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Here we study the analytical performance of label-free optical biosensors with respect to analyte-induced refractive index changes that can be measured by a biosensor (refractive index resolution). We present an analytical model that interrelates the refractive index resolution and the parameters of the optical platform of a biosensor. We demonstrate that the figure of merit (FOM), which has been widely used to design optical platforms of label-free optical biosensors, is not an appropriate metric to guide the design or predict the performance of label-free optical biosensors. Therefore, we propose an extended definition of FOM that addresses its limitations. We confirm the validity of the proposed approach by both numerical simulations and experiments. Finally, we show that the analytical model of the refractive index resolution not only makes it possible to predict the performance of a biosensor but also provides strategies for achieving optimal performance.

The nucleus-encoded 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid β peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17β-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17β-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction (ka 2.0 × 105 M1s−1, kd 5.8 × 104 s−1, and KD 3.5 × 10–10 M). In McGill-R-Thy1-APP rats compared to controls, levels of 17β-HSD10–cypD complexes were decreased and those of total amyloid β increased. Moreover, the levels of 17β-HSD10–cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls (the sensitivity of the complexes to AD dementia was 92.9%, that to FTLD 73.8%, the specificity to AD dementia equaled 91.7% in a comparison with the controls but only 26.2% with FTLD). Our results demonstrate the weakened ability of 17β-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid β. Levels of 17β-HSD10–cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.

This paper reviews fundamentals of optical affinity biosensors based on plasmonic nanostructures and discusses recent advances in the development of this technology, including plasmonic nanostructures and surface plasmon phenomena, advances in sensor instrumentation, and functional coatings. Examples of applications for both the detection of chemical and biological substances as well as the investigation of biomolecular interactions are also given.

This paper describes direct label-free detection of 17beta-hydroxysteroid dehydrogenase type 10 (17β-HSD10) using a surface plasmon resonance (SPR) biosensor. This multifunctional mitochondrial enzyme is involved in pathogenesis of Alzheimer disease (AD) and represents a potential target for AD diagnostics. A multichannel SPR sensor with spectral modulation is functionalized with amyloid beta or polyclonal antibody against 17β-HSD10 peptide using a self-assembled monolayer of alkylthiolates and amino coupling chemistry. Detection of a synthetic peptide of 14 amino acids from the 17β-HSD10 molecule (residues 133–146 aa) and whole enzyme 17β-HSD10 is performed. Detection of 17β-HSD10 enzyme in artificial cerebrospinal fluid (ACSF) buffer is also carried out. It is demonstrated that the reported SPR biosensor is capable of detecting 17β-HSD10 enzyme at ng/ml levels.

In this work, we present an antibody array for the detection of cancer biomarker candidates by a surface plasmon resonance (SPR) imaging sensor with polarization contrast. Responses from the SPR imaging sensor are shown to be similar to those from a conventional spectroscopy-based SPR sensor. Antibodies are spotted onto a self-assembled monolayer (SAM) composed of oligo(ethylene glycol) (OEG)-containing alkanethiol chains. Detection of two cancer biomarker candidates, activated leukocyte cell adhesion molecule/CD 166 (ALCAM) and transgelin-2 (TAGLN2), is demonstrated. Limits of detection for ALCAM and TAGLN2 are established at 6 ng/mL and 3 ng/mL, respectively, in buffer. No cross-reactivity is observed between immobilized antibodies and nonspecific antigen. Biomarker candidates are also detected in a 10% human serum solution.

In this work, we present a novel surface and assay for the simultaneous detection of DNA and protein analytes on a surface plasmon resonance (SPR) imaging sensor. A mixed DNA/oligo (ethylene glycol) (OEG) self-assembled monolayer (SAM) is created using a microarrayer. Thiol-modified single-stranded DNA sequences are spotted onto a gold-coated glass substrate. Backfilling with an OEG-modified alkanethiol creates a protein-resistant surface background. Antibodies conjugated to complementary single-stranded DNA sequences are immobilized on the surface through DNA hybridization. By converting only part of the DNA array into a protein array, simultaneous detections of DNA and protein analytes are possible. A model system of two cDNA sequences and two human pregnancy hormones are used to demonstrate the assay. No cross-reactivity was observed between DNA or protein analytes and nontargeted immobilized cDNA sequence or antibodies. A response from a detection of a single analyte in a mixture of protein and DNA analytes corresponds well with that of a single-analyte solution.

We have examined the noise sources for a surface plasmon resonance (SPR) sensor system to facilitate optimization of SPR sensor instrumentation and data-processing methods for high-resolution SPR sensing. We found detector shot noise to be the dominant source of noise in the SPR sensor, and investigated the propagation of noise through a commonly used SPR sensor data-processing algorithm. Deriving an expression relating the detector noise level to the noise of the output data, we used measurements of the magnitude and distribution of both charge-coupled device and photodiode array detector noise to predict the noise levels observed in the sensor output. We used this method to optimize the parameters of the algorithm and introduced a modified algorithm with enhanced resistance to correlated light level noise. In addition, we evaluated the noise performance of alternative SPR sensor designs, including a sensor with no polarization optics and another with optics producing an inverted SPR absorption feature. Experimental SPR sensor data demonstrated good agreement with predicted noise levels.

Theoretical analysis of the sensitivity of surface plasmon resonance (SPR) sensors with spectral interrogation is presented. Two basic configurations of SPR sensors with spectral interrogation are investigated—for the measurement of variations in the refractive index of bulk media, and for the monitoring of variations in the thickness of thin films. In both cases, analytical expressions allowing the sensitivity of SPR sensors to be calculated are derived and validated. On the basis of the theoretical analysis, the optimization of a spectral SPR sensor in terms of the operation wavelength and the choice of metal layer is carried out. It is demonstrated that spectral SPR sensors may attain higher sensitivity if operating at longer wavelengths and if using metal layers with a higher modulus of the real part of the dielectric constant.

Background: Insulin resistance, as seen in type 2 diabetes, has been shown to increase the risk of late onset Alzheimer’s Disease (AD) by as much as two-fold. In AD patients, characteristic findings are seen with structural magnetic resonance imaging (MRI) including global atrophy as well as regional reductions in gray matter volume (GMV) in the hippocampus, precuneus, posterior cingulate gyrus, parietotemporal, prefrontal, and orbitofrontal cortices. Additionally, negative correlations between peripheral insulin levels and regional GMV are seen in insulin resistant subjects in the middle temporal gyrus.The current study investigated whether elevated fasting glucose (FSG) levels were associated with lower GMV in brain areas that have been preferentially affected by AD. This association difference was further investigated between carrier and non-carrier (NC) groups of the apolipoprotein E (APOE) e4 allele, a genetic risk factor for late onset AD. Methods: Statistical Parametric Mapping (SPM), an automated brain imaging analysis computer package, was used to spatially normalize individual brains to a standard brain template, to obtain GMV maps from structural MRI, and to examine the correlations between higher FSG levels and lower structural MRI GMV measurements in 118 cognitively normal, non-diabetic individuals 6466 years of age (59e4 carriers and 59 NCs). A significance threshold of p 1⁄4 .005 was set for all imaging related analyses. Results: As predicted, significant correlations were seen between higher levels of FSG and lower GMV in AD-affected regions including the left inferior orbitofrontal cortex, parietal, and occipital lobes and the left and right frontal and temporal lobes. Negative correlations between FSG and regional GMV in the middle temporal gyri confirmed findings seen in insulin resistant subjects. Additionally, these associations in AD-related areas are seen in both carriers and NC. Conclusions: Higher FSG levels in cognitively normal, non-diabetic, older adults are associated with lower GMV in AD related brain areas, confirming that elevated FSG may be associated with AD risk, independent of APOEe4 status. Lastly, this study encourages the consideration of elevated FSG and other indicators of glucose control as targets for AD prevention trials, complementing the findings previously established with fluorodeoxyglucose positron emission tomography neuroimaging.

We report a system, which combines electrochemical and surface plasmon resonance (SPR) techniques on the same sensing chip. Each channel of a four-channel laboratory SPR sensor is supplemented with two planar gold electrodes (the reference and the counter electrodes), whereas the gold layer of SPR chip is used as the working electrode. A custom electronics enables to set an arbitrary potential between the reference and working electrodes and to measure the current flow between the counter and the working electrodes. Information from standard electrochemical techniques, i.e. cyclovoltammetry and chronoamperometry can be acquired with the system while simultaneously monitoring the shift in the surface plasmon resonance. The electrochemical SPR biosensor was used to study desorption of thiolated DNA probes with a negative potential. By comparing the acquired electrochemical and SPR signals, we show that DNA probes as well as a monolayer of alkanethiols can be desorbed by applying negative potentials to the SPR chip surface. Moreover, it is shown that the DNA probes can be reabsorbed on the SPR sensor surface and the complementary DNA can be detected without loss in detection sensitivity.

It is suggested that intracellular tau protein (τ), when released extracellularly upon neuron degeneration, could evoke direct toxic effects on the cholinergic neurotransmitter system through muscarinic receptors and thus contribute to the pathogenesis of Alzheimer's disease. In this study, we evaluated the in vitro effects of six naturally occurring monomeric τ isoforms on rat hippocampal synaptosomal choline transporters CHT1 (large transmembrane proteins associated with high-affinity choline transport and vulnerable to actions of amyloid β peptides (Aβ) applied in vitro or in vivo). Some τ isoforms at nM concentrations inhibited choline transport in a dose- and time-dependent saturable manner (352 = 441 > 410 = 383 > 381 = 412) and effects were associated with changes in the Michaelis constant rather than in maximal velocity. Moreover, the actions of τ 352/441 were not influenced by previous depolarisation of synaptosomes or by previous depletion of membrane cholesterol. Specific binding of [3H]hemicholinium-3 was not significantly altered by τ 352/441 at higher nM concentrations. Results of in vitro tests on CHT1 transporters from cholesterol-depleted synaptosomes supported interactions between Aβ 1-40 and τ 352. In addition, we developed surface plasmon resonance biosensors to monitor complexes of Aβ 1-42 and τ 352 using a sandwich detection format. It seems, therefore, that protein τ, similar to Aβ peptides, can contribute to the pathogenesis of Alzheimer's disease through its actions on CHT1 transporters. However, the interaction mechanisms are quite different (τ probably exerts its effects through direct interactions of microtubule binding repeats with extracellular portions of the CHT1 protein without influencing the choline recognition site, Aβ rather through lipid rafts in the surrounding membranes). An N-terminal insert of τ is not necessary but the N-terminal projection domain plays a role. The developed biosensor will be used to detect Aβ-τ complexes in cerebrospinal fluid in order to evaluate them as prospective biomarkers of Alzheimer's disease.

Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders characterized by a high risk of evolution into acute myeloid leukemia (AML). Different processes are involved in its pathogenesis, such as (epi)genetic alterations and immunological dysfunctions. Till now, in plasma several protein markers for MDS were found. One of them is a misfolded receptor protein VEGFR-1 for which we have created a detection assay based on the surface plasmon resonance (SPR) (Pimkova K, Suttnar J, et al., Anal Bioanal Chem. 2012, 402, 381-387). An elevated level of circulating 70-kDa heat shock proteins (Hsp70) has been detected in the blood of patients with myeloid leukemia. The Hsp70 are ATP-dependent molecular chaperones which interacts with unfolded/misfolded and aggregated proteins, thus providing cytoprotective role against various cellular stresses. Hsp70 released from cancer cells can carry peptide antigens including ones specific for a tumour and contribute to the development of anti-cancer immune response. The goal of this study is to identify proteins in HSP70 complexes using SPR biosensor followed by mass spectroscopy (MS) in the blood plasma of patients from MDS subgroups. Methods A six-channel spectroscopic SPR biosensor for detection of protein comlexes in diluted blood plasma samples was used. Preincubated ATP-Hsp70 complex was immobilized on the sensor surface via covalent attachment by dispersionless microfluidics. The biosensor utilizes Hsp70 rate-limiting step of ATPase cycle, ADP/ATP exchange, which allows in an ATP-controlled manner binding of protein substrates and their elution with minimal non specificity. Eluted proteins were subsequently analyzed and identified by using LC-MS/MS (TripleTOF 5600). Patients of MDS (n=10) and MDS patients who progress to AML (n=5) were chosen. Results and Conclusion Using preincubated ATP-Hsp70 complex significant differences in SPR responses of MDS patients in comparison to healthy donors as well as differences among MDS patients were observed. The responses represented hundreds of proteins identified by mass spectrometry. Protein interaction networks were visualized and analyzed by bioinformatics tools which cluster proteins into groups that share the same biological function, are similarly localized in the cell, or are known to be a part of a complex. The identified proteins are known to be involved in several processes including ubiquitinylation, protein degradation, cell signaling, stress response, protein synthesis, and also regulation of the immune system. We used Power Graphs, a novel representation of (protein) networks which provided a valuable insight into the presence of protein complexes, their internal organization and relationships. Interaction networks also indicated possible proteins involved in immunomodulatory functions of MDS. The results showed that SPR biosensors are a promising tool for the diagnosis and follow-up efficiency treatment in complex heterogeneous malignancies. Disclosures No relevant conflicts of interest to declare.

Introduction Myelodysplastic syndromes are clonal hematopoetic stem cell disorders characterized by refractory cytopenias with displasia as a result of ineffective hematopoiesis. Approximately 30 percent of MDS cases progress to acute myeloid leukemia. Excess blasts are the strongest predictors for poor outcome and are associated with disease progression. Because of its heterogeneity and lack of molecular and protein markers that effectively monitor disease progression, clinical management of MDS patients is challenging. The concentrations of several proteins were found elevated during MDS disease. Their interaction with receptors/ligands is a part of signaling pathways that may play a crucial role in pathogenesis of the disease. Serum levels of vascular cell adhesion protein 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) were found higher in MDS patients in comparison with control group and were related to MDS severity. Alpha-2-HS-glycoprotein (FETUA) was analyzed in plasma of MDS patients. Leucine-rich alpha-2-glycoprotein (LRG) was found increased in RAEB-I and RAEB-II patients by proteomic studies. In this work we developed a protein chip for study of molecular interactions in plasma of MDS patients using surface plasmon resonance (SPR) imaging. Interacting proteins were analyzed and identified using LC-MS/MS. Methods Patients with diagnosis of refractory anemia and refractory anemia with ringed sideroblasts (RA/RARS, n=13), refractory cytopenia with multilineage dysplasia (RCMD, n=16), and refractory anemia with excess blasts (RAEB, n=15) as a subgroup of MDS, and MDS patients who progress to acute myeloid leukemia (AML, n=8) were chosen for this study. The high-resolution SPR imaging (SPRi) system with polarization contrast and internal referencing combined with dispersionless microfluidics was used to study protein interactions. Passive mixing structures were employed to further improve the sensing performance of this SPRi biosensor. The respective biomarkers (ICAM1, VCAM1, FETUA and LRG) were covalently attached to the functionalized sensor surface and interaction with their counterparts in MDS plasma samples were monitored. Interacting proteins were removed from the chip surface by diluted NaOH, treated by trypsin, analyzed by nanoLC-MS/MS (TripleTOF 5600) and identified using ProteinPilot database. Results and Conclusion Significant differences were observed between analyzed MDS subtypes for VCAM1, FETUA, and LRG (ANOVA: P = 0,00755, P = 0,0306 and P < 0.001 respectively). Using post hoc Tukey contrasts tests we ascertained that sensor responses were significantly higher in group RCMD and AML with respect to the healthy donors for immobilized VCAM1 and in group RA/RARS, RCMD, RAEB and AML with respect to the healthy donors for LRG. Moreover sensor responses were significantly higher in group AML with respect to the RA, RCMD or RAEB for immobilized LRG. Based on our results LRG seems to be a suitable marker of MDS progression. The identified proteins presented in MDS plasma samples unlike in healthy donor plasma are clusterin, protein S100-A8, serum amyloid A-1 protein, annexin A2, and zinc-alpha-2-glycoprotein among others. S100-A8 and clusterin have been connected with MDS or AML previously; it could be tested as a new protein marker of MDS by SPRi. The results show that SPR biosensors combined with nanoLC-MS/MS are a promising tool for improvement of the diagnosis in complex heterogeneous malignancies. Disclosures No relevant conflicts of interest to declare.

A finite element method based on the full-vectorial H-field formulation has been employed to achieve the maximum field penetration in the sensing medium of the slot-waveguide-based ring resonator biosensor. The use of nanometer scale guiding structure where optical mode is confined in a low-index region permits a very compact sensor with high optical intensity in the region, which makes it possible to detect minimum refractive index change, and offers higher sensitivities. We analyze the change in effective refractive index of mode, sensitivity, and power confinement of the proposed slot-waveguide-based ring resonator biosensor for the detection of DNA hybridization. The biosensor exhibited theoretical sensitivity of 856 nm per refractive index unit (RIU) and a detection limit of 1.43×10(-6) RIU.

Here we present a theoretical study of the sensing capability of a biosensor based on localized surface plasmons (LSP). The sensing structure consists of an array of gold cylinders which support coupling of LSP with Rayleigh anomalies, resulting in a narrow line spectrum. We utilize numerical simulations pertaining to both the photonic and masstransfer aspects of this sensor, where the sensor performance as a function of various fabrication parameters is investigated and compared with a conventional surface plasmon resonance sensor. The simulations predict an improvement in the limit of detection (LOD) of such a device.

Optical affinity biosensors are devices that incorporate a biomolecular recognition element (e.g. antibody) which specifically recognizes a particular analyte and an optical transduction system which allows quantification of the interaction between the analyte and the biomolecular recognition element. In the last decade we have witnessed development of numerous optical transduction methods, including both label-based methods such as fluorescence spectroscopy and label-free methods such as optical interferometry, spectroscopy of guided modes of optical waveguides, and surface plasmon resonance. Label-free optical biosensors present unique technology that enables direct observation of molecular interaction in real-time and offer benefits of rapid, sensitive and label-free detection of chemical and biological species with potential applications in numerous important areas including proteomics, medical diagnostics, environmental monitoring, food analysis, agriculture, and security.

This contribution reviews the present state of the art in the development of surface plasmon resonance (SPR) (bio)sensor technology, discusses emerging trends, and presents recent results of research into SPR biosensors at the Institute of Photonics and Electronics, Prague. The developments discussed in detail include a high-performance SPR sensor for parallelized observation of biomolecular interactions, a miniature fiber optic SPR sensor for localized measurements, and sensing based on localized surface plasmons on gold nanoparticles.

We report a SPR biosensor based on long-range (LR) and short-range (SR) surface plasmon (SP) modes excited simultaneously on a diffraction grating. Employing the LRSP and SRSP in the grating-coupled SPR sensor offers several interesting features such as extended probe depth of the LRSP and ability to distinguish sensor response caused by bulk and surface refractive index changes. Prototype device based on wavelength interrogation of SPs was developed and be tested in model refractometric experiment. This paper presents results of theoretical analysis and experimental characterization of the sensor. Sensitivity of the laboratory prototype of the sensor agrees well with the theory. The sensor is shown to be able to detect changes in the refractive index as small as 3.5 x 10-6 RIU (refractive index unit).

We report a surface plasmon resonance (SPR) sensor based on two-plasmon-spectroscopy on a special bi-diffractive grating and investigate its ability to distinguish contributions to sensor response due to surface refractive index changes (i.e. binding) and due to refractive index changes in the whole sample. Theoretical analysis yielding an estimate of an error of such decomposition is presented and used to compare performance of this sensor to that of an alternative approach based on simultaneous excitation of short-range and long-range surface plasmons on a thin metallic layer.

We have optimized surface plasmon resonance (SPR) biosensor technology for a rapid, direct, and low-consumption label-free multianalyte screening of synthetic oligonucleotides (ONs) with modified internucleotide linkages potentially applicable in antisense therapy. Monitoring of the ONs hybridization is based on the formation of complex between the natural oligonucleotide probe immobilized on the sensor surface and the ON in solution in contact with the sensor surface. An immobilization chemistry utilizing the streptavidin-biotin interaction was employed to obtain desired ligand density and high hybridization efficiency. It was demonstrated that the sensor is capable of detecting complementary 23-mer ONs in concentrations as low as 0.1 nM with high specificity and reproducibility.

A stable and versatile biosensor surface is prepared by site-directed immobilization of protein-DNA conjugates onto a mixed self-assembled monolayer (SAM) composed of ssDNA thiols and oligo(ethylene glycol) (OEG) terminated thiols. The protein conjugates consist of an antibody chemically linked to a ssDNA target with a sequence complementary to the surface-bound ssDNA probes and are immobilized on the surface via sequence-specific hybridization. Compared to standard antibody immobilization techniques, this approach offers many advantages. The exceptional specificity of DNA hybridization combined with the diversity of potential sequences makes this platform perfect for multichannel sensors. Once a surface is patterned with the appropriate probe sequences, sequence-specific hybridization will sort out the target conjugates and direct them to the appropriate spots on the surface. In addition, the DNA SAMs are very stable and well suited to recycling by dehybdridization of the conjugates from the surface-bound probes. In this work, we demonstrate the specificity, sensitivity, and convenience of using protein-DNA conjugates to convert a DNA/OEG SAM surface into a biosensor surface and apply this platform to the detection of human chorionic gonadotropin using surface plasmon resonance.

The simultaneous detection of multiple analytes is an important consideration for the advancement of biosensor technology. Currently, few sensor systems possess the capability to accurately and precisely detect multiple antigens. This work presents a simple approach for the functionalization of sensor surfaces suitable for multichannel detection. This approach utilizes self-assembled monolayer (SAM) chemistry to create a nonfouling, functional sensor platform based on biotinylated single-stranded DNA immobilized via a streptavidin bridge to a mixed SAM of biotinylated alkanethiol and oligo(ethylene glycol). Nonspecific binding is minimized with the nonfouling background of the sensor surface. A usable protein chip is generated by applying protein-DNA conjugates which are directed to specific sites on the sensor chip surface by utilizing the specificity of DNA hybridization. The described platform is demonstrated in a custom-built surface plasmon resonance biosensor. The detection capabilities of a sensor using this protein array have been characterized using human chorionic gonadotropin (hCG). The platform shows a higher sensitivity in detection of hCG than that observed using biotinylated antibodies. Results also show excellent specificity in protein immobilization to the proper locations in the array. The vast number of possible DNA sequences combine with the selectivity of base-pairing makes this platform an excellent candidate for a sensor capable of multichannel protein detection.

In this work, we studied guest-host interactions between various dye molecules and the fifth-generation poly(propylene imine) (PPI-5) dendrimers in aqueous solutions using a surface plasmon resonance (SPR) sensor. The effect of the properties of guest and host molecules (e.g., charge and shape) and media (e.g., pH and ion strength) on affinity between guest and host molecules was investigated. Based on an immobilized homogeneous monolayer of PPI-5 dendrimer tethered to carboxyl-terminal self-assembled monolayers, the adsorption behavior of a group of dye molecules in PPI-5 was obtained. Results show that the strong affinity of PPI-5 to Rose Bengal and erythrosine B is attributed to the good match in charge and shape between the cavities of the dendrimer and the dye molecules. Maximum adsorption around a pH value of 7 was observed. The kinetic behaviors of different dye molecules in dendrimers were also studied. A fundamental understanding of guest-host interactions in dendrimers will guide the design of new-generation sensors and drug delivery carriers.

Introduction The protein microarrays are becoming the leading technology in proteomic research area. They enable to implement both features of proteins that can be altered in disease as quantitative proteomics (levels in biological samples) as well as functional proteomics (determination of their selective interactions with other biomolecules). Protein microarray techniques such as sandwich immunoassays, antigen capture immunoassay and direct immunoassays use labeling and antibodies. However, these labels can interfere with the analyte binding site and thus affect protein activity. Using antibodies requires a prior knowledge of the studied proteins which is in the case of a heterogeneous disease with little knowledge in its proteomic field a huge disadvantage. Surface plasmon resonance (SPR) is a label-free and direct method allowing quantification as well as monitoring of protein-protein interactions simultaneously and in real time. Myelodysplastic syndrome (MDS) is a heterogeneous group of hematological malignancies. It affects pluripotent hematopoietic stem cell and is manifested by variety of clinical symptoms according to predominant involvement of development lineage. The high risk of MDS to transform into acute myeloid leukemia makes it a suitable model for study of biological processes leading to leukemia development. In this work, we use SPR imaging for simultaneous screening of blood plasma of MDS patients followed by mass spectrometry (MS) for identification of interacting partners and analysis of protein network properties. Proteins, whose levels are either elevated during MDS disease or interaction with their receptors/ligands is a part of signaling pathway, were employed. Method SPR imaging system with polarization contrast and internal referencing was combined with dispersionless microfluidics for parallel screening of blood plasma samples. Proteins involved in pathogenesis of MDS and their physiological counterparts were immobilized under flow to create 6x6 sensing spots. Specifically, these include integrin αMβ2 (LFA1), intercellular adhesion molecule 1 (ICAM1), integrin α4β1 (VLA4), vascular cell adhesion protein 1 (VCAM1) and cytotoxic T-lymphocyte protein 4 (CTLA4). The sensor surface functionalization was optimized with respect to its ability to provide a low-fouling sensing surface with biologically active receptors. Plasma samples of controls and MDS patients were flowed along the functionalized surface and differences in individual interactions were evaluated. Selected interacting partners were further identified using 2D-HPLC/ESI-MS/MS. Identified proteins were analyzed by String Networks and Power Graph Analysis. Results and Conclusion Significant differences in the protein profiles among different MDS groups of patients as well as relative to control healthy donors were observed using SPR imaging; tens of interacting proteins were identified by mass spectrometry. Protein interaction networks were explored through clustering of proteins into groups that share the same biological function, are similarly localized in the cell, or are known to be a part of a complex. Identified proteins are involved in several processes; regulation of immune system, ubiquitinylation and protein degradation, cell signaling, hemostasis, protein synthesis, cell adhesion, metastasis, and inhibition of blood coagulation. Using of Power Graphs, a novel representation of (protein) networks, provided valuable insight into the existence of protein complexes, their internal organization, and their relationships. Interaction networks also indicated possible pathways involved in MDS pathogenesis (especially Src tyrosine kinases). The results showed that SPR biosensors are a promising tool for the diagnosis and follow-up efficiency treatment in complex heterogeneous malignancies. Disclosures No relevant conflicts of interest to declare.

We present an optical sensor based on excitation of surface plasma waves in optical fiber structure consisting of a side-polished single-mode polarization-maintaining fiber and a metal overlayer. We describe two modes of operation of the sensor in which variations in the refractive index of the sample are determined by measuring changes in the transmitted optical power at a fixed wavelength (amplitude mode) and by measuring changes in the wavelength at which the resonant attenuation of the fiber mode occurs (spectral mode). We demonstrate that this design allows suppressing sensitivity of the sensor to deformation of the fiber yielding an improved stability and resolution.

We present a novel surface plasmon resonance (SPR) sensor based on serial organization of sensing channels and sequential spectral encoding of responses from sensing channels into an optical spectrum. This approach allows probing interfacial processes by surface plasmons of different field profiles which makes it possible to distinguish surface and bulk contributions to SPR sensor response. We illustrate this unique feature of the presented approach in a model biosensing experiment in which the detection of human chorigonadotropin (hCG) is compensated for background refractive index interference.

A novel approach to multichannel SPR sensing based on encoding information from separate sensing channels into a single optical spectrum is presented. A dual channel SPR sensor using this approach is demonstrated. Attention is given to exploitation of the dual-channel SPR sensor for compensation for background interference and non-specific adsorption of the biomolecules to the surface of the SPR biosensor. Experimental results indicate that background refractive index changes were compensated with accuracy better than 8 X 10-5 RIU (refractive index unit); the effect of a temperature change of 3.6 K was reduced by a factor of 13 by the dual-channel sensor. SPR biosensor-based detection of monoclonal anti-dinitrophenyl antibody (a-DNP) with compensation for non-specific adsorption is demonstrated.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

A novel design of surface plasmon resonance fiber optic sensor is reported which leads to a compact, highly miniaturized sensing element with excellent sensitivity. The sensing device is based on a side-polished single-mode optical fiber with a thin metal overlayer supporting surface plasmon waves. The strength of interaction between a fiber mode and a surface plasmon wave depends strongly on the refractive index near the sensing surface. Therefore, refractive index changes associated with biospecific interaction between antibodies immobilized on the sensor and antigen molecules can be monitored by measuring light intensity variations. Detection of horse radish peroxidase (HRP) of the concentration of 100 ng/ml has been accomplished using the fiber optic sensor with a matrix of monoclonal antibodies against HRP immobilized on the sensor surface.

Optical waveguide surface plasmon resonance (SPR) sensors are presented which exploit the phenomenon of the resonant excitation of surface plasmon waves by guided modes of optical fibers and integrated optical waveguides. Theoretical analysis of the SPR waveguide sensing structures is based on the mode expansion and propagation method. The integrated optical waveguide SPR sensor is composed of an optical waveguide fabricated by the K + --Na + ion exchange in a glass substrate and gold as a surface plasmon resonance overlayer. The optical fiber SPR sensor is realized on a side-polished single-mode optical fiber which is locally coated with a thin gold film. Experimental study of the realized samples of the sensors has shown that variations in the refractive index of the sensed medium below 5X10 -5 can be resolved with the sensors.

The exploitation of surface plasmon resonance (SPR) sensing principle for the study of formation of multilayers of human immunoglobulin (hlgG) and monoclonal antibody against human choriogonadotropin (a-hCG) as well as for monitoring immunoreaction between the immobilized a-hCG and human choriogonadotropin (hCG) is reported. It is demonstrated that even a very small attachment of proteins (due to the adsorption or the specific interaction) may be detected by the presented SPR sensor. An immobilization technique is described which allows to form stable multilayers of antibodies on the gold surface.

The multifunctional mitochondrial enzyme 17beta-hydroxysteroid dehydrogenase type 10 might play a role in the development of Alzheimer disease via its high-affinity binding to amyloid beta peptides and its neuronal over-expression. It is suggested that the cerebrospinal fluid levels of the enzyme, free or bound to amyloid beta peptides, are a potential specific biomarker of Alzheimer disease. However, mitochondrial dysfunction seems to play a role in many neurological diseases including multiple sclerosis. In this study, the specificity of changes in relation to the enzyme over-expression was evaluated using enzyme-linked immunosorbent and surface plasmon resonance sensors. The data indicated pronounced increases in the enzyme levels, specifically to 179% in multiple sclerosis and to 573% in Alzheimer disease when compared to the age-matched controls. Although the differences between both diseases were statistically significant, enzyme levels do not appear to be a highly specific biomarker of Alzheimer disease. On the other hand, enhancement in levels of the enzyme bound to amyloid beta peptides was only observed in people with Alzheimer disease, which suggests that the complex should be further considered as a possible biomarker. In patients with multiple sclerosis, our results are the first to demonstrate significant changes in enzyme expression and to suggest possible alterations in amyloid beta peptides.

A novel dual-channel surface plasmon resonance sensor is described which uses a high refractive index overlayer to spectrally separate sensing channels, allowing for their simultaneous interrogation using a single spectrograph. A theoretical analysis of the sensor is presented as well as experimental results demonstrating its capability as a biosensor.

We present a novel surface plasmon resonance (SPR) biosensor for detection of human interferon gamma (hIFNγ), an important diagnostic marker for several infectious diseases. The biosensor employs engineered proteins derived from albumin binding domain (ABD) and selected with ribosome display to provide high affinity towards hIFNγ. We demonstrate that the biosensor provides rapid, sensitive, and cost-effective platform for the detection of hIFNγ and allows its detection at nanomolar levels both in buffer and diluted human plasma.