ABSTRACT
Fast and high-throughput determination of drugs is a key trend in clinical medicine.Single particles have increasingly been adopted in a variety of photoanalytical and electroanalytical applications,and microscopic analysis has been a hot topic in recent years,especially for electrochemiluminescence (ECL).This paper describes a simple ECL method based on single gold microbeads to image lecithin.Lecithin reacts to produce hydrogen peroxide under the successive enzymatic reaction of phospholipase D and choline oxidase.ECL was generated by the electrochemical reaction between a luminol analog and hydrogen peroxide,and ECL signals were imaged by a camera.Despite the heterogeneity of single gold microbeads,their luminescence obeyed statistical regularity.The average luminescence of 30 gold microbeads is correlated with the lecithin concentration,and thus,a visualization method for analyzing lecithin was established.Calibration curves were constructed for ECL intensity and lecithin concentra-tion,achieving detection limits of 0.05 mM lecithin.This ECL imaging platform based on single gold microbeads exhibits outstanding advantages,such as high throughput,versatility and low cost,and holds great potential in disease diagnostics,environmental monitoring and food safety.
ABSTRACT
Single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) is a new tool that can provide the element composition, size distribution, and number concentration of nanoparticles. Here we discussed the effects of dwell time and settling time on analysis of nanoparticles by SP-ICP-MS. We analyzed standard materials of gold nanoparticles (30, 40 and 60 nm AuNPs, from NIST and NCNST), showing that better signal-to-noise ratio and higher determination efficiency could be achived when using shorter dwell time and settling time. We utilized a nano mode for SP-ICP-MS, in which the dwell time was set as 0. 05 ms and the settling time was 0. The size of NIST AuNP standard material determined here was in accord with the certified size using the developed method. The detection limits of size and number concentration of AuNPs were 8 nm and 1. 1×105 particle / L, respectively.
ABSTRACT
Cryo electron microscopy (cryo-EM) is one of the most important methods in structural biology.In the past five years,cryo-EM has milestone breakthrough due to revolutionary advances in hardware and methodology.The resolution has been pushed to as high as 1.8 （A),which significantly extended the research scope of this method.Single particle reconstruction method has become one of the most exciting fields of structural biology and related subjects.Here we will briefly introduce the history of cryo-EM,recent revolutionary breakthrough,and the facility at Fudan University.
ABSTRACT
The present situation and research progress of the single-particle focusing device and related technologies were introduced in foreign countries and China. The principles and advantages of different single-particle focusing devices were analyzed from the aspects of liquid and gas single-particle focusing, and it's pointed out that rapid detection of the particles such as bacteria could be realized by liquid and gas single-particle focusing measures. The two measures both had brilliant prospects providing the structure and design were optimized to ensure high detection precision and efficiency, which could be promoted for bio-agents detection and fulminating infectious diseases prevention and treatment.
ABSTRACT
Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.
Subject(s)
Animals , Humans , Rats , ADP-Ribosylation Factor 1 , Chemistry , Metabolism , Coatomer Protein , Chemistry , Metabolism , Cytosol , Chemistry , Metabolism , GTPase-Activating Proteins , Chemistry , Metabolism , Membranes, ArtificialABSTRACT
Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macromolecules. An intrinsic assumption in single particle analysis is that all the analyzed particles must have identical composition and conformation. Thus specimen heterogeneity in either composition or conformation has raised great challenges for high-resolution analysis. For particles with multiple conformations, inaccurate alignments and orientation parameters will yield an averaged map with diminished resolution and smeared density. Besides extensive classification approaches, here based on the assumption that the macromolecular complex is made up of multiple rigid modules whose relative orientations and positions are in slight fluctuation around equilibriums, we propose a new method called as local optimization refinement to address this conformational heterogeneity for an improved resolution. The key idea is to optimize the orientation and shift parameters of each rigid module and then reconstruct their three-dimensional structures individually. Using simulated data of 80S/70S ribosomes with relative fluctuations between the large (60S/50S) and the small (40S/30S) subunits, we tested this algorithm and found that the resolutions of both subunits are significantly improved. Our method provides a proof-of-principle solution for high-resolution single particle analysis of macromolecular complexes with dynamic conformations.
Subject(s)
Humans , Algorithms , Computer Simulation , Cryoelectron Microscopy , Methods , Crystallography, X-Ray , Macromolecular Substances , Chemistry , Models, Molecular , Protein Conformation , Ribosomes , ChemistryABSTRACT
Objective To explore the relationship between the spot size and the result of ploidy analysis in the detection technology based on single-particles, and to fix the scope of spot thickness on the basis of the experimental result.Methods The influence of spot thickness on voltage signals produced by single cells was analyzed.The parameters of the beam shap system in the incident field were designed and optimized on ZEMAX.Finally, according to the cells′diameter, the target size of spots was set.A set of spots of different thickness and of Gaussian distribution obtained from the optical experimental platform was used to conduct ploidy detection experiments.Results Target spots were both obtained from ZEMAX simulation and the optical platform.When the spot thickness was larger than both monocytes and coenocytes, the mean fluorescence intensity ratio was 2.03,which met the demand of the index.Conclusion When the height of the pulse is used to represent the fluorescence density, the relative size of spots and cells will affect the result of ploidy detection.Only when spot thickness is larger than cells is the ploidy ratio accurate.
ABSTRACT
OBJECTIVES: Rapid increase in engineered nanoparticles (ENPs) in many goods has raised significant concern about their environmental safety. Proper methodologies are therefore needed to conduct toxicity and exposure assessment of nanoparticles in the environment. This study reviews several analytical techniques for nanoparticles and summarizes their principles, advantages and disadvantages, reviews the state of the art, and offers the perspectives of nanometrology in relation to ENP studies. METHODS: Nanometrology is divided into five techniques with regard to the instrumental principle: microscopy, light scattering, spectroscopy, separation, and single particle inductively coupled plasma-mass spectrometry. RESULTS: Each analytical method has its own drawbacks, such as detection limit, ability to quantify or qualify ENPs, and matrix effects. More than two different analytical methods should be used to better characterize ENPs. CONCLUSIONS: In characterizing ENPs, the researchers should understand the nanometrology and its demerits, as well as its merits, to properly interpret their experimental results. Challenges lie in the nanometrology and pretreatment of ENPs from various matrices; in the extraction without dissolution or aggregation, and concentration of ENPs to satisfy the instrumental detection limit.
Subject(s)
Limit of Detection , Microscopy , Nanoparticles , Spectrum AnalysisABSTRACT
Asingleparticle-inductivelycoupledplasmamassspectrometric(SP-ICP-MS)methodwas established to detect the size distribution and number concentrations of silver nanoparticle ( AgNPs) in dilute aqueous solution. The optimal dwell time was 3 ms to reduce possibility of two or more particles entering into detector simultaneously. An iterative algorithm was applied to distinguish AgNPs as outliers from baseline and dissolved metal ion signal if the measured intensity was beyond five time standard deviation of whole data. Size distribution and number concentration of three commercial silver nanoparticle dispersions ( nominal diameters of 30, 50, 100 nm) were determined using SP-ICP-MS. The result of SP-ICP-MS is accurately similar to the transmission electron microscopy ( TEM) , indicating that SP-ICP-MS is able to size silver nanoparticles. The particle size detection limit is 25 nm and the limit of number concentration is 8 × 104 particles/L in dilute solution. Tap water added with silver nanoparticle was tested to obtain a similar size distribution and number concentration. This method is simple, fast and highly sensitive, which can be used to investigate risk assessment of silver nanoparticle in aqueous environment and monitor silver nanoparticle in drinking water.
ABSTRACT
Volatility can influence the lifetime of particles in the atmosphere, and provide useful information on the formation of secondary aerosol. The previous studies generally utilized thermodenuder ( TD ) to investigate the volatility behavior of particles. Using TD, semivolatile species are vaporized at different temperature, and the vaporized gas is adsorpted by activated charcoal. However, carbon might be emitted from activated charcoal under high temperature or activated charcoal ageing. In this study, a new method was developed for the measurement of particle volatility by coupling a thermodiluter system to an online single particle aerosol mass spectrometer ( SPAMS) . Aerosol particles were passed into two different channels, and then analyzed by SPAMS. Through Channel 1, aerosol particles were heated to different temperature by heating tube, then non-volatile particles and volatile gas entered into the diluter. After diluting and cooling by diluent air, the non-volatile particles were analyzed by SPAMS. Through Channel 2, aerosol particles were analyzed directly by SPAMS without the heating process. Particle volatility was obtained by comparing the information ( particle size, particle number and mass spectrum ) of particles through Channels 1 and 2. Laboratory tests showed that the diluter could avoid the re-condensation of volatiles to the particles. This developed method was applied in the real time measurement of individual particle volatility in the spring of Guangzhou. The results showed that these particles were primarily comprised of highly volatile and moderate volatile species.