Identification Of Individual Molecules Research Articles

In Situ Synthesis and Visualization of Membrane SNAP25 Nano-Organization.

Cryo-electron tomography (cryo-ET) can provide insights into the structure and states of natural membrane environments to explore the role of SNARE proteins at membrane fusion and understand the relationship between their subcellular localization/formation and action mechanism. Nevertheless, the identification of individual molecules in crowded and low signal-to-noise ratio membrane environments remains a significant challenge. In this study, cryo-ET is employed to image near-physiological state 293T cell membranes, specifically utilizing in situ synthesized gold nanoparticles (AuNPs) bound with cysteine-rich protein tags to single-molecularly labeled synaptosomal-associated protein 25 (SNAP25) on the membrane surface. The high-resolution images reveal that SNAP25 is predominantly located in regions of high molecular density within the cell membrane and aggregates into smaller clusters, which may increase the fusion efficiency. Remarkably, a zigzag arrangement of SNAP25 is observed on the cell membrane. These findings provide valuable insights into the functional mechanisms of SNARE proteins.

DNA origami signposts for identifying proteins on cell membranes by electron cryotomography

SummaryElectron cryotomography (cryoET), an electron cryomicroscopy (cryoEM) modality, has changed our understanding of biological function by revealing the native molecular details of membranes, viruses, and cells. However, identification of individual molecules within tomograms from cryoET is challenging because of sample crowding and low signal-to-noise ratios. Here, we present a tagging strategy for cryoET that precisely identifies individual protein complexes in tomograms without relying on metal clusters. Our method makes use of DNA origami to produce “molecular signposts” that target molecules of interest, here via fluorescent fusion proteins, providing a platform generally applicable to biological surfaces. We demonstrate the specificity of signpost origami tags (SPOTs) in vitro as well as their suitability for cryoET of membrane vesicles, enveloped viruses, and the exterior of intact mammalian cells.

Numerical simulation study of surface enhancement coherent anti-Stokes Raman scattering reinforced substrate

Plasma nanostructures are of particular significance for serving as a substrate for spectroscopic detection and identification of individual molecules. By combining the excitation wavelength of the molecule with the resonance wavelength of the nanostructure, the sensitive single-molecule Raman detection can be achieved. A high and stable plasma substrate for coherent anti-Stokes Raman scattering(CARS) is very useful for developing the surface-enhanced coherent anti-Stokes Raman scattering (SECARS). In the plasma nanostructures, the strong coupling of plasmonic nanoparticles with an inter-particle gap smaller than the diameter of the individual nanoparticles results in the hybridization of the optical properties of these individual nanoparticles. There are also the charge transfer plasmons(CTP) appearing in conductive bridging nanoparticles. Their unique properties make linked nanosystems a suitable candidate for building artificial molecules, nanomotors, sensors, and other optoelectronic devices. In this work, we, starting from reality, theoretically design a new linked nanosystem SECARS substrate where Fano resonance can be generated by the plasmon hybridization (PH) model resonance and the charge transfer plasmon resonance. The introduction of charge transfer plasma improves the tunability of structural resonance. By adjusting the conductivity of the conductive junction, the wavelength of the charge transfer plasma resonance can be easily adjusted to change the wavelength position of the Fano resonance. The data obtained by numerical simulation of the Raman mode at 1557 cm<sup>–1</sup> of L-tryptophan when a 1064 nm light source is used as the pump light show that this spatially symmetrical structure can generate multiple high-enhancement hot spots that do not depend on the polarization direction of the incident light. Ordinary CARS signal can generally be enhanced by 10<sup>12</sup>, and its maximum can reach 10<sup>14</sup>. Due to the ultrastrong field enhancement and insensitive-to-polarization, this method of using charge transfer plasma to design a substrate can be used in the practical substrate of SECARS and provide new ideas for designing other nonlinear optical processes such as four wave mixing and stimulated Raman scattering.

Coherent anti-Stokes Raman scattering with single-molecule sensitivity using a plasmonic Fano resonance

Plasmonic nanostructures are of particular interest as substrates for the spectroscopic detection and identification of individual molecules. Single-molecule sensitivity Raman detection has been achieved by combining resonant molecular excitation with large electromagnetic field enhancements experienced by a molecule associated with an interparticle junction. Detection of molecules with extremely small Raman cross-sections (~10(-30) cm(2) sr(-1)), however, has remained elusive. Here we show that coherent anti-Stokes Raman spectroscopy (CARS), a nonlinear spectroscopy of great utility and potential for molecular sensing, can be used to obtain single-molecule detection sensitivity, by exploiting the unique light harvesting properties of plasmonic Fano resonances. The CARS signal is enhanced by ~11 orders of magnitude relative to spontaneous Raman scattering, enabling the detection of single molecules, which is verified using a statistically rigorous bi-analyte method. This approach combines unprecedented single-molecule spectral sensitivity with plasmonic substrates that can be fabricated using top-down lithographic strategies.

Nanotechnologies in Proteomics

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Nanotechnologies in proteomics

Progress in proteomic researches is largely determined by development and implementation of new methods for the revelation and identification of proteins in biological material in a wide concentration range (from 10(-3) M to single molecules). The most perspective approaches to address this problem involve (i) nanotechnological physicochemical procedures for the separation of multicomponent protein mixtures; among these of particular interest are biospecific nanotechnological procedures for selection of proteins from multicomponent protein mixtures with their subsequent concentration on solid support; (ii) identification and counting of single molecules by use of molecular detectors. The prototypes of biospecific nanotechnological procedures, based on the capture of ligand biomolecules by biomolecules of immobilized ligate and the concentration of the captured ligands on appropriate surfaces, are well known; these are affinity chromatography, magnetic biobeads technology, different biosensor methods, etc. Here, we review the most promising nanotechnological approaches for selection of proteins and kinetic characterization of their complexes based on these biospecific methods with subsequent MS/MS identification of proteins and protein complexes. Two major groups of methods for the analysis and identification of individual molecules and their complexes by use of molecular detectors will be reviewed: scanning probe microscopy (SPM) (including atomic-force microscopy) and cryomassdetector technology.

STM Study of trans-Stilbene Self-Organized on the Ag/Ge(111)-(√3 × √3)R30° Surface

The interfacial structures of trans-stilbene (TSB) on Ag/Ge(111)-( × )R30° were studied by low-temperature scanning tunneling microscopy (LT-STM) in ultrahigh vacuum (UHV). Stilbene overlayers were prepared by vapor deposition at a substrate temperature of 200 K and imaged after the samples were cooled to 100 K. High-resolution images allow the identification of individual molecules, with TSB appearing with a distinctive dumbbell shape. From in situ observation of the substrate lattice, the TSB monolayers were found to form a (2 × 1) structure. Due to the excellent matching of unit cell length for Ag/Ge(111)-( × )R30° to the molecular length of TSB, the interaction between TSB and the substrate surface plays the controlling role in influencing the structure of the TSB overlayers. A model for the unit cell of TSB monolayers is proposed and discussed.

Scanning tunneling microscopy studies of diazo dye monolayers on HOPG

We report on scanning tunneling microscopy (STM) studies of monolayers of the diazo dye 4-[4-(N,N-dimethylamino)phenylazo]azobenzene (D2, summation formula C 20H 19N 5) on the basal plane of highly oriented pyrolytic graphite (HOPG). Monolayers of the dye were prepared by vapour deposition or by dissolving the molecules in the liquid crystal octylcyanobiphenyl (8CB). The STM images show a double-row structure exhibiting different types of lattice defects and various domains. High-resolution images allow the identification of individual molecules and the observation of intramolecular contrast. The different orientations of the rows can be explained by a commensurate registry of the molecules with the substrate. A model for the unit cell is proposed.

Computer-Aided Identification of Organic Molecules by their Molecular Spectra

I. INTRODUCTION In recent years the different branches of analytical chemistry, especially those dealing with the identification of individual molecules, as well as qualitative and quantitative analysis of molecular mixtures, have become increasingly important. Questions in these areas often arise in solving a number of global problems. For example, the analysis of compounds produced in chemical reactions under ordinary conditions is common, and more work is being done under conditions of plasma, photochemistry under different interaction energies, laser synthesis, and so on. The analysis of molecules and molecular associates is also encountered with regard to environmental pollution: organic impurities in water, the state of the atmosphere, the state of biological objects, and many others. It should be remembered that molecules in natural media enter into various reactions and change with time, thus forming innumerable derivatives. Moreover, a problem for a number of industries is automatic protection of the final product from contamination by different kinds of by-products formed in the course of production. At some stage in the research, all these problems call for exact and express analyses aimed at investigating both individual molecular systems and their associates as well as mixtures. The need for mass-scale analysis inevitably leads to the creation of automatic indentification systems. In some respects, this reminds us of the situation which existed a few decades back when the rapid advances in metallurgy and the creation of diverse alloys called for the development of highly reliable and express methods of analyzing metals and alloys at the final stage and during production. This problem was essentially solved after an effective quantitative analysis method had been developed on the basis of emission spectral analysis and a series of automatic analyzers (of which the best is the quantometer) capable of performing quantitative analysis of metals and alloys (often simultaneously for several dozen compounds) in a few seconds and sometimes in a fraction of a second.

Concatemers in DNA replication: Electron microscopic studies of partially denatured intracellular lambda DNA

We have verified, by identification of individual molecules in the electron microscope, that λ DNA concatemers are long linear molecules containing repeats of the mature phage DNA sequence. The molecules are not made up of whole multiples of the length of mature λ DNA and do not seem to contain specific start or end points. The concatemers, comprising about 20% of the molecular forms extracted late in infection, can be found in the absence of genetic recombination and in the presence or absence of maturation defects. It may be concluded that concatemers are normal intermediates in the late stage of λ replication. A second form of λ DNA, comprising from 40 to 60% of the late intracellular molecules and having the same length as DNA from mature phages, has been observed. This form seems to contain circular permutations of the mature sequence, as though derived by random breakage of circular DNA molecules. We propose that both molecular forms could be generated from a rolling circle (earlier proposed for late replication) which has fallen apart at its growing point. Evidence from sucrose-gradient sedimentation analyses supports the view that some intracellular λ molecules were preferentially broken during purification.