Hana Šípová

Surface Plasmon Resonance Biosensor for Rapid Label-Free Detection of Microribonucleic Acid at Subfemtomole Level

Microribonucleic acids (miRNAs) have been linked with various regulatory functions and disorders, such as cancers and heart diseases. They, therefore, present an important target for detection technologies for future medical diagnostics. We report here a novel method for rapid and sensitive miRNA detection and quantitation using surface plasmon resonance (SPR) sensor technology and a DNA*RNA antibody-based assay. The approach takes advantage of a novel high-performance portable SPR sensor instrument for spectroscopy of surface plasmons based on a special diffraction grating called a surface plasmon coupler and disperser (SPRCD). The surface of the grating is functionalized with thiolated DNA oligonucleotides which specifically capture miRNA from a liquid sample without amplification. Subsequently, an antibody that recognizes DNA*RNA hybrids is introduced to bind to the DNA*RNA complex and enhance sensor response to the captured miRNA. This approach allows detection of miRNA in less than 30 min at concentrations down to 2 pM with an absolute amount at high attomoles. The methodology is evaluated for analysis of miRNA from mouse liver tissues and is found to yield results which agree well with those provided by the quantitative polymerase chain reaction (qPCR).

Ultrasensitive broadband probing of molecular vibrational modes with multifrequency optical antennas.

Optical antennas represent an enabling technology for enhancing the detection of molecular vibrational signatures at low concentrations and probing the chemical composition of a sample in order to identify target molecules. However, efficiently detecting different vibrational modes to determine the presence (or the absence) of a molecular species requires a multispectral interrogation in a window of several micrometers, as many molecules present informative fingerprint spectra in the mid-infrared between 2.5 and 10 μm. As most nanoantennas exhibit a narrow-band response because of their dipolar nature, they are not suitable for such applications. Here, we propose the use of multifrequency optical antennas designed for operating with a bandwidth of several octaves. We demonstrate that surface-enhanced infrared absorption gains in the order of 10(5) can be easily obtained in a spectral window of 3 μm with attomolar concentrations of molecules, providing new opportunities for ultrasensitive broadband detection of molecular species via vibrational spectroscopy techniques.

Novel high‐affinity binders of human interferon gamma derived from albumin‐binding domain of protein G

Recombinant ligands derived from small protein scaffolds show promise as robust research and diagnostic reagents and next generation protein therapeutics. Here, we derived high‐affinity binders of human interferon gamma (hIFNγ) from the three helix bundle scaffold of the albumin‐binding domain (ABD) of protein G from Streptococcus G148. Computational interaction energy mapping, solvent accessibility assessment, and in silico alanine scanning identified 11 residues from the albumin‐binding surface of ABD as suitable for randomization. A corresponding combinatorial ABD scaffold library was synthesized and screened for hIFNγ binders using in vitro ribosome display selection, to yield recombinant ligands that exhibited Kd values for hIFNγ from 0.2 to 10 nM. Molecular modeling, computational docking onto hIFNγ, and in vitro competition for hIFNγ binding revealed that four of the best ABD‐derived ligands shared a common binding surface on hIFNγ, which differed from the site of human IFNγ receptor 1 binding. Thus, these hIFNγ ligands provide a proof of concept for design of novel recombinant binding proteins derived from the ABD scaffold. Proteins 2011.

Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin‐23 receptor (IL‐23R), which is a key element of proinflammatory IL‐23‐mediated signaling. A library of variants derived from the three‐helix bundle scaffold of the albumin‐binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high‐affinity binders of recombinant extracellular IL‐23R. A collection of 34 IL‐23R‐binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL‐23, or the biologically active human IL‐23 cytokine, to the recombinant IL‐23R or soluble IL‐23R‐IgG chimera. The strongest competitors for IL‐23R binding in ELISA were confirmed to recognize human IL‐23R‐IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub‐ to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K‐562, THP‐1 and Jurkat, and this binding correlated with IL‐23R cell‐surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP‐1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL‐23‐driven expansion of IL‐17‐producing primary human CD4+ T‐cells. Thus, we conclude that unique IL‐23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti‐inflammatory biologicals. Proteins 2014; 82:975–989.

Analytical value of detecting an individual molecular binding event: the case of the surface plasmon resonance biosensor.

We explore the ultimate limits of the performance of bioanalytical approaches based on the detection of individual molecular binding events taking place at the sensor surface interfaced with a microfluidic flow-through cell. As a case study, we investigate and compare the bioanalytical performance of flow-through surface plasmon resonance (SPR) sensors based on (1) localized surface plasmons (LSP) which detect a single binding event and (2) propagating surface plasmons (PSP) which integrate a great number of simultaneously occurring binding events. We demonstrate that for the biomolecular interactions most relevant to biosensing the single-binding-event LSP approach is inferior to the integrating PSP approach. We estimate that the number of biorecognition elements available to interact with the analyte molecules would need to be, depending on the size of the analyte and parameters of the molecular interaction, in the order of 10 to 10(3) to increase the probability of the positive response of the LSP-based sensor to that of the PSP-based sensor.

Ambiguous Refractive Index Sensitivity of Fano Resonance on an Array of Gold Nanoparticles

We investigate the optical response to refractive index changes of a Fano resonance occurring in a random array of gold nanoparticles supported on a glass substrate. The Fano resonance results from the interference between localized surface plasmon on a gold nanoparticle and the light reflected at the boundary of the glass substrate. We demonstrate that the sensitivity of the resonance to the refractive index of the surrounding medium is highly dependent on the excitation geometry and can assume either positive or negative values. We furthermore present a theoretical analysis explaining this behavior based on the rigorous coupled wave analysis (RCWA) as well as the island film theory.

Superior LSPR substrates based on electromagnetic decoupling for on-a-chip high-throughput label-free biosensing

Localized surface plasmon resonance (LSPR) biosensing based on supported metal nanoparticles offers unparalleled possibilities for high-end miniaturization, multiplexing and high-throughput label-free molecular interaction analysis in real time when integrated within an opto-fluidic environment. However, such LSPR-sensing devices typically contain extremely large regions of dielectric materials that are open to molecular adsorption, which must be carefully blocked to avoid compromising the device readings. To address this issue, we made the support essentially invisible to the LSPR by carefully removing the dielectric material overlapping with the localized plasmonic fields through optimized wet-etching. The resulting LSPR substrate, which consists of gold nanodisks centered on narrow SiO2 pillars, exhibits markedly reduced vulnerability to nonspecific substrate adsorption, thus allowing, in an ideal case, the implementation of thicker and more efficient passivation layers. We demonstrate that this approach is effective and fully compatible with state-of-the-art multiplexed real-time biosensing technology and thus represents the ideal substrate design for high-throughput label-free biosensing systems with minimal sample consumption.

High index dielectric metasurfaces and colloidal solutions: from fabrication to application

High index dielectric nanoparticles and meta-materials have been proposed for many different applications, including light harvesting, sensing and metalenses. However, widespread utilization in practice also requires large-scale fabrication methods able to produce homogeneous structures with engineered optical properties in a cost effective manner. Here, it is presented a facile fabrication method for silicon nanoparticles which is scalable to 4-inch wafers and can produce a wide range of nanoparticle shapes on demand. We also show that the fabricated nanoparticles can be detached from their support using a simple substrate removal technique and then transferred to colloidal suspension. We will finally discuss some uses of the fabricated systems. For the metasurfaces, we will demonstrate complete absorption due to far field interference effects. For the nanoparticles colloids we will show the possibility of realizing an intrinsically chiral structure composed of a low-loss dielectric resonator and we will study optical trapping phenomena for different particle sizes and shapes.