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.

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.

Ultrasensitive SPR detection of miRNA-93 using antibody-enhanced and enzymatic signal amplification.

MiRNAs are endogenous noncoding RNA molecules. They play important gene-regulatory roles by binding to the mRNA of target genes thereby leading to either transcript degradation or translational repression. In virtually all diseases, distinct alterations of miRNA expression profiles have been found thus suggesting miRNAs as interesting biomarkers. Here, we present an SPR biosensor that utilizes disposable, injection-molded sensor chip/microfluidic hybrids combined with a lateral imaging optical system for parallel analysis of three one-dimensional spot arrays to detect miRNA-93. To increase the sensitivity of the biosensor we used two different amplification strategies. By adding an RNA-DNA-hybrid antibody for primary signal amplification, a limit of detection of 10 pmol/L was achieved. Based on that method we demonstrate the detection of miRNA-93 in total RNA lysate from HEK-293 cells. Utilizing an enzymatic signal amplification with Poly(A) polymerase, the sensitivity could be increased even further leading to a limit of detection of 1 fmol/L.

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.

Optimization of angularly resolved Bloch surface wave biosensors.

Bloch surface wave (BSW) sensors to be used in biochemical analytics are discussed in angularly resolved detection mode and are compared to surface plasmon resonance (SPR) sensors. BSW supported at the surface of a dielectric thin film stack feature many degrees of design freedom that enable tuning of resonance properties. In order to obtain a figure of merit for such optimization, the measurement uncertainty depending on resonance width and depth is deduced from different numerical models. This yields a limit of detection which depends on the sensor's free measurement range and which is compared to a figure of merit derived previously. Stack design is illustrated for a BSW supporting thin film stack and is compared to the performance of a gold thin film for SPR sensing. Maximum sensitivity is obtained for a variety of stacks with the resonance positioned slightly above the TIR critical angle. Very narrow resonance widths of BSW sensors require sufficient sampling but are also associated with long surface wave propagation lengths as the limiting parameter for the performance of this kind of sensors.

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.

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.

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.

'Phytochip': on-chip detection of phytopathogenic RNA viruses by a new surface plasmon resonance platform.

The surface plasmon resonance (SPR) based 'Phytochip' was developed to distinguish virus-infected plants from non-infected plants. The system detects DNA-RNA hybridization to show the presence of phytopathogenic viruses such as the RNA virus barley stripe mosaic virus (BSMV) in wheat leaves. To achieve this BSMV and wheat specific oligonucleotides, and a negative control yeast oligonucleotide, were immobilized on a SPR gold surface chip. After optimization of the hybridization parameters with purified wheat samples, wheat infected with BSMV resulted in detectable signals with both the BSMV and the wheat probes. In contrast, a hybridization reaction was not be detected with the negative probe. The method is fast and sensitive with a detection time of 3000s (50min), a detection limit of 14.7pgµl(-1) BSMV RNA and a measuring range of 14.7-84pgµl(-1) BSMV RNA (1.323-7.56ng BSMV RNA per 90µl sample). These characteristics, combined with the high throughput design, make it suitable for application in plant breeding and virus control.

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.

Hydrogenated amorphous silicon nitride photonic crystals for improved-performance surface electromagnetic wave biosensors.

We exploit the properties of surface electromagnetic waves propagating at the surface of finite one dimensional photonic crystals to improve the performance of optical biosensors with respect to the standard surface plasmon resonance approach. We demonstrate that the hydrogenated amorphous silicon nitride technology is a versatile platform for fabricating one dimensional photonic crystals with any desirable design and operating in a wide wavelength range, from the visible to the near infrared. We prepared sensors based on photonic crystals sustaining either guided modes or surface electromagnetic waves, also known as Bloch surface waves. We carried out for the first time a direct experimental comparison of their sensitivity and figure of merit with surface plasmon polaritons on metal layers, by making use of a commercial surface plasmon resonance instrument that was slightly adapted for the experiments. Our measurements demonstrate that the Bloch surface waves on silicon nitride photonic crystals outperform surface plasmon polaritons by a factor 1.3 in terms of figure of merit.

Detection of sub-10 nm emission profile features in organic light-emitting diodes using destructive interference.

The position of light-emitting molecules can be identified using interferometric approaches. Standard schemes utilize constructive interference to obtain a sectioned area of interest with high detection efficiency. The examination of organic light-emitting diodes (OLED) removes the common constraint of low light levels and enables a more generalized analysis. The OLED emitters are located in the front of a metal mirror, giving rise to an approximate two-wave fringe pattern in the far field. It is demonstrated theoretically and experimentally that positions around the field nodes enable the extraction of emitter distribution details within an electroluminescent layer of only 10 nm thickness.