scholarly journals Fast and easy single-molecule pulldown assay based on agarose microbeads

2020 ◽  
Author(s):  
Qirui Zhao ◽  
Yusheng Shen ◽  
Xiaofen Li ◽  
Fang Tian ◽  
Xiaojie Yu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Original Method ◽  
Auditory Hair Cells ◽  
Single Molecule Level ◽  
Cell Extracts ◽  
Binding Partners ◽  
Pulldown Assay ◽  
Ultrahigh Sensitivity ◽  
First Time

SUMMARYThe recently developed single-molecule pulldown (SiMPull) assay by Jain and colleagues is a highly innovative technique but its wide application is hindered by the high technical barrier and time consumption. We report an innovative, agarose microbead-based approach for SiMPull. We used commercially available, pre-surface-functionalized agarose microbeads to capture the protein of interest together with its binding partners specifically from cell extracts and observed these interactions under a microscope at the single-molecule level. Relative to the original method, microbead-based SiMPull is considerably faster, easier to use, and more reproducible and yet provides similar sensitivity and signal-to-noise ratio; specifically, with the new method, sample-preparation time is substantially decreased (from ∼10 to ∼3 h). These crucial features should facilitate wide application of powerful and versatile SiMPull in common biological and clinical laboratories. Notably, by exploiting the simplicity and ultrahigh sensitivity of microbead-based SiMPull, we used this method in the study of rare auditory hair cells for the first time.

On the way to supramolecular photochemistry at the single-molecule level

2006 ◽  
Vol 78 (12) ◽  
pp. 2247-2259 ◽  
Author(s):  
Christian Schäfer ◽  
Björn Decker ◽  
Matthias Letzel ◽  
Francesca Novara ◽  
Rainer Eckel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Pocket ◽  
Ion Cyclotron Resonance ◽  
Dynamic Force ◽  
Fticr Mass Spectrometry ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force ◽  
Molecular Length ◽  
First Time

Two examples of artificial supramolecular host-guest systems derived from resorc[4]arenes (calix[n]arenes based on resorcinol) and ammonium ions as guests have been studied by atomic force microscopy (AFM). For the first time, real single-molecule events have been determined for this type of supramolecular complexes and off-rates as well as molecular parameters of single-molecule aggregates such as the depths of the binding pocket (molecular length parameter) could be measured by applying the methods of dynamic force spectroscopy. In addition, this technique was also applied to differentiate between the two states (open and closed) of a photoswitchable resorc[4]arene-anthracene tweezer. An investigation of the exchange rates of various complexes in the gas phase by means of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry confirmed the results of the AFM study.

scholarly journals Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy

2018 ◽  
Author(s):  
Gerti Beliu ◽  
Andreas Kurz ◽  
Alexander Kuhlemann ◽  
Lisa Behringer-Pliess ◽  
Natalia Wolf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Super Resolution ◽  
Alder Reaction ◽  
Cell Permeability ◽  
High Signal ◽  
Noncanonical Amino Acids ◽  
Single Molecule Level ◽  
Genetic Code Expansion ◽  
Super Resolution Microscopy

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

scholarly journals Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

2020 ◽  
Author(s):  
Sven A. Szilagyi ◽  
Moritz Burmeister ◽  
Q. Tyrell Davis ◽  
Gero L. Hermsdorf ◽  
Suman De ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Living Cells ◽  
High Signal ◽  
Active Optics ◽  
Single Molecule Level ◽  
Light Sheet Microscopy ◽  
Reflected Light

AbstractObservation of highly dynamic processes inside living cells at the single molecule level is key for a quantitative understanding of biological systems. However, imaging of single molecules in living cells usually is limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination have recently been developed. For example, a reflected light-sheet design combines the illumination by a thin light-sheet with a high numerical aperture objective for single-molecule detection. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-color acquisition of z-stacks. We demonstrate fast volume scanning by imaging a two-color giant unilamellar vesicle (GUV) hemisphere. In addition, the high signal-to-noise ratio enabled the imaging and tracking of single lipids in the cap of a GUV. In the long term, the enhanced reflected scanning light sheet microscope enables fast 3D scanning of artificial membrane systems and cells with single-molecule sensitivity and thereby will provide quantitative and molecular insight into the operation of cells.

scholarly journals Tethered multifluorophore motion reveals equilibrium transition kinetics of single DNA double helices

2018 ◽  
Vol 115 (32) ◽  
pp. E7512-E7521 ◽  
Author(s):  
Matthias Schickinger ◽  
Martin Zacharias ◽  
Hendrik Dietz
Keyword(s):  
Single Molecule ◽  
Bimolecular Reaction ◽  
Fluorescent Reporter ◽  
Single Molecule Level ◽  
Cation Concentration ◽  
Binding Partners ◽  
Equilibrium Transition ◽  
Dna Strand ◽  
Dynamics Simulations ◽  
Actual Target

We describe a tethered multifluorophore motion assay based on DNA origami for revealing bimolecular reaction kinetics on the single-molecule level. Molecular binding partners may be placed at user-defined positions and in user-defined stoichiometry; and binding states are read out by tracking the motion of quickly diffusing fluorescent reporter units. Multiple dyes per reporter unit enable singe-particle observation for more than 1 hour. We applied the system to study in equilibrium reversible hybridization and dissociation of complementary DNA single strands as a function of tether length, cation concentration, and sequence. We observed up to hundreds of hybridization and dissociation events per single reactant pair and could produce cumulative statistics with tens of thousands of binding and unbinding events. Because the binding partners per particle do not exchange, we could also detect subtle heterogeneity from molecule to molecule, which enabled separating data reflecting the actual target strand pair binding kinetics from falsifying influences stemming from chemically truncated oligonucleotides. Our data reflected that mainly DNA strand hybridization, but not strand dissociation, is affected by cation concentration, in agreement with previous results from different assays. We studied 8-bp-long DNA duplexes with virtually identical thermodynamic stability, but different sequences, and observed strongly differing hybridization kinetics. Complementary full-atom molecular-dynamics simulations indicated two opposing sequence-dependent phenomena: helical templating in purine-rich single strands and secondary structures. These two effects can increase or decrease, respectively, the fraction of strand collisions leading to successful nucleation events for duplex formation.

scholarly journals Resolving Active Ion Transport at the Single Molecule Level for the First Time

2016 ◽  
Vol 110 (3) ◽  
pp. 179a
Author(s):  
Salome Veshaguri ◽  
Sune M. Christensen ◽  
Gerdi C. Kemmer ◽  
Mads P. Møller ◽  
Garima Ghale ◽  
...  
Keyword(s):  
Ion Transport ◽  
Single Molecule ◽  
Single Molecule Level ◽  
Active Ion Transport ◽  
First Time

scholarly journals Visualization of the dynamics of fibrin clot growth 1 molecule at a time by total internal reflection fluorescence microscopy

Blood ◽  
2013 ◽  
Vol 121 (8) ◽  
pp. 1455-1458 ◽  
Author(s):  
Alina Hategan ◽  
Kathryn C. Gersh ◽  
Daniel Safer ◽  
John W. Weisel
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Fibrin Clot ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Fibrin Polymerization ◽  
Single Molecule Level ◽  
Key Points ◽  
First Time

Key PointsFibrin polymerization was observed for the first time at the single-molecule level by total internal reflection fluorescence microscopy. Live observation of fibrin polymerization with a single-molecule fluorescence intensity calibration revealed the real-time growth kinetics.

Ultra-rapid and highly efficient enrichment of organic pollutants via magnetic nanoparticles/mesoporous nanosponge compounds for ultrasensitive nanosensors

2021 ◽  
Author(s):  
Lingling Zhang ◽  
Rui Hao ◽  
Hongjun You ◽  
Hu Nan ◽  
Yanzhu Dai ◽  
...  
Keyword(s):  
Magnetic Nanoparticles ◽  
Organic Pollutants ◽  
Single Molecule ◽  
Limit Of Detection ◽  
Organic Micropollutants ◽  
Single Molecule Level ◽  
Fast Detection ◽  
Enrichment Protocol ◽  
Ultrahigh Sensitivity ◽  
Enrichment Strategy

Abstract Developing advanced sensing and detection technologies to effectively monitor organic micropollutants in water is under urgent demand in both scientific and industrial communities. Currently, owing to the ultrahigh sensitivity on the single-molecule level with highly informative spectra characteristics, SERS technique is regarded as the most direct and effective detection technique. However, some weakly adsorbed molecules, such as most of persistent organic pollutants, cannot exhibit strong SERS signals, which is a long-standing key challenge that has not been solved. Here, we show an enrichment-typed sensing strategy based on a powerful porous composite material, call mesoporous nanosponge. The nanosponge consists of magnetic nanoparticles immobilized porous β-cyclodextrin polymers, demonstrating remarkable capability of effective and fast removal of organic micropollutants, e.g. ~90% removal efficiency within ~1 min. With the anchoring of magnetic nanoparticles, the current new polymer adsorbent can be easily recycled from water and re-dispersed in ethanol so that the target molecules in the cavity of adsorbent is concentrated, with an enrichment factor up to ~103. By means of the current enrichment strategy, the limit of detection (LOD) of the typical organic pollutants can be significantly improved, i.e. increasing 2~3 orders of magnitude, compared with the detection without molecule enrichment protocol. Consequently, the current enrichment strategy is proved to be applicable in a variety of fields for portable and fast detection, such as Raman and fluorescent.

scholarly journals Polymerization of fibrin: specificity, strength, and stability of knob-hole interactions studied at the single-molecule level

Blood ◽  
2005 ◽  
Vol 106 (9) ◽  
pp. 2944-2951 ◽  
Author(s):  
Rustem I. Litvinov ◽  
Oleg V. Gorkun ◽  
Scott F. Owen ◽  
Henry Shuman ◽  
John W. Weisel
Keyword(s):  
Single Molecule ◽  
Specific Inhibitor ◽  
Strong Interactions ◽  
Laser Tweezers ◽  
Fibrin Polymerization ◽  
Binding Strength ◽  
Single Molecule Level ◽  
Peptide Mimic ◽  
Highly Sensitive ◽  
First Time

Abstract Using laser tweezers, we measured for the first time the forces of individual knob-into-hole interactions underlying fibrin polymerization. Exposure of A-knobs in desA-fibrin or its fragment from the central part of the molecule (N-terminal disulphide knot, NDSK) resulted in strong interactions with fibrinogen or fragment D (containing only a- and b-holes), producing a binding strength of approximately 125 to 130 pN. The interactions were not present in the absence of either knobs or holes and were abrogated by a specific inhibitor of fibrin polymerization, a peptide mimic of the A-knob (GPRPam). Exposure of both the A- and B-knobs in desAB-fibrin or desAB-NDSK did not change the rupture force spectra compared with the desA molecules, and their interactions with fibrinogen remained highly sensitive to GPRPam but not to GHRPam (B-knob), suggesting that neither A:b nor B:b nor B:a contacts contributed significantly to binding strength in addition to A:a contacts. The A:a interactions had a relatively small zero-force off-rate of approximately 10–4 s–1 and tight knob-to-hole contacts characterized by a transition state distance of approximately 0.3 nm. The results demonstrate that the knob-hole binding during thrombin-induced fibrin polymerization is driven by strong, stable, and highly specific A:a bonding, whereas A:b, B:b, or B:a interactions were not detected.

Probing molecular properties and the role of the environment at the single-molecule level

2006 ◽  
Vol 78 (12) ◽  
pp. 2261-2266 ◽  
Author(s):  
J. Hofkens ◽  
T. D. M. Bell ◽  
A. Stefan ◽  
E. Fron ◽  
K. Müllen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Local Environment ◽  
Coupled System ◽  
Strongly Coupled ◽  
Single Molecule Level ◽  
Weakly Coupled ◽  
Weakly Coupled System ◽  
Donor Acceptor ◽  
First Time

Evidence for intramolecular photoinduced electron transfer (ET) in synthetic systems consisting of a triphenylamine-perylenediimide donor-acceptor dendrimer or a triphenylamine-peryleneimide dendrimer at the ensemble and single-molecule (SM) level is presented. Moreover, for the first time a direct observation of the forward as well as the backward ET step is made in a single emitting entity. Fluctuations in the values of the rate constants for forward and backward ET were observed, induced by the local environment as well as by conformational changes of the dendrimer itself. The results obtained in a weakly coupled system can also be extended to a strongly coupled donor-acceptor system based on peryleneimide and penta-phenylene.

Application of Novel Low Current OBIRCH Amplifier and Nanoprobing to Identify Subtle Leakages in Advanced Node Technologies

2018 ◽  
Author(s):  
Satish Kodali ◽  
Liangshan Chen ◽  
Yuting Wei ◽  
Tanya Schaeffer ◽  
Chong Khiam Oh
Keyword(s):  
Signal To Noise Ratio ◽  
Semiconductor Industry ◽  
Fault Isolation ◽  
Ring Oscillator ◽  
High Signal ◽  
The Novel ◽  
Resistance Change ◽  
Three Samples ◽  
Relative Threshold ◽  
First Time

Abstract Optical beam induced resistance change (OBIRCH) is a very well-adapted technique for static fault isolation in the semiconductor industry. Novel low current OBIRCH amplifier is used to facilitate safe test condition requirements for advanced nodes. This paper shows the differences between the earlier and novel generation OBIRCH amplifiers. Ring oscillator high standby leakage samples are analyzed using the novel generation amplifier. High signal to noise ratio at applied low bias and current levels on device under test are shown on various samples. Further, a metric to demonstrate the SNR to device performance is also discussed. OBIRCH analysis is performed on all the three samples for nanoprobing of, and physical characterization on, the leakage. The resulting spots were calibrated and classified. It is noted that the calibration metric can be successfully used for the first time to estimate the relative threshold voltage of individual transistors in advanced process nodes.

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