ABSTRACT
Current interest in melanin films derived from the autoxidation of dopamine stems from their use as a universal adhesion layer. Here we report chemical and physical characterization of polydopamine films deposited on gold surfaces from stirred basic solutions at times ranging from 2 to 60 min, with a focus on times ≤10 min. Data from Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and electrochemical methods suggest the presence of starting (dopamine) and intermediate (C=N-containing tautomers of quinone and indole) species in the polydopamine films at all deposition times. A uniform overlayer analysis of the XPS data indicates that film thickness increased linearly at short deposition times of ≤10 min. At deposition times ≥10 min, the films appeared largely continuous with surface roughness ≈ ≤ 2 nm, as determined by atomic force microscopy (AFM). Pinhole-free films, as determined by anionic redox probe measurements, required deposition times of 60 min or greater.
Subject(s)
Dopamine/chemistry , Indoles/chemistry , Polymers/chemistry , Electrochemical Techniques , Gold/chemistry , Microscopy, Atomic Force , Molecular Structure , Oxidation-Reduction , Spectrophotometry , Spectroscopy, Fourier Transform Infrared , Surface Properties , Time Factors , X-RaysABSTRACT
We report a glycoanalysis method in which lectins are used to probe the glycans of therapeutic glycoproteins that are adsorbed on gold nanoparticles. A model mannose-presenting glycoprotein, ribonuclease B (RNase B), and the therapeutic monoclonal antibody (mAb) rituximab, were found to adsorb spontaneously and non-specifically to bare gold nanoparticles such that glycans were accessible for lectin binding. Addition of a multivalent binding lectin, such as concanavalin A (Con A), to a solution of the modified gold nanoparticles resulted in cross-linking of the nanoparticles. This phenomenon was evidenced within 1 min by a change in the hydrodynamic diameter, D(H), measured by dynamic light scattering (DLS) and a shift and increase in absorbance of the plasmon resonance band of the gold nanoparticles. By combining the sugar-binding specificity and the cross-linking capabilities of lectins, the non-specific adsorption of glycoproteins to gold surfaces, and the unique optical reporting properties of gold nanoparticles, a glycosylation pattern of rituximab could be generated. This assay provides advantages over currently used glycoanalysis methods in terms of short analysis time, simplicity of the conjugation method, convenience of simple spectroscopic detection, and feasibility of providing glycan characterization of the protein drug product by using a variety of binding lectins.
Subject(s)
Glycoproteins/analysis , Gold/chemistry , Metal Nanoparticles/chemistry , Plant Lectins/chemistry , Adsorption , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal, Murine-Derived/chemistry , Biotechnology/methods , Flocculation Tests , Glycoproteins/chemistry , Glycoproteins/metabolism , Glycosylation , Light , Models, Chemical , Plant Lectins/metabolism , Ribonucleases/chemistry , Rituximab , Scattering, Radiation , Spectrophotometry, UltravioletABSTRACT
Glycoanalysis is important in the manufacture and quality control of protein therapeutics. An emerging method for glycoanalysis is the use of lectin arrays. Critical to the performance of these arrays is the immobilization of lectin molecules. Polydopamine has recently been shown to adsorb to a wide variety of surfaces. In this study, polydopamine (pDA) was used to modify gold, indium, and iridium surfaces and promote the adhesion of the alpha-mannose-specific lectin concanavalin A (Con A). The activity of the surface-bound lectin was demonstrated with the alpha-mannose-presenting glycoprotein ribonuclease B (RNase B). Surface plasmon resonance spectroscopy (SPRS) was used to demonstrate the selective affinity of RNase B for Con A. Surface-MALDI-TOF MS experiments revealed that the affinity of polydopamine-immobilized Con A for the glycoforms of RNase B is significantly affected by slight variations in oligosaccharide structure and composition. Specifically, surface-bound Con A binds certain Man7, Man8, and Man9 RNase B glycoforms more strongly than Man5 and Man6 glycoforms.
Subject(s)
Concanavalin A/chemistry , Dopamine/chemistry , Ribonucleases/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Glycosylation , Protein Binding , Ribonucleases/chemistry , Surface Plasmon ResonanceABSTRACT
Meckel's diverticulum is a relatively uncommon cause of small bowel obstruction in the adolescent population. Here we discuss a patient with clinical and radiographic evidence of a complete bowel obstruction and describe the subsequent operative management. Finally, current literature concerning the diagnosis and management of Meckel's diverticulum is reviewed.
