scholarly journals Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays

2016 ◽  
Vol 11 (3) ◽  
pp. 031001 ◽  
Author(s):  
Norbert Orgovan ◽  
Rita Ungai-Salánki ◽  
Szilvia Lukácsi ◽  
Noémi Sándor ◽  
Zsuzsa Bajtay ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Cell Adhesion ◽  
Optical Biosensor ◽  
Label Free ◽  
End Point ◽  
Dynamic Cell ◽  
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.

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 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 Glycocalyx regulates the strength and kinetics of cancer cell adhesion revealed by biophysical models based on high resolution label-free optical data

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Nicolett Kanyo ◽  
Kinga Dora Kovacs ◽  
Andras Saftics ◽  
Inna Szekacs ◽  
Beatrix Peter ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
High Resolution ◽  
Cancer Cell ◽  
Kinetic Data ◽  
Dermatan Sulfate ◽  
Cellular Adhesion ◽  
Label Free ◽  
Cancer Cell Adhesion ◽  
Kinetics Of ◽  
Adhesion Process

AbstractThe glycocalyx is thought to perform a potent, but not yet defined function in cellular adhesion and signaling. Since 95% of cancer cells have altered glycocalyx structure, this role can be especially important in cancer development and metastasis. The glycocalyx layer of cancer cells directly influences cancer progression, involving the complicated kinetic process of cellular adhesion at various levels. In the present work, we investigated the effect of enzymatic digestion of specific glycocalyx components on cancer cell adhesion to RGD (arginine–glycine–aspartic acid) peptide motif displaying surfaces. High resolution kinetic data of cell adhesion was recorded by the surface sensitive label-free resonant waveguide grating (RWG) biosensor, supported by fluorescent staining of the cells and cell surface charge measurements. We found that intense removal of chondroitin sulfate (CS) and dermatan sulfate chains by chondroitinase ABC reduced the speed and decreased the strength of adhesion of HeLa cells. In contrast, mild digestion of glycocalyx resulted in faster and stronger adhesion. Control experiments on a healthy and another cancer cell line were also conducted, and the discrepancies were analysed. We developed a biophysical model which was fitted to the kinetic data of HeLa cells. Our analysis suggests that the rate of integrin receptor transport to the adhesion zone and integrin-RGD binding is strongly influenced by the presence of glycocalyx components, but the integrin-RGD dissociation is not. Moreover, based on the kinetic data we calculated the dependence of the dissociation constant of integrin-RGD binding on the enzyme concentration. We also determined the dissociation constant using a 2D receptor binding model based on saturation level static data recorded at surfaces with tuned RGD densities. We analyzed the discrepancies of the kinetic and static dissociation constants, further illuminating the role of cancer cell glycocalyx during the adhesion process. Altogether, our experimental results and modelling demonstrated that the chondroitin sulfate and dermatan sulfate chains of glycocalyx have an important regulatory function during the cellular adhesion process, mainly controlling the kinetics of integrin transport and integrin assembly into mature adhesion sites. Our results potentially open the way for novel type of cancer treatments affecting these regulatory mechanisms of cellular glycocalyx.

scholarly journals Non-Langmuir kinetics of DNA surface hybridization

2020 ◽  
Author(s):  
L. Vanjur ◽  
T. Carzaniga ◽  
L. Casiraghi ◽  
M. Chiari ◽  
G. Zanchetta ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Steric Hindrance ◽  
Optical Biosensor ◽  
Bulk Solution ◽  
Electrostatic Repulsion ◽  
Surface Immobilization ◽  
Label Free ◽  
Fundamental Biological Process ◽  
Effective Models ◽  
Kinetics Of

AbstractHybridization of complementary single strands of DNA represents a very effective natural molecular recognition process widely exploited for diagnostic, biotechnology and nanotechnology applications. A common approach relies on the immobilization on a surface of single stranded DNA probes that bind complementary targets in solution. However, despite the deep knowledge on DNA interactions in bulk solution, the modelling of the same interactions on a surface are still challenging and perceived as strongly system-dependent. Here we show that a two dimensional analysis of the kinetics of hybridization, performed at different target concentration and probe surface density by a label-free optical biosensor, reveals peculiar features inconsistent with an ideal Langmuir-like behaviour. We propose a simple non-Langmuir kinetic model accounting for an enhanced electrostatic repulsion originating from the surface immobilization of nucleic acids and for steric hindrance close to full hybridization of the surface probes. The analysis of the kinetic data by the model enables to quantify the repulsive potential at the surface, as well as to retrieve the kinetic parameters of isolated probes. We show that the strength and the kinetics of hybridization at large probe density can be improved by a 3D immobilization strategy of probe strands with a double stranded linker.Statement of SignificanceHybridization of nucleic acids strands with complementary sequences is a fundamental biological process and is also widely exploited for diagnostic purposes. Despite the availability of effective models for the equilibrium strength of freely diffusing strands, a general predictive model for surface hybridization is still missing. Moreover, the kinetics of hybridization is not fully understood neither in solution nor on a surface. In this work we show that the analysis of the kinetics of hybridization on a surface reveals and enables to quantify two main additional contributions: electrostatic repulsion and steric hindrance. These are general effects expected to occur not only on a surface but in any condition with large density of nucleic acids, comparable to that of the cellular nucleus.

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

Label-free optical biosensor based on a dual-core microstructured polymer optical fiber

Optik ◽  
2015 ◽  
Vol 126 (21) ◽  
pp. 2930-2933 ◽  
Author(s):  
Nai-Fei Ren ◽  
Bing Sun ◽  
Ming-Yang Chen
Keyword(s):  
Optical Fiber ◽  
Optical Biosensor ◽  
Label Free ◽  
Polymer Optical Fiber ◽  
Dual Core
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