Direct competitive assay for HER2 detection in human plasma using Bloch surface wave-based biosensors.

The overexpression and/or amplification of the HER2/neu oncogene has been proposed as a prognostic marker in breast cancer. The detection of the related peptide HER2 remains a grand challenge in cancer diagnosis and for therapeutic decision-making. Here, we used a biosensing device based on Bloch Surface Waves excited on a one-dimensional photonic crystal (1DPC) as valid alternative to standard techniques. The 1DPC was optimized to operate in the visible spectrum and the biosensor optics has been designed to combine label-free and fluorescence operation modes. This feature enables a real-time monitoring of a direct competitive assay using detection mAbs conjugated with quantum dots for an accurate discrimination in fluorescence mode between HER2-positive/negative human plasma samples. Such a competitive assay was implemented using patterned alternating areas where HER2-Fc chimera and reference molecules were bio-conjugated and monitored in a multiplexed way. By combining Label-Free and fluorescence detection analysis, we were able to tune the parameters of the assay and provide an HER2 detection in human plasma in less than 20 min, allowing for a cost-effective assay and rapid turnaround time. The proposed approach offers a promising technique capable of performing combined label-free and fluorescence detection for both diagnosis and therapeutic monitoring of diseases.

Enhanced fluorescence detection of miRNA by means of Bloch surface wave-based biochips.

We report on the use of biochips based on one-dimensional photonic crystals sustaining Bloch surface waves to specifically detect target miRNA that is characteristic of hemorrhagic stroke (miR-16-5p) at low concentration in a buffer solution. The biochips were functionalized with streptavidin and ssDNA oligonucleotides to enable miRNA detection. To discriminate the target miRNA from a non-specific control (miR-101a-3p), we made use of an optical platform developed to work both in label-free and fluorescence detection modes. We demonstrate that the limit of detection provided when operating in the fluorescence mode allows us to specifically detect the target miRNA down to 1 ng mL-1 (140 pM), which matches the recommendations for diagnostic miRNA assays, 5 ng mL-1. The low costs open the way towards the application of these disposable optical biochips based on 1DPC sustaining Bloch surface waves as a promising tool for early disease detection in a liquid biopsy format.

Spectral analysis of organic LED emitters' orientation in thin layers by resonant emission on dielectric stacks.

Purposely tailored thin film stacks sustaining surface waves have been utilized to create a unique link between emission angle and wavelength of fluorescent dye molecules. The knowledge of the thin film stack's properties allows us to derive the intrinsically emitted luminescence spectrum as well as to gain information about the orientation of fluorophores from angularly resolved experiments. This corresponds to replacing all the equipment necessary for polarized spectroscopy with a single smart thin film stack, potentially enabling single shot analyses in the future. The experimental results agree well with those from other established techniques, when analyzing the Rubrene derivative in a 2,4,6-tris(biphenyl-3-yl)-1,3,5-triazine (T2T) host used for the fabrication of optimized organic light-emitting diodes. The findings illustrate how resonant layered stacks can be applied to integrated spectroscopic analyses.

Bioassay engineering: a combined label-free and fluorescence approach to optimize HER2 detection in complex biological media.

We report on the combined label-free/fluorescence use of one-dimensional photonic crystals to optimize cancer biomarker detection in complex biological media. The optimization of the assay working parameters permits us to maximize the final response of the biosensor. The detection approach utilizes a sandwich assay, in which one-dimensional photonic crystals sustaining Bloch surface waves are modified with monoclonal antibodies in order to guarantee high specificity during biological recognition. The multiple outcomes generated by such optimization experiments permitted us to determine the effective capture efficiency and the repeatability of the immobilization process, which was estimated to be close to 5%. By exploiting the resolution of the fluorescence operation mode, we studied non-specific interactions in different blocking agents, different analyte diluting buffers, and diverse concentrations of the detection antibody. As a clinically relevant biomarker, we selected the trans-membrane receptor tyrosine kinase HER2. HER2 regulates a variety of cell proliferation, growth, and differentiation pathways and its over-expression occurs in approximately 20-30% of breast cancer worldwide. As a final application, we transferred all the optimized working parameters to HER2 cancer biomarker assays in a complex biological environment. The label-free and fluorescence results obtained by analyzing MCF-7 (HER2 low positive) and 32D (HER2 negative) cell lysates demonstrate that we can successfully discriminate the two lysates.

Ultraviolet-transparent low-index layers for antireflective coatings.

Nanostructured low-index layers are useful as the last layers of antireflective (AR) coatings because they can broaden their spectral ranges and improve the performance for oblique light incidence. Structuring of evaporated organic layers by plasma opens a route to produce inorganic interference stacks and low-index layers in the same vacuum process. The organic material uracil has been investigated as a template material for AR nanostructures. An additional plasma-treatment step was added to the manufacturing process, which decreases the organic fraction of the coating substantially. As a result, a better environmental stability and higher transmission in the ultraviolet range was achieved.

Vortex Beam Generation by Spin-Orbit Interaction with Bloch Surface Waves.

Axis-symmetric grooves milled in metallic slabs have been demonstrated to promote the transfer of Orbital Angular Momentum (OAM) from far- to near-field and vice versa, thanks to spin-orbit coupling effects involving Surface Plasmons (SP). However, the high absorption losses and the polarization constraints, which are intrinsic in plasmonic structures, limit their effectiveness for applications in the visible spectrum, particularly if emitters located in close proximity to the metallic surface are concerned. Here, an alternative mechanism for vortex beam generation is presented, wherein a free-space radiation possessing OAM is obtained by diffraction of Bloch Surface Waves (BSWs) on a dielectric multilayer. A circularly polarized laser beam is tightly focused on the multilayer surface by means of an immersion optics, such that TE-polarized BSWs are launched radially from the focused spot. While propagating on the multilayer surface, BSWs exhibit a spiral-like wavefront due to the Spin-Orbit Interaction (SOI). A spiral grating surrounding the illumination area provides for the BSW diffraction out-of-plane and imparts an additional azimuthal geometric phase distribution defined by the topological charge of the spiral structure. At infinity, the constructive interference results into free-space beams with defined combinations of polarization and OAM satisfying the conservation of the Total Angular Momentum, based on the incident polarization handedness and the spiral grating topological charge. As an extension of this concept, chiral diffractive structures for BSWs can be used in combination with surface cavities hosting light sources therein.

Label-Free Monitoring of Human IgG/Anti-IgG Recognition Using Bloch Surface Waves on 1D Photonic Crystals.

Optical biosensors based on one-dimensional photonic crystals sustaining Bloch surface waves are proposed to study antibody interactions and perform affinity studies. The presented approach utilizes two types of different antibodies anchored at the sensitive area of a photonic crystal-based biosensor. Such a strategy allows for creating two or more on-chip regions with different biochemical features as well as studying the binding kinetics of biomolecules in real time. In particular, the proposed detection system shows an estimated limit of detection for the target antibody (anti-human IgG) smaller than 0.19 nM (28 ng/mL), corresponding to a minimum surface mass coverage of 10.3 ng/cm². Moreover, from the binding curves we successfully derived the equilibrium association and dissociation constants (KA = 7.5 × 107 M-1; KD = 13.26 nM) of the human IgG⁻anti-human IgG interaction.

Bloch surface wave enhanced biosensor for the direct detection of Angiopoietin-2 tumor biomarker in human plasma.

Quantitative detection of angiogenic biomarkers provides a powerful tool to diagnose cancers in early stages and to follow its progression during therapy. Conventional tests require trained personnel, dedicated laboratory equipment and are generally time-consuming. Herein, we propose our developed biosensing platform as a useful tool for a rapid determination of Angiopoietin-2 biomarker directly from patient plasma within 30 minutes, without any sample preparation or dilution. Bloch surface waves supported by one dimensional photonic crystal are exploited to enhance and redirect the fluorescence arising from a sandwich immunoassay that involves Angiopoietin-2. The sensing units consist of disposable and low-cost plastic biochips coated with the photonic crystal. The biosensing platform is demonstrated to detect Angiopoietin-2 in plasma samples at the clinically relevant concentration of 6 ng/mL, with an estimated limit of detection of approximately 1 ng/mL. This is the first Bloch surface wave based assay capable of detecting relevant concentrations of an angiogenic factor in plasma samples. The results obtained by the developed biosensing platform are in close agreement with enzyme-linked immunosorbent assays, demonstrating a good accuracy, and their repeatability showed acceptable relative variations.

Bloch Surface Waves Biosensors for High Sensitivity Detection of Soluble ERBB2 in a Complex Biological Environment.

We report on the use of one-dimensional photonic crystals to detect clinically relevant concentrations of the cancer biomarker ERBB2 in cell lysates. Overexpression of the ERBB2 protein is associated with aggressive breast cancer subtypes. To detect soluble ERBB2, we developed an optical set-up which operates in both label-free and fluorescence modes. The detection approach makes use of a sandwich assay, in which the one-dimensional photonic crystals sustaining Bloch surface waves are modified with monoclonal antibodies, in order to guarantee high specificity during the biological recognition. We present the results of exemplary protein G based label-free assays in complex biological matrices, reaching an estimated limit of detection of 0.5 ng/mL. On-chip and chip-to-chip variability of the results is addressed too, providing repeatability rates. Moreover, results on fluorescence operation demonstrate the capability to perform high sensitive cancer biomarker assays reaching a resolution of 0.6 ng/mL, without protein G assistance. The resolution obtained in both modes meets international guidelines and recommendations (15 ng/mL) for ERBB2 quantification assays, providing an alternative tool to phenotype and diagnose molecular cancer subtypes.

Design rules for combined label-free and fluorescence Bloch surface wave biosensors.

We report on the fabrication and physical characterization of optical biosensors implementing simultaneous label-free and fluorescence detection and taking advantage of the excitation of Bloch surface waves at a photonic crystal's truncation interface. Two types of purposely designed one-dimensional photonic crystals on molded organic substrates with micro-optics were fabricated. These crystals feature either high or low finesse of the Bloch surface wave resonances and were tested on the same optical readout system. The experimental results show that designing biochips with a large resonance quality factor does not necessarily lead in the real case to an improvement of the biosensor performance. The conditions for optimal biochip design and operation of the complete bio-sensing platform are established.

Detection of soluble ERBB2 in breast cancer cell lysates using a combined label-free/fluorescence platform based on Bloch surface waves.

We report on the use of one-dimensional photonic crystals to detect clinically relevant concentrations of ERBB2/neu/Her2 in cell lysates. ERBB2 is a pivotal breast cancer biomarker and targetable oncogenic driver associated with aggressive breast cancer subtypes. To quantitate soluble ERBB2, we developed an optical platform that combines label-free and fluorescence detection modes. Such platform makes use of a sandwich assay in which the one-dimensional photonic crystals sustaining Bloch surface waves are tailored with a monoclonal antibody for highly specific biological recognition (BSW biochip). In a second step, a second antibody to ERBB2 quantitatively detects the bound analyte. The strategy of the present approach takes advantage of the combination of label-free and fluorescence techniques, making bio-recognition more robust and sensitive. In the fluorescence operation mode, the platform can attain the limit of detection 0.3ng/mL (1.5pM) for ERBB2 in cell lysates. Such resolution meets the international guidelines and recommendations (15ng/mL) for diagnostic ERBB2 assays that in the future may help to more precisely assign therapies counteracting cancer cell proliferation and metastatic spread.

Mechanical, structural, and optical properties of PEALD metallic oxides for optical applications.

Structural, optical, and mechanical properties of Al2O3, SiO2, and HfO2 materials prepared by plasma-enhanced atomic layer deposition (PEALD) were investigated. Residual stress poses significant challenges for optical coatings since it may lead to mechanical failure, but in-depth understanding of these properties is still missing for PEALD coatings. The tensile stress of PEALD alumina films decreases with increasing deposition temperature and is approximately 100 MPa lower than the stress in thermally grown films. It was associated with incorporation of -OH groups in the film as measured by infrared spectroscopy. The tensile stress of hafnia PEALD layers increases with deposition temperature and was related to crystallization of the film. HfO2 nanocrystallites were observed even at 100°C deposition temperature with transmission electron microscopy. Stress in hafnia films can be reduced from approximately 650 MPA to approximately 450 MPa by incorporating ultrathin Al2O3 layers. PEALD silica layers have shown moderate stress values and stress relaxation with the storage time, which was correlated to water adsorption. A complex interference coating system for a dichroic mirror (DCM) at 355 nm wavelength was realized with a total coating thickness of approximately 2 µm. Severe cracking of the DCM coating was observed, and it propagates even into the substrate material, showing a good adhesion of the ALD films. The reflectance peak is above 99.6% despite the mechanical failure, and further optimization on the material properties should be carried out for demanding optical applications.

Effect of thickness disorder on the performance of photonic crystal surface wave sensors.

We investigated experimentally and numerically the robustness of optical sensors based on Bloch waves at the surface of periodic one-dimensional photonic crystals. The distributions of sensor characteristics caused by the fabrication uncertainties in dielectric layer thicknesses have been analyzed and robustness criteria have been set forth and discussed. We show that the performance of the surface wave sensors is sufficiently robust with respect to the changes of the photonic crystal layer thicknesses. Layer thickness optimization of the photonic crystal, carried out to achieve low limit of detection, leads to an improvement of the robustness of the surface wave sensors that is attributed to Bloch states lying deeper in the photonic band gap.

Angularly resolved ellipsometric optical biosensing by means of Bloch surface waves.

In label-free biosensing, a continuous improvement of the limit of detection is necessary to resolve the small change of the surface refractive index produced by interacting biomolecules at a very small concentration. In the present work, optical sensors based on one-dimensional photonic crystals supporting Bloch surface waves are proposed and adopted for label-free optical biosensing. We describe the implementation of an angularly resolved ellipsometric optical sensing scheme based on Bloch surface waves sustained by tantala/silica multilayers. The angular operation is obtained using a focused beam at fixed wavelength and detection of the angular reflectance spectrum by means of an array detector. The results show that the experimental limit of detection for a particular photonic crystal design is 6.5 × 10(-7) refractive index units (RIU)/Hz(1/2) and further decrease could be obtained. For the first time, we report on the practical application of this technique to a cancer biomarker protocol that aims at the detection of a specific glycoprotein (angiopoietin 2) involved in angiogenesis and inflammation processes.

Combining label-free and fluorescence operation of Bloch surface wave optical sensors.

We report on the design, fabrication, and characterization of optical sensors based on Bloch surface waves propagating at the truncation edge of one-dimensional photonic crystals. The sensors can be simultaneously operated in both a label-free mode, where small refractive index changes at the surface are detected, and a fluorescence mode, where the fluorescence from a novel heptamethyne dye label in the proximity of the surface is collected. The two modes operate in the near-infrared spectral range with the same configuration of the optical reading apparatus. The limit of detection is shown to be smaller than that of equivalent surface plasmon sensors and the fluorescence collection efficiency is such that it can be efficiently analyzed by the same camera sensor used for label-free operation.

Focusing and extraction of light mediated by Bloch surface waves.

The control of emission from localized light sources is an objective of outstanding relevance in nanophotonics. In a recent past, a large number of metallic nanostructures has been proposed to this end, wherein plasmonic modes are exploited as energy carriers on a subwavelength scale. As an interesting alternative, we present here the use of surface modes on patterned dielectric multilayers to deliver electromagnetic power from free-space to localized volumes and vice versa. Thanks to this low-loss energy transfer, proper periodic ring structures are shown to provide a subwavelength focusing of an external radiation onto the multilayer surface. By reciprocity, the radiated power from emitters within the ring center is shown to be efficiently beamed in the free-space, with a well-controlled angular divergence. This mechanism overcomes some important limitations involved in the all-plasmonic approach, while opening new opportunities for hybrid devices in photon management applications such as optical sensing and lighting.

A full ellipsometric approach to optical sensing with Bloch surface waves on photonic crystals.

We report on the investigation on the resolution of optical sensors exploiting Bloch surface waves sustained by one dimensional photonic crystals. A figure of merit is introduced to quantitatively assess the performance of such sensors and its dependency on the geometry and materials of the photonic crystal. We show that the figure of merit and the resolution can be improved by adopting a full ellipsometric phase-sensitive approach. The theoretical predictions are confirmed by experiments in which, for the first time, such type of sensors are operated in the full ellipsometric scheme.

Leakage radiation interference microscopy.

We present a proof of principle for a new imaging technique combining leakage radiation microscopy with high-resolution interference microscopy. By using oil immersion optics it is demonstrated that amplitude and phase can be retrieved from optical fields, which are evanescent in air. This technique is illustratively applied for mapping a surface mode propagating onto a planar dielectric multilayer on a thin glass substrate. The surface mode propagation constant estimated after Fourier transformation of the measured complex field is well matched with an independent measurement based on back focal plane imaging.

Probing losses of dielectric multilayers by means of Bloch surface waves.

We exploit the excitation of electromagnetic surface waves on high-quality dielectric multilayers to measure the very low extinction coefficient of the structures, with a resolution down to 4·10(-7) and in a simple optical configuration. The effect of exposition to a rhodamine 6G solution in water and ethanol is also reported, including dye adsorption in the layers and bleaching upon resonant excitation.

A fluorescent one-dimensional photonic crystal for label-free biosensing based on BLOCH surface waves.

A one-dimensional photonic crystal (1DPC) based on a planar stack of dielectric layers is used as an optical transducer for biosensing, upon the coupling of TE-polarized Bloch Surface Waves (BSW). The structure is tailored with a polymeric layer providing a chemical functionality facilitating the covalent binding of orienting proteins needed for a subsequent grafting of antibodies in an immunoassay detection scheme. The polymeric layer is impregnated with Cy3 dye, in such a way that the photonic structure can exhibit an emissive behavior. The BSW-coupled fluorescence shift is used as a means for detecting refractive index variations occurring at the 1DPC surface, according to a label-free concept. The proposed working principle is successfully demonstrated in real-time tracking of protein G covalent binding on the 1DPC surface within a fluidic cell.