SPR Method Research Articles

Quantification of protein interaction kinetics in a micro droplet

Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule.

Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer.

스테레오 영상을 이용한 비행 중인 항공기 날개의 변위 및 진동 측정

The present study was conducted by using stereo pattern recognition method(SPR method) to measure the displacement and vibration of an airplane wing in flight condition. A SPR based measurement system was developed using two visible light stereo cameras. The visible light stereo images were processed to obtain marker points by adaptive threshold method and marker filtering technique. The marker points were used to reconstruct 3D point, displacement, and vibration data. The SPR system was installed on F-16 fighter. The wing displacement and vibration were measured in flight condition. Therefore, this paper presents a possibility that SPR based measurement system using visible light stereo camera can be very useful for measuring displacement and vibration of an airplane in flight condition.

Optimization of Nanoporus Anodic Aluminum Sensor-Based Lspr for Methicillin-Resistant Staphylococcus Aureus detection

Methicillin-resistant Staphylococcus aureus (MRSA) causes foodborne illnesses in approximately 241,000 persons in the United States annually, despite of greatly underreported (about 25 fold) and underdiagnosed (about 29 fold) ratio. MRSA has considered as one of the major foodborne pathogens, and it is considered as one of the most significant threats to public health. Recently, MRSA has gained substantial attention due to its increasing resistance against a broad range of antibiotics. The timely detection and quantification of MRSA are of key importance for the prevention of MRSA outbreaks. Considerable efforts have been directed towards the development of rapid and practical biosensor methods for MRSA detection. To keep up this trend, SPR method has been combined with a nanoporous anodic aluminum (NAA) sensor in this study, which is called as NAA sensor -based localized surface plasmon resonance (LSPR) system (Figure 1). This method will overcome the limitations of SPR method due to larger surface-to-volume ratio of NAA sensor. This study aims to optimize the pore size of NAA prior to employment of the NAA-based LSPR system for MRSA detection. For the fabrication of three different sizes of NAA sensor, a two-step anodizing method was employed using various anodization voltage and time, and electrolyte solution. The topology of NAA sensors were observed by employing FE-SEM for measuring the pore diameter, inter-pore distance, and pore length. The reflectance spectrum and fringe pattern of each NAA sensor were measured by using LSPR for the selection of optimum NAA sensor for MRSA detection. The NAA sensor was modified with 11-mercaptoundecanoic acid and immobilized with anti-S. aureus polyclonal antibody. The NAA immunosensors were then exposed to serially diluted MRSA culture starting from 101 CFU/mL to 108 CFU/mL for the measurement of wavelength shift. The attachment of MRSA on the surface of NAA immunosensor was confirmed by SEM observation. Three different pore sizes of sensors were successfully fabricated in the range of 50 to 200 nm by the modification of fabrication conditions. The inter-pore distance and pore length of each sensor with different pore size were 99.7 ± 4.13 nm and 1.02 ± 0.01 µm for 50 nm size sensor, 201.5 ± 2.12 nm and 1.01 ± 0.01 µm for 100 nm size sensor, and 260 ± 1.40 nm and 1.00 ± 0.01 µm for 200 nm size sensor, respectively. The reflectance spectrum and fringe pattern of 200 nm size of sensor showed clearer and greater reflectance spectrum than those of 50 nm and 100 nm pore size of sensor. In addition, the sensitivity of 50, 100 and 200 nm pore size of NAA sensors were determined to be 747 nm/RIU, 1000 nm/RIU, and 1534 nm/RIU, respectively. The wavelength shift of MRSA on the NAA immunosensor was increased as the concentration of MRSA increased. The confirmed numbers of MRSA captured on the NAA immunosensor were increased in a concentration-dependent manner. Detection limit of NAA-based LSPR method was determined to be 102 ± 0.79 CFU/mL. Taking the excellent advantage in cost-effectiveness and simple fabrication of NAA sensor, an optimized NAA sensor-based LSPR method could be applied to detect MRSA in food with successful practicability. Figure 1

Quantitative eeg applying the statistical pattern recognition method: A useful tool in the dementia diagnostic work-up

To diagnose a dementia disorder clinical criteria are applied based on findings from a comprehensive clinical assessment. However, most of these clinical criteria are not very helpful when patients seek care services in a very early phase of a dementia disorder. The aim of this study was to examine the discriminatory power of the qEEG to separate patients with AD in a very early phase of the disease from elderly persons without dementia using the SPR method. In this study 422 controls and 734 clinical patients, were recruited. The EEGs were recorded using 19 electrodes within the IS 10-20 placement system. The EEG was recorded for three minutes at rest with eyes closed. The SPR technique was used to construct a healthy control classifier by contrasting the 422 healthy controls against the 734 clinical patients from the Mentis Cura EEG database. Subsequently each qEEG data was classified by the healthy control classifier. A receiver operating curve (ROC) analysis was performed on the resulting healthy control indices. The result of the ROC analysis of the healthy control index. 1) For the separation of healthy controls and all clinical patients AUC = 0.89, ACC = 0.85, SS = 0.81, SP = 0.86, LR+ = 5.8, LR- = 0.22 2) For the separation of healthy controls with SCI and other clinical patients AUC = 0.87, ACC = 0.81, SS = 0.82, SP = 0.80, LR+ = 4.1, LR- = 0.23 3) For the separation of healthy controls and AD AUC = 0.93, ACC = 0.87, SS = 0.87, SP = 0.88, LR+ = 7.3, LR- = 0.15 AUC: area under the corresponding ROC curve; ACC: accuracy; SS: sensitivity; SP: specificity; LR: likelihood ratio; SCI: patients with subjective cognitive impairment. Using the healthy control index the SPR method separated well the controls from the patient groups and the AD group. The result can be seen as a “proof of concept” of the SPR method, and may be used in order to increase to diagnostic accuracy when diagnosing the causes of dementia.

Improved smoothness and homogeneity of icosahedral grids using the spring dynamics method

An icosahedral grid that has both high smoothness and homogeneity is proposed. The grid-generation method is based on the combination of the spring dynamics (SPR) method with zero natural spring length (SPR0) and transformation by a smooth analytic function around the 12 vertices of an icosahedron. As a preliminary step, we first showed that the grid interval of the grid generated by SPR0 was inversely proportional to a Lambert conformal conic projection map factor, with a map angle of 300° around the vertices. Then, the transformation function was analytically determined, such that the resolution for the azimuthal direction became constant. In order to estimate cost-efficiency of numerical simulation with the newly proposed grid, we introduced an index defined as the ratio between the minimum grid interval and the squared maximum grid interval. It showed a 2.5% improvement from a recursive grid, and a 0.3–12% improvement from the best cases of the original SPR grid proposed by Tomita et al. (2002) [23] [hereafter, T02] dependent on global resolution. We also re-examined the original SPR method and found that the natural spring length proposed in T02 should be shortened to avoid instability when the global resolution is higher than grid-level 8. Finally, we examined the grids using advection/shallow water simulations.

Measurement by SPR of Very Low Dissociation Rates: Oxidation‐Mediated Loss of Biotin–Streptavidin Affinity

Long-term relationship: biotin labels on RNAs, and possibly other biomacromolecules, are easily oxidized causing a dramatic loss of affinity for streptavidin and adversely affecting the measurement of high-affinity interactions. A new SPR method has been developed for measuring the very low rate-dissociation constants of biotin- and biotin oxide-conjugated RNAs with streptavidin.

Sensitive and continuous screening of inhibitors of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) at single SPR chips.

The development of new methods that meet the demand of high-throughput, high-fidelity screening of hit compounds is important to researching modalities of important diseases such as neurological disorders, HIV, and cancer. A surface plasmon resonance- (SPR-) based method capable of continuously screening enzyme inhibitors at a single chip with antibody-amplified signal enhancement has been developed. The proof of concept is demonstrated by monitoring the cleavage of chip-confined peptide substrates [a segment of the amyloid precursor protein (APP) with the Swiss mutation] by β-site APP-cleaving enzyme 1 (BACE1). In the presence of a noninhibitor, BACE1 clips the peptide substrate at the cleavage site, detaching a fragment that is homologous to the N-terminus of the amyloid beta (Aβ) peptide. Consequently, a subsequent injection of the Aβ antibody does not lead to any molecular recognition or SPR signal change at the chip. In contrast, suppression of the BACE1 activity by a strong inhibitor leaves the peptide substrate intact, and the subsequent antibody attachment produces an easily detectable SPR signal. Compared to the widely used FRET (fluorescence resonance energy transfer) assay, the method reported here is more cost-effective, as unlabeled peptide is used as the BACE1 substrate. Furthermore, the assay is faster (each screening cycle lasts for ca. 1.5 h) and can be continuously carried out at a single, regenerable SPR chip for more than 30 h. Consequently, excellent reproducibility (RSD < 5%) and throughput can be attained. Two inhibitors were screened, and their half-maximal inhibitory concentrations (IC50) determined by the SPR method were in excellent agreement with values deduced from ELISA and mass spectrometry.

Detection of Sulfamethazine Residues in Milk by Surface Plasmon Resonance-Based Biosensor Inhibition Immunoassay

A method for detection of sulfamethazine（SMT）in milk by coupling indirect inhibition assay with a self-assembled monolayer was developed. The method of immobilization of SMT-BSA on the chip surface was also introuduced. A series of milk samples with spiked 0, 5, 10, 20, 30, 40 ng/mL of SMT were analyzed by the SPR biosensor and the standard calibration curve of SMT in milk was constructed. The intra-assay relative standard deviation (RSD) and recovery were 6.3% and 102.8%, respectively. The inter-assay deviation (7 days) was less than 6 IU, and demonstrated the method’s stability. The high stability of the SPR sensor underlies the potential of the SPR method as a stable and high-precision tool for detection of SMT.

Several approaches for vitamin D determination by surface plasmon resonance and electrochemical affinity biosensors

This work has been focused on the development of novel optical (Surface Plasmon Resonance) and electrochemical based biosensors for the determination of 25-OH vitamin D (25OHD) which is an important factor involved in avoiding both skeletal damage and a variety of pathological conditions, and to evaluate their potential use in clinical practice.Different approaches to the determination of vitamin D using affinity based biosensors, are described herein; firstly, an immunosensor based on SPR transduction was realized for direct determination of vitamin D, obtaining a LOD of 2μg/ml which unfortunately is too far from the needs in clinical analysis. In order to enhance the sensitivity, the vitamin D was modified with gold nanoparticles (AuNPs): the binding of 25OHD with AuNPs determines the amplification of SPR signal, allowing to lower the LOD down to 1μg/ml, doubling the sensitivity. An alternative SPR method, based on the indirect determination of vitamin D by means of Vitamin D Binding Protein (VDBP), led to a further sensitivity increase reaching a LOD of 45ng/ml which is really close to the fixed accomplishment.Finally, an electrochemical transduced biosensor has been realized, based on the reaction of vitamin D with 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD): once derivatized, the determination of 25OHD was possible in the range 20–200ng/ml with a LOD of 10ng/ml. The latter proposed system fits the requirement of determining vitamin D in a concentration range which is of significance for clinical applications; moreover, since a screen printed electrode has been used, this opens the possibility to miniaturize the sensor and developing a portable and easy-to-automate point-of-care testing device. The proposed devices provide an improvement with respect to traditional methods that are time and reagents consuming and require radioactive compounds, pretreatment procedures and expensive instrumentation.

Topical anesthesia for transnasal esophagogastroduodenoscopy: Spray, applicator, or both?

Transnasal esophagogastroduodenoscopy (EGD) has become widely accepted in Japan. Better performance and compliance for the procedure are expected with appropriate nasal anesthesia.We aimed to elucidate the effectiveness, the safety and the compliance of nasal anesthesia for transnasal EGD. Subjects were 372 asymptomatic patients examined by transnasal EGD for gastric cancer screening. They were pretreated with topical anesthesia either with a nasal spray method (Spr group, n = 127), cotton-tipped applicator method (Cot group, n = 115), or both (Com group, n = 130). Lidocaine (4%) was applied with atomizer in the Spr method, whereas a cotton-tipped applicator soaked in 4% lidocaine was placed in the nasal cavity for 5 min in the Cot method. After transnasal EGD, each subject rated procedure-related discomfort according to visual analogue scales. In subjects who had prior experience of transoral EGD, tolerance against EGD was compared between transnasal and transoral routes. There were no severe adverse events related to topical anesthesia.Transnasal EGD was completed in 94.9% of the patients.Age, gender or insertion failure rate were not different among Spr, Cot and Com groups. Pain scores for anesthesia and examination were significantly lower in the Spr group, whereas the pain score for insertion was not different among the three groups.The rate of patients who deemed transnasal EGD to be more tolerable than transoral EGD was highest in the Spr group. Topical nasal spray seems to be appropriate for topical anesthesia in transnasal EGD.

Remapping Method for Transferring Data between Two Meshes Using a Modified Iterative SPR Approach for Parallel Resolution

In metal forming simulations, the transfer of data from one mesh to another can be very often required such as, in ALE formulation at each time step or in Lagrangian formulation at each remeshing step. Its accuracy is one of the main concerns for researchers, since the accumulation of generated diffusion can lead to larger numerical error and create convergence problems. Since 1992, the Super convergent Patch Recovery method (SPR) introduced by Zienkiewicz &amp; Zhu [1], was a major breakthrough for the methods to estimate errors in FE solution. Later, it has also been used to recover nodal fields from integration points, in order to transfer data between two meshes [10]. The original method raises some difficulties to treat the domain boundaries. They have not quite properly been handled, in particular in the frame of parallel implementation, despite the fact that, surface phenomena, such as contact and friction, play such an important role in metal forming applications. In the present paper, it is presented a modified iterative SPR method which deals with boundary points with the same order of accuracy as the interior points. It does not require increasing the patch size and it is easier to implement in parallel environment. Also, when interpolating the field on new mesh, a new and consistent technique (P0+ transport) of enriched field has been used. It involves building a P1+ field by mixing the recovered SPR nodal field with the known field at integration points. Results are presented in form of convergence rate and L2 error norm, for several analytical functions for a SPR based transfer operator against a simple volumetric based transfer operator, before being applied to an actual metal forming problem. All computations presented here have been done by using four processors in parallel environment.

地中に埋設された管路を掘り起こさずにリニューアルするSPR工法(&lt;メカライフ特集&gt;発想の転換)

地中に埋設された管路を掘り起こさずにリニューアルするSPR工法(&lt;メカライフ特集&gt;発想の転換)

Assessing the Performance of Methods to Detect and Quantify African Dust in Airborne Particulates

African dust (AD) contributions to particulate matter (PM) levels may be reported by Member States to the European Commission during justification of exceedances of the daily limit value (DLV). However, the detection and subsequent quantification of the AD contribution to PM levels is complex, and only two measurement-based methods are available in the literature: the Spanish-Portuguese reference method (SPR), and the Tel Aviv University method (TAU). In the present study, both methods were assessed. The SPR method was more conservative in the detection of episodes (71 days identified as AD by SPR, vs 81 by TAU), as it is less affected by interferences with local dust sources. The mean annual contribution of AD was lower with the TAU method than with SPR (2.7 vs 3.5 ± 1.5 μg/m(3)). The SPR and TAU AD time series were correlated with daily aluminum levels (a known tracer of AD), as well as with an AD source identified by the Positive Matrix Factorization (PMF) receptor model. Higher r(2) values were obtained with the SPR method than with TAU in both cases (r(2) = 0.72 vs 0.56, y = 0.05x vs y = 0.06x with aluminum levels; r(2)=0.79 vs 0.43, y = 0.8x vs y = 0.4x with the PMF source). We conclude that the SPR method is more adequate from an EU policy perspective (justification of DLV exceedances) due to the fact that it is more conservative than the TAU method. Based on our results, the TAU method requires adaptation of the thresholds in the algorithm to refine detection of low-impact episodes and avoid misclassification of local events as AD.

Characterization of PEGylated Anti-VEGF aptamers using surface plasmon resonance

SPR analysis for the assessment of anti-VEGF RNA aptamer binding affinity was successfully carried out before and after PEGylation. Anti-VEGF 2′-OMe RNA aptamer was synthesized and PEGylated through the amide bond formation between the succinimidyl group of mPEG-SPA and the terminal amine group of anti-VEGF 2′-OMe RNA aptamer. According to the SPR analysis using a BIAcore 2000 instrument, the binding affinity of anti-VEGF 2′-OMe-RNA aptamer to the VEGF (K d =1.87×10−9 M) decreased significantly after PEGylation (K d =8.×10−8 M). The PEGylated anti-VEGF RNA aptamer showed a slow adsorption and a rapid desorption. Interestingly, the anti-VEGF DNA aptamer having the same sequence with the anti-VEGF 2′-OMe RNA aptamer showed no binding affinity at all. The SPR method was thought to be usefully applied for the assessment of bio-conjugated aptamer therapeutics.

Comparison of artificial synthesis methods of gene

The comparative study of the methods for constructing the artificial genes have done. Equine interferon alpha-1 gene and porcine interferon alpha-1 gene were synthesized respectively by several methods from short DNA oligomers and each gene was evaluated about the accuracy of the gene sequence. The gene made by the SPR method was the most accurate.

Modulation of biorecognition of glucoamylases with Concanavalin A by glycosylation via recombinant expression

Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 × 10 −6 M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl-α- d-mannopyranoside.

State of the industry: Where do we stand?: Looking back, ahead at issues reshaping surface finishing

In this work, thin films of chloroaluminium phthalocyanine (ClAlPc), fluoroaluminium phthalocyanine (FAlPc) and fluorochromium phthalocyanine (FCrPc), which are insoluble in conventional solvents, were deposited by spin coating of their solutions in trifluoroacetic acid. The sensing response of these films versus acetic acid, three alcohols (methanol, ethanol, butanol) and three amines (methylamine, dimethylamine, trimethylamine) have been investigated using surface plasmon resonance as the sensing method. It has been shown that the sensor response of the investigated films decreases in the following order: acetic acid > alcohols > amines. The optical changes as monitored by SPR method have been used in conjunction with Fick's second law of diffusion to determine the diffusion coefficients of analyte vapor during the films’ swelling process. The obtained results showed that the diffusion coefficients and the swelling characteristics of the films are dependent on the functional group of the phthalocyanine molecule and the molecular size of the analyte.

Rehabilitation of Canal Tunnel by Use of SPR Method

Rehabilitation of Canal Tunnel by Use of SPR Method

A new adaptive remeshing scheme based on the sensitivity analysis of the SPR point wise error estimation

This paper presents a formulation for the obtainment of the sensitivity analysis of a point wise error estimator with respect to the nodal coordinates using the adjoint state method. The proposed point wise error estimator is based on the SPR method. The numerical accuracy of the presented sensitivity analysis has been tested by perturbing a mesh. The capability of the presented sensitivity analysis for the detection of pollution error has also been tested. A new adaptive remeshing strategy based on the sensitivity analysis of the point wise error estimation has been developed and tested. This strategy produces very cheap meshes for the accurate evaluation of stresses at specific points.