A. Tsargorodskaya

Detection of β-amyloid peptide (1–16) and amyloid precursor protein (APP770) using spectroscopic ellipsometry and QCM techniques: A step forward towards Alzheimers disease diagnostics

A highly sensitive method of spectroscopic ellipsometry in total internal reflection mode (TIRE) was exploited for detecting β-amyloid peptide (Aβ(1-16)) in the direct immune reaction with monoclonal DE2 antibodies (raised against Aβ(1-16)) electrostatically immobilised on the surface of gold. A rapid detection of Aβ(1-16) in a wide range of concentrations from 5 μg/ml down to 0.05 ng/ml was achieved using a cost-effective and label-free direct immunoassay format. TIRE dynamic spectral measurements proved that the immune reaction between DE2 monoclonal antibodies and Aβ(1-16) is highly specific with the affinity constant K(D)=1.46×10(-8) mol/l. The same DE2 antibodies were utilised for detection of amyloid precursor protein APP(770), a larger protein containing Aβ(1-16) domain, using the quartz crystal microbalance (QCM) measurements in liquid. A combination of QCM and TIRE kinetics results allowed the evaluation of the originally unknown concentration of APP(770) in complete medium solution containing other proteins, salts, and amino acids.

Ellipsometric study of the adsorption of bovine serum albumin into porous silicon

The method of spectroscopic ellipsometry has been applied to study in situ the adsorption of bovine serum albumin (BSA). The porosity and amount of adsorbed BSA were determined by fitting the ellipsometric data to the Bruggeman effective medium approximation model. The presence of intermediate adsorbed layers of polyelectrolytes was found to increase protein adsorption.

Hybridization of Genomic DNA Adsorbed Electrostatically onto Cationic Surfaces

Our previous study revealed an intriguing phenomenon of partial hybridization of two single strands of genomic DNA, with one of them being electrostatically adsorbed on a solid surface. Although the effect was confirmed with different methods and even recommended for a crude DNA analysis, the exact mechanism of hybridization was not clear. This work presents the results of more detailed study of adsorption and hybridization of two genomic DNA, of salmon and herring, using the experimental techniques of total internal reflection ellipsometry (TIRE), ATR FTIR spectroscopy, and AFM. The in situ TIRE study of the hybridization kinetics allowed the evaluation of the association constant. It appeared to be in the range of 10(5) mol(-1) L for binding complementary ss-DNA in comparison to 10(4) mol(-1) L for binding of noncomplementary ss-DNA. FTIR study directly confirmed the effect of partial binding of complementary ss-DNA by monitoring the 1650 and 1690 cm(-1) spectral bands. AFM showed the transformation from clearly resolved images of separate chains of ss-DNA molecules adsorbed on the surface of mica to an inhomogeneous layer of tangled and overlapping DNA molecules following binding of another complementary ss-DNA.

Study of receptor-chaperone interactions using the optical technique of spectroscopic ellipsometry

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.

Registration of low molecular weight environmental toxins with total internal reflection ellipsometry

A very sensitive optical method of total internal reflection ellipsometry (TIRE) in conjunction with an immune assay approach were exploited for the registration of low molecular weight environmental toxins, such as simazine, atrazine and T2 mycotoxin in a wide range of concentrations down to 0.1 ng/ml. QCM impedance measurements provided additional information on the mechanism of T2 binding.

Analysis of Protein Interactions at Native Chloroplast Membranes by Ellipsometry

Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE). We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.