Real-time Surface Plasmon Resonance Research Articles

Estimation of Serum YKL-40 by Real-Time Surface Plasmon Resonance Technology in North-Indian Asthma Patients.

Many studies reported for estimating serum YKL-40 using ELISA or RIA methods. This study introduces the plausible utilization of real-time surface plasmon resonance (SPR) technology in investigating the expression of serum YKL-40 protein levels and ELISA method for serum IgE in bronchial asthma. A commercially available BIAcore 2000 instrument, based on SPR technology, was utilized for assessing serum YKL-40 levels in a control sample size of 45 and active sample size of 97. Antibody immobilization was optimized to obtain the best sensor performance and a sensitive analytic detection. A commercially available ELISA kit was utilized for detecting serum IgE to estimate allergic condition-associated asthma. The results of SPR technology could distinctly classify with highly statistical significance, the asthma severities by estimating the elevated levels of YKL-40 in blood sera of minute quantities (up to 0.33 ng/ml), and thus differentiates superior utility in comparison with ELISA method. No statistically significant correlation of YKL-40 and IgE was observed. Serum YKL-40 may be used as a protein marker in classifying asthma severity by applying SPR technology as a reliable, label-free, highly sensitive, and cost-effective tool.

Feasibility Study of the Permeability and Uptake of Mesoporous Silica Nanoparticles across the Blood-Brain Barrier.

Drug delivery into the brain is impeded by the blood-brain-barrier (BBB) that filters out the vast majority of drugs after systemic administration. In this work, we assessed the transport, uptake and cytotoxicity of promising drug nanocarriers, mesoporous silica nanoparticles (MSNs), in in vitro models of the BBB. RBE4 rat brain endothelial cells and Madin-Darby canine kidney epithelial cells, strain II, were used as BBB models. We studied spherical and rod-shaped MSNs with the following modifications: bare MSNs and MSNs coated with a poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) block copolymer. In transport studies, MSNs showed low permeability, whereas the results of the cellular uptake studies suggest robust uptake of PEG-PEI-coated MSNs. None of the MSNs showed significant toxic effects in the cell viability studies. While the shape effect was detectable but small, especially in the real-time surface plasmon resonance measurements, coating with PEG-PEI copolymers clearly facilitated the uptake of MSNs. Finally, we evaluated the in vivo detectability of one of the best candidates, i.e. the copolymer-coated rod-shaped MSNs, by two-photon in vivo imaging in the brain vasculature. The particles were clearly detectable after intravenous injection and caused no damage to the BBB. Thus, when properly designed, the uptake of MSNs could potentially be utilized for the delivery of drugs into the brain via transcellular transport.

Estimation of Tau and Phosphorylated Tau181 in Serum of Alzheimer's Disease and Mild Cognitive Impairment Patients.

The elevated level of cerebrospinal fluid (CSF) Tau and phosphorylated Tau181 (p-Tau181) proteins are well established hallmarks of Alzheimer’s disease (AD). Elevated level of p-Tau181 can differentiate AD from other neurodegenerative disease. However, the expression level of these proteins in serum of AD patient is not well set up. This study sought to evaluate the level of Tau and p-Tau181 in serum of AD, and mild cognitive impairment (MCI) patients for an alternative approach to establish protein-based markers by convenient way. Blood samples were collected from 39 AD patients, 37 MCI patients and 37 elderly individuals as controls. The levels of Tau and p-Tau181 in the serum of the different groups were measured by label free real time Surface Plasmon Resonance technology by using specific antibodies, and were further confirmed by the conventional western blot method. An appropriate statistical analysis, including Receiver Operating Characteristic (ROC), was performed. The concentrations of serum Tau and p-Tau181 were significantly higher (p<0.00001) in AD (Tau; 47.49±9.00ng/μL, p-Tau181; 0.161±0.04 ng/μL) compared to MCI (Tau; 39.26±7.78 ng/μL, p-Tau181; 0.135±0.02 ng/μL) and were further higher compared to elderly controls (Tau; 34.92±6.58 ng/μL, p-Tau181; 0.122±0.01 ng/ μL). A significant (p<0.0001) downhill correlation was found between Tau as well as p-Tau181 levels with HMSE and MoCA score. This study for the first time reports the concentration of Tau and p-Tau181 in serum of AD and MCI patients. The cutoff values of Tau and p-Tau181 of AD and MCI patients with sensitivity and specificity reveal that serum level of these proteins can be used as a predictive marker for AD and MCI.

Measuring Protein Binding to Individual Hydrogel Nanoparticles with Single-Nanoparticle Surface Plasmon Resonance Imaging Microscopy

The specific binding and uptake of protein molecules to individual hydrogel nanoparticles is measured with real-time single-nanoparticle surface plasmon resonance imaging (SPRI) microscopy. Nanoparticles that adsorb onto chemically modified gold thin films interact with traveling surface plasmon polaritons and create individual point diffraction patterns in the SPRI microscopy differential reflectivity images. The intensity of each point diffraction pattern depends on the integrated refractive index of the nanoparticle; an increase in this single nanoparticle point diffraction intensity (Δ%RNP) is observed for nanoparticles that bind proteins. SPRI adsorption measurements can be used to measure an average increase in Δ%RNP that can be correlated with bulk dynamic light scattering measurements. Moreover, the distribution of Δ%RNP values observed for individual nanoparticles can be used to learn more about the nature of the protein–nanoparticle interaction. As a first example, the binding of the lectin Concanavalin A to 180 nm N-Isopropylacrylamide hydrogel nanoparticles that incorporate a small percentage of mannose sugar monomer units is characterized.

High-affinity Anticalins with aggregation-blocking activity directed against the Alzheimer β-amyloid peptide.

Amyloid beta (Aβ) peptides, in particular Aβ42 and Aβ40, exert neurotoxic effects and their overproduction leads to amyloid deposits in the brain, thus constituting an important biomolecular target for treatments of Alzheimer's disease (AD). We describe the engineering of cognate Anticalins as a novel typeof neutralizing protein reagent based on the human lipocalin scaffold. Phage display selection from a genetic random library comprising variants of the human lipocalin 2 (Lcn2) with mutations targeted at 20 exposed amino acid positions in the four loops that form the natural binding site was performed using both recombinant and synthetic target peptides and resulted in three different Anticalins. Biochemical characterization of the purified proteins produced by periplasmic secretion in Escherichia coli revealed high folding stability in a monomeric state, with Tm values ranging from 53.4°C to 74.5°C, as well as high affinities for Aβ40, between 95pM and 563pM, as measured by real-time surface plasmon resonance analysis. The central linear VFFAED epitope within the Aβ sequence was mapped using a synthetic peptide array on membranes and was shared by all three Anticalins, despite up to 13 mutual amino acid differences in their binding sites. All Anticalins had the ability-with varying extent-to inhibit Aβ aggregation invitro according to the thioflavin-T fluorescence assay and, furthermore, they abolished Aβ42-mediated toxicity in neuronal cell culture. Thus, these Anticalins provide not only useful protein reagents to study the molecular pathology of AD but they also show potential as alternative drug candidates compared with antibodies.

Protein immobilization and fluorescence quenching on polydopamine thin films

Mussel inspired polydopamine (PDA) film has attracted great interest as a versatile functional coating for biomolecule immobilization in various bio-related devices. However, the details regarding the interaction between a protein and PDA film remain unclear. Particularly, there is very limited knowledge regarding the protein immobilization on PDA film, even though it is of essential importance in various fields. The situation is even more complicated if considering the fact that quite a number of approaches (e.g., different oxidizing reagent, buffer pH, grown time, grown media, etc.) have been developed to grow PDA films. In this work, protein attachment on PDA film was systematically investigated by using the real-time and label-free surface plasmon resonance (SPR) technique. The kinetics of protein-PDA interaction was explored and the influence of buffer pH and deposition media on the protein attachment was studied. Fluorescent protein microarray was further printed on PDA-coated glass slides for quantitative investigations and together with SPR data, the interesting fluorescence quenching phenomenon of PDA film was revealed. This work may deepen our understanding on the PDA-protein interaction and offer a valuable guide for efficient protein attachment on PDA film in various bio-related applications.

Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.

Real-Time Surface Plasmon Resonance (SPR) for the Analysis of Interactions Between SUMO Traps and Mono- or PolySUMO Moieties.

Isolating endogenous SUMOylated proteins is a challenging task due to the high reversibility of this posttranslational modification. We have shown that SUMO traps are useful tools for the enrichment and isolation of proteins modified by SUMO in vitro and in vivo. To characterize the affinity and specificity of different SUMO chains for these traps, that are based on SUMO-interacting motifs, we have used real-time surface plasmon resonance (SPR), which allows a label-free analysis of protein/protein interactions. Here, a protocol to determine the affinities of multivalent SUMO traps for polySUMO chains or mono-SUMO molecules by SPR is presented.

Overcoming low yields of plant-made antibodies by a protein engineering approach.

The commercial development of plant-based antibody production platforms is often limited by low and variable yields, but little is known about the factors that affect antibody accumulation during and after translation. Here, we present a strategy to identify yield-limiting regions in the transcript and protein. We exchanged variable heavy chain (VH) domain sequences between two human antibodies at structurally conserved positions, thus creating ten chimeric VH domains containing sequences from M12 (∼1000 μg/g leaf fresh weight [FW]) and 4E10 (∼100 μg/g FW). After transient expression in Nicotiana benthamiana leaves, we measured mRNA and protein levels by quantitative real-time PCR and surface plasmon resonance spectroscopy, respectively. Transcript levels were similar for all constructs, but antibody levels ranged from ∼250 μg/g to over 2000 μg/g FW. Analysis of the expression levels showed that: i) 4E10 yields were only marginally increased by suppression of post-transcriptional gene silencing; ii) the CDR3 of 4E10 contains a protease site; and iii) a bipartite, yield-limiting region exists in the CDR2/CDR3. Our findings highlight the strong impact of cotranslational and posttranslational events on antibody yields and show that protein engineering is a powerful tool that can be used to overcome the remaining limitations affecting antibody production in plants.

Development of novel methodologies for the identification of CFTR-targeted drugs: a multidisciplinary approach using Real Time Surface Plasmon Resonance interaction assay supported by bioinformatics

Development of novel methodologies for the identification of CFTR-targeted drugs: a multidisciplinary approach using Real Time Surface Plasmon Resonance interaction assay supported by bioinformatics

In vitro selection of RNA aptamers against CA125 tumor marker in ovarian cancer and its study by optical biosensing

Early identification of neoplastic diseases is essential to achieve timely therapeutic interventions and significantly reduce the mortality of patients. A well-known biomarker is the Cancer Antigen 125 (CA125) or 16 mucin (MUC 16), a glycoprotein of the human family of mucins, already used for the diagnostic and prognostic evaluation of ovarian cancer. Therefore, the detection of CA125 to now remains a promising tool in the early diagnosis of this tumor. In this paper, we describe the development of RNA aptamers that bind with high affinity the tumor antigen CA125. We performed eight cycles of selection against CA125 purified protein. The selected aptamers were cloned and sequenced and the binding properties of the most promising sequences were studied by Real Time PCR and Surface Plasmon Resonance (SPR) to evaluate their ability in targeting CA125 protein with perspective applications in aptamer-based bioassays.

NFκB-p50 as a blood based protein marker for early diagnosis and prognosis of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the major health concern in Indian population. Despite of advanced treatment the mortality rate for this disease has not been improved very much. Current research focused on development of protein marker for the diagnosis and prognosis of HNSCC. The case control study was performed with 125 HNSCC patients and 104 control cases. The level of p50 and IκBα proteins in serum were evaluated at pre and post therapy by label free real time surface plasmon resonance (SPR) and western blot analysis. The serum p50 concentration were significantly (P < 0.0001) higher at the time of diagnosis i.e. pre therapy (Mean ± SD = 27.06 ± 4.88 ng/μl) as compared to controls (Mean ± SD = 16.96 ± 4.04 ng/μl) while it decline at post therapy (Mean ± SD = 21.01 ± 4.98 ng/μl). Similarly, the concentration of IκBα protein in serum were slightly higher at pre therapy (Mean ± SD = 8.33 ± 1.85 ng/μl) as compared to controls (Mean ± SD = 7.27 ± 1.84 ng/μl) and declined at post therapy (Mean ± SD = 7.09 ± 1.24 ng/μl). The level of p50 was also high at the early stage of the disease. The specificity and sensitivity of p50 proteins obtained from ROC analysis revealed the potentiality to be diagnostic protein marker for HNSCC for its accuracy in the study cohort.

A quantitative binding study of fibrinogen and human serum albumin to metal oxide nanoparticles by surface plasmon resonance.

The interaction of plasma proteins with metal oxide nanoparticles (NPs) is important due to the potential biomedical application of these NPs. In this study, new approaches were applied to measure quantitatively the kinetics and affinities of fibrinogen and human serum albumin (HSA) for TiO2, CeO2, Al2O3 and ZnO NPs immobilized on a sensor chip. Real-time surface plasmon resonance (SPR) measurements showed that fibrinogen interacted with TiO2 and CeO2 NPs with high affinity (135 and 40 pM, respectively) and to Al2O3 NPs with moderate affinity (15 nM). The data fitted well to the Langmuir model describing a 1:1 interaction. In contrast, HSA interacted with TiO2, CeO2 and Al2O3 NPs with lower affinity (80 nM, 37 nM and 2 µM, respectively) with the data fitting better to the conformational change model. TiO2 and CeO2 NPs had fast association rate constants with fibrinogen (1×10(6) M(-1) s(-1)) and Al2O3 NPs had a slower association rate constant (1×10(4) M(-1) s(-1)). By contrast, HSA had markedly slower association rate constants (1×10(3)-1×10(4) M(-1) s(-1)). The binding of the proteins was reversible, thus allowing the rapid capture of data for replicates. The occurrence of matrix effects was evaluated by using surfaces with different chemistries to capture the NPs, namely alginate, NeutrAvidin and bare gold. The affinity values determined for the NP-protein interactions were largely independent of the underlying surface used to capture the NPs.

ISCOMATRIX™ adjuvant promotes epitope spreading and antibody affinity maturation of influenza A H7N9 virus like particle vaccine that correlate with virus neutralization in humans

In a previously reported phase I clinical trial, subjects vaccinated with two doses of an unadjuvanted H7N9 virus like particle (VLP) vaccine responded poorly (15.6% seroconversion rates with 45μg hemagglutinin (HA) dose). In contrast, 80.6% of subjects receiving H7N9 VLP vaccine (5μg HA) with ISCOMATRIX™ adjuvant developed hemagglutination-inhibition (HI) responses. To better understand the role of adjuvant, complete antibody epitope repertoires of post-vaccination sera were investigated using Whole Genome Fragment Phage Display Library (GFPDL). In addition, antibody affinity maturation following vaccination was measured against HA1 and HA2 antigenic domains using real time Surface Plasmon Resonance (SPR) based kinetic assays. Unadjuvanted H7N9-VLP vaccine generated primarily antibodies targeting the C-terminus of the HA1 domain, predicted to be mostly buried on the native HA spikes, while adjuvanted VLP vaccine generated antibodies against large epitopes in the HA1 spanning the receptor binding domain (RBD). SPR analysis using a functional H7-HA1 domain demonstrated that sera from adjuvanted H7N9-VLP vaccine induced higher total binding antibodies and significantly higher antibody affinity maturation to HA1 compared to sera from unadjuvanted vaccine. Total antibody binding and affinity to the HA1 (but not HA2) domain correlated with HI and neutralization titers. This study demonstrates that ISCOMATRIX™ adjuvanted vaccine promotes higher quality antibody immune response against avian influenza in naïve humans.

Psd1 binding affinity toward fungal membrane components as assessed by SPR: The role of glucosylceramide in fungal recognition and entry.

Psd1 is a plant defensin that has antifungal activity against several pathogenic and nonpathogenic fungi. Previous analysis of Psd1 chemical shift perturbations by nuclear magnetic resonance (NMR) spectroscopy demonstrated that this defensin interacts with phospholipids and the sphingolipid glucosylceramide isolated from Fusarium solani (GlcCer(Fusarium solani)). In this study, these interactions were evaluated by real-time surface plasmon resonance (SPR) analysis. The data obtained demonstrated that Psd1 could bind more strongly to small unilamellar vesicles (SUV)-containing GlcCer(Fusarium solani) than to SUV that was composed of phosphatidylcholine (PC) alone or was enriched with GlcCer that had been isolated from soybeans. An increase in the SPR response after cholesterol or ergosterol incorporation in PC-SUV was detected; however, SUV composed of PC:Erg (7:3; molar:molar) became unstable in the presence of Psd1, suggesting membrane destabilization. We also observed a lack of Psd1 internalization in Candida albicans strains that were deficient in the glucosyl ceramide synthase gene. Together, these data indicate that GlcCer is essential for Psd1 anchoring in the fungal plasma membrane as well as internalization.

Real-time surface plasmon resonance study of biomolecular interactions between polymerase and bulky mutagenic DNA lesions.

Surface plasmon resonance (SPR) was used to measure polymerase-binding interactions of the bulky mutagenic DNA lesions N-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl (FABP) or N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) in the context of two unique 5′-flanking bases (CG*A and TG*A). The enzymes used were exo-nuclease-deficient Klenow fragment (Kf-exo–) or polymerase β (pol β). Specific binary and ternary DNA binding affinities of the enzymes were characterized at subnanomolar concentrations. The SPR results showed that Kf-exo– binds strongly to a double strand/single strand template/primer junction, whereas pol β binds preferentially to double-stranded DNA having a one-nucleotide gap. Both enzymes exhibited tight binding to native DNA, with high nucleotide selectivity, where the KD values for each base pair increased in the order dCTP ≪ dTTP ∼ dATP ≪ dGTP. In contrast to that for pol β, Kf-exo– binds tightly to lesion-modified templates; however, both polymerases exhibited minimal nucleotide selectivity toward adducted DNA. Primer steady-state kinetics and 19F NMR results support the SPR data. The relative insertion efficiency fins of dCTP opposite FABP was significantly higher in the TG*A sequence compared to that in CG*A. Although Kf-exo– was not sensitive to the presence of a DNA lesion, FAAF-induced conformational heterogeneity perturbed the active site of pol β, weakening the enzyme’s ability to bind to FAAF adducts compared to FABP adducts. The present study demonstrates the effectiveness of SPR for elucidating how lesion-induced conformational heterogeneity affects the binding capability of polymerases and ultimately the nucleotide insertion efficiency.

Dual nanoparticle amplified surface plasmon resonance detection of thrombin at subattomolar concentrations.

A novel dual nanoparticle amplification approach is introduced for the enhanced surface plasmon resonance (SPR) detection of a target protein at subattomolar concentrations. Thrombin was used as a model target protein as part of a sandwich assay involving an antithrombin (anti-Th) modified SPR chip surface and a thrombin specific DNA aptamer (Th-aptamer) whose sequence also includes a polyadenine (A30) tail. Dual nanoparticle (NP) enhancement was achieved with the controlled hybridization adsorption of first polythymine-NP conjugates (T20-NPs) followed by polyadenine-NPs (A30-NPs). Two different nanoparticle shapes (nanorod and quasi-spherical) were explored resulting in four different NP pair combinations being directly compared. It was found that both the order and NP shape were important in optimizing the assay performance. The use of real-time SPR measurements to detect target concentrations as low as 0.1 aM is a 10-fold improvement compared to single NP-enhanced SPR detection methods.

Non-scan and real-time multichannel angular surface plasmon resonance imaging method

We describe a non-scan and real-time multichannel angular surface plasmon resonance (SPR) imaging method. We demonstrate experimentally, with multiple line shaped light illuminations to construct multi-imaging channels, that an image captured with an area detector can probe the surface plasmons with different field distributions. Thus, it provides a fixed optical-sensing module measuring the spatial variations of the refractive index in the flow system in real time without scanning. This technique has the advantages of high system stability and similar optical arrangement to the conventional parallel-channel SPR sensors and could have potential applications in multianalyte detection and reference-compensated biosensing.

Rapid microfluidic solid-phase extraction system for hyper-methylated DNA enrichment and epigenetic analysis

Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva, sample enrichment and amplification is typically required before detection. We present a rapid microfluidic solid phase extraction (μSPE) system for the capture and elution of low concentrations of hm-DNA (≤1 ng ml(-1)), based on a protein-DNA capture surface, into small volumes using a passive microfluidic lab-on-a-chip platform. All assay steps have been qualitatively characterized using a real-time surface plasmon resonance (SPR) biosensor, and quantitatively characterized using fluorescence spectroscopy. The hm-DNA capture/elution process requires less than 5 min with an efficiency of 71% using a 25 μl elution volume and 92% efficiency using a 100 μl elution volume.

Swelling behavior of pyrene-labelled polystyrene LB thin film exposed to various volatile organic vapors

In this work thin films of pyrene-labelled polystyrene (PS) prepared Langmuir–Blodgett (LB) film technique are characterized by UV–visible spectroscopy, Atomic Force Microscopy (AFM) and Surface Plasmon Resonance (SPR). The sensing behaviors of the films were investigated with respect to volatile organic compounds (VOCs) at room temperature. The sensing responses of the films against VOCs (chloroform, benzene, toluene and ethanol) were measured by SPR method. In addition, the effects of film morphology and gas sensing properties were discussed. It was found that the PS films exhibited good response, reversibility, stability and faster response and recovery characteristic to VOCs. The changes in reflectivity implied the swelling behavior of PS thin film during adsorption and can be explained by the capture of organic vapor molecules. Fick's law for early-time diffusion was adopted to quantify real time SPR data for the swelling processes. It was observed that diffusion coefficients (D) for swelling obeyed the t1/2 law and could be correlated with the VOCs used. The response of PS films to the choosed VOCs has been investigated in conditions of physical properties of the solvents, and the films were obtain to be largely sensitive to chloroform vapor compared to other studied vapors.