RESUMEN
Compared with traditional tedious organic solvent-assisted separation process in natural medicinal chemistry, cytomembrane (CM) fishing technique became a more appealing and greener choice for screening bioactive components from natural products. However, its large-scale practical value was greatly weakened by the easy fall-off of CMs from magnetic supports, rooted in the instability of common Fe3O4 particles and their insufficient interaction with CMs. In this research, a new green biostable platform was developed for drug screening through the integration of hyperbranched quaternized hydrothermal magnetic carbon spheres (HQ-HMCSs) and CMs. The positive-charged HQ-HMCSs were constructed by chitosan-based hydrothermal carbonization onto Fe3O4 nanospheres and subsequent aqueous hyperbranching quaternization with 1,4-butanediol diglycidyl ether and methylamine. The strong interaction between HQ-HMCSs and CMs was formed via electrostatic attraction of HQ-HMCSs to negative-charged CMs and covalent linkage derived from the epoxy-amine addition reactions. The chemically stable HMCSs and its integration with CMs contributed to dramatically higher stability and recyclability of bionic nanocomposites. With the fishing of osteoblast CMs integrated HQ-HMCSs, two novel potential anti-osteoporosis compounds, narcissoside and beta-ionone, were discovered from Hippophae rhamnoides L. Enhanced osteoblast proliferation, alkaline phosphatase, and mineralization levels proved their positive osteogenesis effects. Preliminary pharmacological investigation demonstrated their potential action on membrane proteins of estrogen receptor alpha and insulin-like growth factor 1. Furthermore, beta-ionone showed apparent therapeutic effects on osteogenic lesions in zebrafish. These results provide a green, stable, cost-efficient, and reliable access to rapid discovery of drug leads, which verifiably benefits the design of nanocarbon-based biocomposites with increasingly advanced functionality.
Asunto(s)
Productos Biológicos , Quitosano , Nanosferas , Norisoprenoides , Animales , Quitosano/química , Nanosferas/química , Pez Cebra , Carbono/química , Fenómenos MagnéticosRESUMEN
Miniaturized systems, such as integrated microarray and microfluidic devices, are constantly being developed to satisfy the growing demand for sensitive and high-throughput biochemical screening platforms. Owing to its recyclability, and robust mechanical and optical properties, poly(methyl methacrylate) (PMMA) has become the most sought after material for the large-scale fabrication of these platforms. However, the chemical inertness of PMMA entails the use of complex chemical surface treatments for covalent immobilization of proteins. In addition to being hazardous and incompatible for large-scale operations, conventional biofunctionalization strategies pose high risks of compromising the biomolecular conformations, as well as the stability of PMMA. By exploiting radio frequency (RF) air plasma and standard 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry in tandem, we demonstrate a simple yet scalable PMMA functionalization strategy for covalent immobilization (chemisorption) of proteins, such as green fluorescent protein (GFP), while preserving the structural integrities of the proteins and PMMA. The surface density of chemisorbed GFP is shown to be highly dependent on the air plasma energy, initial GFP concentration, and buffer pH, where a maximum GFP surface density of 4 × 10-7 mol/m2 is obtained, when chemisorbed on EDC-NHS-activated PMMA exposed to 27 kJ of air plasma, at pH 7.4. Furthermore, antibody-binding studies validate the preserved biofunctionality of the chemisorbed GFP molecules. Finally, the coupled air plasma and EDC-NHS PMMA biofunctionalization strategy is used to fabricate microfluidic antibody assay devices to detect clinically significant concentrations of Chlamydia trachomatis specific antibodies. By coupling our scalable and tailored air plasma-enhanced PMMA biofunctionalization strategy with microfluidics, we elucidate the potential of fabricating sensitive, reproducible, and sustainable high-throughput protein screening systems, without the need for harsh chemicals and complex instrumentation.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Inmovilizadas/química , Dispositivos Laboratorio en un Chip , Polimetil Metacrilato/química , Aire , Proteínas Fluorescentes Verdes/química , Análisis por Micromatrices/métodos , Gases em Plasma/química , Ondas de Radio , Propiedades de SuperficieRESUMEN
Intracellular detection of caspase-3 activity is crucial for the study of cell apoptosis and caspase-3 related diseases. Although various nanomaterials-based biosensors have been constructed for this purpose, they often suffer from poor stability or complicated construction due to the lack of a facile and efficient biofunctionalization method, which decreases their sensing performance and limits their use in the complex physiological environments. As novel two-dimentional (2D) nanomaterials, MoS2 nanosheets (NSs) have shown great potential for biosensing due to their unique properties. Herein, we develop a versatile yet facile covalent biofucntionalization strategy of MoS2 NSs by utilizing polydopamine (PDA) as nano-bio interface, and construct an intracellular fluorescent biosensor (MoS2@PDA-PEG-Peptide, MPPP) for the determination of caspase-3 activity. This covalent biofunctionalization of MoS2 NSs can significantly improve the conjugation efficiency of biomolecules and enhance their stability in complicated environments, which is much better than conventional biofunctionalization by using thiol-metal coordination. Furthermore, this novel caspase-3 biosensor based on peptides biofunctionalized MoS2 NSs shows high sensitivity and selectivity for the detection of caspase-3 with a limit of detection (LOD) of 0.33â¯ng/mL, and can be used for high-contrast fluorescent imaging of cell apoptosis.
Asunto(s)
Técnicas Biosensibles , Caspasa 3/análisis , Disulfuros/química , Molibdeno/química , Nanopartículas/química , Péptidos/química , Caspasa 3/metabolismo , Humanos , Tamaño de la Partícula , Espectrometría de Fluorescencia , Propiedades de SuperficieRESUMEN
Silk fibroin isolated from Bombyx mori cocoons is a promising material for a range of biomedical applications, but it has no inherent cell-interactive domains, necessitating functionalization with bioactive molecules. Here we demonstrate significantly enhanced cell interactions with silk fibroin biomaterials in the absence of biofunctionalization following surface modification using plasma immersion ion implantation (PIII). Further, PIII treated silk fibroin biomaterials supported direct covalent immobilization of proteins on the material surface in the absence of chemical cross-linkers. Surface analysis after nitrogen plasma and PIII treatment at 20 kV revealed that the silk macromolecules are significantly fragmented, and at the higher fluences of implanted ions, surface carbonization was observed to depths corresponding to that of the ion penetration. Consistent with the activity of radicals created in the treated surface layer, oxidation was observed on contact with atmospheric oxygen and the PIII treated surfaces were capable of direct covalent immobilization of bioactive macromolecules. Changes in thickness, amide and nitrile groups, refractive index, and extinction coefficient in the wavelength range 400-1000 nm as a function of ion fluence are presented. Reactions responsible for the restructuring of the silk surface under ion beam treatment that facilitate covalent binding of proteins and a significant improvement in cell interactions on the modified surface are proposed.