could specifically target αvß3 integrin-expressed human glioblastoma (U87) cells. The pH-responsiveness and low-fouling properties of PDPA@PCBMA NPs are comparable to PDPA@poly(ethylene glycol) (PDPA@PEG) NPs, which indicates that PCBMA is an alternative to PEG for low-fouling coatings. The advantage of PDPA@PCBMA NPs lies in the presence of carboxyl groups on their surfaces for further modification (e.g., RGD functionalization for cell targeting). The reported polymer NPs represent a new carrier that have the potential for targeted therapeutic delivery.

A Magnetic Dynamic Microbiointerface with Biofeedback Mechanism for Cancer Cell Capture and Release.

Dynamic biointerfaces with reversible surface bioactivities enable dynamic modulation of cell-material interactions, thus attracting great attention in biomedical science. Herein, we demonstrated a paradigm shift of dynamic biointerfaces from macroscopical substrates to micron-sized particles by reversible engineering of a phenylboronic acid (PBA)-functionalized magnetic microbead with mussel-inspired cancer cell-targeting peptide. Due to reversible catechol-boronate interactions between the peptides and microbeads, the micron-sized dynamic biointerface exhibited sugar-responsive cancer-targeting activity, showing the potential as a microplatform for magnetic and noninvasive isolation of cancer cells through natural biofeedback mechanism (e.g., human glycemic volatility). Our results demonstrated that the dynamic magnetic platform was capable of selective cancer cell capture (∼85%) and sugar-triggered release of them (>93%) in cell culture medium with high efficiency. More importantly, by using this platform, a decent number of target cells (∼23 on average) could be magnetically isolated and identified from artificial CTC blood samples (1 mL) spiked with 100 cancer cells. In view of the biomimetic nature, high capture efficiency, excellent selectivity, and superiority in cell separation and purification processes, the dynamic magnetic microplatform reported here would be a promising and general tool for rare cell detection and separation and cell-based disease diagnosis.

Lipidoid-polymer hybrid nanoparticles loaded with TNF siRNA suppress inflammation after intra-articular administration in a murine experimental arthritis model.

Rheumatoid arthritis (RA) is a common autoimmune disease, which is characterized by painful chronic inflammation in the joints, and novel safe and efficacious treatments are urgently needed. RNA interference (RNAi) therapy based on small interfering RNA (siRNA) is a promising approach for silencing specific genes involved in inflammation. However, delivery of siRNA to the target site, i.e. the cytosol of immune cells, is a challenge. Here, we designed lipid-polymer hybrid nanoparticles (LPNs) composed of lipidoid and poly(DL-lactic-co-glycolic acid) loaded with a therapeutic cargo siRNA directed against the proinflammatory cytokine tumor necrosis factor (TNF), which plays a key role in the progression of RA. We compared their efficacy and safety with reference lipidoid-based stable nucleic acid lipid particles (SNALPs) in vitro and in vivo. Cryogenic transmission electron microscopy, atomic force microscopy and small-angle X-ray scattering revealed that the mode of loading of siRNA in lamellar structures differs between the two formulations. Thus, siRNA was tightly packed in LPNs, while LPNs displayed lower adhesion than SNALPs. The LPNs mediated a higher TNF silencing effect in vitro than SNALPs in the RAW 264.7 macrophage cell line activated with lipopolysaccharide. For both types of delivery systems, macropinocytosis was involved in cellular uptake. In addition, clathrin-mediated endocytosis contributed to uptake of SNALPs. LPNs loaded with TNF siRNA mediated sequence-specific suppression of inflammation in a murine experimental arthritis model upon intra-articular administration. Hence, the present study demonstrates that LPN-mediated TNF knockdown constitutes a promising approach for arthritis therapy of TNF-mediated chronic inflammatory conditions.

Disassembly of Alzheimer's amyloid fibrils by functional upconversion nanoparticles under near-infrared light irradiation.

Alzheimer's disease (AD) is a common neurodegenerative disease characterized by cognitive and memory function impairment. Studies have shown that the overproduction and further misfolding of amyloid polypeptide (Aß) are the main causes of AD. Therefore, how to reduce Aß species, especially to disassemble the preformed amyloid aggregates, has become the focus of related research. Photodynamic therapy (PDT) using normal photosensitizers eg. porphyrins, is a traditional way of inhibiting amyloid aggregation or degrading the amyloid aggregates, but UV light irradiation presents a side effect for the damage of normal tissue, which limits its medical application. Upconversion nanoparticles (UCNPs) is a luminous material which can be modified by photodynamic agent to form the complex generating reactive oxygen species (ROS) with noninvasive light irradiation. It presents a good advantage in the disassembly of amyloid aggregates via ROS in noninvasive light irradiation. Herein, we prepared ß-NaYF4:Yb/Er@SiO2@RB by combing UCNPs with photosensitizer to disassemble preformed Aß aggregates under near-infrared light. The results displayed upconversion nanomaterials could degrade Aß1-42 fibrils effectively by photo-generating ROS, which demonstrated that the functional UCNPs may have potential applications in the treatment of amyloid diseases in future.

Synchronous delivery of hydroxyapatite and connective tissue growth factor derived osteoinductive peptide enhanced osteogenesis.

In bone tissue engineering, electrospun fibrous scaffolds can provide excellent mechanical support, extracellular matrix mimicking components, such as 3D spacial fibrous environment for cell growth and controlled release of signaling molecules for osteogenesis. Here, a facile strategy comprising the incorporation of an osteogenic inductive peptide H1, derived from the cysteine knot (CT) domain of connective tissue growth factor (CTGF), in the core of Silk Fibroin (SF) was developed for osteogenic induction, synergistically with co-delivering hydroxyapatite (HA) from the shell of poly(l-lactic acid-co-ε-caprolactone) (PLCL). The core-shell nanofibrous structure was confirmed by transmission electron microscopy (TEM). Furthermore, the sustained released H1 has effectively promoted proliferation and osteoblastic differentiation of human induced pluripotent stem cells-derived mesenchymal stem cells (hiPS-MSCs). Moreover, after 8 weeks implantation in mice, this SF-H1/PLCL-HA composite induced bone tissue formation significantly faster than SF/PLCL as indicated by µCT. The present study is the first to demonstrate that release of short hydrophilic peptides derived from CTGF combined with HA potentiated the regenerative capacity for healing critical sized calvarial defect in vivo.

Enhanced Photoresponsive Graphene Oxide-Modified g-C3N4 for Disassembly of Amyloid ß Fibrils.

Protein misfolding and abnormal self-assembly lead to the aggregates of oligomers, fibrils, or senior amyloid ß (Aß) plaques, which are associated with the pathogenesis of many neurodegenerative diseases. Progressive cerebral accumulation of Aß protein was widely proposed to explain the cause of Alzheimer's disease, for which one promising direction of the preclinical study is to convert the preformed ß-sheet structure of Aß aggregates into innocent structures. However, the conversion is even harder than the modulation of the amyloidosis process. Herein, a graphene oxide/carbon nitride composite was developed as a good photocatalyst for irreversibly disassembling the Aß aggregates of Aß(33-42) under UV. Quartz crystal microbalance, circular dichroism spectrum, atomic force microscopy, fluorescent spectra, and mechanical property analysis were performed to analyze this photodegradation process from different aspects for fully understanding the mechanism, which may provide an important enlightenment for the relevant research in this field and neurodegenerative disease study.

Resolving protein mixtures using microfluidic diffusional sizing combined with synchrotron radiation circular dichroism.

Circular dichroism spectroscopy has become a powerful tool to characterise proteins and other biomolecules. For heterogeneous samples such as those present for interacting proteins, typically only average spectroscopic features can be resolved. Here we overcome this limitation by using free-flow microfluidic size separation in-line with synchrotron radiation circular dichroism to resolve the secondary structure of each component of a model protein mixture containing monomers and fibrils. To enable this objective, we have integrated far-UV compatible measurement chambers into PDMS-based microfluidic devices. Two architectures are proposed so as to accommodate for a wide range of concentrations. The approach, which can be used in combination with other bulk measurement techniques, paves the way to the study of complex mixtures such as the ones associated with protein misfolding and aggregation diseases including Alzheimer's and Parkinson's diseases.

Nickel nanoparticles encapsulated in porous carbon and carbon nanotube hybrids from bimetallic metal-organic-frameworks for highly efficient adsorption of dyes.

Nickel nanoparticles encapsulated in porous carbon/carbon nanotube hybrids (Ni/PC-CNT) were successfully prepared by a facile carbonization process using Ni/Zn-MOF as the precursor. Distinct from previous studies, Ni/Zn-MOF precursors were prepared via a direct precipitation method at room temperature for only 5min. After the carbonization, magnetic Ni nanoparticles were well embedded in the porous carbon and carbon nanotube. The obtained Ni/PC-CNT composites had a high surface area (999m2 g-1), large pore volume (0.86cm3 g-1) and well-developed graphitized wall. The Ni/PC-CNT composites showed excellent adsorption capacity for removal of malachite green (MG), congo red (CR), rhodamine B (Rh B), methylene blue (MB) and methyl orange (MO) from aqueous solution. The maximum adsorption capacity of Ni/PC-CNT composites were about 898, 818, 395, 312 and 271mg/g for MG, CR, RB, MB and MO dyes, respectively, which were much higher than most of the previously reported adsorbents. Moreover, the Ni/PC-CNT composites could be easily regenerated by washing it with ethanol and easy magnetic separation.

Differential Modulating Effect of MoS2 on Amyloid Peptide Assemblies.

The abnormal fibrillogenesis of amyloid peptides such as amyloid fibril and senior amyloid plaques, is associated with the pathogenesis of many amyloid diseases. Hence, modulation of amyloid assemblies is related to the possible pathogenesis of some diseases. Some two-dimensional nanomaterials, that is, graphene oxide, tungsten disulfide, exhibit strong modulation effects on the amyloid fibrillogenesis. Herein, the modulation effect of molybdenum disulfide on two amyloid peptide assemblies based on the label-free techniques is presented, including quartz crystal microbalance (QCM), AFM, and CD spectroscopy. MoS2 presents different modulating effects on the assembly of amyloid-ß peptide (33-42) [Aß (33-42)] and amylin (20-29), mainly owing to the distinct affinity between amyloid peptides and MoS2 . This is to our knowledge the first report of MoS2 as a modulator for amyloid aggregation. It enriches the variety of 2D nanomodulators of amyloid fibrillogenesis and explains the mechanism for the self-assembly of amyloid peptides, and expands the applications of MoS2 in biology.

Thermal effect on the degradation of hIAPP20-29 fibrils.

Uncontrolled misfolding of proteins resulting in the formation of amyloid deposits is associated with over 40 types of diseases, for instance, type-2 diabetes. The human Islet amyloid polypeptide (hIAPP) amyloid formation is thought to be the cause of type-2 diabetes occurrence. A possible strategy to the current challenge of reducing the toxicity of its aggregates to pancreatic ß-cell is the discovery of an efficient way to degrading amyloid deposits. In this work, hIAPP20-29, a core fibrillating fragment of hIAPP, was selected as model system to explore the thermal effect at different temperature on the degradation of hIAPP20-29 mature fibrils. Insights on the degradation mechanism are obtained by analyzing the morphologies, the mechanical properties, the interactions between the peptides, and the secondary structure of amyloid aggregates. In addition, thermal degradation displayed a possible way to breaking the interaction of peptides and further disassembling the amyloid fibrils. These findings may initiate a new avenue to degrade the amyloid peptide aggregates and enrich and update the current selection of nanostructure modulations.