Evgenia G. Matveeva

Advances in Surface-Enhanced Fluorescence

We report recent achievements in metal-enhanced fluorescence from our laboratory. Several fluorophore systems have been studied on metal particle-coated surfaces and in colloid suspensions. In particular, we describe a distance dependent enhancement on silver island films (SIFs), release of self-quenching of fluorescence near silver particles, and the applications of fluorescence enhancement near metalized surfaces to bioassays. We discuss a number of methods for various shaped silver particle deposition on surfaces.

Enhanced Fluorescent Immunoassays on Silver Fractal-like Structures

Using the effect of the fluorescence enhancement in close proximity to metal nanostructures, we have been able to demonstrate ultrasensitive immunoassays suitable for the detection of biomarkers. Silver fractal-like structures have been grown by electrochemical reduction of silver on the surface of glass slides. A model immunoassay was performed on the slide surface with rabbit IgG (antigen) noncovalently immobilized on the slide, and rhodamine red-X-labeled antirabbit IgG conjugate was subsequently bound to the immobilized antigen. The fluorescence signal was measured from the glass-fractals surface using a confocal microscope, and the images were compared to the images from the same surface not coated with fractals. Our results showed significant enhancement (more than 100-fold) of the signal detected on fractals compared to bare glass. We thus demonstrate that such fractal-like structures can assist in improving the signals from assays used in medical diagnostics, especially those for analytes with molecular weight under 100 kDa.

Single Molecule Studies of Multiple-Fluorophore Labeled Antibodies. Effect of Homo-FRET on the Number of Photons Available Before Photobleaching

Advancements in single molecule detection (SMD) continue to unfold powerful ways to study the behavior of individual and complex molecular systems in real time. SMD enables the characterization of complex molecular interactions and reveals basic physical phenomena underlying chemical and biological processes. We present here a systematic study of the quenching efficiency of Förster-type energy-transfer (FRET) for multiple fluorophores immobilized on a single antibody. We simultaneously monitor the fluorescence intensity, fluorescence lifetime, and the number of available photons before photobleaching as a function of the number of identical emitters bound to a single IgG antibody. The detailed studies of FRET between individual fluorophores reveal complex through-space interactions. In general, even for two or three fluorophores immobilized on a single protein, homo-FRET interactions lead to an overall non-linear intensity increase and shortening of fluorescence lifetime. Over-labeling of protein in solution (ensemble) results in the loss of fluorescence signal due to the self-quenching of fluorophores making it useless for assays applications. However, in the single molecule regime, over-labeling may bring significant benefits in regards to the number of available photons and the overall survival time. Our investigation reveals possibilities to significantly increase the observation time for a single macromolecule allowing studies of macromolecular interactions that are not obscured by ensemble averaging. Extending the observation time will be crucial for developing immunoassays based on single-antibody.

Nanostructured Silver-Based Surfaces: New Emergent Methodologies for an Easy Detection of Analytes

In this work, we describe how to realize a new sensing platform for an easy and fast detection of analytes. In particular, we utilized enhanced fluorescence emission on silver island films (SIFs) coupled to the total internal reflection fluorescence mode (TIRF) to develop a new assay format for the detection of target analytes. Here, as an example, we report on the detection of the toxic peptides present in gliadin (Gli). Our assay was performed as follows: (1) gliadin was first captured on surfaces coated with anti-Gli antibodies; (2) the surfaces were then incubated with fluorophore-labeled anti-Gli antibodies; (3) the signal from the fluorophore-labeled anti-Gli antibody bound to the antigen was detected by TIRF. The system was examined on glass surfaces and on SIFs. We observed a relevant enhancement of the signal from SIFs compared to the signal from the glass substrate not modified with a SIF. In addition, the estimated detection limit (EDL) of our methodology was 60 ng/mL (or lower). This limit is therefore lower than the clinical cut-off for Gli presence in food for celiac patients. The advantage of our method is a reduced number of testing steps, which allows for easy detection of residual toxic peptides in food labeled as gluten free. The proposed technology can be easily expanded to the determination of different target analytes.

Molecular Fluorescence Enhancement on Fractal-Like Structures

In this report we discuss strong fluorescence enhancements on electrochemically grown silver nanostructures examined through fluorescence lifetime imaging microscopy (FLIM). Silver fractal-like nanostructures were deposited on three different substrates: glass, plastic, and silicon. For all of the surfaces the same dye was tested, DyLight 649, deposited in the form of a model immunoassay through excitation from a 635 nm pulsed solid-state laser. The brightness improvement in hot spots exceeded 300 fold, which is about two times higher than was observed previously on similar surfaces. The strongest enhancements correspond to the shortest lifetimes, indicating a strong interaction between excited molecules and silver nanostructures. Additionally, the photostability of the fluorescence dye was dramatically increased in the presence of electrochemically deposited silver nanostructures. The production of silver fractals is easy, very controllable, and can be applied to any surface. We therefore believe that silver fractal-like nanostructures can be used successfully in ultrasensitive assays and fluorophore trace detection.

Tryptophan Fluorescence Quenching by Enzyme Inhibitors As a Tool for Enzyme Active Site Structure Investigation: Epoxide Hydrolase

We present the strong fluorescence effect, a new 392 nm emission peak appearing after binding of a naphtol-urea inhibitor XIIa to the enzyme epoxide hydrolase (EH), along with the quenching of the EH tryptophan fluorescence. We have studied the quenching of the 392-nm peak (attributed to XIIa bound inside the active center of the enzyme) of the mixture EH +XIIa by various strong transparent inhibitors (competing with XIIa for binding to EH), and measured the corresponding values of the Stern-Volmer constants, K(mix)(SV). Strong EH inhibitors demonstrate different replacement behavior which can be used to distinguish them. We further demonstrate a novel fluorescent assay which allows to distinguish highly potent inhibitors and to visualize the strongest among them. We generated our assay calibration curve based on the quenching data, by plotting quenching strength K(mix)(SV) versus inhibiting strength, IC(50) values. We used moderate inhibitors for the assay plot generation. We then applied this curve to determine IC(50) values for several highly potent inhibitors, with IC(50) values at the limit of the IC(50) detection sensitivity by colorimetric enzyme assay. IC(50) values determined from our quenching assay show correlation with IC(50) values determined in the literature by more sensitive radioactive-based assay and allow differentiating the inhibitors potency in this group. To our knowledge, this is the first inhibitor assay of such kind. Chemical inhibition of EH is an important technology in the treatment of various cardiovascular diseases, therefore, this tool may play a crucial role in discovering new inhibitor structures for therapeutic EH inhibition.

Plastic Versus Glass Support for an Immunoassay on Metal-Coated Surfaces in Optically Dense Samples Utilizing Directional Surface Plasmon-Coupled Emission

We compared plastic (polycarbonate) and high-quality glass support materials for gold-coated slides, when performing a model immunoassay against rabbit IgG using fluorescently labeled (AlexaFluor-647) anti-rabbit IgG, and detecting surface plasmon-coupled emission (SPCE) signals. Both, glass and plastic slides were simultaneously coated with a 48-nm layer of gold and protected with a 10-nm layer of silica. The maximum SPCE signal of AlexaFluor-647 was only two- to three-fold smaller on plastic slides than on glass slides. A small difference in the SPCE angles on glass (θF = 55°) and plastic (θF = 52.5°) slides was observed and can be explained with a slightly smaller refractive index of the plastic. We have not found any difference in the angle distribution (sharpness of the fluorescence signal at optimal SPCE angle) for the plastic slide compared to the glass slide. The kinetics of binding was monitored on the plastic slide as well as on the glass slide. Optically dense samples, a 4% red blood cell suspension and a 15% hemoglobin solution, are causing a reduction in the immunoassay SPCE signal by approximately 15% and three times, respectively, and the percentage of the reduction is the same for plastic and for glass slides. We believe that plastic substrates can be readily used in any SPCE assay, with only marginally lower total signal compared to high-quality glass slides.

Amyloid beta modulation of neuronal network activity in vitro.

In vitro assays offer a means of screening potential therapeutics and accelerating the drug development process. Here, we utilized neuronal cultures on planar microelectrode arrays (MEA) as a functional assay to assess the neurotoxicity of amyloid-β 1-42 (Aβ42), a biomolecule implicated in the Alzheimer׳s disease (AD). In this approach, neurons harvested from embryonic mice were seeded on the substrate-integrated microelectrode arrays. The cultured neurons form a spontaneously active network, and the spiking activity as a functional endpoint could be detected via the MEA. Aβ42 oligomer, but not monomer, significantly reduced network spike rate. In addition, we demonstrated that the ionotropic glutamate receptors, NMDA and AMPA/kainate, play a role in the effects of Aβ42 on neuronal activity in vitro. To examine the utility of the MEA-based assay for AD drug discovery, we tested two model therapeutics for AD, methylene blue (MB) and memantine. Our results show an almost full recovery in the activity within 24h after administration of Aβ42 in the cultures pre-treated with either MB or memantine. Our findings suggest that cultured neuronal networks may be a useful platform in screening potential therapeutics for Aβ induced changes in neurological function.

DEVELOPMENT OF A HOMOGENEOUS PHOSPHORESCENT IMMUNOASSAY FOR THE DETECTION OF POLYCHLORINATED DIBENZO-p-DIOXINS

A homogeneous immunoassay for dioxins was developed using a phosphorescent label for detection. A dioxin derivative was conjugated to Pt-coproporphyrin. In the assay, when the antibodies against dioxin (DD3) were bound to the phosphorescent conjugate, the signal from Pt-coproporphyrin was quenched. The interaction between antibody and the conjugate was studied by time-resolved luminescence spectroscopy. As concentrations of dioxin standards increased from 39 pg/well to 2.5 ng/well the lifetime of the phosphorescence of the shortlived component increased from 25.6 microseconds to 58.9 microseconds. This increase in half-life was associated with a dose dependent quenching of the phosphorescence. The inhibition obtained is similar to that for a reported enzyme-based immunoassay, but the data were obtained in minutes instead of hours.

Long Wavelength Fluorescence Ratiometric Zinc Biosensor

A protein-based emission ratiometric fluorescence biosensor is described that exhibits sensitivity to free zinc ion in solution down to picomolar concentrations. Ratiometric measurements are widely used to assure accurate quantitation, and emission ratios are preferred for laser scanning microscopes such as confocal fluorescence microscopes. The relatively long emission wavelengths used are well suited to studies in tissues and other matrices which exhibit significant fluorescence background, and the apo-carbonic anhydrase moiety recognizes zinc ion with high and controllable specificity.