Files
Abstract
1. A simple two-step protocol for modification of atomic force microscopy (AFM) tip and substrate by using a "click reaction" has been developed. The modified tip and substrate would be applied to detect trace amounts of ricin by using atomic force microscopy. A key feature of the approach is the use of a PEG (polyethylene glycol) derivative functionalized with one thiol and one azide ending group. One end of the PEG was attached to the gold-coated AFM tip by a strong Au-thiol bond. The azide group hanging at the other end of the immobilized PEG was used for the attachment of an antiricin antibody modified with an alkyne group using a "click reaction". The latter reaction is highly efficient, compatible with the presence of many functional groups and could proceed under mild reaction conditions. In a separate step, ricin was immobilized on the gold substrate surface that was modified by active esters. For this process, a novel bifunctional reagent was employed containing an active ester and a thioctic acid moiety. By these modification processes, AFM recognition imaging was used to detect the toxin molecules and the results show fg/mL detection sensitivity, surpassing the existing detection techniques. With measurement of the unbinding force between the antiricin antibody and ricin, which was statistically determined to be 64.89 +/- 1.67 pN, the single molecular specificity of this sensing technique is realized.2. The authors report on a study of detecting ricin molecules immobilized on chemically modified Au (111) surface by chemomechanically mapping the molecular interactions with a chemically modified atomic force microscopy (AFM) tip. AFM images resolved the different fold-up conformations of single ricin molecule as well as their intramolecule structure of A- and B-chains. AFM force spectroscopy study of the interaction indicates that the unbinding force has a linear relation with the logarithmic force loading rate, which agrees well with calculations using one-barrier bond dissociation model.3. Gold self-assembly: Understanding protein adsorption on gold surface bears increasing importance because of surface-induced changes in conformation and bioactivity. Nanofibril structures of protein fibrinogen (fg) molecules, playing paramount role in blood coagulation, are found self-assembled on a Au(1,1,1) surface without any addition of thrombin, growing in two orientations (longitude and transverse, see figure).4. The biomedical applications of gold nanoparticle (GNP) are extraordinarily promising due to its special optical properties. However, before transforming into real clinical test, a systematic understanding of the physiological interactions of GNP becomes imperative. For example, protein-GNP interactions and their biological consequences are the most fundamental and exigent for the related studies in cell level. In this study, we report on our findings that the interaction of GNP and fibrinogen (fg) could induce blood clot, one important blood protein, under near-physiological conditions. Firstly, through different characterization methods, namely, UV spectrum, dynamic lighter scattering (DLS) and atomic force microscopy (AFM), fg-GNP clots with the m size were found to be formed and their average size is time- and concentration dependent. Besides, the dissociation constant was calculated to be 1.362.05 g/mL (nM level), suggesting that the interaction between fg and GNP is very strong. Finally, by scrutinizing the fg sequences, this strong binding was found to originate from many Cys residues distributed in , , and chains of fg through Au-S bond. Most of these Cys residues are in the form of disulfide bonds, which locate at the central E domain and flank parts of C-terminal and N-terminal in the coil-coil region.