scholarly journals Validation of the Slow Off‐Kinetics of Sirtuin‐Rearranging Ligands (SirReals) by Means of Label‐Free Electrically Switchable Nanolever Technology

ChemBioChem ◽  
2020 ◽  
Vol 21 (8) ◽  
pp. 1161-1166 ◽  
Author(s):  
Matthias Schiedel ◽  
Herwin Daub ◽  
Aymelt Itzen ◽  
Manfred Jung
Keyword(s):  
Label Free ◽  
Kinetics Of

scholarly journals High-Resolution Adhesion Kinetics of EGCG-Exposed Tumor Cells on Biomimetic Interfaces: Comparative Monitoring of Cell Viability Using Label-Free Biosensor and Classic End-Point Assays

ACS Omega ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. 3882-3891 ◽  
Author(s):  
Beatrix Peter ◽  
Rita Ungai-Salanki ◽  
Bálint Szabó ◽  
Agoston G. Nagy ◽  
Inna Szekacs ◽  
...  
Keyword(s):  
High Resolution ◽  
Cell Viability ◽  
Tumor Cells ◽  
Label Free ◽  
End Point ◽  
Biomimetic Interfaces ◽  
Kinetics Of

scholarly journals Single-cell adhesion force kinetics of cell populations from combined label-free optical biosensor and robotic fluidic force microscopy

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Milan Sztilkovics ◽  
Tamas Gerecsei ◽  
Beatrix Peter ◽  
Andras Saftics ◽  
Sandor Kurunczi ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Single Cell ◽  
Temporal Resolution ◽  
Adhesion Force ◽  
Optical Biosensor ◽  
Cell Populations ◽  
Label Free ◽  
Kinetic Evaluation ◽  
Force Microscopy ◽  
Kinetics Of

AbstractSingle-cell adhesion force plays a crucial role in biological sciences, however its in-depth investigation is hindered by the extremely low throughput and the lack of temporal resolution of present techniques. While atomic force microcopy (AFM) based methods are capable of directly measuring the detachment force values between individual cells and a substrate, their throughput is limited to few cells per day, and cannot provide the kinetic evaluation of the adhesion force over the timescale of several hours. In this study a high spatial and temporal resolution resonant waveguide grating based label-free optical biosensor was combined with robotic fluidic force microscopy to monitor the adhesion of living cancer cells. In contrast to traditional fluidic force microscopy methods with a manipulation range in the order of 300–400 micrometers, the robotic device employed here can address single cells over mm-cm scale areas. This feature significantly increased measurement throughput, and opened the way to combine the technology with the employed microplate-based, large area biosensor. After calibrating the biosensor signals with the direct force measuring technology on 30 individual cells, the kinetic evaluation of the adhesion force and energy of large cell populations was performed for the first time. We concluded that the distribution of the single-cell adhesion force and energy can be fitted by log-normal functions as cells are spreading on the surface and revealed the dynamic changes in these distributions. The present methodology opens the way for the quantitative assessment of the kinetics of single-cell adhesion force and energy with an unprecedented throughput and time resolution, in a completely non-invasive manner.

Label-free detection of early oligomerization of α-synuclein and its mutants A30P/E46K through solid-state nanopores

Nanoscale ◽  
2019 ◽  
Vol 11 (13) ◽  
pp. 6480-6488 ◽  
Author(s):  
Xiaoqing Li ◽  
Xin Tong ◽  
Wenlong Lu ◽  
Dapeng Yu ◽  
Jiajie Diao ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Solid State ◽  
Time Dependent ◽  
Label Free ◽  
Label Free Detection ◽  
Kinetics Of

Time-dependent kinetics of early oligomerization of Parkinson's disease-related α-synuclein and its mutants A30P/E46K have been studied through solid-state nanopores.

scholarly journals Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hajnalka Jankovics ◽  
Boglarka Kovacs ◽  
Andras Saftics ◽  
Tamas Gerecsei ◽  
Éva Tóth ◽  
...  
Keyword(s):  
Binding Sites ◽  
Kinetic Data ◽  
Specific Binding ◽  
Genetically Engineered ◽  
Binding Kinetics ◽  
Titration Calorimetry ◽  
Affinity Binding ◽  
Label Free ◽  
Low Protein ◽  
Kinetics Of

AbstractReliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the binding kinetics of Ni(II) ions to genetically modified flagellin layers, we demonstrate that: (1) Grating-Coupled Interferometry (GCI) is well suited to resolve the binding of ions, even at very low protein immobilization levels; (2) it supplies high quality kinetic data from which the number and strength of available binding sites can be determined, and (3) the rate constants of the binding events can also be obtained with high accuracy. Experiments were performed using a flagellin variant incorporating the C-terminal domain of the nickel-responsive transcription factor NikR. GCI results were compared to affinity data from titration calorimetry. We found that besides the low-affinity binding sites characterized by a micromolar dissociation constant (Kd), tetrameric FliC-NikRC molecules possess high-affinity binding sites with Kd values in the nanomolar range. GCI enabled us to obtain real-time kinetic data for the specific binding of an analyte with molar mass as low as 59 Da, even at signals lower than 1 pg/mm2.

scholarly journals Design and Optimization of a Rapid, Multiplex miRNA Assay without Washing Steps

Proceedings ◽  
2020 ◽  
Vol 60 (1) ◽  
pp. 32
Author(s):  
Giuliano Zanchetta ◽  
Thomas Carzaniga ◽  
Luka Vanjur ◽  
Luca Casiraghi ◽  
Giovanni Tagliabue ◽  
...  
Keyword(s):  
Early Stage ◽  
Optical Biosensor ◽  
Label Free ◽  
Time Intervals ◽  
One Pot ◽  
Design And Optimization ◽  
Individual Interaction ◽  
Equilibrium And Kinetics ◽  
Kinetics Of

MicroRNAs are widely studied as circulating biomarkers for early stage diagnosis of several diseases, but the procedures for their detection and quantification are currently complex and time consuming. We demonstrate a rapid, multiplex, one-pot detection method based on two-step amplification of the signal measured by a recent label-free optical biosensor, Reflective Phantom Interface (RPI). The specific capture with surface DNA probes is combined with mass amplification by an antibody targeting DNA–RNA hybrids and polyclonal secondary antibody, all performed without washing steps. Through this method, we achieved linear, sub-pM quantification of different miRNAs in 1.5 h. The RPI enabled the characterization of equilibrium and kinetics of each individual interaction involved in this multi-step process, which allowed us to model and optimize the relative concentrations and the time intervals of the assay.

scholarly journals Validation of Slow Off-Kinetics of Sirtuin Rearranging Ligands (SirReals) by Means of the Label-Free Electrically Switchable Nanolever Technology

2019 ◽  
Author(s):  
Matthias Schiedel ◽  
Herwin Daub ◽  
Aymelt Itzen ◽  
Manfred Jung
Keyword(s):  
Residence Time ◽  
Dissociation Rate ◽  
The Novel ◽  
Label Free ◽  
X Ray ◽  
Cofactor Binding ◽  
Lysine Deacetylase ◽  
Slow Dissociation ◽  
Kinetics Of ◽  
Binding Loop

Recently, we have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase Sirt2. In previous studies, using a biotinylated SirReal analogue in combination with biolayer interferometry, we observed a slow dissociation rate of the inhibitor-enzyme complex, which had been postulated to be the key to the high affinity and selectivity of SirReals. However, for the attachment of biotin to the SirReal core, we introduced a triazole as a linking moiety, which was shown by X-ray co-crystallography to interact with Arg97 of the cofactor binding loop. This study now is directed to answer the question, whether the observed long residence time of the SirReals is induced mainly by triazole incorporation or is an inherent characteristic of the SirReal inhibitor core. Therefore, we used the novel label-free switchSENSE® technology, based on electrically switchable DNA nanolevers, to validate that the long residence time of the SirReals is caused by the core scaffold.<br>

scholarly journals Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

2012 ◽  
Vol 4 (10) ◽  
pp. 846-853 ◽  
Author(s):  
Wei Wang ◽  
Yunze Yang ◽  
Shaopeng Wang ◽  
Vinay J. Nagaraj ◽  
Qiang Liu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Living Cells ◽  
Binding Kinetics ◽  
Label Free ◽  
Single Living Cells ◽  
Kinetics Of ◽  
Single Living

Nanomechanical Microcantilevers for In-Vitro Biomolecular Detection

2007 ◽  
Author(s):  
Kilho Eom ◽  
Tae Yun Kwon ◽  
Jinsung Park ◽  
Sungsoo Na ◽  
Dae Sung Yoon ◽  
...  
Keyword(s):  
Real Time ◽  
Biological Molecules ◽  
Label Free ◽  
Biomolecular Detection ◽  
Label Free Detection ◽  
Detection Of Biomolecules ◽  
Kinetics Of ◽  
Insight Into

Nanomechanical microcantilevers have enabled the sensitive label-free detection of chemical and/or biological molecules. In recent years, resonating microcantilevers have achieved the unprecedented sensitivity in detecting molecules. In this article, we review our current works on the label-free detection of biomolecules based on resonating microcantilevers. Our piezoelectric thick film microcantilevers exhibit the relatively high quality factor in a viscous liquid, indicating the potential of our cantilever to in situ biosensor applications for real-time detection of biomolecular interactions. It is shown that our microcantilevers allow the noise-free real-time monitoring of biomolecular recognitions, providing the insight into kinetics of biomolecular recognitions.

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