Surface Plasmon Resonance Method Research Articles

Capabilities of Double-Resonance LPG and SPR Methods for Hypersensitive Detection of SARS-CoV-2 Structural Proteins: A Comparative Study.

The danger of the emergence of new viral diseases and their rapid spread demands apparatuses for continuous rapid monitoring in real time. This requires the creation of new bioanalytical methods that overcome the shortcomings of existing ones and are applicable for point-of-care diagnostics. For this purpose, a variety of biosensors have been developed and tested in proof-of-concept studies, but none of them have been introduced for commercial use so far. Given the importance of the problem, in this study, long-period grating (LPG) and surface plasmon resonance (SPR) biosensors, based on antibody detection, were examined, and their capabilities for SARS-CoV-2 structural proteins detection were established. Supersensitive detections of structural proteins in the order of several femtomoles were achieved by the LPG method, while the SPR method demonstrated a sensitivity of about one hundred femtomoles. The studied biosensors are compatible in sensitivity with ELISA and rapid antigen tests but, in contrast, they are quantitative, which makes them applicable for acute SARS-CoV-2 infection detection, especially during the early stages of viral replication.

Application of SPR Method as an Approach to Gas Phase Sensing of Volatile Compound Profile in Mezcal Spirits Conferred by Agave Species

Mezcal is a traditional Mexican spirit produced by distilling fermented agave, with a unique taste directly related to its volatile compound composition. Thus, the present research proposed the surface plasmon resonance (SPR) technique as a potential method to differentiate mezcals, studying several parameters at angular interrogations and at a fixed angle. The study evaluated eight mezcals from different agave species using SPR and gas chromatography-mass spectrometry (GC-MS). Despite the similarities in mezcal spirits corresponding to the same ethanol content and the same artisanal method, it was possible to obtain well-differentiated characteristics by SPR parameters, such as the width of the curve, the resonant angle, and reflectance intensities. Therefore, it was possible to demonstrate the potential use of the SPR technique as a rapid first approach to a screening test to differentiate types of spirits.

The nanomolar affinity of C-phycocyanin from virtual screening of microalgal bioactive as potential ACE2 inhibitor for COVID-19 therapy

The global pandemic of COVID-19 caused by SARS-CoV-2 has caused more than 400 million infections with more than 5.7 million deaths worldwide, and the number of validated therapies from natural products for treating coronavirus infections needs to be increased. Therefore, the virtual screening of bioactive compounds from natural products based on computational methods could be an interesting strategy. Among many sources of bioactive natural products, compounds from marine organisms, particularly microalgae and cyanobacteria, can be potential antiviral agents. The present study investigates bioactive antiviral compounds from microalgae and cyanobacteria as a potential inhibitor of SARS-CoV-2 by targeting Angiotensin-Converting Enzyme II (ACE2) using integrated in silico and in vitro approaches. Our in silico analysis demonstrates that C-Phycocyanin (CPC) can potentially inhibit the binding of ACE2 receptor and SARS-CoV-2 with the docking score of −9.7 kcal mol−1. This score is relatively more favorable than the native ligand on ACE2 receptor. Molecular dynamics simulation also reveals the stability interaction between both CPC and ACE2 receptor with a root mean square deviation (RMSD) value of 1.5 Å. Additionally, our in vitro analysis using the surface plasmon resonance (SPR) method shows that CPC has a high affinity for ACE2 with a binding affinity range from 5 to 125 µM, with KD 3.37 nM. This study could serve as a reference to design microalgae- or cyanobacteria-based antiviral drugs for prophylaxis in SARS-CoV-2 infections.

Characterization of Small Molecule-Protein Interactions Using SPR Method.

Surface plasmon resonance (SPR) is an optical phenomenon being used to monitor molecular binding events. With the advantages of being label-free, real-time, and sensitive, SPR assays have become one of the most commonly used techniques to measure binding kinetics, affinity, specificity, and concentration of molecular interactions. In an SPR experiment to measure small molecule-protein interactions, the protein is immobilized on the biosensor surface, while the small molecule flows through the surface of the sensor chip. The interactions between the small molecules and proteins are monitored by subsequent changes in the refractive index and quantified with resonance units. In this chapter, we have utilized an SPR assay to study the interaction of flavonoids and the glucose-regulated protein 78. Assay steps are detailed for immobilization optimization, SPR instrument setup, operation, sampleinjection, and affinity data analysis.

Interaction of Host Proteins with Cell Surface Molecules of the Pathogenic Yeast Trichosporon asahii.

Trichosporon asahii is an invasive pathogenic yeast that infects immunocompromised hosts. Several virulence factors contribute to the fungal infection; however, the factors that contribute to the occurrence of T. asahii infections remain unclear. Since adhesins are typical virulence factors reported for pathogenic fungi, we looked for host proteins that interact with the T. asahii cell surface. T. asahii and Candida albicans were used for screening using a pull-down assay with fetal bovine serum. Serum albumin and elongation factor 2 were identified as the yeast-binding serum proteins. Additionally, we investigated the interactions of the cell surface-associated molecules (CSM) of T. asahii with vitronectin (VTN), fibronectin, fetuin-A, and alpha-1antitrypsin (AAT). The surface plasmon resonance (SPR) method was used to examine the interaction between CSM and human proteins. On the other hand, the pull-down assay was used to examine the interaction between human proteins and the T. asahii cell surface. Serum albumin, AAT, and VTN were found to interact with T. asahii in both SPR and pull-down assays. This study identified several proteins that interact with T. asahii, suggesting that these proteins play a role in infection mechanisms.

ВИЗНАЧЕННЯ ТОВЩИНИ ПЛАЗМОН-НОСІЙНИХ ШАРІВ ФОТОМЕТРИЧНИМ МЕТОДОМ

The influence of design and technological factors, in particular, the deviation of the thickness of the plasma-carrying layer from the nominal value, introduces uncertainty into the measurement by the method of surface plasmon resonance. The effect of the thickness of nanoscale films of surface plasmon resonance sensors on the characteristics of these sensors is shown in the work using the method of numerical modeling. An experimental study of the transmission spectra and reflection characteristics of gold films of different thicknesses was conducted and consistency with theoretical calculations was established. Transmittance coefficients were used as an informative parameter for estimating the thickness of nanosized gold films of surface plasmon resonance sensors. Not only the difference in the angular positions of the resonance and the intensity of the reflected light in it, but also the different width of the resonance characteristics in the region of the minimum and the shape of the intensity graph at angles smaller than the critical one are characteristic for samples with different thicknesses. The correlation coefficient between the thickness of the studied samples and the corresponding transmission coefficients at a wavelength of 532 nm is r = - 0.943. This means that there is a correlation between the parameters, there is a linear functional dependence between them. An empirical dependence of d=1100.7712·T-0.3328 with a high degree of correlation (r = - 0.943) was established between the thickness and the corresponding transmittance coefficients of gold films for λ=532 nm. With this relative empirical error of thickness determination does not exceed 3%. The dependence of the maximum transmission spectrum Tλmax of nanosized gold films on the thickness of these films was experimentally established. At this step, the thickness determination did not exceed 1 Ǻ. The research data show the prospects of using the monochromatic photometric method to determine the thickness of plasma-carrying nanoscale layers.

Intercalation of anticancer drug mitoxantrone into DNA: Studied by spectral and surface plasmon resonance methods

Studying DNA interaction with different drugs is interesting and important in discovering the interaction mechanism and developing novel drugs. Mitoxantrone (MTX) is a potent antitumor drug used to treat certain types of cancer. In this work, we evaluated the binding strength of MTX to 16 base pair double strands (ds) DNA. For the first time, this work investigates the intercalation of anticancer drugs into DNA by spectral fluorescence spectroscopy and SPR (surface plasmon resonance) based methods. Furthermore, the thermodynamic and kinetic parameters, including KD, KA, entropy change (ΔS), enthalpy change (ΔH), and ΔG, were calculated using different concentrations of ds DNA at three different temperatures. The results indicated that MTX binding to DNA was an exothermic and spontaneous process. The negative value of both ΔH and ΔS calculated by SPR and fluorescence data analysis showed that hydrogen bonding was the major binding force for the complexation of MTX to DNA. In addition, fluorescence data analysis revealed that MTX could quench the DNA fluorescence via the static quenching mechanism. This study shows that the investigation of intercalation anticancer drugs into DNA can be studied by spectral methods and can be investigated using SPR based method, and obtained data from the SPR method is as reliable as the fluorescence method. The present work shows hydrogen bonding is the major binding force in intercalating MTX to DNA.

Subcellular Interactomes Revealed by Merging APEX with Cross-Linking Mass Spectrometry.

Subcellular protein-protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical cross-linking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation. APEX-CXMS was first applied to mitochondria and identified 653 pairs of interprotein cross-links. Six pairs of new interactions were selected and verified by coimmunoprecipitation, the mammalian two-hybrid system, and surface plasmon resonance method. Besides, our approach was further applied to the nucleus, capturing 336 pairs of interprotein cross-links with approximately 94% nuclear specificity. APEX-CXMS thus provides a simple, fast, and general alternative to map diverse subcellular PPIs.

Broadband differential interference in a waveguide with a gradient refractive index distribution

The paper presents a model of a planar broadband differential waveguide interferometer with a gradient refractive index distribution. Its response to the change in the refractive index of the waveguide cover layer is presented. The analysis was performed for the wavelength range from 0.5um to 0.7um. The orthogonal TE0 and TM0 modes propagating in this wavelength range are considered. The influence of the coverage refractive index change on the output characteristics of the system is shown. Full Text: PDF ReferencesP. Kozma, F. Kehl, E.Ehrentreich-Forster, C. Stamm and F.F. Bier, "Integrated planar optical waveguide interferometer biosensors: A comparative review", Biosens. Bioelectron. 58, 287 (2014), CrossRef M. Kitsara, K. Misiakos, I. Raptis, and E. Makarona, "Integrated optical frequency-resolved Mach-Zehnder interferometers for label-free affinity sensing", Opt. Express 18, 8193 (2010). CrossRef K. Misiakos, I. Raptis, A. Salapatas, E. Makarona, A. Bostials, et al., "Broad-band Mach-Zehnder interferometers as high performance refractive index sensors: Theory and monolithic implementation", Opt. Express 22, 8856 (2014). CrossRef K. Misiakos, I. Raptis, E. Makarona, A. Botsialas, A. Salapatas, et al, "All-silicon monolithic Mach-Zehnder interferometer as a refractive index and bio-chemical sensor", Opt. Express 22, 26803 (2014) CrossRef K. Misiakos, E. Makarona, M. Hoekman, R. Fyrogenis, K. Tukkiniemi, et al., "All-Silicon Spectrally Resolved Interferometric Circuit for Multiplexed Diagnostics: A Monolithic Lab-on-a-Chip Integrating All Active and Passive Components", ACS Photonics 6, 1694 (2019). CrossRef E. Makarona, A. Salapatas, I. Raptis, P. Petrou, S. Kakabakos, et al., "Broadband Young interferometry for simultaneous dual polarization bioanalytics", J Opt Soc Am B 34, 1691 (2017). CrossRef K. Gut, "Broad-band difference interferometer as a refractive index sensor", Opt. Express 25, 3111 (2017), CrossRef K. Gut, "Study of a Broadband Difference Interferometer Based on Low-Cost Polymer Slab Waveguides", Nanomaterials 9, 729 (2019), CrossRef T. Pustelny, J. Ignac-Nowacka and Z. Opilski, "Optical investigations on layered metalphthalocyanine nanostructures affected by NO2 applying the surface plasmon resonance method", Opt. Appl. 34, 563 (2004). CrossRef W. Lukosz, Sensor Actuat. B-Chem. "Integrated optical chemical and direct biochemical sensors", 29, 37 (1995). CrossRef Z. Qi, S. Xia and N. Matsuda, "Spectropolarimetric interferometer based on single-mode glass waveguides", Opt. Express, 16, 2245 (2008). CrossRef K. Gut, A. Zakrzewski, T. Pustelny, "Sensitivity of Polarimetric Waveguide Interferometer for Different Wavelengths", Acta Phys. Pol. 118, 1140 (2010). CrossRef J.E. Broquin, S. Honkanen, "Integrated Photonics on Glass: A Review of the Ion-Exchange Technology Achievements", Appl.Sci. 11, 4472 (2021). CrossRef G.C. Righini, J. Linares, "Active and Quantum Integrated Photonic Elements by Ion Exchange in Glass", Appl.Sci. 11, 5222 (2021). CrossRef

Advanced microstructure, morphology and CO gas sensor properties of Cu/Ni bilayers at nanoscale

In this study, we investigated the morphology of synthesized Cu/Ni nanoparticles in trace of carbon sources by the co-deposition process of RF sputtering and RF-PECVD methods and localized surface plasmon resonance of CO gas sensing of Cu/Ni nanoparticles. The surface morphology was studied by analyzing 3D micrographs of atomic force microscopy using image processing techniques and fractal/multifractal analyses. The MountainsMap® Premium software with the two-way ANOVA (Variance analysis) and least-significant differences tests were used for statistical analysis. The surface nano-patterns have a local and global particular distribution. Experimental and simulated Rutherford backscattering spectra confirm the quality of nanoparticles. Then, prepared samples were exposed to CO gas flue to study their gas sensor application using the localized surface plasmon resonance method. Increasing the Ni layer over Cu one shows an interesting result in both morphology and gas sensing sides. Advanced stereometric analyses for the surface topography of thin films in conjunction with Rutherford backscattering spectrometry and Spectroscopic analysis make a unique study in the field.

Recent Advances in the Recognition Elements of Sensors to Detect Pyrethroids in Food: A Review.

The presence of pyrethroids in food and the environment due to their excessive use and extensive application in the agriculture industry represents a significant threat to public health. Therefore, the determination of the presence of pyrethroids in foods by simple, rapid, and sensitive methods is warranted. Herein, recognition methods for pyrethroids based on electrochemical and optical biosensors from the last five years are reviewed, including surface-enhanced Raman scattering (SERS), surface plasmon resonance (SPR), chemiluminescence, biochemical, fluorescence, and colorimetric methods. In addition, recognition elements used for pyrethroid detection, including enzymes, antigens/antibodies, aptamers, and molecular-imprinted polymers, are classified and discussed based on the bioreceptor types. The current research status, the advantages and disadvantages of existing methods, and future development trends are discussed. The research progress of rapid pyrethroid detection in our laboratory is also presented.

Surface Plasmon Resonance (SPR) Determination of Adenosine Triphosphate (ATP) Using Silver (I) induced Configuration Changes of a Single Stranded DNA Probe with Cytosine (C)

ATP is related to the occurrence of many diseases, its facile, sensitive and accurate determination in real samples is needed for disease screening and treatment. Although some methods realized the sensitive detection of ATP using the signal amplification of cyclic reactions or nanoparticles, complex experimental processes limit their applications. In this paper, we report a simple surface plasmon resonance (SPR) method based on a single stranded DNA probe. The probe has dual recognition sites of ATP and Ag+; namely the ATP aptamer located in the middle of the probe and five cytosine (C) at both ends of the probe. In the absence of ATP, the probe transforms into a rigid hairpin structure by forming C-Ag+-C structures and produces a large SPR signal. However, once the probe interacts with ATP, three-dimensional structures are formed, and then the Ag+-induced probe configuration change is smaller, resulting in an small SPR signal. The signal difference before and after the probe interacts with ATP is proportional to the analyte concentration. The probe configuration change is in the optimum area for SPR detection, ensuring the sensitive ATP determination. The signal difference has a linear response with the logarithm of ATP concentration from 0.05 to 500 nM with a detection limit of 15 pM. Moreover, the strategy was demonstrated to determine ATP in serum samples with satisfactory recoveries. The results show that this simple and sensitive method has potential applications for clinical ATP determination.

An SPR-based method for Hill coefficient measurements: the case of insulin-degrading enzyme

Insulin-degrading enzyme (IDE) is a highly conserved zinc metallopeptidase and is capable to catalytically cleave several substrates besides insulin, playing a pivotal role in several different biochemical pathways. Although its mechanism of action has been widely investigated, many conundrums still remain, hindering the possibility to rationally design specific modulators which could have important therapeutical applications in several diseases such as diabetes and Alzheimer's disease. In this scenario, we have developed a novel surface plasmon resonance (SPR) method which allows for directly measuring the enzyme cooperativity for the binding of insulin in the presence of different IDE activity modulators: carnosine, ATP, and EDTA. Results indicate that both positive and negative modulations of the IDE activity can be correlated to an increase and a decrease of the measured Hill coefficient, respectively, giving a new insight into the IDE activity mechanism. The use of the IDE R767A mutant for which oligomerization is hindered confirmed that the positive allosteric modulation of IDE by carnosine is due to a change in the enzyme oligomeric state occurring also for the enzyme immobilized on the gold SPR chip.

Dynamic features of tapasin as revealed by structures of two tapasin/Fab complexes

Abstract Intracellular loading of major histocompatibility complex class I (MHC-I) molecules occurs within the peptide loading complex and is accomplished through the concerted action of several proteins, including the chaperone/catalyst tapasin. In the absence of tapasin, peptide loading is compromised, resulting in decreased cell surface expression of some, though not all, MHC-I allelomorphs. To gain a mechanistic understanding of tapasin’s function we have produced recombinant Fab fragments of two anti-tapasin antibodies, PaSta1 and PaSta2, and investigated their binding to human recombinant soluble tapasin by surface plasmon resonance methods and X-ray crystallography. Both antibody Fabs bind with nanomolar affinities characterized by slow off rates. PaSta1 and PaSta2 recognize distinct epitopes as tapasin captured on a surface immobilized with one PaSta Fab binds the other PaSta Fab with nanomolar affinity. However, tapasin captured on a PaSta2 Fab surface is not able to bind peptide/HLA-B*44:05/beta-2 microglobulin complexes while tapasin captured on a PaSta1 Fab surface can, indicating that PaSta2 sterically competes for the MHC-I binding site. This finding was echoed in the structural characterization of PaSta Fab-tapasin complexes by X-ray crystallography. PaSta1 binds to an extended N-terminal region of tapasin, a site accessible even in the tapasin/ERp57 complex. By contrast, PaSta2 binds at the junction of the N-terminal and C-terminal domains of tapasin, where tapasin may interact with HLA-I molecules. Comparison of conformations of tapasin in complex with PaSta1, PaSta2, HLA-B*44:05, and ERp57 reveals the dynamic nature of tapasin, a versatile chaperone and catalyst. Supported by the intramural research program of the NIAID, NIH

Regenerable and high-throughput surface plasmon resonance assay for rapid screening of anti-SARS-CoV-2 antibody in serum samples

Current serological antibody tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require enzyme or fluorescent labels, and the titer well plates cannot be reused. By immobilizing histidine (His)-tagged SARS-CoV-2 spike (S1) protein onto tris‒nitrilotriacetic acid (tris-NTA) sensor and using the early association phase for mass-transfer-controlled concentration determination, we developed a rapid and regenerable surface plasmon resonance (SPR) method for quantifying anti-SARS-CoV-2 antibody. On a five-channel SPR instrument and with optimized S1 protein immobilization density, each of the four analytical channels is sequentially used for multiple measurements, and all four channels can be simultaneously regenerated once they have reached a threshold value. Coupled with a programmable autosampler, each sensor can be regenerated at least 20 times, enabling uninterrupted assays of more than 800 serum samples. The accuracy and speed of our method compare well with those of the enzyme-linked immunosorbent assay (ELISA), and the detection limit (0.057 μg mL−1) can easily meet the requirement for screening low antibody levels such as those in convalescent patients. In addition, our method exhibits excellent channel-to-channel (RSD = 1.9%) and sensor-to-sensor (RSD = 2.1%) reproducibility. Obviation of an enzyme label drastically reduced the assay cost, rending our method (<60 cents) much more cost effective than those of commercial ELISA kits ($4.4–11.4). Therefore, our method offers a cost-effective and high-throughput alternative to the existing methods for serological measurements of anti-SARS-CoV-2 antibody levels, holding great promise for rapid screening of clinical samples without elaborate sample pretreatments and special reagents.

Comparison of Hydrogen Peroxide Secretion From Living Cells Cultured in Different Formats Using Hydrogel-Based LSPR Substrates.

Reactive oxygen species (ROS), a number of reactive molecules and free radicals derived from molecular oxygen, are generated as by-products during mitochondrial electron transport within cells. Physiologically, cells are capable of metabolizing the ROS exploiting specific mechanisms. However, if excessive ROS accumulate inside the cells, it will cause the cells apoptosis or necrosis. Hydrogen peroxide (H2O2) is one of the essential ROS often participating in chemical reactions in organisms and regulating homeostasis in the body. Therefore, rapid and sensitive detection of H2O2 is a significant task in cell biology research. Furthermore, it has been found that cells cultured in different formats can result in different cellular responses and biological activities. In order to investigate the H2O2 secretion from the cells cultured in different formats, a hydrogel-based substrate is exploited to separate relatively large molecular (e.g., proteins) for direct measurement of H2O2 secreted from living cells in complete cell culture medium containing serum. The substrate takes advantage of the localized surface plasmon resonance (LSPR) method based on enzyme immunoprecipitation. In addition, the H2O2 secreted from the cells cultured in different dimensions (suspension of single cells and three-dimensional cell spheroids) treated with identical drugs is measured and compared. The spheroid samples can be prepared with ample amount using a designed microfluidic device with precise control of size. The results show that the H2O2 secretion from the cells are great affected by their culture formats.

Real-Time Detection of LAMP Products of African Swine Fever Virus Using Fluorescence and Surface Plasmon Resonance Method

African swine fever (ASF) is a swine disease with a very high fatality rate caused by a complex double-stranded DNA virus. The fluorescence PCR detection method is widely used for virus nucleic acid detection. Surface plasmon resonance (SPR) is a label-free and real-time detection method, unlike the fluorescence PCR detection method. In this research, we detected the loop-mediated isothermal amplification (LAMP) products of the African swine fever virus by using the SPR and fluorescence methods separately and simultaneously. By comparing the positive and negative control results, we found that the SPR response unit is completely different before and after the LAMP process. In addition, the fluorescence results on a chip showed that with an increase in the concentration of the sample, the cycle threshold (CT) value decreased, which is consistent with commercial instruments. Both the decline rate of the SPR response unit and the CT value of the fluorescence realized were used to distinguish the positive control from the negative control and water, which indicates that the SPR method can be combined with fluorescence to detect LAMP products. This research provides a label-free and simple method for detecting LAMP products.

Highly sensitive gold-film surface plasmon resonance (SPR) sensor employing germanium selenide (GeSe) nanosheets

Although surface plasmon resonance (SPR) methods are frequently employed due to their remarkable advantages such as reduced sample requirements, they confront a significant challenge in terms of sensitivity improvement. GeSe is an appropriate material for resolving this issue. According to the theoretical basis of the research, GeSe is a two-dimensional material which is a direct band gap semiconductor that may be utilized to promote electron transport and stimulate greater SPR phenomena. The first use of GeSe in an optical fiber sensor is reported in this study. A U-shaped optical fiber-GeSe-Au sensor is proposed. As a two-dimensional material with comparable physical characteristics to GeSe, MoS2 is compared to GeSe to characterize its influence upon the sensitivity. The sensitivity of fiber-GeSe-Au sensor is 7398.1 nm/refractive index unit (RIU), which is nearly twice as sensitive as a gold film SPR sensor and 1.5 times as sensitive as a sensor utilizing MoS2 nanosheets with the same structure. This study introduces a novel approach for enhancing the sensitivity, which gives a new option for monitoring biological substances at low concentrations with high sensitivity and stability and demonstrates that GeSe nanosheets have significant promise for future applications.

Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70.

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.

Magnetic Nanoparticles Enhanced Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Romaine Lettuce.

Salmonella is one of the major foodborne pathogens responsible for many cases of illnesses, hospitalizations and deaths worldwide. Although different methods are available to timely detect Salmonella in foods, surface plasmon resonance (SPR) has the benefit of real-time detection with a high sensitivity and specificity. The purpose of this study was to develop an SPR method in conjunction with magnetic nanoparticles (MNPs) for the rapid detection of Salmonella Typhimurium. The assay utilizes a pair of well-characterized, flagellin-specific monoclonal antibodies; one is immobilized on the sensor surface and the other is coupled to the MNPs. Samples of romaine lettuce contaminated with Salmonella Typhimurium were washed with deionized water, and bacterial cells were captured on a filter membrane by vacuum filtration. SPR assays were compared in three different formats—direct assay, sequential two-step sandwich assay, and preincubation one-step sandwich assay. The interaction of flagellin and MNPs with the antibody-immobilized sensor surface were analyzed. SPR signals from a sequential two-step sandwich assay and preincubation one-step sandwich assay were 7.5 times and 14.0 times higher than the direct assay. The detection limits of the assay were 4.7 log cfu/mL in the buffer and 5.2 log cfu/g in romaine lettuce samples.