Magdalena Stobiecka

Ligand exchange effects in gold nanoparticle assembly induced by oxidative stress biomarkers: Homocysteine and cysteine

The interactions of oxidative stress biomarkers: homocysteine (Hcys) and cysteine (Cys) with the multifunctional gold nanoparticles, important in view of novel biomedical applications in diagnostics and therapy, have been investigated using resonance elastic light scattering (RELS), UV-Vis plasmonic spectroscopy, and high-resolution TEM imaging. The Hcys-induced assembly of gold nanoparticles has been observed for non-ionic surfactant-capped gold nanoparticles as well as for negatively-charged citrate-capped gold nanoparticles. We have observed for the first time the de-aggregation of citrate-capped gold nanoparticle ensembles followed by their conversion to citrate-linked Hcys-capped nanoparticle assemblies. The Cys molecules, which are smaller than Hcys by only one CH(2) group, show much less activity. The mechanisms leading to this intriguing disparity in the abilities of these two thioaminoacids to ligand exchange with surfactant- or citrate-capping molecules of the gold nanoparticle shells are proposed on the basis of the experimental evidence, molecular dynamics simulations, and quantum mechanical calculations. For citrate-capped gold nanoparticles, we postulate the formation of surface complexes facilitated by electrostatic attractions and formation of double hydrogen bonds for both Hcys and Cys. The conformational differences between these two kinds of complexes result in marked differences in the distance between -SH groups of the biomarkers to the gold surface and different abilities to induce nanoparticle assembly. Analytical implications of these mechanistic differences are discussed.

Resonance elastic light scattering (RELS) spectroscopy of fast non-Langmuirian ligand-exchange in glutathione-induced gold nanoparticle assembly

The interactions of a biomolecule glutathione (GSH) with citrate-capped gold nanoparticles (AuNP) have been investigated to evaluate the viability of a rapid GSH-capture by gold nanoparticle carriers, as a model system for applications ranging from designing nanoparticle-enhanced functional biosensor interfaces to nanomedicine. The measurements, performed using resonance elastic light scattering (RELS) spectroscopy, have shown a strong dependence of GSH-induced scattering cross-section on gold nanoparticle size. A large increase in RELS intensity after injection of GSH, in a short reaction time (tau=60 s), has been observed for small AuNP (5nm dia.) and ascribed to the fast ligand-exchange followed by AuNP assembly. The unexpected non-Langmuirian concentration dependence of scattering intensity for AuNP (5 nm) indicates on a 2D nucleation and growth mechanism of the ligand-exchange process. The ligand-exchange and small nanoparticle ensemble formation followed by relaxation have been observed in long term (10 h) monitoring of GSH-AuNP interactions by RELS. The results of molecular dynamics and quantum mechanical calculations corroborate the mechanism of the formation of hydrogen-bonded GSH-linkages and interparticle interactions and show that the assembly is driven by multiple H-bonding between GSH-capped AuNP and electrostatic zwitterionic interactions. The RELS spectroscopy has been found as a very sensitive tool for studying interparticle interactions. The application of RELS can be expanded to monitor reactivities and assembly of other monolayer-protected metal clusters, especially in very fast processes which cannot be followed by other techniques.

Effect of buried potential barrier in label-less electrochemical immunodetection of glutathione and glutathione-capped gold nanoparticles.

The influence of potential barriers, introduced to the immunoglobulin-based sensory films, on voltammetric signals of a redox ion probe has been investigated. Films with positive and negative barriers have been examined by depositing charged self-assembled thiol monolayers as the basal layers of a sensory film. The studies performed with monoclonal anti-glutathione antibody-based sensors using ferricyanide ion probe have shown stronger sensor response to the layer components, as well as to the glutathione-capped gold nanoparticles acting as the antigen, for films with positive potential barrier buried deep in the film than for negative barrier films. The larger changes in differential resistance, peak separation and peak heights observed for films with positive barrier have been attributed to different depth and width of the charge distributions in these films. A buried positive barrier with narrow charge distribution width provides the best conditions for film stability and prevents fouling (less ion-exchanges with the medium). This conclusion has been confirmed by calculations of the electric field distribution and potential profiles in immunosensing films performed by numerical integration of Poisson equation for Gaussian distributions of fixed charges of covalently bound components. The proposed fixed-charge model can aid in rapid evaluation of sensory films in sensor development work. The implications of potential barriers in sensory film design are discussed.

Intervention of glutathione in pre-mutagenic catechol-mediated DNA damage in the presence of copper(II) ions.

The catechol-mediated DNA damage in the presence of Cu(II) ions involves oxidation of guanine to 8-oxoguanine (8-oxoG) and DNA strand scission. It proceeds through the reactive oxygen species (ROS) generation. The mutagenicity of 8-oxoG lesions is due to its miscoding propensity reflected in GC→TA transversion taking place during the DNA repair process. To gain new insights into the nature of catechol-mediated DNA damage and its prevention, we have investigated the changes in DNA melting characteristics and 8-oxoG formation as the indicators of DNA damage in a model calf-thymus DNA system. A novel fluorescence method for DNA melting temperature determination, based on DAPI fluorescent-probe staining, has been proposed. The DNA melting-onset temperature has been found to be more sensitive to DNA damage than the standard melting temperature due to the increased width of the melting transition observed in oxidatively damaged DNA. We have found that the efficiency of Fenton cascade in generating DNA-damaging ROS is higher for catechol than for GSH, two strong antioxidants, mainly due to the much longer distance between ROS-generating radical group in GS to nucleobases than that of semiquinone radical group to nucleobases (2.1nm vs. 0.27nm), making the ROS transport from GSH an order of magnitude less likely to damage DNA because of short lifetime of HO radicals. The antioxidant and DNA-protecting behaviors of GSH have been elucidated. We have found that the redox potential of GSH/GSSG couple is lower than that of catechol/semiquinone couple. Hence, GSH keeps catechol in the reduced state, thereby shutting down the initial step of the catechol-mediated Fenton cascade. The catechol-induced DNA damage in the presence of Cu(II) ions has also been confirmed in studies of ON-OFF hairpin-oligonucleotide beacons.

Modulation of Plasmon-Enhanced Resonance Energy Transfer to Gold Nanoparticles by Protein Survivin Channeled-Shell Gating.

The resonance energy transfer (RET) from excited fluorescent probe molecules to plasmonic gold nanoparticles (AuNPs) can be gated by modulating the width of channels (gates) in submonolayer protein shells surrounding AuNPs. We have explored the gated-RET (gRET) processes using an antiapoptotic protein survivin (Sur) as the gating material, citrate-capped gold nanoparticles (AuNP@Cit), and fluorescein isothiocyanate as the fluorescent probe. Despite the electrostatic repulsive forces between these components, a strong modulation of RET efficiency by Sur down to 240 pM (S/N = 3) is possible. Using piezometric measurements, we have confirmed the Sur adsorbability on Cit-coated Au surfaces with monolayer coverage: γSur = 5.4 pmol/cm(2) and Langmuirian adsorption constant KL,Sur = 1.09 × 10(9) M(-1). The AuNP@Cit/Sur stability has been corroborated using resonance elastic light scattering. The quantum mechanical calculations indicate that multiple hydrogen bonding between Cit ligands and -NH3(+), =NH2(+), and -NH2 groups of lysines and arginines of Sur have likely facilitated Sur bonding to nanoparticles. A theoretical model of gated-RET has been developed, enabling predictions of the system behavior. In contrast to the positive slope of the Stern-Volmer quenching dependence (F0/F) = f(QA), a negative slope has been obtained for gRET relationship (F0/F) = f(cP), attributed to the dequenching.

DNA Strand Replacement Mechanism in Molecular Beacons Encoded for the Detection of Cancer Biomarkers.

Signaling properties of a fluorescent hairpin oligonucleotide molecular beacon (MB) encoded to recognize protein survivin (Sur) mRNA have been investigated. The process of complementary target binding to SurMB with 20-mer loop sequence is spontaneous, as expected, and characterized by a high affinity constant (K = 2.51 × 10(16) M(-1)). However, the slow kinetics at room temperature makes it highly irreversible. To understand the intricacies of target binding to MB, a detailed kinetic study has been performed to determine the rate constants and activation energy Ea for the reaction at physiological temperature (37 °C). Special attention has been paid to assess the value of Ea in view of reports of negative activation enthalpy for some nucleic acid reactions that would make the target binding even slower at increasing temperatures in a non-Arrhenius process. The target-binding rate constant determined is k = 3.99 × 10(3) M(-1) s(-1) at 37 °C with Ea = 28.7 ± 2.3 kcal/mol (120.2 ± 9.6 kJ/mol) for the temperature range of 23 to 55 °C. The positive high value of Ea is consistent with a kinetically controlled classical Arrhenius process. We hypothesize that the likely contribution to the activation energy barrier comes from the SurMB stem melting (tm = 53.7 ± 0.2 °C), which is a necessary step in the completion of target strand hybridization with the SurMB loop. A low limit of detection (LOD = 2 nM) for target tDNA has been achieved. Small effects of conformational polymorphs of SurMB have been observed on melting curves. Although these polymorphs could potentially cause a negative Ea, their effect on kinetic transients for target binding is negligible. No toehold preceding steps in the mechanism of target binding were identified.

Detection of Oxidative Stress Biomarkers Using Functional Gold Nanoparticles

Biomarkers of oxidative stress are biomolecules that can be utilized in the diagnosis of diminished capacity of a biological system to counteract an overproduction or invasion of reactive oxygen species and other radicals. In this Chapter, the detection methods of the oxidative stress biomarkers, such as glutathione (GSH), homocysteine (Hcys) and cysteine (Cys), based on their interactions with monolayer-protected gold nanoparticles are described. The nanoparticle utilization in a solution-phase analysis as well as in a multifunctional sensory film preparation is presented. The interactions of AuNP with GSH, a tripeptide maintaining the redox potential level in eukaryotic cells, have been investigated using resonance elastic light scattering (RELS) and plasmonic UV–vis spectroscopy. The high sensitivity of the RELS measurements enables monitoring of ligand exchanges and the biomarker-induced AuNP assembly. The viability of designing simple and rapid assays for the detection of GSH and Hcys is discussed. The surface plasmon band broadening and bathochromic shift are consistent with the biomarker-induced AuNP assembly and corroborate the RELS measurements and HR-TEM imaging. The results of molecular dynamics and quantum mechanical calculations support the mechanism of the formation of GSH- and Hcys-linkages in the interparticle interactions and show that multiple H-bonding can occur. The functionalized gold nanoparticles have also been shown to enhance the design of molecularly-templated conductive polymer films for the detection of biomolecules and to amplify the analytical signal by AuNP labeling. Novel designs of molecularly imprinted orthophenylenediamine (oPD) sensor films are presented. The biolyte-induced assembly of monolayer protected gold nanoparticles is evaluated in view of bioanalytical applications and the design of novel sensory films for molecularly-templated conductive polymers for microsensor arrays.

Novel DNA-Hybridization Biosensors for Studies of DNA Underwinding Caused by Herbicides and Pesticides

Among sensors developed for the detection of environmental pollutants, the biosensors based on DNA recognition abilities play a special role. In this work, a DNA biosensor for the evaluation of DNA damage by toxic agents has been designed which is not based on the DNA biorecognition properties but rather on the sensitivity of the intercalator-probe uptake to the conformational alterations of DNA double-helix. The sensors have been tested to assess DNA alterations caused by an herbicide atrazine. We have found that incubation of the DNA biosensor in atrazine solutions results in the increased capacity of DNA towards binding of an intercalator probe Nile Blue. On the basis of this and other findings, the mechanism of atrazine interactions with DNA double-helix has been elucidated.

Systematic study of interaction of the neutral form of anilines with undecylcalix[4]resorcinarene derivatives by means of potentiometry

The elucidation of the mechanism of potentiometric signals generation by liquid membrane electrodes incorporating undecylcalix[4]resorcinarene derivatives upon stimulation by uncharged aniline derivatives is the main aim of the research presented. A series of molecules which play the role of host and guests in the molecular recognition process occurring at the aqueous–organic membrane interface were explored. It was proved that in the conditions where all aniline derivatives exist as uncharged molecules, cationic potentiometric signals were observed for all undecylcalix[4]resorcinarene liquid membrane electrodes. The main parameters crucial for this phenomenon are the acidity/basicity of the hosts as well as the guests. The lipophilicity of the guests plays a secondary role.

Detection of Oxidative Stress Biomarkers Using Novel Nanostructured Biosensors

The oxidative stress is associated with the diminished capacity of a biological system to counteract an overproduction or invasion of reactive oxygen species and other radicals. Since oxidative stress is the leading cause of DNA damage, genetic disorders, cancer, and many environmental pollution related diseases, there is an urging need for oxidative stress screening and its prevention. There is growing evidence that oxidative stress may cause autism in children. The oxidative stress has also been implicated in the development of diabetes. Several biomarkers of oxidative stress have been identified, including glutathione (GSH), 3-nitrotyrosine (NT), homocysteine (Hcys), and cysteine (Cys). The tripeptide glutathione and its oxidized form, glutathione disulphide (GSSG), form a redox potential maintenance system in all eukaryotic cells. Since glutathione efficiently protects the DNA, proteins and lipid membranes from radical attacks, its diminished level is signaling an oxidative stress and the increased vulnerability of a biological entity to the environmental influences. An increased level of 3-nitrotyrosine, which is formed under oxidative stress in the presence of nitric oxide, has been found in diabetic patients. Homocysteine is a biomarker and an active agent leading to cardiovascular deterioration. While these biomarkers can be accurately determined using advanced instrumental assays, a wide screening would require the development of small, inexpensive, rapid, and simple in operation platforms for biomarker analysis. In this Chapter, the detection methods for the oxidative stress biomarkers based on their interactions with monolayer-protected gold nanoparticles (AuNP) are described. The nanoparticle utilization in a solution-phase analysis as well as in a multifunctional sensory film preparation is presented. The interactions of AuNP with glutathione and homocysteine have been investigated using resonance elastic light scattering (RELS) and plasmonic UV-Vis spectroscopy. The high sensitivity of the RELS measurements enables monitoring of ligand exchanges and the biomarker-induced AuNP assembly. The viability of designing simple and rapid assays for the detection of glutathione and homocysteine is discussed. The surface plasmon band broadening and bathochromic shift are consistent with the biomarker-induced AuNP assembly and corroborate the RELS measurements and HR-TEM imaging. The results of molecular dynamics and quantum mechanical calculations support the mechanism of the formation of GSHand Hcys-linkages in the interparticle interactions and show that multiple H-bonding can occur. In contrast to homocysteine and glutathione that induce gold nanoparticle assembly in specific pH ranges, no aggregation of nanoparticles has been