Surface Plasmon Resonance Measurements Research Articles

SORL1 is a receptor for tau that promotes tau seeding

Sortilin-related receptor 1 (SORL1) is an intracellular sorting receptor genetically implicated in Alzheimer's disease (AD) that impacts amyloid precursor protein trafficking. The objective of these studies was to test the hypothesis that SORL1 binds tau, modulates its cellular trafficking and impacts the aggregation of cytoplasmic tau induced by pathological forms of tau. Using surface plasmon resonance measurements, we observed high-affinity binding of tau to SORL1 and the vacuolar protein sorting 10 (VPS10) domain of SORL1. Interestingly, unlike LRP1, SORL1 binds tau at both pH 7.4 and pH 5.5, revealing its ability to bind tau at endosomal pH. Immunofluorescence studies confirmed that exogenously added tau colocalized with SORL1 in H4 neuroglioma cells, while overexpression of SORL1 in LRP1-deficient Chinese hamster ovary (CHO) cells resulted in a marked increase in the internalization of tau, indicating that SORL1 can bind and mediate the internalization of monomeric forms of tau. We further demonstrated that SORL1 mediates tau seeding when tau RD P301S FRET biosensor cells expressing SORL1 were incubated with high molecular weight forms of tau isolated from the brains of patients with AD. Seeding in H4 neuroglioma cells is significantly reduced when SORL1 is knocked down with siRNA. Finally, we demonstrate that the N1358S mutant of SORL1 significantly increases tau seeding when compared to WT SORL1, identifying for the first time a potential mechanism that connects this specific SORL1 mutation to Alzheimer's disease. Together, these studies identify SORL1 as a receptor that contributes to trafficking and seeding of pathogenic tau.

Glyceraldehyde 3-Phosphate Dehydrogenase on the Surface of Candida albicans and Nakaseomyces glabratus Cells-A Moonlighting Protein That Binds Human Vitronectin and Plasminogen and Can Adsorb to Pathogenic Fungal Cells via Major Adhesins Als3 and Epa6.

Candida albicans and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this "moonlighting" role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of C. albicans and Nakaseomyces glabratus. GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins-vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10-8 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall-agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)-were suggested to serve as the docking platforms for GAPDH in C. albicans and N. glabratus, respectively.

The effect of electrode composition on bimetallic AgAu nanoparticles produced by spark ablation

A flexible way to generate bimetallic nanoparticles with high control of their composition is to use spark ablation of alloyed electrodes. It has been generally accepted and stated that particles produced using spark ablation of alloyed electrodes obtain the same chemical composition as the electrodes. However, we identify a lack of studies fully supporting the connection between electrode and particle composition, presented in a small literature survey. The aim of the study is, hence, to explore the validity of the statement by analysing the relation between alloyed electrodes and their resulting particle composition using three sets of AgAu electrodes containing Au and 25, 50, and 75 atomic % Ag, respectively. The resulting composition is thoroughly investigated using both single particle (scanning- and transmission electron microscopy) and ensemble particle techniques (inductive coupled plasma-mass spectroscopy, x-ray photoelectron spectroscopy, x-ray fluorescence, and optical measurements of surface plasmon resonance. We also investigate how sample size (e.g., the number of particles analysed) affects the reliability of the resulting sample mean. For single-particle measurements of a sample with a compositional standard deviation of a few atomic percentage points, a sample size of 20 particles is a good benchmark for obtaining reliable results of the sample mean. Furthermore, this article aims to challenge the practice in which the composition of nanoparticles is measured, presented, and interpreted, to improve and facilitate future research related to this topic. From the results of this study, it could be concluded that for the investigated Ag–Au material system, the particles obtained a composition very similar to the alloyed AgAu electrodes.

Comparison of Fluidic and Non-Fluidic Surface Plasmon Resonance Biosensor Variants for Angular and Intensity Modulation Measurements.

Fluidic and non-fluidic surface plasmon resonance measurements were realized for the same type of sensory layer and using the same mouse IgG antibody and anti-mouse IgG antibody biomolecular system. A comparison of the thicknesses of the anti-mouse IgG antibody layers bound to the ligand at increasing analyte concentrations ranging from 0.0 μg mL-1 to 5.0 μg mL-1 in the non-fluidic and the fluidic variant showed that the thickness of the bound anti-mouse antibody layers in the fluidic variant was approximately 1.5-3 times larger than in the non-fluidic variant. The greater thicknesses of the deposited layers were also reflected in the larger increment of the resonant angle in the fluidic variant compared to the non-fluidic variant in the considered range of analyte concentrations. The choice between fluidic and non-fluidic surface plasmon resonance biosensors may be justified by the availability of analyte volume and the intended modulation technique. When working with limited analyte, non-fluidic biosensors with intensity modulation are more advantageous. For larger analyte quantities, fluidic biosensors with angular modulation are recommended, primarily due to their slightly higher sensitivity in this measurement mode.

Wax appearance temperature in crude oils measured by surface plasmon resonance

To predict and quantify the wax appearance temperature in crude oils, we proposed and implemented a well-explored sensing method known as surface plasmon resonance. This phenomenon occurs when an incident light beam couples into a metallic surface. As sensing method, this phenomenon has been widely used in biosensing applications. In this research, we showed the effectiveness of the surface plasmon resonance sensing technique in hydrocarbon sensing in general and in crude oil wax appearance prediction in particular. To the authors knowledge, this is the first exploration of monitoring wax deposition events from crude oil using surface plasmon resonance measurements. A compact optical sensor was developed from an ultra-thin layer of gold deposited on a sapphire prism and supported by a new data analyzing metric to analyze the acquired reflected beam intensity profile and detect the precipitation of crude oil wax or other hydrocarbon particles under varying-temperature and low-pressure conditions. The obtained wax appearance temperatures using the introduced on-site method were found to be comparable with the literature measurements using the common in-lab Cross-Polarization Microscopy method.

Monitoring levodopa oxidation and reduction reactions using surface plasmon resonance on a nanohole array electrode

The traditional method of monitoring the oxidation and reduction of biomedical materials usually relies on electrochemical (EC) measurement techniques. Here, we demonstrate a surface plasmon resonance (SPR) method to monitor the oxidation process. Using levodopa L-dopa as the target analyte, a nanohole sensing plate is embedded in the EC electrode to enhance the oxidation signal and generate SPR. Cyclic voltammetry (CV) measurement was first conducted to understand the baseline of EC response of L-Dopa. Then, the redox reactions were simultaneously monitored through SPR measurements during the CV voltage scan. The results showed that the limit of detection using traditional CV reached 1.47 μM while using EC-SPR, the limit of detection improved to 1.23 μM. Most importantly, we found a strong correlation between CV current profiles and the SPR reflection spectra. Our results facilitate detecting electrochemical reactions using an optical probing method.

Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second-Generation.

Bispecific antibodies (BisAbs) are biotherapeutics that amalgamate the specificities of two distinct antibodies into one molecule, however, their engineering requires genetic modification and remains time-consuming. Therefore, we used AJICAP second-generation technology, which drives the production of site-specific conjugation without genetic modification requirements, to generate BisAbs. Using haloketone chemistry as an alternative to maleimide chemistry, we successfully produced site-specific antibody conjugates. Pharmacokinetic studies revealed that the haloketone-based antibody conjugate was stable in the rat plasma. The resultant BisAbs were rigorously evaluated, and surface plasmon resonance measurements and flow cytometry analyses confirmed that the antigen binding remained intact. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. Further cytotoxicity evaluation emphasized the pronounced activity of the generated BisAbs. This novel approach introduces a fully chemical, site-specific strategy capable of producing BisAbs, heralding a new era in the field of biotherapeutics.

Chrysin 7-O-β-D-glucuronide, a dual inhibitor of SARS-CoV-2 3CLpro and PLpro, for the prevention and treatment of COVID-19

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) resulted in the coronavirus disease 2019 (COVID-19) pandemic. Given the advent of subvariants, there is an urgent need to develop novel drugs. The aim of this study was to find SARS-CoV-2 inhibitors from Scutellaria baicalensis Georgi targeting the proteases 3CLpro and PLpro. After screening 25 flavonoids, chrysin 7-O-β-D-glucuronide was found to be a potent inhibitor of SARS-CoV-2 on Vero E6 cells, with half-maximal effective concentration of 8.72 µM. Surface plasmon resonance assay, site-directed mutagenesis and enzymatic activity measurements indicated that chrysin-7-O-β-D-glucuronide inhibits SARS-CoV-2 by binding to H41 of 3CLpro, and K157 and E167 of PLpro. Hydrogen-deuterium exchange mass spectrometry analysis showed that chrysin-7-O-β-D-glucuronide changes the conformation of PLpro. Finally, chrysin 7-O-β-D-glucuronide was shown to have anti-inflammatory activity, mainly due to reduction of the levels of the pro-inflammatory cytokines interleukin (IL)-1β and IL-6.

An Analytical Tooth Model Based on Surface Plasmon Resonance Chips Coated with Hydroxyapatite Used for Investigation of the Acquired Dental Pellicle

One of the main targets in modern dentistry is the prevention of caries. Not only daily nutrition plays an important role, but also the so‐called “pellicle” on the outer surface of teeth, which is mainly formed by saliva proteins. Moreover, numerous bacteria are part of this pellicle, which might cause caries by acidic metabolites. In the here‐presented study, a method for electrophoretic deposition (EPD) of hydroxyapatite (HAP) nanoparticles on gold surfaces, suitable for surface plasmon resonance (SPR) measurements, is developed. An “artificial tooth” is created and loaded by natural saliva in different concentrations (to form a natural pellicle), while the influence of an environment with different pH values has been studied. It could be demonstrated that even slight acid solutions damage the pellicle significantly within seconds, exposing the HAP to acidic degradation, which would lead to caries in the human oral cavities. This model allows to study pellicle formation as well as degradation in real time. As a practical example, the influence of beverages on the pellicle could be demonstrated.

Simultaneous detection method for two cardiac disease protein biomarkers on a single chip modified with mixed aptamers using surface plasmon resonance

A simultaneous detection method for two cardiac disease protein biomarkers present in serum samples on a single planar gold chip using surface plasmon resonance (SPR) is described. The detection of N-terminal pro-brain natriuretic peptide (NT-proBNP) and tumor necrosis factor α (TNF-α), which are known as acute myocardial infarction (AMI) biomarkers, with predetermined clinically relevant concentrations was performed using mixed aptamers specific to each protein tethered on a single gold surface. After the binding of NT-proBNP and/or TNF-α to the mixed aptamers, an antibody specific to each target protein was injected to form a surface sandwich complex to improve selectivity. In order to adjust the dynamic ranges in the known clinically relevant concentration significantly different for NT-proBNP (0.13–0.24 nM) and TNF-α (0.5–3 pM), the surface density ratios of the corresponding pair of aptamer and antibody were first systematically determined, which were the 1:1 mixed aptamer chip with 40 nM anti-NT-proBNP and 100 nM anti-TNF-α. This allowed to establish the distinct dynamic ranges of 0.05–0.5 nM for NT-proBNP and 0.1–5 pM for TNF-α in a buffer, along with detection and quantification limits of 0.03 and 0.19 nM for NT-proBNP and 0.06 and 0.21 pM for TNF-α, respectively. The changes in refractive unit (RU) values observed when exposing both proteins at different concentrations alongside the corresponding fixed concentration of antibodies onto the 1:1 mixed aptamer chip were then correlated to the sum of RU values measured when using the injection of individual protein for evaluating each protein concentration. With a complete characterization of the simultaneous quantification of two protein concentrations in the buffer, the mixed aptamer chip was finally employed for direct measurements of NT-proBNP and TNF-α concentrations in undiluted serum samples from healthy controls and AMI patients. The results of simultaneous SPR measurements for the two proteins in the serum samples were further compared to the individual protein concentration results using an enzyme-linked immunosorbent assay.

Directed Evolution of Material Binding Peptide for Polylactic Acid-specific Degradation in Mixed Plastic Wastes.

In order to preserve our livelihood for future generations, responsible use of plastics in a climate-neutral and circular economy has to be developed so that plastics can be used in an environmentally friendly way by future generations. The prerequisite is that bioplastic polymers such as polylactic acid (PLA) can be efficiently recycled from petrochemical based plastic. Here, a concept in which accelerated PLA degradation in the mixed suspension of PLA and polystyrene (PS) nanoparticles has been achieved through an engineered material binding peptide. After comparison of twenty material binding peptides, Cg-Def is selected due to its PLA binding specificity. Finally, a suitable high-throughput screening system is developed for enhancing material-specific binding toward PLA in presence of PS. Through KnowVolution campaign, a variant Cg-Def YH (L9Y/S19H) with 2.0-fold improved PLA binding specificity compared to PS is generated. Contact angle and surface plasmon resonance measurements validated higher surface coverage of Cg-Def YH on PLA surface and the fusion of Cg-Def YH with PLA degrading enzyme confirmed the accelerated PLA depolymerization (two times higher than only enzyme) in mixed PLA/PS plastics.

Separation of shifts in surface plasmon resonance into refractive index and thickness of deposited layers

Routine Surface Plasmon Resonance (SPR) measurements, which are widely applied in affinity (bio)sensors and surface science, do not allow one to separate the obtained SPR signals into the change of the refractive index and thickness of deposited layers. For thin layers the signal is proportional to the product of the layer thickness and the difference in the refractive indices of this layer and of the aqueous media. In this study, we suggest an approach to separate these parameters. It is performed by subsequent measurements in the presence and in the absence of polyethylene glycol (PEG), an inert additive which modifies the refractive index of the liquid phase but do not penetrate into the adsorbed layer. Both parameters were successively determined for human serum albumin (HSA) as well as for anti-HSA IgG in three different settings: i) HSA adsorbed directly onto a gold surface, ii) HSA chemically immobilized on a gold surface coated with a self-assembled monolayer of 1,16 mercaptohexadecanoic acid and iii) chemically immobilized HSA with additional monomolecular layer of anti-HSA IgG antibodies bond to this protein layer. The suggested approach can be applied in most SPR devices with a flow cell for analysis of various adsorbed layers.

Affinity Maturated Transferrin Receptor Apical Domain Blocks Machupo Virus Glycoprotein Binding

Transferrin receptor 1 (TfR) delivers iron across cellular membranes by shuttling the ion carrier protein transferrin. This ability to deliver large protein ligands inside cells is taken advantage of by pathogens to infiltrate human cells. Notably, the receptor’s outermost ectodomain, the apical domain, is used as a point of attachment for several viruses including hemorrhagic arenaviruses. To better understand interactions with the receptor it would be advantageous to probe sequence determinants in the apical domain with viral spike proteins. Here, we carried out affinity maturation of our computationally designed apical domain from human TfR to identify underlying driving forces that lead to better binding. The improved variants were confirmed by in vitro surface plasmon resonance measurements with dissociation constants obtained in the lower nanomolar range. It was found that the strong binding affinities for the optimized variants matched the strength of interactions with the native receptor. The structure of the best variant was determined experimentally indicating that the conformational change in the hairpin binding motif at the protein–protein interface plays a crucial role. The experimental methodology can be straightforwardly applied to other arenavirus or pathogens that use the apical domain. It can further be useful to probe host–virus compatibility or therapeutic strategies based on the transferrin receptor decoys.

Inferring the Interfacial Reactivity of Gold Nanoparticles by Surface Plasmon Resonance Measurements.

Gold nanoparticles (GNPs) require a functionalization step in most cases to be suitable for applications. Optimizing this step in order to maintain both the stability and the plasmonic properties of the GNPs is a demanding process. Indeed, multiple analyses are required to get sufficient information on the grafting rate and the stability of the obtained suspension, leading to material and time waste. In this study, we propose to investigate ligand reactivity on a gold surface with surface plasmon resonance (SPR) measurements as a way to simulate the reactivity in GNP suspensions. We consider two thiolated ligands in this work: thioglycolic acid (TA) and 6-mercaptohexanoic acid (MHA). These thiols are grafted using different conditions on GNPs (monitored by optical absorption) and on a gold surface (monitored by SPR) and the grafting efficiency and stability are compared. The same conclusions are reached in both cases regarding the best protocol to implement, namely, the thiol molecules should be introduced in a water solution at a low concentration. This demonstrates the suitability of SPR to predict the reactivity on a GNP surface.

Design of 8-mer peptides that block Clostridioides difficile toxin A in intestinal cells

Infections by Clostridioides difficile, a bacterium that targets the large intestine (colon), impact a large number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can be delivered to the gut and inhibit the biocatalytic activity of these toxins represent a promising therapeutic strategy to prevent and treat C. diff. infection. We describe an approach that combines a Peptide Binding Design (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in colon epithelial cells. One peptide, SA1, is found to block TcdA toxicity in primary-derived human colon (large intestinal) epithelial cells. SA1 binds TcdA with a KD of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR).

A Competitive High-Throughput Screening Platform for Designing Polylactic Acid-Specific Binding Peptides.

Among biobased polymers, polylactic acid (PLA) is recognized as one of the most promising bioplastics to replace petrochemical-based polymers. PLA is typically blended with other polymers such as polypropylene (PP) for improved melt processability, thermal stability, and stiffness. A technical challenge in recycling of PLA/PP blends is the sorting/separation of PLA from PP. Material binding peptides (MBPs) can bind to various materials. Engineered MBPs that can bind in a material-specific manner have a high potential for material-specific detection or enhanced degradation of PLA in mixed PLA/PP plastics. To obtain a material-specific MBP for PLA binding (termed PLAbodies ), protein engineering of MBP Cg-Def for improved PLA binding specificity is reported in this work. In detail, a 96-well microtiter plate based high-throughput screening system for PLA specific binding (PLABS) was developed and validated in a protein engineering (KnowVolution) campaign. Finally, the Cg-Def variant V2 (Cg-Def S19K/K10L/N13H) with a 2.3-fold improved PLA binding specificity compared to PP was obtained. Contact angle and surface plasmon resonance measurements confirmed improved material-specific binding of V2 to PLA (1.30-fold improved PLA surface coverage). The established PLABS screening platform represents a general methodology for designing PLAbodies for applications in detection, sorting, and material-specific degradation of PLA in mixed plastics.

A new approach for the adsorption kinetics using surface plasmon resonance results

This work is focused on a new approach to determine the adsorption kinetics of Surface Plasmon Resonance (SPR) measurement data yielded from Calix[4]arene Langmuir-Blodgett (LB) thin films upon exposure to a range of volatile organic vapors such as benzene, chloroform, toluene and ethyl acetate. Experimental results were analyzed using the well known Pseudo first-order and second-order, Ritchie and Elovich adsorption models. The validity of these models was assessed by using the correlation coefficient (R2) as an indicator of the conformity between the model prediction and experimental adsorption data. Sorption capacity, adsorption rate constant and the amount of vapor molecules were calculated and discussed. The values of R2 = 0.96, R2 = 0.94, and R2 = 0.98 for chloroform vapor were calculated for Pseudo-first order, Pseudo-second order and Elovich models respectively. Pseudo-second order model gave the best fit values for benzene (R2 =0.9991), toluene (R2 =0.999) and ethanol (R2 =0.993).

Potentiating Tweezer Affinity to a Protein Interface with Sequence-Defined Macromolecules on Nanoparticles.

Survivin, a well-known member of the inhibitor of apoptosis protein family, is upregulated in many cancer cells, which is associated with resistance to chemotherapy. To circumvent this, inhibitors are currently being developed to interfere with the nuclear export of survivin by targeting its protein-protein interaction (PPI) with the export receptor CRM1. Here, we combine for the first time a supramolecular tweezer motif, sequence-defined macromolecular scaffolds, and ultrasmall Au nanoparticles (us-AuNPs) to tailor a high avidity inhibitor targeting the survivin-CRM1 interaction. A series of biophysical and biochemical experiments, including surface plasmon resonance measurements and their multivalent evaluation by EVILFIT, reveal that for divalent macromolecular constructs with increasing linker distance, the longest linkers show superior affinity, slower dissociation, as well as more efficient PPI inhibition. As a drawback, these macromolecular tweezer conjugates do not enter cells, a critical feature for potential applications. The problem is solved by immobilizing the tweezer conjugates onto us-AuNPs, which enables efficient transport into HeLa cells. On the nanoparticles, the tweezer valency rises from 2 to 16 and produces a 100-fold avidity increase. The hierarchical combination of different scaffolds and controlled multivalent presentation of supramolecular binders was the key to the development of highly efficient survivin-CRM1 competitors. This concept may also be useful for other PPIs.

Development of Nano-Micro Fused LSPR Chip for In Situ Single-Cell Secretion Analysis.

Single-cell analysis has become increasingly important in uncovering cell heterogeneity, which has great implications in medicine and biology for a deep understanding of cell characteristics. Owing to its significance, it is vital to create novel devices that can reveal special or unique cells. In this work, we developed a single-cell secretion detection chip consisting of microwells that can trap single cells. Each well is surrounded by Au nanopillars capable of localized surface plasmon resonance (LSPR) measurement. Using microfabrication and nanofabrication techniques, Au nanopillar and microwell structures were fabricated on a COP film. The Au nanopillar was modified with IL-6 antibodies for the direct detection of single-cell secreted IL-6 via LSPR absorbance peak shift. Specific IL-6 detection was successfully demonstrated using a null and IL-6 oversecreting Jurkat cell. A high single-cell trapping efficiency of over 80% was also achieved. Overall, the development of this single-cell secretion detection chip with a simple LSPR measurement setup represents a significant development in the field of cell biology and immunology, providing researchers with a powerful tool for studying individual cells and their secreted cytokines, and is useful for point-of-care testing (POCT) diagnostics.

Interaction studies of cannabidiol with human serum albumin by surface plasmon resonance, spectroscopy, and molecular docking

The binding interaction of cannabidiol (CBD) and human serum albumin (HSA) under physiological blood pH conditions (pH 7.4) was conducted by surface plasmon resonance (SPR), fluorescence spectroscopy, UV-Visible spectrophotometry, and molecular docking. The responses from SPR measurement increased with the increase in CBD concentration until equilibrium was reached at the equilibrium dissociation constant (KD) of 9.8 × 10−4 M. The results from fluorescence and UV-Visible spectroscopy showed that CBD bound to HSA at one site in a spontaneous manner to form protein-CBD complexes. The quenching process involved both static and dynamic mechanisms while the static mechanism contributed predominantly to the binding between CBD and albumin. The binding constants estimated from the fluorescence studies were from 0.16 × 103 to 8.10 × 103 M−1, which were calculated at different temperature conditions using Stern-Volmer plots. The thermodynamic parameters demonstrated that the binding interaction was a spontaneous reaction as Gibbs free energy had negative values (ΔG = −12.57 to −23.20 kJ.mol−1). Positive ΔH and ΔS values (ΔH = 2.46 × 105 J.mol−1 and ΔS = 869.81 J.mol−1K−1) indicated that the hydrophobic force was the major binding interaction. Finally, confirmation of the type and extent of interaction was provided using UV-spectroscopy and molecular docking studies. The outcomes of this study are expected to serve as a platform to conduct future studies on binding interactions and toxicological research of CBD. Communicated by Ramaswamy H. Sarma