Surface Plasmon Resonance Measurements Research Articles

Large-area grain-boundary-free copper films for plasmonics

Ultrasmooth single-crystalline metallic thin films provide several key advantages for the fabrication of well-defined and high-resolution plasmonic nanostructures, particularly complex integrated nanocircuits. For this purpose, copper is generally regarded as a poor plasmonic material compared to gold and silver because of its notorious oxidation issues when subjected to air exposure. Here, we report on the use of large-area grain-boundary-free copper films grown epitaxially on sapphire substrates in combination with focused ion beam milling to pattern plasmonic nanostructures with superior quality. The copper surfaces prepared using a single-crystalline copper sputtering target exhibit a very low roughness without any grain boundaries for varying film thicknesses and a strong resistance to oxidation, overcoming the bottleneck in conventional copper film fabrication. Surface plasmon resonance measurements show that improved dielectric constants with higher conductivity and long-term stability can be achieved using the single-crystalline copper films. Plasmonic nanohole arrays patterned from these high-quality films are found to display a stronger field enhancement compared to those made from polycrystalline copper films, thus resulting in an enhanced extraordinary optical transmission performance. This study suggests that our fabrication method is ideally suited for applications in copper-based plasmonic and nanophotonic devices as well as integrated nanocircuits on a large scale.

Functional Tolerance to Cysteine Mutations in Human α7 Nicotinic Acetylcholine Receptors.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is involved in various intracellular signaling pathways that mediate addiction, chronic pain, and other diseases, but its intracellular domain structures remain undetermined. The presence of 17 native cysteines in α7 nAChR provides opportunities for extracting structural information through site-directed labeling of chemical probes in strategic locations, but it also creates uncertainties in channel function when those native cysteines must be mutated. Using site-directed mutagenesis and two-electrode voltage clamp electrophysiology measurements, we found that α7 nAChR's function was well tolerated for mutations of all 13 cysteines as long as two pairs of disulfide-bond cysteines remained in the extracellular domain. Furthermore, surface plasmon resonance measurements showed that the cysteine mutations did not affect α7 nAChR binding to the intracellular protein PICK1. The study suggests that a high native cysteine content does not necessarily preclude the use of single cysteine labeling for acquiring structural information on functional proteins.

Novel fatty chain-modified GLP-1R G-protein biased agonist exerts prolonged anti-diabetic effects through targeting receptor binding sites.

Here, we design and evaluate novel long-lasting GLP-1R G-protein-biased agonists with promising pharmacological virtues. Firstly, six GLP-1R G-protein-biased peptides (named PX01–PX06), screened by using a previous reported high-throughput autocrine-based method, were fused to the N-terminus of GLP-1(9-37) to generate six fusion peptides (PX07–PX012). In vitro surface plasmon resonance (SPR) measurements showed that PX09 exerts the highest binding affinity for both human and mouse GLP-1R extracellular domains (ECD). We further used the PX09 as a starting point to conduct site-specific modifications yielding twelve lysine-modified conjugates, termed PX13–PX24. Of these conjugates, PX17 retained relatively better in vitro GLP-1R activation potency and plasma stability compared with other ones. Preclinical studies in db/db mice demonstrated that acute treatment of PX17 exerts enhanced hypoglycemic and insulinotropic activities in a dosage dependent model within the range of 0.1–0.9 mg kg−1. Similarly, prolonged glucose-lowering abilities were exhibited in modified multiple oral glucose tolerance tests (OGTTs) and a hypoglycemic duration test. Apparently prolonged in vivo half-lives of ∼96 and ∼141 h were observed after a single subcutaneous administration of PX17 at 0.1 and 0.3 mg kg−1, respectively, in healthy cynomolgus monkeys. In addition, twice-weekly treatment of PX17 in db/db mice for 8 weeks obviously improved the hemoglobin A1C (HbA1C), and was more effective at improving the insulin resistance, glucose tolerance as well as function of pancreatic beta cells compared with Semaglutide. Furthermore, subcutaneously dosed PX17 in diet induced obese (DIO) mice achieved long-term beneficial effects on food intake and body weight control, HbA1C and inflammation-related factor level lowering. The above results indicate that PX17, as a novel GLP-1R G-protein-biased agonist, may be a promising candidate for antidiabetic therapies.

Everestmab, a novel long-acting GLP-1/anti GLP-1R nanobody fusion protein, exerts potent anti-diabetic effects

In the present study, a novel single domain antibody (sdAb) fusion protein, named everestmab, composing of a mutated GLP-1(A8G) fused to the tandem bispecific humanized GLP-1R-targeting and albumin-binding nanobodies was designed and characterized for the therapies for type 2 diabetes mellitus (T2DM). Surface plasmon resonance (SPR) measurements demonstrated everestmab associates with serum albumins of rat and monkey species with high affinity, and tends to be cross-reactive with rat and monkey species. In vitro GLP-1R binding and activation assays revealed that everestmab can specifically activate the GLP-1R, and the antagonist exendin-4 (9–39) did not inhibit the activation yet. In vivo multiple oral glucose tolerance tests (OGTTs) and hypoglycaemic efficacy tests proved that a single injection of everestmab reduced the blood glucose for at least 144 h in Goto-Kakizaki (GK) rats. The plasma half-lives of 4.1 and 7.8 days were observed after a single s.c. administration of everestmab in SD rats and cynomolgus monkeys, respectively. Chronic treatment of everestmab to GK and diet induced obese (DIO) rats achieved beneficial effects on weight reducing, HbA1c lowering, glucose tolerance, liver and pancreas islet function impairment. In summary, everestmab is a unique G-protein-coupled receptor-targeted nanobody fusion protein and exerts potential as a therapeutic treatment for T2DM.

Design and biological evaluation of novel BF-30 analogs for the treatment of malignant melanoma.

Aims: To evaluate anti-tumour effects and mechanism of novel BF-30 derivative via cell-based assays and melanoma-bearing model mice.Main methods: BF-30 derivatives were designed by fusing heptapeptide-palmitic tags to native BF-30 via a protease-cleavable linker and prepared by F-moc solid-phase synthesis. Albumin binding affinity test and in vitro control-released assay were performed to screen these BF-30 derivatives and explore the mechanism of anti-tumour effects. The selected BF-30 derivative was further subjected to the preclinical efficacy study and chronic evaluation of anti-tumour effects melanoma-bearing model mice.Key findings: Twenty-one BF-30 derivatives, termed LBF-1 to LBF-21, were obtained with high purity and accurate molecular weight. Surface plasmon resonance (SPR) measurements, plasma stability test and in vitro control-released assay all showed that LBF-14 exerted better druggability compared with the others. Moreover, LBF-14 was proved to inhibit the proliferation of B16F10 melanoma cell by disrupting the cytoplasmic membrane and binding to genomic DNA to prevent transcription. Furthermore, half-life of intact LBF-14 and released BF-30 in rhesus monkeys were approximately 120.9 h and 136.4 h, respectively, after a single subcutaneous injection of 0.9 mg/kg LBF-14. In addition, chronic treatment of LBF-14 significantly suppressed melanoma growth and improved the survival rate of B16F10-bearing mice with the observed inhibition of 63.5% for 0.3mg/kg and 91.5% for 0.9 mg/kg. Furthermore, results of H&E staining prove that chronic treatment of LBF-30 effectively suppressed metastasis and invasion of B16F10 cells.Significance: LBF-14 holds potential to be developed as a promising once-weekly candidate for the treatment of malignant melanoma.

High-affinity binding of plasminogen-activator inhibitor 1 complexes to LDL receptor–related protein 1 requires lysines 80, 88, and 207

It is well-established that complexes of plasminogen-activator inhibitor 1 (PAI-1) with its target enzymes bind tightly to low-density lipoprotein (LDL) receptor-related protein 1 (LRP1), but the molecular details of this interaction are not well-defined. Furthermore, considerable controversy exists in the literature regarding the nature of the interaction of free PAI-1 with LRP1. In this study, we examined the binding of free PAI-1 and complexes of PAI-1 with low-molecular-weight urokinase-type plasminogen activator to LRP1. Our results confirmed that uPA:PAI-1 complexes bind LRP1 with ∼100-fold increased affinity over PAI-1 alone. Chemical modification of PAI-1 confirmed an essential requirement of lysine residues in PAI-1 for the interactions of both PAI-1 and uPA:PAI-1 complexes with LRP1. Results of surface plasmon resonance measurements supported a bivalent binding model in which multiple sites on PAI-1 and uPA:PAI-1 complexes interact with complementary sites on LRP1. An ionic-strength dependence of binding suggested the critical involvement of two charged residues for the interaction of PAI-1 with LRP1 and three charged residues for the interaction of uPA:PAI-1 complexes with LRP1. An enhanced affinity resulting from the interaction of three regions of the uPA:PAI-1 complex with LDLa repeats on LRP1 provided an explanation for the increased affinity of uPA:PAI-1 complexes for LRP1. Mutational analysis revealed an overlap between LRP1 binding and binding of a small-molecule inhibitor of PAI-1, CDE-096, confirming an important role for Lys-207 in the interaction of PAI-1 with LRP1 and of the orientations of Lys-207, -88, and -80 for the interaction of uPA:PAI-1 complexes with LRP1.

Estimating Detection Limits of Potentiometric DNA Sensors Using Surface Plasmon Resonance Analyses.

As the signals of potentiometric-based DNA ion-selective field effect transistor (ISFET) sensors differ largely from report to report, a systematic revisit to this method is needed. Herein, the hybridization of the target and the probe DNA on the sensor surface and its dependence on the surface probe DNA coverage and the ionic strength were systematically investigated by surface plasmon resonance (SPR). The maximum potentiometric DNA hybridization signal that could be registered by an ISFET sensor was estimated based on the SPR measurements, without considering buffering effects from any side interaction on the sensing electrode. We found that under physiological solutions (200 to 300 mM ionic strength), the ISFET sensor could not register the DNA hybridization events on the sensor surface due to Debye screening. Lowering the salt concentration to enlarge the Debye length would at the same time reduce the surface hybridization efficiency, thus suppressing the signal. This adverse effect of low salt concentration on the hybridization efficiency was also found to be more significant on the surface with higher probe coverage due to steric hindrance. With the method of diluting buffer, the maximum potentiometric signal generated by the DNA hybridization was estimated to be only around 120 mV with the lowest detection limit of 30 nM, occurring on a surface with optimized probe coverage and in the tris buffer with 10 mM NaCl. An alternative method would be to achieve high-efficiency hybridization in the buffer with high salt concentration (1 M NaCl) and then to perform potentiometric measurements in the buffer with low salt concentration (1 mM NaCl). Based on the characterization of the stability of the hybridized DNA duplexes on the sensor surface in low salt concentration buffer solutions, the estimated maximum potentiometric signal could be significantly higher using the alternative method. The lowest detection limit for this alternative method was estimated to be around 0.6 nM. This work can serve as an important quantitative reference for potentiometric DNA sensors.

A simple and inexpensive surface plasmon resonance setup for phase detection using rotating analyser ellipsometric method

In the present work we have developed an inexpensive surface plasmon resonance (SPR) setup for the detection of phase modulation. The developed setup is based on Kretschmann’s prism based configuration and uses the principle of rotating analyser ellipsometry. Angular modulation and phase modulation of a self-assembled monolayer of 1-Octanethiol on an Au film surface have been investigated with the developed setup. A sensitivity of 10−4 refractive index unit (RIU) for angular modulation and 10−6 RIU for phase modulation was obtained. Although the obtained sensitivity is an order of magnitude lower compared to other angle and phase modulation based studies, the phase modulation still provides a sensitivity two orders better than that of angular modulation. The advantage of the developed setup is that it uses inexpensive optical components yet provides a good sensitivity for static SPR measurement.

Design of comb-like poly(2-methyl-2-oxazoline) and its rapid co-deposition with dopamine for the study of antifouling properties

In this work, the comb-like poly(2-methyl-2-oxazoline) copolymer, poly(2-aminoethyl methacrylate-random-poly(2-methyl-2-oxazoline) (PMOXA-r-AEMA, PMA) is synthesized, and the CuSO4/H2O2-triggered dopamine/PMA co-deposition process is investigated. Ellipsometry, water contact angle (WCA), and X-ray photoelectron spectroscopy (XPS) are used to characterize the thickness, hydrophilicity, and surface composition of PMA-based coatings. PMA is facilely anchored to substrates within 60 min with the assistance of dopamine/polydopamine triggered by CuSO4/H2O2, and the coating thickness can achieve about 13 nm. Anti-protein adsorption and anti-blood platelet adhesion measurements are also studied to verify their antifouling properties. The adsorption capacity of FITC-BSA can be reduced to 5% of the original surface, and PMA-based coatings present excellent protein-resistant properties (∼95% reduction relative to gold surface) according to surface plasmon resonance (SPR) measurement, and it can resist adhesion of almost all blood platelets. Moreover, it is applied to a capillary inner wall for surface modification. An alkaline protein mixture and egg white proteins are successfully separated, and present excellent stability. The relative standard deviation (RSD) of migration time is lower than 0.71%. The practicability of this promising PMA-based coatings with this preparation strategy can be used for further proteomics analysis.

Toward the Mode of Action of the Clinical Stage All-d-Enantiomeric Peptide RD2 on Aβ42 Aggregation.

The aggregation of amyloid-β (Aβ) into oligomers and fibrillary structures is critical for the pathogenesis of Alzheimer's disease (AD). Recently, research effort has been focused on developing novel agents that can preferentially suppress Aβ oligomer mediated toxicities, for example, by directly targeting these toxic assemblies. The compound RD2 has been developed and optimized for Aβ42 monomer binding and stabilization of the monomer in its native intrinsically disordered conformation. It has been demonstrated to improve and even reverse the cognitive and behavioral deficits in AD mouse models, while the detailed mechanism of action is not fully clarified. Here we focused on exploring the interaction between RD2 and Aβ42 monomers and its consequences for the fibrillation of Aβ42. RD2 binds to Aβ42 monomers with nanomolar affinities, according to microscale thermophoresis and surface plasmon resonance measurements. Complexes between RD2 and Aβ42 monomers are formed at 1:1 and other stoichiometries, as revealed by analytical ultracentrifugation. At substoichiometric levels, RD2 slows down the secondary structure conversion of Aβ42 and significantly delays the fibril formation. Our research provides experimental evidence in supporting that RD2 eliminates toxic Aβ assemblies by stabilizing Aβ monomers in their native intrinsically disordered conformation. The study further supports the promising application of RD2 in counteracting Aβ aggregation related pathologies.

Development of a sensitive DMD-based 2D SPR sensor array using single-point detection strategy for multiple aptamer screening

In this paper, we report a novel single-point detection strategy for 2D solid-state angular interrogated SPR (surface plasmon resonance) sensor array for multiple aptamer screening. The scheme operates with two DMDs (digital micro-mirror devices), which suffices a single-point detector for high-throughput 2D SPR measurements without sacrificing the sensitivity. After having verified the biosensing capability of the system, in terms of sensitivity and repeatability, using standard BSA-anti-BSA antibody interaction, we further conducted experiments to perform rapid screening of aptamer candidates for binding with tumor necrosis factor-α (TNF-α). The selected anti-TNF-α aptamers were subsequently evaluated as an in vitro cell protection agent against TNF-α-induced cell death. Our experiments confirm that the anti-TNF-α aptamer screening approach offers high potential as an alternative to that based on antibodies for diagnostic and therapeutic applications.

Calorimetric approach for comparison of Angiopoietin-like protein 4 with other pancreatic lipase inhibitors

Pancreatic lipase (PNLIP) is a digestive enzyme that is a potential drug target for the treatment of obesity. A better understanding of its regulation mechanisms would facilitate the development of new therapeutics. Recent studies indicate that intestinal lipolysis by PNLIP is reduced by Angiopoietin-like protein 4 (ANGPTL4), whose N-terminal domain (nANGPTL4) is a known inactivator of lipoprotein lipase (LPL) in blood circulation and adipocytes. To elucidate the mechanism of PNLIP inhibition by ANGPTL4, we developed a novel approach, using isothermal titration calorimetry (ITC). The obtained results were compared with those of well-described inhibitors of PNLIP – ε-polylysine (EPL), (−)-epigallocatechin-3-gallate (EGCG) and tetrahydrolipstatin. We demonstrate that ITC allows to investigate PNLIP inhibition mechanisms in complex substrate emulsions and that the ITC-based assay is highly sensitive - the lowest concentration for quantification of PNLIP is 1.5 pM. Combining ITC with surface plasmon resonance and fluorescence measurements, we present evidence that ANGPTL4 is a lipid-binding protein that influences PNLIP activity through interactions with components of substrate emulsions (bile salts, phospholipids and triglycerides), and this promotes the aggregation of triglyceride emulsions similarly to the PNLIP inhibitors EPL and EGCG. In the absence of substrate emulsion, unlike in the case of LPL, ANGPTL4 did not induce the inactivation of PNLIP. Our data also prove that due to various interactions with components of substrate systems, the effect of a PNLIP inhibitor depends on whether its effect is measured in a complex substrate emulsion or in a simple substrate system.

Enhanced Biosensing Activity of Bimetallic Surface Plasmon Resonance Sensor

Surface plasmon resonance (SPR) sensors present a challenge when high sensitivity and small FWHM (full width at half maximum) are required to be achieved simultaneously. FWHM is defined by the difference between the two extreme values of the independent variable at which the value of the dependent variable is equal to half of its maximum. A smaller value of FWHM indicates better accuracy of SPR measurements. Theoretically, many authors have claimed the possibility of simultaneously achieving high sensitivity and small FWHM, which in most of the cases has been limited by experimental validation. In this report, an experimental study on the improved surface plasmon resonance (SPR) characteristics of gold over silver bimetallic sensor chips of different film thicknesses is presented. A comparative study of antigen–antibody interaction of the bimetallic chip using a custom-made, low-cost, and portable SPR device based on an angular interrogation scheme of Kretschmann configuration is performed. Pulsed direct current (DC) magnetron-sputtered bimetallic films of gold over silver were used in the construction of the SPR chip. The FWHM and sensitivity of the bimetallic sensors were firstly characterized using standard solutions of known refractive index which were later immobilized with monoclonal anti-immunoglobulin G (IgG) in the construction of the SPR biochip. Spectroscopic measurements such as ultraviolet–visible light spectroscopy (UV–Vis) and Fourier-transform infrared spectroscopy (FTIR) were used for the confirmation of the immobilization of the antibody. The performance of the bimetallic SPR biochip was investigated by exposing the sensor to various concentrations of the target protein. The results indicated that the bimetallic sensors of silver/gold had a 3.5-fold reduced FWHM compared to pure gold-based sensors, indicating a higher detection accuracy. In addition, they exhibited a significant shift in resonance angle as high as 8.5 ± 0.2 due to antigen–antibody interaction, which was ~1.42-fold higher than observed for pure silver-based sensors.

Unraveling Compositional Changes of the Double Layer upon Sulfate Adsorption on Au(111) by Surface Plasmon Resonance

The potential-induced sulfate adsorption on (111)-textured Au films in 0.1 M sulfuric acid was studied by surface plasmon resonance (SPR) measurements. Full SPR curves were recorded with millisecon...

Preparation of single-chain Fv antibodies in the cytoplasm of Escherichia coli by simplified and systematic chaperone optimization.

A single-chain variable fragment (scFv) antibody is a recombinant protein in which a peptide linker connects the variable regions of the heavy chain and light chain. Due to its smaller molecular size, an scFv can be expressed using Escherichia coli. The presence of two disulphide bonds in the molecule often prevents expression of correctly folded scFv in the E. coli cytoplasm, making a refolding process necessary to regenerate scFv activity. The refolding process is time-consuming and requires large amounts of expensive reagents, such as guanidine hydrochloride, l-arginine and glutathione. Here, to conveniently obtain scFv proteins, we devised a simple and systematic method to optimize the co-expression of chaperone proteins and to combine them with specially engineered E. coli strains that permit the formation of stable disulphide bonds within the cytoplasm. Several scFv proteins were successfully obtained in a soluble form from E. coli cytoplasm. Thermal denaturation experiments and/or surface plasmon resonance measurements revealed that the thus-obtained scFvs possessed a stable tertiary structure and antigen-binding activity. The combined use of engineered E. coli with the simplified and systematic chaperone optimization can be useful for the production of scFv proteins.

Determining the Subnanometer Thickness of the Water-Depletion Layer at the Interface between Water and the Hydrophobic Substrate.

Surface plasmon resonance (SPR) is one of the most popular and powerful techniques for label-free detecting and quantitatively analyzing the interfacial refractive index (RI). So far, most of the SPR measurements are mainly applied to detect the relative change of RI upon biological and chemical events occurring at the interface, while the determinations on the absolute value of RI remains challenging. However, the absolute value of RI has become increasingly urgent in some cases, such as the existence and physical properties of the water depletion layer (WDL). WDL refers to a subnanometer-thick layer with reduced density between water and the hydrophobic substrate. The detailed explanations of how water meets hydrophobic surface have been studied by several kinds of techniques for decades but it remains under debate. In this work, we successfully established a method to measure the absolute RI at a gold-liquid interface by surface plasmon resonance microscopy (SPRM) and 2D Fourier transformation image processing and further applied this method to study the existence and physical nature of WDL. It was found that a 0.6 nm thick WDL existed at the interface of water and the hydrophobic substrate, leading to a reduced refractive index of 1.3295 ± 0.0006 compared with the standard value of 1.3325. Our results further indicated that the WDL consisted of a uniform layer rather than numerous isolated surface nanobubbles that distributed at the interface with high density.

Immunoglobulin G-Encapsulated Gold Nanoclusters as Fluorescent Tags for Dot-Blot Immunoassays.

Few-atom gold nanoclusters (AuNCs) have been fabricated and used for various fields owing to their remarkable optical and photophysical features. However, the rational design for the antibody-mediated synthesis of fluorescent AuNCs for direct antigen-antibody reactions remains unexplored. In this work, immunoglobulin G (IgG)-functionalized AuNCs (IgG-AuNCs) were successfully prepared via a facile and fast biomineralization process. The generated IgG-AuNCs can emit intense red fluorescence with a high photoluminescence quantum yield. Besides strong emission, the bioactivity of IgG on the IgG-AuNCs can be retained. Surface plasmon resonance measurements suggested that IgG-AuNCs can bind to goat anti-human IgG with an affinity constant of 6.21 × 10-8 M. A simple detection method was then developed using a dot-blot immunoassay with IgG-AuNCs as fluorescent tags. Experimental results confirmed that the IgG-AuNC-based fluorescent reporters had many advantages such as low nonspecific adsorption and good photostability, offering immense potential for the development of efficient biosensors. This work can be extended to other specific antibodies to produce multifunctional AuNCs and utilized to detect and monitor targeted analytes and biological events of interest.

ZIKV Envelope Domain-Specific Antibodies: Production, Purification and Characterization.

Infection with Zika virus (ZIKV) came first to public attention after it was found to be associated with congenital microcephaly during the outbreak in Brazil (2015–2016). Diagnosis of ZIKV suffers from extensive cross-reactivity with other Flaviviruses, which are circulating in many ZIKV epidemic areas. Due to the fatal outcome of ZIKV infection during pregnancy, detailed knowledge about neutralizing and non-neutralizing epitopes is crucial for the development of robust detection systems of protective antibodies. Therefore, additional information about ZIKV immunogenicity and antibody response is required. In this project, we report the production, purification and characterization of six different polyclonal antibodies against ZIKV envelope (E) protein. The produced antibodies bind to isolated ZIKV E protein as well as to the surface of ZIKV particles, interestingly without being potently neutralizing. Surface plasmon resonance measurement showed that these antibodies bind with high affinity to ZIKV E protein. Epitope mapping revealed that the epitopes are distributed among the three ZIKV E domains with seven binding sites. These identified binding sites overlap only partially with the previously described epitopes recognized by neutralizing antibodies, which is in accordance with their lack of potent neutralizing activity. Additionally, these antibodies showed neither cross-reactivity nor potent neutralizing activity against West Nile virus, a related flavivirus. The gained set of data helps to extend our understanding about the distribution of neutralizing and non-/weak-neutralizing epitopes in ZIKV E protein, and provides a rationale for ZIKV vaccine design and development of robust detection assays for neutralizing antibodies.

Binding of Monovalent and Bivalent Ligands by Transthyretin Causes Different Short- and Long-Distance Conformational Changes.

The wild type protein, transthyretin (TTR), and over 120 genetic TTR variants are amyloidogenic and cause, respectively, sporadic and hereditary systemic TTR amyloidosis. The homotetrameric TTR contains two identical thyroxine binding pockets, occupation of which by specific ligands can inhibit TTR amyloidogenesis in vitro. Ligand binding stabilizes the tetramer, inhibiting its proteolytic cleavage and its dissociation. Here, we show with solution-state NMR that ligand binding induces long-distance conformational changes in the TTR that have not previously been detected by X-ray crystallography, consistently with the inhibition of the cleavage of the DE loop. The NMR findings, coupled with surface plasmon resonance measurements, have identified dynamic exchange processes underlying the negative cooperativity of binding of “monovalent” ligand tafamidis. In contrast, mds84, our prototypic “bivalent” ligand, which is a more potent stabilizer of TTR in vitro that occupies both thyroxine pockets and the intramolecular channel between them, has greater structural effects.

Multimodal Miniature Surface Forces Apparatus (μSFA) for Interfacial Science Measurements.

Advances in the research of intermolecular and surface interactions result from the development of new and improved measurement techniques and combinations of existing techniques. Here, we present a new miniature version of the surface forces apparatus-the μSFA-that has been designed for ease of use and multimodal capabilities with the retention of the capabilities of other SFA models including accurate measurements of the surface separation distance and physical characterization of dynamic and static physical forces (i.e., normal, shear, and friction) and interactions (e.g., van der Waals, electrostatic, hydrophobic, steric, and biospecific). The small physical size of the μSFA, compared to previous SFA models, makes it portable and suitable for integration into commercially available optical and fluorescence light microscopes, as demonstrated here. The large optical path entry and exit ports make it ideal for concurrent force measurements and spectroscopy studies. Examples of the use of the μSFA in combination with surface plasmon resonance (SPR) and Raman spectroscopy measurements are presented. Because of the short working distance constraints associated with Raman spectroscopy, an interferometric technique was developed and applied to calculate the intersurface separation distance based on Newton's rings. The introduction of the μSFA will mark a transition in SFA usage from primarily physical characterization to concurrent physical characterization with in situ chemical and biological characterization to study interfacial phenomena, including (but not limited to) molecular adsorption, fluid flow dynamics, the determination of surface species and morphology, and (bio)molecular binding kinetics.