Mapping Single Molecular Binding Kinetics of Carbohydrate-Binding Module with Crystalline Cellulose by Atomic Force Microscopy Recognition Imaging

We studied the binding kinetics of family 3 carbohydrate-binding module (CBM3a) molecules to crystalline cellulose fibrils extracted from the poplar cell wall by atomic force microscopy (AFM) recognition imaging. The free CBM3a molecules of different concentrations were added to the buffer solution to bind to the crystalline cellulose sample immobilized on the AFM substrate. During <i>in-situ</i> AFM imaging, the CBM molecules were observed to bind to cellulose efficiently and regularly, especially in the first 60–120 min. A 1:1 single-molecule binding model was used to study the kinetics of the CBM3a–cellulose interaction. The saturation time when the concentration of occupied binding sites is 99% of the maximum bound CBM3a concentration at the end of reaction, <i>t</i>_(0.99), was determined by fitting different concentrations of CBM3a against reaction time using the high resolution AFM images and the single-molecule kinetics equations. Based on the experimental data and kinetics calculations, the minimal effective initial CBM3a concentration was estimated to be 5.1 × 10^–7 M at 287 min reaction time. This study provides an in-depth understanding of the binding mechanism of CBM with crystalline cellulose at single molecule level

Photoconductance from Exciton Binding in Molecular Junctions

We report on a theoretical analysis and experimental verification of a mechanism for photoconductance, the change in conductance upon illumination, in symmetric single-molecule junctions. We demonstrate that photoconductance at resonant illumination arises due to the Coulomb interaction between the electrons and holes in the molecular bridge, so-called exciton-binding. Using a scanning tunneling microscopy break junction technique, we measure the conductance histograms of perylene tetracarboxylic diimide (PTCDI) molecules attached to Au-electrodes, in the dark and under illumination, and show a significant and reversible change in conductance, as expected from the theory. Finally, we show how our description of the photoconductance leads to a simple design principle for enhancing the performance of molecular switches

Mapping the Details of Contact Effect of Modulated Au-Octanedithiol-Au Break Junction by Force–Conductance Cross-Correlation

We have measured the force and conductance of Au-octanedithiol-Au junctions using a modified conducting atomic force microscopy break junction technique with sawtooth modulations. Force–conductance two-dimensional cross-correlation histogram (FC-2DCCH) analysis for the single-molecule plateaus is demonstrated. Interestingly, four strong correlated regions appear in FC-2DCCHs consistently when modulations with different amplitudes are applied, in sharp contrast to the results under no modulation. These regions reflect the conductance and force changes during the transition of two molecule/electrode contact configurations. As the modulation amplitude increases, intermediate transition states of the contact configurations are discerned and further confirmed by comparing individual traces. This study unravels the relation between force and conductance hidden in the data of a modulated single-molecule break junction system and provides a fresh understanding of electron transport properties at molecule/electrode interfaces

Imaging and Measuring Single-Molecule Interaction between a Carbohydrate-Binding Module and Natural Plant Cell Wall Cellulose

The affinitive interaction between a carbohydrate-binding module (CBM3a) and natural crystalline cellulose was visualized and measured at the single-molecule level. Noncontact high resolution imaging by atomic force microscopy (AFM) was used to follow the binding process, in real time, of CBM3a-functionalized 6 nm gold nanoparticles (GNPs) to the cell wall polymers on poplar stem sections. The GNP–CBM3a complexes were found to bind to the cellulose surface, closely aligning along the cellulose fibril axis. The binding details were further confirmed and studied by single-molecule recognition imaging and AFM single-molecule dynamic force spectroscopy (SMDFS) using a CBM3a-functionalized AFM tip. The unbinding force was measured to be 44.96 ± 18.80 pN under a loading rate of 67.2 nN/s. This research provides a radical method for the study of single-molecule affinity between CBM and cellulose that is critical to the engineering of novel cellulolytic enzymes

High-Resolution Single-Molecule Recognition Imaging of the Molecular Details of Ricin–Aptamer Interaction

We studied the molecular details of DNA aptamer–ricin interactions. The toxic protein ricin molecules were immobilized on a Au(111) surface using a <i>N</i>-hydroxysuccinimide (NHS) ester to specifically react with lysine residues located on the ricin B chains. A single ricin molecule was visualized in situ using the AFM tip modified with an antiricin aptamer. Computer simulation was used to illustrate the protein and aptamer structures, the single-molecule ricin images on a Au(111) surface, and the binding conformations of ricin–aptamer and ricin–antibody complexes. The various ricin conformations on a Au(111) surface were caused by the different lysine residues reacting with the NHS ester. It was also observed that most of the binding sites for aptamer and antibody on the A chains of ricin molecules were not interfered by the immobilization reaction. The different locations of the ricin binding sites to aptamer and antibody were also distinguished by AFM recognition images and interpreted by simulations

MOESM1 of Real-time single molecular study of a pretreated cellulose hydrolysis mode and individual enzyme movement

Additional file 1. Supporting information

Direct Optical Detection of Viral Nucleoprotein Binding to an Anti-Influenza Aptamer

We have demonstrated label-free optical detection of viral nucleoprotein binding to a polyvalent anti-influenza aptamer by monitoring the surface-enhanced Raman (SERS) spectra of the aptamer-nucleoprotein complex. The SERS spectra demonstrated that selective binding of the aptamer-nucleoprotein complex could be differentiated from that of the aptamer alone based solely on the direct spectral signature for the aptamer-nucleoprotein complex. Multivariate statistical methods, including principal components analysis, hierarchical clustering, and partial least squares, were used to confirm statistically significant differences between the spectra of the aptamer-nucleoprotein complex and the spectra of the unbound aptamer. Two separate negative controls were used to evaluate the specificity of binding of the viral nucleoproteins to this aptamer. In both cases, no spectral changes were observed that showed protein binding to the control surfaces, indicating a high degree of specificity for the binding of influenza viral nucleoproteins only to the influenza-specific aptamer. Statistical analysis of the spectra supports this interpretation. AFM images demonstrate morphological changes consistent with formation of the influenza aptamer-nucleoprotein complex. These results provide the first evidence for the use of aptamer-modified SERS substrates as diagnostic tools for influenza virus detection in a complex biological matrix

Direct Optical Detection of Viral Nucleoprotein Binding to an Anti-Influenza Aptamer

We have demonstrated label-free optical detection of viral nucleoprotein binding to a polyvalent anti-influenza aptamer by monitoring the surface-enhanced Raman (SERS) spectra of the aptamer-nucleoprotein complex. The SERS spectra demonstrated that selective binding of the aptamer-nucleoprotein complex could be differentiated from that of the aptamer alone based solely on the direct spectral signature for the aptamer-nucleoprotein complex. Multivariate statistical methods, including principal components analysis, hierarchical clustering, and partial least squares, were used to confirm statistically significant differences between the spectra of the aptamer-nucleoprotein complex and the spectra of the unbound aptamer. Two separate negative controls were used to evaluate the specificity of binding of the viral nucleoproteins to this aptamer. In both cases, no spectral changes were observed that showed protein binding to the control surfaces, indicating a high degree of specificity for the binding of influenza viral nucleoproteins only to the influenza-specific aptamer. Statistical analysis of the spectra supports this interpretation. AFM images demonstrate morphological changes consistent with formation of the influenza aptamer-nucleoprotein complex. These results provide the first evidence for the use of aptamer-modified SERS substrates as diagnostic tools for influenza virus detection in a complex biological matrix

From Ring-in-Ring to Sphere-in-Sphere: Self-Assembly of Discrete 2D and 3D Architectures with Increasing Stability

Directed by increasing the density of coordination sites (DOCS) to increase the stability of assemblies, discrete 2D ring-in-rings and 3D sphere-in-sphere were designed and self-assembled by one tetratopic pyridyl-based ligand with 180° diplatinum­(II) acceptors and naked Pd­(II), respectively. The high DOCS resulted by multitopic ligand provided more geometric constraints to form discrete structures with high stability. Compared to reported supramolecular hexagons and polyhedra by ditotpic ligands, the self-assembly of such giant architectures using multitopic ligands with all rigid backbone emphasized the structural integrity with precise preorganization of entire architecture, and required elaborate synthetic operations for ligand preparation. In-depth structural characterization was conducted to support desired structures, including multinuclear NMR (¹H, ³¹P, and ¹³C) analysis, 2D NMR spectroscopy (COSY and NOESY), diffusion-ordered NMR spectroscopy (DOSY), multidimensional mass spectrometry, TEM and AFM. Furthermore, a quantitative definition of DOCS was proposed to compare 2D and 3D structures and correlate the DOCS and stability of assemblies in a quantitative manner. Finally, ring-in-rings in DMSO or DMF could undergo hierarchical self-assembly into the ordered nanostructures and generated translucent supramolecular metallogels

Supersnowflakes: Stepwise Self-Assembly and Dynamic Exchange of Rhombus Star-Shaped Supramolecules

With the goal of increasing the complexity of metallo-supramolecules, two rhombus star-shaped supramolecular architectures, namely, supersnowflakes, were designed and assembled using multiple 2,2′:6′,2″-terpyridine (tpy) ligands in a stepwise manner. In the design of multicomponent self-assembly, ditopic and tritopic ligands were bridged through Ru­(II) with strong coordination to form metal–organic ligands for the subsequent self-assembly with a hexatopic ligand and Zn­(II). The combination of Ru­(II)–organic ligands with high stability and Zn­(II) ions with weak coordination played a key role in the self-assembly of giant heteroleptic supersnowflakes, which encompassed three types of tpy-based organic ligands and two metal ions. With such a stepwise strategy, the self-sorting of individual building blocks was prevented from forming the undesired assemblies, e.g., small macrocycles and coordination polymers. Furthermore, the intra- and intermolecular dynamic exchange study of two supersnowflakes by NMR and mass spectrometry revealed the remarkable stability of these giant supramolecular complexes