Strong Coupling between Surface Plasmon Resonance and Exciton of Labeled Protein-Dye Complex for Immunosensing Applications.

In this study, we present an analysis of the optical response of strong coupling between SPR and labeled proteins. We demonstrate a sensing methodology that allows to evaluate the protein mass adsorbed to the gold's surface from the Rabi gap, which is a direct consequence of the strong light-matter interaction between surface plasmon polariton and dye exciton of labeled protein. The total internal reflection ellipsometry optical configuration was used for simulation of the optical response for adsorption of HSA-Alexa633 dye-labeled protein to a thin gold layer onto the glass prism. It was shown that Rabi oscillations had parabolic dependence on the number of labeled proteins attached to the sensor surface; however, for photonic-plasmonic systems in real experimental conditions, the range of the Rabi energy is rather narrow, thus it can be linearly approximated. This approach based on the strong coupling effect paves the alternative way for detection and monitoring of the interaction of the proteins on the transducer surface through the change of coupling strengths between plasmonic resonance and the protein-dye complex.

High Spectral Sensitivity of Strongly Coupled Hybrid Tamm-Plasmonic Resonances for Biosensing Application.

In this study, the sensitivity to the refractive index changes of the ambient was studied on the uniform gold film (~50 nm) with a 1D photonic crystal (PC) from periodic five TiO2 (~110 nm)/SiO2 (~200 nm) bilayers and gold nano-bumps array produced by direct laser writing on the same sample. The optical signal sensitivity of hybrid plasmonic resonances was compared with traditional surface plasmon resonance (SPR) on a single gold layer. The influence of the strong coupling regime between Tamm plasmon polariton (TPP) and propagated plasmon polaritons in the hybrid plasmonic modes on the sensitivity of the optical was discussed. Recent studies have shown very high hybrid plasmonic mode sensitivity SHSPP ≈ 26,000 nm/RIU to the refractive index on the uniform gold layer; meanwhile, the introduction of gold lattice reduces the signal sensitivity, but increases the Q-factor of the plasmonic resonances. Despite this, the sensitivity to the ellipsometric parameters Ψ and Δ on the gold lattice was rather high due to the increased Q-factor of the resonances. The comparison of plasmonic resonance sensitivity to the refractive index changes of hybrid TPP-SPP mode on the uniform gold layer and traditional SPR have shown that hybrid plasmonic mode, due to a strong coupling effect, overcomes the SPR by about 27%.

Photonic Sensors in Chemical and Biological Applications.

Biosensors are described as analytical devices in which biological substances are detected by using various physicochemical detection systems [...].

Total Internal Reflection Ellipsometry Approach for Bloch Surface Waves Biosensing Applications.

A one-dimensional photonic crystal with an additional TiO2 layer, supporting Bloch surface waves (BSW), was used for enhanced signal sensitivity for the detection of protein interaction. To compare the optical response of BSW and photonic crystals (PC), bovine serum albumin and specific antibodies against bovine serum were used as a model system. The results obtained show the enhanced sensitivity of p- and s-BSW components for the 1D PC sample with an additional TiO2 layer. Furthermore, a higher sensitivity was obtained for the BSW component of p-polarization in the PC sample with an additional TiO2 layer, where the sensitivity of the ellipsometric parameter Ψ was five times higher and that of the Δ parameter was eight times higher than those of the PC sample. The capabilities of BSW excitations are discussed from the sensitivity point of view and from the design of advanced biosensing.

Investigation of SARS-CoV-2 nucleocapsid protein interaction with a specific antibody by combined spectroscopic ellipsometry and quartz crystal microbalance with dissipation.

Detailed evaluations of the antigen and antibody interaction rate and strength of the immune complex formed are very important for medical and bioanalytical applications. These data are crucial for the development of sensitive and fast immunosensors suitable for continuous measurements. Therefore, combined spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation (QCM-D) technique (SE/QCM-D) was used for the evaluation: (i)of covalent immobilization of SARS-CoV-2 nucleocapsid protein (SCoV2-N) on QCM-D sensor disc modified by self-assembled monolayer based on 11-mercaptoundecanoic acid and (ii)interaction of immobilized SCoV2-N with specific polyclonal anti-SCoV2-N antibodies followed by immune complex formation process. The results show that the SCoV2-N monolayer is rigid due to the low energy dissipation registered during the QCM-D measurement. In contrast, the anti-SCoV2-N layer produced after interaction with the immobilized SCoV2-N formed a soft and viscous layer. It was determined, that the sparse distribution of SCoV2-N on the surface affected the spatial arrangement of the antibody during the formation of immune complexes. The hinge-mediated flexibility of the antibody Fab fragments allows them to reach the more distantly located SCoV2-N and establish a bivalent binding between proteins in the formed SCoV2-N/anti-SCoV2-N complex. It was noted that the SE/QCM-D method can provide more precise quantitative information about the flexibility and conformational changes of antibody during the formation of the immune complex on the surface over time.

Application of Tamm Plasmon Polaritons and Cavity Modes for Biosensing in the Combined Spectroscopic Ellipsometry and Quartz Crystal Microbalance Method.

Low-cost 1D plasmonic photonic structures supporting Tamm plasmon polaritons and cavity modes were employed for optical signal enhancement, modifying the commercially available quartz crystal microbalance with dissipation (QCM-D) sensor chip in a combinatorial spectroscopic ellipsometry and quartz microbalance method. The Tamm plasmon optical state and cavity mode (CM) for the modified mQCM-D sample obtained sensitivity of ellipsometric parameters to RIU of ΨTPP = 126.78 RIU-1 and ΔTPP = 325 RIU-1, and ΨCM = 264 RIU-1 and ΔCM = 645 RIU-1, respectively. This study shows that Tamm plasmon and cavity modes exhibit about 23 and 49 times better performance of ellipsometric parameters, respectively, for refractive index sensing than standard spectroscopic ellipsometry on a QCM-D sensor chip. It should be noted that for the optical biosensing signal readout, the sensitivity of Tamm plasmon polaritons and cavity modes are comparable with and higher than the standard QCM-D sensor chip. The different origin of Tamm plasmon polaritons (TPP) and cavity mode (CM) provides further advances and can determine whether the surface (TPP) or bulk process (CM) is dominating. The dispersion relation feature of TPP, namely the direct excitation without an additional coupler, allows the possibility to enhance the optical signal on the sensing surface. To the best of our knowledge, this is the first study and application of the TPP and CM in the combinatorial SE-QCM-D method for the enhanced readout of ellipsometric parameters.

Evaluation of kinetics and thermodynamics of interaction between immobilized SARS-CoV-2 nucleoprotein and specific antibodies by total internal reflection ellipsometry.

During the pandemic, different methods for SARS-CoV-2 detection and COVID-19 diagnostics were developed, including antibody and antigen tests. For a better understanding of the interaction mechanism between SARS-CoV-2 virus proteins and specific antibodies, total internal reflection ellipsometry based evaluation of the interaction between SARS-CoV-2 nucleoprotein (SCoV2-rN) and anti-SCoV2-rN antibodies was performed. Results show that the appropriate mathematical model, which takes into account the formation of an intermediate complex, can be applied for the evaluation of SCoV2-rN/anti-SCoV2-rN complex formation kinetics. The calculated steric factor indicated that SCoV2-rN/anti-SCoV2-rN complex formation has very strict steric requirements. Estimated Gibbs free energy (ΔGAssoc) for SCoV-rN and anti-SCoV-rN binding was determined as -34 kJ/mol. The reported findings are useful for the design of new analytical systems for the determination of anti-SCoV2-rN antibodies and for the development of new anti-SARS-CoV-2 medications.

Human granulocyte-colony stimulating factor (G-CSF)/stem cell factor (SCF) fusion proteins: design, characterization and activity.

BACKGROUND: Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are well-characterized vital hematopoietic growth factors that regulate hematopoiesis. G-CSF and SCF synergistically exhibit a stimulatory effect on hematopoietic progenitors. The combination of G-CSF and SCF has been used for mobilization of peripheral blood progenitor cells in cancer and non-cancerous conditions. To overcome challenges connected with the administration of two cytokines, we developed two fusion proteins composed of human SCF and human G-CSF interspaced by an alpha-helix-forming peptide linker. METHODS: The recombinant proteins SCF-Lα-GCSF and GCSF-Lα-SCF were purified in three steps using an ion-exchange and mixed-mode chromatography. The purity and quantity of the proteins after each stage of purification was assessed using RP-HPLC, SDS-PAGE, and the Bradford assays. Purified proteins were identified using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and the Western blot analyses. The molecular weight was determined by size exclusion HPLC (SE-HPLC). The activity of heterodimers was assessed using cell proliferation assays in vitro. The capacity of recombinant fusion proteins to stimulate the increase of the absolute neutrophil count in rats was determined in vivo. The binding kinetics of the proteins to immobilized G-CSF and SCF receptors was measured using total internal reflection ellipsometry and evaluated by a standard Langmuir kinetics model. RESULTS: The novel SCF-Lα-GCSF and GCSF-Lα-SCF proteins were synthesized in Escherichia coli. The purity of the heterodimers reached >90% as determined by RP-HPLC. The identity of the proteins was confirmed using the Western blot and HPLC/ESI-MS assays. An array of multimeric forms, non-covalently associated dimers or trimers were detected in the protein preparations by SE-HPLC. Each protein induced a dose-dependent proliferative response on the cell lines. At equimolar concentration, the heterodimers retain 70-140% of the SCF monomer activity (p ≤ 0.01) in promoting the M-07e cells proliferation. The G-CSF moiety in GCSF-Lα-SCF retained 15% (p ≤ 0.0001) and in SCF-Lα-GCSF retained 34% (p ≤ 0.01) of the monomeric G-CSF activity in stimulating the growth of G-NFS-60 cells. The obtained results were in good agreement with the binding data of each moiety in the fusion proteins to their respective receptors. The increase in the absolute neutrophil count in rats caused by the SCF-Lα-GCSF protein corresponded to the increase induced by a mixture of SCF and G-CSF.

Evaluation of affinity sensor response kinetics towards dimeric ligands linked with spacers of different rigidity: Immobilized recombinant granulocyte colony-stimulating factor based synthetic receptor binding with genetically engineered dimeric analyte derivatives.

The modelling of protein-protein binding kinetics is important for the development of affinity-sensors and the prediction of signaling protein based drug efficiency. Therefore, in this research we have evaluated the binding kinetics of several genetically designed protein models: (i) three different ligands based on granulocyte colony-stimulating factor GCSF homo-dimeric derivatives linked by differed by linkers of different length and flexibility; (ii) an antibody-like receptor (GCSF-R) based on two GCSF-receptor sites immobilized to Fc domains, which are common parts of protein structures forming antibodies. Genetically engineered GCSF-R is similar to an antibody because it, like the antibody, has two binding sites, which both selectively bind with GCSF ligands. To design the affinity sensor model studied here, GCSF-R was immobilized on a thin gold layer via self-assembled monolayer conjugated with Protein-G. Binding kinetics between immobilized GCSF-R and all three different recombinant GCSF-based homo-dimeric derivatives were evaluated by total internal reflection ellipsometry. Association constants were determined by fitting mathematical models to the experimental data. It was clearly observed that both (i) affinity and (ii) binding kinetics depend on the length and flexibility of the linker that connects both domains of a GCSF-based ligand. The fastest association between immobilized GCSF-R and GCSF-based ligands was observed for ligands whose GCSF domains were interconnected by the longest and the most flexible linker. Here we present ellipsometry-based measurements and models of the interaction kinetics that advance the understanding of bidentate-receptor-based immunosensor action and enables us to predict the optimal linker structure for the design of GCSF-based medications.

Optical Dispersions of Bloch Surface Waves and Surface Plasmon Polaritons: Towards Advanced Biosensors.

The total internal reflection ellipsometry (TIRE) method was used for the excitation and study of the sensitivity features of surface plasmon polariton (SPP) and Bloch surface waves (BSWs) resonances. For the BSWs generation distributed Bragg gratings were formed on the tops of the substrates (BK7 glass substrate), which had six bilayers of ~120 nm SiO2 and ~40 nm TiO2 and 40 nm of TiO2 on the top. The SPP sample consisted of the BK7 glass prism and a gold layer (45 nm). Numerical calculations of the optical dispersions and the experimental TIRE data have shown that SPP resonance overtake the BSWs in wavelength scanning by a factor of about 17. However, for the ellipsometric parameters Ψ and Δ in the vicinity of excitations, the BSW sensitivity is comparable with SPP. The obtained resolutions were Δ S P P = 7.14 × 10 - 6 R I U , Ψ S P P = 1.7 × 10 - 5 R I U for the SPP and Δ B S W = 8.7 × 10 - 6 R I U , Ψ B S W = 2.7 × 10 - 5 R I U for the BSW. The capabilities of both surface excitations are discussed from the sensitivity point of view in the design of these advanced biosensors.

Hybrid Tamm-surface plasmon polaritons mode for detection of mercury adsorption on 1D photonic crystal/gold nanostructures by total internal reflection ellipsometry.

Spectroscopic ellipsometry was used for the generation and study of the hybrid TPP-SPP mode as a sensor probe for the real-time formation of amalgam structures on the surface of a plasmon active gold layer. The Au/Hg amalgam formation features and the mercury atoms' penetration into the gold layer were determined by means of the experimental TIRE data and a regression analysis of a multi-layer model containing the index-profile amalgam layer. The hybrid TPP-SPP mode behavior of the coupled excitations provided more information about the mercury atoms' penetration into the gold layer than the single TPP and SPP resonances did. The present study demonstrated the possibility of using the hybrid TPP-SPP mode to design advanced optical gas sensor technologies.

Tunable Bloch surface waves in anisotropic photonic crystals based on lithium niobate thin films.

We present an original type of one-dimensional photonic crystal that includes one anisotropic layer made of a lithium niobate thin film. We demonstrate the versatility of such a device sustaining different Bloch surface waves (BSWs), depending on the orientation of the incident wave. By varying the orientation of the illumination of the multilayer, we measured an angle variation of 7° between the BSWs corresponding to the extraordinary and the ordinary index of the lithium niobate thin film. The potential of such a platform opens the way to novel tunable and active planar optics based on the electro- and thermo-optical properties of lithium niobate.

Synthesis of polypyrrole within the cell wall of yeast by redox-cycling of [Fe(CN)6](3-)/[Fe(CN)6](4-).

Yeast cells are often used as a model system in various experiments. Moreover, due to their high metabolic activity, yeast cells have a potential to be applied as elements in the design of biofuel cells and biosensors. However a wider application of yeast cells in electrochemical systems is limited due to high electric resistance of their cell wall. In order to reduce this problem we have polymerized conducting polymer polypyrrole (Ppy) directly in the cell wall and/or within periplasmic membrane. In this research the formation of Ppy was induced by [Fe(CN)6](3-)ions, which were generated from K4[Fe(CN)6], which was initially added to polymerization solution. The redox process was catalyzed by oxido-reductases, which are present in the plasma membrane of yeast cells. The formation of Ppy was confirmed by spectrophotometry and atomic force microscopy. It was confirmed that the conducting polymer polypyrrole was formed within periplasmic space and/or within the cell wall of yeast cells, which were incubated in solution containing pyrrole, glucose and [Fe(CN)6](4-). After 24h drying at room temperature we have observed that Ppy-modified yeast cell walls retained their initial spherical form. In contrast to Ppy-modified cells, the walls of unmodified yeast have wrinkled after 24h drying. The viability of yeast cells in the presence of different pyrrole concentrations has been evaluated.

Crowding enhances lipase turnover rate on surface-immobilized substrates.

Utilizing surface-immobilized synthetic lipid substrates containing the redox-active ferrocene groups, the enzymatic activity of lipase from Thermomyces lanuginosus was measured by the cyclic voltammetry method. The activity was correlated with the surface density of the protein by the ATR-IR spectroscopy and the total internal reflection ellipsometry. It was found that the lipase turnover rate significantly increases with its surface density. Despite expected hindrance effects due to the crowding of the enzyme molecules in the near surface-saturation range of concentrations, the turnover rate was consistently higher compared with the values measured at low concentrations. The effect was explained by the change in the surface arrangement of the enzyme. In the low concentration range, lipase adsorbs onto a surface adopting a predominantly horizontal position. At high concentrations, as the surface density approaches saturation, the enzyme molecules due to crowding are forced into the predominantly vertical position, which is more favorable for the activation of the lipase through the interaction between the "hydrophobic lid" of the lipase and the hydrophobic adsorbate surface.

Spectroellipsometric characterization and modeling of plasmonic diamond-like carbon nanocomposite films with embedded Ag nanoparticles.

Diamond-like carbon nanocomposite films with embedded silver nanoparticles are considered experimentally (spectroellipsometric characterization) and theoretically (modeling of optical properties). Metallic nanocomposite films were synthesized by reactive magnetron sputtering and were studied by transmission electron microscope (TEM) and atomic force microscope (AFM). The optical constants of the films were determined from spectroscopic ellipsometry measurements and were modeled using the Maxwell-Garnett approximations. Comparison between the extended and renormalized Maxwell-Garnett theory was conducted. Surface plasmon resonance peak have been found to be strongly dependent on the shape of nanoparticles and interaction between them.

Study of antibody/antigen binding kinetics by total internal reflection ellipsometry.

Total internal reflection ellipsometry (TIRE) has been applied for the investigation of (i) kinetics of biosensing layer formation, which was based on the immobilization of fragmented and intact antibodies, and (ii) kinetics of antigen interaction with the immobilized antibodies. It has been demonstrated that ellipsometric parameter Δ(t) showed much higher sensitivity at the initial phase of Au-protein and protein-protein interaction, while the parameter Ψ(t) was more sensitive when the steady-state conditions were established. A new method, which taking into consideration this feature and nonlinear change of Δ(t) and Ψ(t) parameters during various stages of biological layer formation process, was used for the calculation of antibody and antigen adsorption/interaction kinetics. The obtained results were analyzed using a model, which took into account partial reversibility during the formation of both antibody and antigen based monolayers. It was shown that the immobilization rate of antibody during the preparation of the sensing layer was similar for the formation of both intact and fragmented antibody based layers; however, the residence time was 25 times longer for intact antibody based layer formation in comparison to that of fragmented antibody based layer formation. On the contrary, residence time of antigen interaction with immobilized antibodies was about 8 times longer for the sensor based on fragmented antibodies. Moreover, it has been determined that the structural differences of immobilized antibodies (fragmented or intact) significantly influence antibody-antigen interaction rate, the major difference being in the residence time of antigen interaction with both types of immobilized antibodies.