"Vanishing mass" in the Sauerbrey world: quartz crystal microbalance study of self-assembled monolayers based on a tripod-branched structure with tuneable molecular flexibility.

Complex loadings that appear on a surface with flexible spatial organisation can reveal anti-Sauerbrey behaviour due to their variable interfacial architecture even for an ultrathin monomolecular sensitive layer. The presented results demonstrate that compounds with branched and flexible structures may be organised in various spatial architectures with different viscoelastic characteristics. They act not only as a mass loading (Sauerbrey behaviour) but also as a viscous damping (anti-Sauerbrey one) of the QCM resonator at the same time. The concept is illustrated by SAM formed on a QCM silver electrode surface with a tripod-like hydroxamic thioligand (S3HX). The obtained data indicate that the interfacial layer was a combination of two S3HX surface structures - namely, the ordered α-(alpha)- and the disordered σ-(sigma)-conformation of SAM. The sigma-conformation is likely to belong to Sauerbrey-like materials with a negative frequency shift: such a system is sufficiently rigid because of multiple interactions and spatial restrictions due to the so-called "tangle of tendrils". The alfa-conformation with a labile structure and highly ordered flexible spatial organisation is likely to be predominant in anti-Sauerbrey-like samples. Sigma- and alpha-based interfacial ensembles demonstrate different behaviour under gaseous analyte exposure. The possibility of anti-Sauerbrey-like behaviour opens the way for the formation of a unique (oppositely shifted) signal in analytical applications, especially ones that require a false-free detection of highly dangerous xenobiotics.

In vivo characterization of protein uptake by yeast cell envelope: single cell AFM imaging and µ-tip-enhanced Raman scattering study.

Direct detection of biological transformations of single living cells in vivo has been performed by the advanced combination of local topographic imaging by Atomic Force Microscopy (AFM) and label-free sub-surface chemical characterization using new µ-Tip-Enhanced Raman Spectroscopy (µ-TERS). The enhancing mechanism for µ-TERS tips with micrometre range radius differs significantly to that of the conventional tapered structures terminated by a sharp apex and conditioned by the effects of propagating instead of localizing surface plasmon resonance phenomena. Sub-wavelength light confinement in the form of a nonradiative evanescent wave near the tip surface with penetration depth in the sub-micrometre range opens the way for monitoring of subsurface processes near or within the cell wall, inaccessible by other methods. The efficiency of the approach has been demonstrated by the analysis of the cell envelope of genetically modified (by glucose dehydrogenase (GDH) gene bearing Kluyveromyces lactis toxin signal sequence) yeast cells enriched by GDH protein. The presence of trans-membrane fragments in GDH together with the tendency to form active dimers and tetramers causes the accumulation of the proteins within the periplasmic space. These results demonstrate that the advanced combination of AFM imaging and subsurface chemical characterization by the novel µ-TERS technique provides a new analytical tool for the investigation of single living cells in vivo.

A simple SPR-based method for the quantification of the effect of potential virus inhibitors.

Here, we describe a highly sensitive method that allows for the correct quantification of inhibition effect with a higher degree of accuracy directly at the molecular level. The protocol involves two stages, namely serological virus titration in comparison with the same procedure for virus-effector mixture. Owing to the robustness of the analysis this assay can be performed on crude cellular and plant extracts, and therefore it may be suitable for the routine analysis of clinical samples, or in the field. The efficiency of the approach to the quantification of the inhibition effect of polysaccharide glucuronoxylomannan (GXM) on the infection efficiency of the tobacco mosaic virus (TMV) was investigated using advanced serological approaches based on label-free surface plasmon resonance technique. It was shown that GXM drastically decreases the efficiency of TMV infection by blocking up to 70% of the virus shell. The obtained results are in conformity with the method of indicator plant infection.

Analysis of protein-protein interactions in a complex environment: capture of an analyte-receptor complex with standard additions of the receptor (CARSAR) approach.

Traditional methods of analytical chemistry to detect an interaction between certain proteins in multicomponent mixtures (e.g. cell lysates, etc.) have limitations. This is due to difficulties in identification of a specific signal of an analyte (a molecule to be detected) against the background. In the present work, we propose the new analytical protocol for transducer-based sensors with a restricted sensitive area. It uses a combination of analyte-receptor complex precipitation with serial additions of the receptor (CARSAR). To test this new analytical strategy, we used a surface plasmon resonance technique to confirm an interaction between the Epstein-Barr virus-encoded nuclear antigen 6 and the mitochondrial ribosomal protein MRPS18-2.

Graphene-enhanced Raman imaging of TiO2 nanoparticles.

The interaction of anatase titanium dioxide (TiO(2)) nanoparticles with chemical vapour deposited graphene sheets transferred on glass substrates is investigated by using atomic force microscopy, Raman spectroscopy and imaging. Significant electronic interactions between the nanoparticles of TiO(2) and graphene were found. The changes in the graphene Raman peak positions and intensity ratios indicate that charge transfer between graphene and TiO(2) nanoparticles occurred, increasing the Raman signal of the TiO(2) nanoparticles up to five times. The normalized Raman intensity of TiO(2) nanoparticles per their volume increased with the disorder of the graphene structure. The complementary reason for the observed enhancement is that due to the higher density of states in the defect sites of graphene, a higher electron transfer occurs from the graphene to the anatase TiO(2) nanoparticles.

Epstein-Barr virus-encoded EBNA-5 forms trimolecular protein complexes with MDM2 and p53 and inhibits the transactivating function of p53.

We report that MDM2, a negative regulator of p53, can bind to EBNA-5. Using GST pull-down assay, immunoprecipitation, surface plasmon resonance and immunostaining of lymphoblastoid cells, we found that trimolecular complexes are formed between EBNA-5, MDM2 and p53, where MDM2 serves as a bridge. The EBNA-5 binding to MDM2 counteracted destabilizing effect of the latter on the p53. In ubiquitination and degradation assays in vitro, EBNA-5 inhibited p53 polyubiquitination (but not monoubiquitination) in a concentration-dependent manner. This resembles the effect of p14ARF on p53. Moreover, EBNA-5 was found to inhibit the degradation of p53 in vitro. High levels of p53 expression were maintained in LCLs. The binding of EBNA-5 to MDM2 also could impair the functional activity of p53. The p53-dependent genes P21 and VDR were not induced in EBV-infected, in contrast to mitogen-activated cells. This may explain the tolerance of established LCLs to high levels of p53 without undergoing apoptosis.

EBV-encoded EBNA-6 binds and targets MRS18-2 to the nucleus, resulting in the disruption of pRb-E2F1 complexes.

Epstein-Barr virus (EBV), like other DNA tumor viruses, induces an S-phase in the natural host cell, the human B lymphocyte. This is linked with blast transformation. It is believed that the EBV-encoded nuclear antigen 6 (EBNA-6) is involved in the regulation of cell cycle entry. However, the possible mechanism of this regulation is not approached. In our current study, we found that EBNA-6 binds to a MRPS18-2 protein, and targets it to the nucleus. We found that MRPS18-2 binds to both hypo- and hyperphosphorylated forms of Rb protein specifically. This binding targets the small pocket of pRb, which is a site of interaction with E2F1. The MRPS18-2 competes with the binding of E2F1 to pRb, thereby raising the level of free E2F1. Our experimental data suggest that EBNA-6 may play a major role in the entry of EBV infected B cells into the S phase by binding to and raising the level of nuclear MRPS18-2, protein. This would inhibit pRb binding to E2F1 competitively and lift the block preventing S-phase entry.

Study on the spatial architecture of p53, MDM2, and p14ARF containing complexes.

We have developed a surface plasmon resonance (SPR)-based immunocapture approach to study multimeric protein-protein complexes. A composition and spatial architecture of protein complexes that contained GST-tagged p53, p14ARF, and MDM2 was examined by the developed approach. Obtained results verified that the p53 protein possesses two binding sites for MDM2. Ternary complexes containing p14ARF, MDM2, and p53 proteins could only be formed when MDM2 protein functions as a bridging molecule. That was confirmed by immunoprecipitation and immunostaining.

Evanescent-field-induced Raman scattering for bio-friendly fingerprinting at sub-cellular dimension.

Evanescent field induced chemical imaging concept has been realized in analytical platform based on the µ-tip-enhanced Raman scattering spectroscopy (µ-TERS). The technique aimed to minimize thermal decomposition of dried biological sample as the result of huge concentration of optical field near the tip by increasing the size of an aperture-less "excitation source". µ-TERS technique is similar to classical biosensor systems based on propagating surface plasmon resonance phenomenon but with sensitive elements a few micrometers in size that can be targeted to the area of interest. The utility of the concept is exemplified by the analysis of dried single cell envelope of genetically modified Saccharomyces cerevisiae yeast cells, which do not have any heat-removing pathways, by water as in the case of the living cell. Practical excitation conditions effective for µ-TERS Raman observation of single layer dried biological samples without photodamage-related spectral distortion have been determined - the allowable limit is above 30s at 13 µW/µm(2). Finally, potential of µ-TERS spectroscopy as new bio-friendly instrumental platform for chemical fingerprinting and analytical characterization of buried nanoscale features is discussed.

Generation of diversiform gold nanostructures inspired by honey's components: growth mechanism, characterization, and shape separation by the centrifugation-assisted sedimentation.

The green synthesis of irregular-shaped nanomaterials used for various applications in nanoplasmonics, medicine, and biotechnology creates an economical and environmental challenge. We describe the rapid wet-chemical approach to synthesis of stable and water-soluble gold nanostructues at room temperature. In addition to spherical and road-like nanoparticles, gold decahedra and triangular plates were grown using the one-step synthesis process of HAuCl(4) in the presence of honey, in which main components act as reducing (glucose) and stabilizing (fructose) agents; the mechanism of the process is discussed in details. The requirements for anisotropic phase boundaries for generation of polyhedral gold nanocrystals in solutions are highlighted. The synthesis, morphology, and separation procedure of gold nanoparticles are examined using the techniques of optical spectroscopy, transmission electron microscopy, and atomic force microscopy. We demonstrate that centrifugation can be used for efficient separation of nanoparticles with different shapes from a mixture. It was found that while centrifuging, the spheres sediment at the bottom of the tube, segregating from rods that form a deposit on the side wall, whereas polygons remain in the solution.

The effect of low pH on the glycitein-BSA conjugate interaction with specific antiserum: competitive inhibition study using surface plasmon resonance technique.

Competitive inhibition serological assay for detection of the phytoestrogen glycitein (Glyc) was developed using surface plasmon resonance (SPR) technique with protein conjugates and polyclonal antibodies initially designed for the enzyme-linked immunosorbent assays (ELISA). The efficiency of the approach to the quantification of the soy isoflavone glycitein in water was investigated using the competitive reaction of analyte (free Glyc)and immobilized Glyc-BSA-conjugate with polyclonal antibodies. It was shown that the efficiency to detect Glyc drastically depends on the pH level of the probe solution. With the decrease in pH from 7.4 to 4.0, (i) the affinity of the specific reaction increases and (ii) the level of unspecific sorption becomes saturated. Non-specific adsorption to a SPR sensor surface obscures the specific component and shaded specific response at higher pH (6.0-7.4) when used serum for the quantification of specific analytes. The standard curves obtained in acidic solutions (pH 4-5) indicate that the linear part of the dependence completely covers the range between detection limit (0.1 µg/ml) and Glyc solubility in water (0.9 µg/ml). The difference in SPR- and ELISA-based analytical protocols as well as the requirements for increasing the efficiency in quantitative SPR analysis using purified antibodies is discussed.

Measurement uncertainty in analytical studies based on surface plasmon resonance.

The sensitivity of surface plasmon resonance (SPR) transducers depends on the thickness and spatial organization of interfacial structures at their surfaces. This is because the response of the SPR sensor is determined by integrating the distance-dependent refractive index (spatial interfacial architectures), weighted by the square of the electromagnetic field, from zero to infinite distance. The effect of SPR transducer sensitivity variation on the accuracy of SPR analysis is considered. Our quantitative estimation (based on the results of refractometric studies) gave a value for sensitivity variation of about 3% for the formation of a self-assembled thiocyanate layer or a trypsin-soybean trypsin inhibitor surface complex. The estimated accuracy in measured variation (i.e., by 0.01) for the refractive index of the external medium was 3x10(-4). This restriction, which follows immediately from the physical mechanism of the SPR phenomenon, should be taken into account when analyzing data obtained with the above technique.

Imaging technique for the screening of protein-protein interactions using scattered light under surface plasmon resonance conditions.

We propose a novel technique to detect protein-protein interactions in microarray format. The technique involves measuring scattered light under surface plasmon resonance (SPR) conditions. We have shown that the maximum scattering angle correlates with the traditionally employed reflection minimum. Panoramic scanning of scattered light under SPR conditions has all the functional advantages of the SPR technique. In addition, the proposed technique simplifies device design, increases the dynamic range of analysis, and integrates data with those from surface-plasmon field-enhanced fluorescence spectroscopy. We demonstrate the technique by showing direct protein-protein interaction between protein A and either rabbit antibodies or human serum.

SPR-based immunocapture approach to creating an interfacial sensing architecture: Mapping of the MRS18-2 binding site on retinoblastoma protein.

Biosensor technologies based on optical readout are widely used in protein-protein interaction studies. Here we describe a fast and simple approach to the creation of oriented interfacial architectures for surface plasmon resonance (SPR) transducers, based on conventional biochemical procedures and custom reagents. The proposed protocol permits the oriented affinity-capture of GST fusion proteins by a specific antibody which is bound to protein A, which in turn has been immobilized on the transducer surface (after the surface has been modified by guanidine thiocyanate). The applicability of the method was demonstrated by studying the interaction between retinoblastoma tumor suppressor protein (pRb) and MRS18-2 proteins. The formation of the pRb-MRS18-2 protein complex was examined and the pRb binding site (A-box-spacer-B-box) was mapped. We have also shown that MRS18-2, which was detected as the Epstein-Barr virus-encoded EBNA-6 binding partner using the yeast two-hybrid system, binds to pRb in GST pull-down assays.