The interaction affinity between vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4) analyzed by quantitative FRET.

Very late antigen-4 (VLA-4), a member of integrin superfamily, interacts with its major counter ligand vascular cell adhesion molecule-1 (VCAM-1) and plays an important role in leukocyte adhesion to vascular endothelium and immunological synapse formation. However, irregular expressions of these proteins may also lead to several autoimmune diseases and metastasis cancer. Thus, quantifying the interaction affinity of the VCAM-1/VLA-4 interaction is of fundamental importance in further understanding the nature of this interaction and drug discovery. In this study, we report an 'in solution' steady state organic fluorophore based quantitative fluorescence resonance energy transfer (FRET) assay to quantify this interaction in terms of the dissociation constant (Kd). We have used, in our FRET assay, the Alexa Fluor 488-VLA-4 conjugate as the donor, and Alexa Fluor 546-VCAM-1 as the acceptor. From the FRET signal analysis, Kd of this interaction was determined to be 41.82 ± 2.36 nM. To further confirm our estimation, we have employed surface plasmon resonance (SPR) technique to obtain Kd = 39.60 ± 1.78 nM, which is in good agreement with the result obtained by FRET. This is the first reported work which applies organic fluorophore based 'in solution' simple quantitative FRET assay to obtain the dissociation constant of the VCAM-1/VLA-4 interaction, and is also the first quantification of this interaction. Moreover, the value of Kd can serve as an indicator of abnormal protein-protein interactions; hence, this assay can potentially be further developed into a drug screening platform of VLA-4/VCAM-1 as well as other protein-ligand interactions

Amorphous Carbon Films with Embedded Well-Dispersed Nanodiamonds: Plasmon-Enhanced Analysis and Possible Antimicrobial Applications

An amorphous carbon film with embedded detonation nanodiamond (DND) particles (a-C:ND) was produced by magnetron sputtering of nanodiamond powder. An Ag film was deposited on the carbon structure by radiofrequency magnetron sputtering. The silver film was irradiated with a 150 eV Ar+ to form plasmonic-active nanoparticles (NP) on the surface of the a-C:ND. The structure of the obtained a-C:ND and a-C:ND/Ag structures were studied by scanning and transmission electron microscopy, electron energy-loss spectroscopy, UV–Visible absorption spectroscopy, Raman spectroscopy, and fluorescence lifetime imaging at two-photon excitation. The analysis revealed 76% of sp3-carbon and a good dispersion of diamond nanoparticles in the a-C. Surface-enhanced Raman scattering (SERS) was applied to investigate the a-C:ND/Ag structure, allowing for the observation of SERS from the sp2-carbon species and the absence of significant a-C:ND damage after Ar+ irradiation of the Ag overlayer. A plasmonic-metal-enhanced luminescence was observed at one- and two-photon excitations, revealing a two- to five-fold intensity increase. The activity of the used DNDs was tested using the agar diffusion method and observed against the bacteria of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli and the fungi of Aspergillus niger, Aspergillus fumigatus, and the yeast of Candida albicans, showing DND activity against all the test strains of fungi

Combined use of optical tweezers and scanning electron microscopy to reveal influence of nanoparticles on red blood cells interactions

Abstract As a promising drug delivery system, itself or coupled with red blood cells (RBC), nanoparticles (NP) should be studied in frames of their interaction at the cellular level. Experiments were performed on RBC in autologous blood plasma incubated with different NP — TiO₂, ZnO, nanodiamonds and polymeric nanocapsules. RBC aggregates formation in RBC suspension was observed with conventional microscopy, while quantitative interaction force measurements between individual RBC was assessed with optical tweezers. Scanning electron microscopy (SEM) imaging demonstrated NP localization and RBC membrane modifications upon binding with NP. Among tested NP, nanodiamonds caused increasing the size of aggregates in RBC suspensions, RBC interaction force increase and strong membrane surface modifications, comparing to other tested NP and control sample. Nanocapsules do not cause any adverse effects on RBC properties, confirming biocompatibility and applicability for drug delivery purposes. Optical tweezers combined with SEM imaging serves as fast informative assessment of NP effects on RBC

The FRET assay.

<p>(a) A schematic illustration of FRET with AF488-VLA-4 as the donor, and AF546-VCAM-1 as the acceptor conjugates. Upon excitation at 470 nm, two emission peaks, one at 520 nm, and the other at 570 nm were observed; the former originates from the free (unbounded) AF488-VLA-4, while the latter is due to FRET from the binding of the two protein conjugates. Proper amount of Mg²⁺ was added to the FRET mixture to induce conformational change of VLA-4 to enhance the binding affinity of VLA-4 and VCAM-1. (b) Normalized absorbance and emission spectra of AF488 and AF546 in PBS, pH 7.4.</p

Amorphous Carbon Films with Embedded Well-Dispersed Nanodiamonds: Plasmon-Enhanced Analysis and Possible Antimicrobial Applications

An amorphous carbon film with embedded detonation nanodiamond (DND) particles (a-C:ND) was produced by magnetron sputtering of nanodiamond powder. An Ag film was deposited on the carbon structure by radiofrequency magnetron sputtering. The silver film was irradiated with a 150 eV Ar+ to form plasmonic-active nanoparticles (NP) on the surface of the a-C:ND. The structure of the obtained a-C:ND and a-C:ND/Ag structures were studied by scanning and transmission electron microscopy, electron energy-loss spectroscopy, UV&ndash;Visible absorption spectroscopy, Raman spectroscopy, and fluorescence lifetime imaging at two-photon excitation. The analysis revealed 76% of sp3-carbon and a good dispersion of diamond nanoparticles in the a-C. Surface-enhanced Raman scattering (SERS) was applied to investigate the a-C:ND/Ag structure, allowing for the observation of SERS from the sp2-carbon species and the absence of significant a-C:ND damage after Ar+ irradiation of the Ag overlayer. A plasmonic-metal-enhanced luminescence was observed at one- and two-photon excitations, revealing a two- to five-fold intensity increase. The activity of the used DNDs was tested using the agar diffusion method and observed against the bacteria of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli and the fungi of Aspergillus niger, Aspergillus fumigatus, and the yeast of Candida albicans, showing DND activity against all the test strains of fungi

Determination of Kd through quantitative FRET and SPR.

<p>(a) Curve fitting of the experimental data representing the absolute FRET emission signals (FRET_emission) with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121399#pone.0121399.e003" target="_blank">Equation (3)</a>. Each experiment was repeated three times under identical condition, and the error bar indicates the standard deviation; the results are shown as arithmetic mean ± standard deviation. The maximum FRET emission signal (maxFRET_emission) and the corresponding equilibrium dissociation constant (K_d) of the VLA-4/VCAM-1 interaction were determined to be 3184 RFU and 41.82 ± 2.36 nM, respectively. (b) Sensorgrams from the SPR sensor chip in BIAcore indicating the interaction of VCAM-1 (immobilized on the surface of the chip; details can be found in the text) with different concentrations (100, 150, 400, 450, to 500 nM) of VLA-4. The equilibrium dissociation constant (K_d) of the VLA-4/VCAM-1 interaction was determined to be 39.60 ± 1.78 nM.</p

Determining the “μ” and “η”.

<p>(a) Fluorescence emission spectrum of AF488-VLA-4 (350 nM) alone when excited at 470 nm. The ratio constant “μ”, defined as the ratio of the emission signal at 570 nm to that at 520 nm of the AF488-VLA-4 emission spectrum was determined to be 0.128 ± 0.031. (b) Fluorescence emission spectra of AF546-VCAM-1 (850 nM) alone when excited at 470 and 520 nm. The ratio constant “η”, defined as the ratio of emission signal at 570 nm upon excitation at 470 nm to that at 570 nm when excited at 520 nm, was determined to be 0.143 ± 0.058. All the experiments were done in triplicate. The results are shown as arithmetic mean ± standard deviation.</p