Early determination of potential critical quality attributes of therapeutic antibodies in developability studies through surface plasmon resonance-based relative binding activity assessment.

Precise measurement of the binding activity changes of therapeutic antibodies is important to determine the potential critical quality attributes (CQAs) in developability assessment at the early stage of antibody development. Here, we report a surface plasmon resonance (SPR)-based relative binding activity method, which incorporates both binding affinity and binding response and allows us to determine relative binding activity of antibodies with high accuracy and precision. We applied the SPR-based relative binding activity method in multiple forced degradation studies of antibody developability assessment. The current developability assessment strategy provided comprehensive, precise characterization of antibody binding activity in the stability studies, enabling us to perform correlation analysis and establish the structure-function relationship between relative binding activity and quality attributes. The impact of a given quality attribute on binding activity could be confidently determined without isolating antibody variants. We identified several potential CQAs, including Asp isomerization, Asn deamidation, and fragmentation. Some potential CQAs affected binding affinity of antibody and resulted in a reduction of binding activity. Certain potential CQAs impaired antibody binding to antigen and led to a loss of binding activity. A few potential CQAs could influence both binding affinity and binding response and cause a substantial decrease in antibody binding activity. Specifically, we identified low abundance Asn33 deamidation in the light chain complementarity-determining region as a potential CQA, in which all the stressed antibody samples showed Asn33 deamidation abundances ranging from 4.2% to 27.5% and a mild binding affinity change from 1.76 nM to 2.16 nM.

A protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors.

Sensing, transport, and utilization of glucose is pivotal to the maintenance of energy homeostasis in animals. Although transporters involved in mobilizing glucose across different cellular compartments are fairly well known, the receptors that bind glucose to mediate its effects independently of glucose metabolism remain largely unrecognized. Establishing precise and reproducible methods to identify glucose receptors in the brain or other peripheral organs will pave the way for comprehending the role of glucose signaling pathways in maintaining, regulating, and reprogramming cellular metabolic needs. Identification of such potential glucose receptors will also likely lead to development of effective therapeutics for treatment of diabetes and related metabolic disorders. Commercially available biotin or radiolabeled glucose conjugates have low molecular weight; therefore, they do not provide enough sensitivity and density to isolate glucose receptors. Here, we describe a protocol to isolate, identify, and verify glucose-binding receptor/s using high molecular weight glucose (or other carbohydrate) conjugates. We have produced 30 kDa glucose- (or other carbohydrate-) biotin-polyacrylamide (PAA) conjugates with mole fractions of 80:5:15% respectively. These conjugates are used with biotin-streptavidin biochemistry, In-cell ELISA, and surface plasmon resonance (SPR) methods to isolate, identify, and verify glucose- or carbohydrate-binding receptors. We first demonstrate how streptavidin-coated magnetic beads are employed to immobilize glucose-biotin-PAA conjugates. Then, these beads are used to enrich and isolate glucose-binding proteins from tissue homogenates or from single-cell suspensions. The enriched or isolated proteins are subjected to mass spectrometry/proteomics to reveal the identity of top candidate proteins as potential glucose receptors. We then describe how the In-cell ELISA method is used to verify the interaction of glucose with its potential receptor through stable expression of the receptor in-vitro. We further demonstrate how a highly sensitive SPR method can be used to measure the binding kinetics of glucose with its receptor. In summary, we describe a protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors using magnetic beads, In-cell ELISA, and SPR. This protocol will form the future basis of studying glucose or carbohydrate receptor signaling pathways in health and in disease.

Efficient characterization of multiple binding sites of small molecule imaging ligands on amyloid-beta, tau and alpha-synuclein.

PURPOSE: There is an unmet need for compounds to detect fibrillar forms of alpha-synuclein (αSyn) and 4-repeat tau, which are critical in many neurodegenerative diseases. Here, we aim to develop an efficient surface plasmon resonance (SPR)-based assay to facilitate the characterization of small molecules that can bind these fibrils. METHODS: SPR measurements were conducted to characterize the binding properties of fluorescent ligands/compounds toward recombinant amyloid-beta (Aß)42, K18-tau, full-length 2N4R-tau and αSyn fibrils. In silico modeling was performed to examine the binding pockets of ligands on αSyn fibrils. Immunofluorescence staining of postmortem brain tissue slices from Parkinson's disease patients and mouse models was performed with fluorescence ligands and specific antibodies. RESULTS: We optimized the protocol for the immobilization of Aß42, K18-tau, full-length 2N4R-tau and αSyn fibrils in a controlled aggregation state on SPR-sensor chips and for assessing their binding to ligands. The SPR results from the analysis of binding kinetics suggested the presence of at least two binding sites for all fibrils, including luminescent conjugated oligothiophenes, benzothiazole derivatives, nonfluorescent methylene blue and lansoprazole. In silico modeling studies for αSyn (6H6B) revealed four binding sites with a preference for one site on the surface. Immunofluorescence staining validated the detection of pS129-αSyn positivity in the brains of Parkinson's disease patients and αSyn preformed-fibril injected mice, 6E10-positive Aß in arcAß mice, and AT-8/AT-100-positivity in pR5 mice. CONCLUSION: SPR measurements of small molecules binding to Aß42, K18/full-length 2N4R-tau and αSyn fibrils suggested the existence of multiple binding sites. This approach may provide efficient characterization of compounds for neurodegenerative disease-relevant proteinopathies.

Highly sensitive detection of low-concentration sodium chloride solutions based on a gold-coated prism in Kretschmann setup.

A gold-coated Kretschmann setup has been constructed and explored as a surface plasmon resonance (SPR) platform, specifically tailored for the detection of low-concentration sodium chloride (NaCl) solutions. The setup employs a BK7 prism coated with a 50 nm gold layer, serving as a plasmonic layer, to induce resonance. This resonance arises from the interplay between light waves and free electrons propagating at the interface of two media. The experimental findings reveal a notable resonance angle shift of 10° when the NaCl concentration is varied from 0 to 2.5 %. Furthermore, angle interrogation provides insightful details about the sensor's response to changes in the refractive index, showcasing a commendable sensitivity of 2400°/RIU, a high level of linearity at 0.9771, and an impressive resolution of 0.217 %. The demonstrated capabilities of this sensor underscore its potential for widespread applications, particularly in the monitoring of salt concentration across diverse domains such as seawater analysis, food processing, and fermentation processes. The robust performance and precision of this proposed sensor position it as a valuable tool with promising prospects for addressing the needs of various industries dependent on accurate salt concentration measurements.

Enhanced organic photovoltaic-based retinal prosthesis using a cathode-modified structure with plasmonic silver nanoparticles: a computational study.

Introduction: Organic interfaces have recently emerged as a breakthrough trend in biomedical applications, demonstrating exceptional performance in stimulating retinal neuronal cells owing to their high flexibility and compatibility with tissues. However, the primary challenge associated with organic photovoltaics is their low efficiency compared to that of their inorganic counterparts. Among different approaches, embedding plasmonic metal nanoparticles (NPs) in active or buffer layers can efficiently improve photovoltaic cell performance. Methods: A cathode decorated with silver nanoparticles is introduced to increase the absorption Phenomenon and improve the interface performance as a computational study. In addition to embedding spherical silver nanoparticles in the active layer (A-AgNPs), a monolayer array of spherical AgNPs in the cathode electrode (K-AgNPs) is incorporated. In this configuration, the large K-AgNPs play dual roles: acting as cathode electrode and serving as plasmonic centers to increase light trapping and absorption. The bulk heterojunction PCPDTBT:PCBM is chosen as the active layer due to its favorable electronic properties. Results: Our computational analysis demonstrates a notable 10% enhancement in the photovoltaic cell current density for the developed structure with K-AgNPs in contrast to without them. Additionally, the simulation results reveal that the modeled device achieves a two-fold efficiency of the bare photovoltaic cell (without A-AgNPs and K-AgNPs), which is particularly evident at a low intensity of 0.26 mW/mm2. Discussion: This study aims to propose an efficient epiretinal prosthesis structure using a different strategy for plasmonic effects rather than conventional methods, such as incorporating NPs into the active or buffer layer. This structure can prevent the harmful side effects of using large metal NPs (r > 10 nm) in the active layer during exciton quenching, charge trapping, and recombination, which deteriorate the power conversion efficiency (PCE).

Insights into Photopolymerization at the Nanoscale Using Surface Plasmon Resonance Imaging.

Near-field photopolymerization (NFPP) driven by surface plasmon resonance has attracted increasing attention in nanofabrication. This interest comes from the nanometer-scale control of polymer thickness, due to the confinement of the evanescent wave within a highly restricted volume at the surface. In this study, a novel approach using a multi-spectral surface plasmon resonance instrument is presented that gives access to real-time images of polymer growth during NFPP with nanometer sensitivity. Using the plasmonic evanescent wave for both polymerization and real-time sensing, the influence of irradiance, concentration of dye, and initiator are investigated on the threshold energy and kinetics of NFPP. How oxygen inhibition in the near field strongly affects photopolymerization is highlighted, more than in the far field.

Artificial Mitochondria Nanoarchitectonics via a Supramolecular Assembled Microreactor Covered by ATP Synthase.

Abiotic stress tends to induce oxidative damage to enzymes and organelles that in turns hampers the phosphorylation process and decreases the adenosine triphosphate (ATP) productivity. Artificial assemblies can alleviate abiotic stress and simultaneously provide nutrients to diminish the oxidative damage. Here, we have integrated natural acid phosphatase (ACP) and ATP synthase with plasmonic Au clusters in a biomimetic microreactor. ACP immobilized on the Au clusters is harnessed to generate proton influx to drive ATP synthase and concurrently supply phosphate to improve phosphorus availability to combat phosphorus-deficiency stress. In tandem with the reactive oxygen species (ROS) scavenging and the photothermal functionality of Au clusters, such an assembled microreactor exhibits an improved abiotic stress tolerance and achieves plasmon-accelerated ATP synthesis. This innovative approach offers an effective route to enhance the stress resistance of ATP synthase-based energy-generating systems, opening an exciting potential of these systems for biomimicking applications.

Fiber Optic LSPR Sensing AFM1 in Milk with Enhanced Sensitivity by the Hot Spot Effect Based on Nanogap Construction.

The detection of the amount of aflatoxin M1 (AFM1) in milk is crucial for food safety. Here, we utilize a fiber optic (FO) localized surface plasmon resonance (LSPR) biosensor by constructing gold nanoparticle (AuNP) multimers, in which the nanogaps amplified the LSPR signal by the hot spot effect, and achieved a highly sensitive detection of f AFM1. Through the optimization of parameter conditions for the fabrication of the sensor and detection system, a high performance result from the FO LSPR biosensor was obtained, and the method for AFM1 detection was established, with a wide detection range of 0.05-100 ng/mL and a low limit of detection (LOD) of 0.04 ng/mL, and it has been successfully validated with the actual sample milk. Therefore, it is a good strategy to fabricate highly sensitive FO LSPR sensors for detecting AFM1 by constructing AuNP multimers, and this approach is suitable for developing other biosensors.

Wavelength Division Multiplexing-Based High-Sensitivity Surface Plasmon Resonance Imaging Biosensor for High-Throughput Real-Time Molecular Interaction Analysis.

In this study, we report the successful development of a novel high-sensitivity intensity-based Surface Plasmon Resonance imaging (SPRi) biosensor and its application for detecting molecular interactions. By optimizing the excitation wavelength and employing a wavelength division multiplexing (WDM) algorithm, the system can determine the optimal excitation wavelength based on the initial refractive index of the sample without adjusting the incidence angle. The experimental results demonstrate that the refractive index resolution of the system reaches 1.77×10-6 RIU. Moreover, it can obtain the optimal excitation wavelength for samples with an initial refractive index in the range of 1.333 to 1.370 RIU and accurately monitor variations within the range of 0.0037 RIU without adjusting the incidence angle. Additionally, our new SPRi technique realized real-time detection of high-throughput biomolecular binding processes, enabling analysis of kinetic parameters. This research is expected to advance the development of more accurate SPRi technologies for molecular interaction analysis.

Anisotropic Sensing Performance in a High-Sensitivity Surface Plasmon Resonance Sensor Based on Few-Layer Black Phosphorus.

Few-layer black phosphorus (FLBP) is a highly promising material for high sensitivity label-free surface plasmon resonance (SPR) sensors due to its exceptional electrical, optical, and mechanical properties. FLBP exhibits inherent anisotropy with different refractive indices along its two main crystal orientations, the zigzag and armchair axes. However, this anisotropic property is often overlooked in FLBP-based sensors. In this study, we conducted a comprehensive investigation of the SPR reflectivity and phase in a BK7-Ag-FLBP structure to understand the influence of the stacking sequence and the number of FLBP layers on the sensing performance. Clear resonant angle shifts caused by different stacking sequences of FLBP could be observed both theoretically and experimentally. In the theoretical study, the highest reflective and phase sensitivities were achieved with a 12-layer black phosphorus (BP) structure. The reflectivity sensitivity reached 287.9°/refractive index units (RIU) with the zz stacking 12-layer BP film exhibiting a sensitivity 76°/RIU higher than the ac stacking structure. Similarly, the phase sensitivity reached 1162°/RIU with the zz stacking 12-layer BP structure showing a sensitivity 276.9°/RIU higher than the ac stacking structure. The electric field distribution of the 12-layer BP structure with four different stacking sequences has also been analyzed. In the experiment study, the well-known Attenuated Total Reflection (ATR) θ-2θ SPR setup is utilized to detect the reflectivity and phase of BK7-Ag-FLBP structures. The FLBP samples with the same thickness but different stacking sequences show significant resonant angle shift (0.275°) and maximum phase difference variation (34.6°). The FLBP sample thickness and crystal orientations have been demonstrated using the angular-resolved polarized Raman spectroscopy (ARPRS). These theoretical and experimental results provide strong evidence that the stacking sequences of FLBP have a significant impact on the sensing performance of SPR sensors. By harnessing the anisotropic properties of materials like FLBP, novel structures of anisotropic-2D material-based SPR sensors could open up exciting possibilities for innovative applications.

Advances in Nanoplasmonic Biosensors: Optimizing Performance for Exosome Detection Applications.

The development of sensitive and specific exosome detection tools is essential because they are believed to provide specific information that is important for early detection, screening, diagnosis, and monitoring of cancer. Among the many detection tools, surface-plasmon resonance (SPR) biosensors are analytical devices that offer advantages in sensitivity and detection speed, thereby making the sample-analysis process faster and more accurate. In addition, the penetration depth of the SPR biosensor, which is <300 nm, is comparable to the size of the exosome, making the SPR biosensor ideal for use in exosome research. On the other hand, another type of nanoplasmonic sensor, namely a localized surface-plasmon resonance (LSPR) biosensor, has a shorter penetration depth of around 6 nm. Structural optimization through the addition of supporting layers and gap control between particles is needed to strengthen the surface-plasmon field. This paper summarizes the progress of the development of SPR and LSPR biosensors for detecting exosomes. Techniques in signal amplification from two sensors will be discussed. There are three main parts to this paper. The first two parts will focus on reviewing the working principles of each sensor and introducing several methods that can be used to isolate exosomes. This article will close by explaining the various sensor systems that have been developed and the optimizations carried out to obtain sensors with better performance. To illustrate the performance improvements in each sensor system discussed, the parameters highlighted include the detection limit, dynamic range, and sensitivity.

Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays.

The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody-antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.

Investigating UV-Irradiation Parameters in the Green Synthesis of Silver Nanoparticles from Water Hyacinth Leaf Extract: Optimization for Future Sensor Applications.

Silver nanoparticles (AgNPs) can be produced safely and greenly using water hyacinth, an invasive aquatic plant, as a reducing agent. This study aimed to optimize the UV-irradiation parameters for the synthesis of AgNPs from water hyacinth leaf extract. The study varied the reaction time and pH levels and added a stabilizing agent to the mixture. The synthesized AgNPs were characterized using UV-visible spectroscopy (UV-vis), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and inductively coupled plasma optical emission spectroscopy (ICP-OES). The findings revealed that the optimal conditions for synthesizing AgNPs were achieved by adjusting the pH level to 8.5, adding starch as a stabilizing agent, and exposing the mixture to UV-A radiation for one hour. These conditions resulted in the smallest size and highest quantity of AgNPs. Furthermore, the synthesized AgNP colloids remained stable for up to six months. This study highlights the potential of utilizing water hyacinth as a sustainable and cost-effective reducing agent for AgNP synthesis, with potential applications in pharmaceuticals, drug development, catalysis, and sensing detection.

An Efficient Method for Vault Nanoparticle Conjugation with Finely Adjustable Amounts of Antibodies and Small Molecules.

Vaults are eukaryotic ribonucleoproteins consisting of 78 copies of the major vault protein (MVP), which assemble into a nanoparticle with an about 60 nm volume-based size, enclosing other proteins and RNAs. Regardless of their physiological role(s), vaults represent ideal, natural hollow nanoparticles, which are produced by the assembly of the sole MVP. Here, we have expressed in Komagataella phaffi and purified an MVP variant carrying a C-terminal Z peptide (vault-Z), which can tightly bind an antibody's Fc portion, in view of targeted delivery. Via surface plasmon resonance analysis, we could determine a 2.5 nM affinity to the monoclonal antibody Trastuzumab (Tz)/vault-Z 1:1 interaction. Then, we characterized the in-solution interaction via co-incubation, ultracentrifugation, and analysis of the pelleted proteins. This showed virtually irreversible binding up to an at least 10:1 Tz/vault-Z ratio. As a proof of concept, we labeled the Fc portion of Tz with a fluorophore and conjugated it with the nanoparticle, along with either Tz or Cetuximab, another monoclonal antibody. Thus, we could demonstrate antibody-dependent, selective uptake by the SKBR3 and MDA-MB 231 breast cancer cell lines. These investigations provide a novel, flexible technological platform that significantly extends vault-Z's applications, in that it can be stably conjugated with finely adjusted amounts of antibodies as well as of other molecules, such as fluorophores, cell-targeting peptides, or drugs, using the Fc portion as a scaffold.

Determining the Affinity and Kinetics of Small Molecule Inhibitors of Galectin-1 Using Surface Plasmon Resonance.

The beta-galactoside-binding mammalian lectin galectin-1 can bind, via its carbohydrate recognition domain (CRD), to various cell surface glycoproteins and has been implicated in a range of cancers. As a consequence of binding to sugar residues on cell surface receptors, it has been shown to have a pleiotropic effect across many cell types and mechanisms, resulting in immune system modulation and cancer progression. As a result, it has started to become a therapeutic target for both small and large molecules. In previous studies, we used fluorescence polarization (FP) assays to determine KD values to screen and triage small molecule glycomimetics that bind to the galectin-1 CRD. In this study, surface plasmon resonance (SPR) was used to compare human and mouse galectin-1 affinity measures with FP, as SPR has not been applied for compound screening against this galectin. Binding affinities for a selection of mono- and di-saccharides covering a 1000-fold range correlated well between FP and SPR assay formats for both human and mouse galectin-1. It was shown that slower dissociation drove the increased affinity at human galectin-1, whilst faster association was responsible for the effects in mouse galectin-1. This study demonstrates that SPR is a sound alternative to FP for early drug discovery screening and determining affinity estimates. Consequently, it also allows association and dissociation constants to be measured in a high-throughput manner for small molecule galectin-1 inhibitors.

An "off-on-enhanced on" electrochemiluminescence biosensor based on resonance energy transfer and surface plasmon coupled 3D DNA walker for ultra-sensitive detection of microRNA-21.

In this study, a novel electrochemiluminescence (ECL) biosensor was developed to detect microRNA-21 (miRNA-21) with high sensitivity by leveraging the combined mechanisms of resonance energy transfer (RET) and surface plasmon coupling (SPC). Initially, the glassy carbon electrode (GCE) were coated with Cu-Zn-In-S quantum dots (CZIS QDs), known for their defect-related emission suitable for ECL sensing. Subsequently, a hairpin DNA H3 with gold nanoparticles (Au NPs) attached at the end was modified over the surface of the quantum dots. The Au NPs could effectively quench the ECL signals of CZIS QDs via RET. Further, a significant amount of report DNA was generated through the action of a 3D DNA walker. When the report DNA opened H3-Au NPs, the hairpin structure experienced a conformational change to a linear shape, increasing the gap between the CZIS QDs and the Au NPs. Consequently, the localized surface plasmon resonance ECL (LSPR-ECL) effect replaced ECL resonance energy transfer (ECL-RET). Moreover, the report DNA was released following the addition of H4-Au NPs, resulting in the formation of Au dimers and a surface plasma-coupled ECL (SPC-ECL) effect that enhanced the ECL intensity to 6.97-fold. The integration of new ECL-RET and SPC-ECL biosensor accurately quantified miRNA-21 concentrations from 10-8 M to 10-16 M with a limit of detection (LOD) of 0.08 fM, as well as successfully applied to validate human serum samples.

A novel biomimetic nanoplasmonic sensor for rapid and accurate evaluation of checkpoint inhibitor immunotherapy.

Immune checkpoint inhibitors (ICIs) emerged as promising immunotherapies for cancer treatment, harnessing the patient's immune system to fight and eliminate tumor cells. However, despite their potential and proven efficacies, checkpoint inhibitors still face important challenges such as the tumor heterogeneity and resistance mechanisms, and the complex in vitro testing, which limits their widespread applicability and implementation to treat cancer. To address these challenges, we propose a novel analytical technique utilizing biomimetic label-free nanoplasmonic biosensors for rapid and reliable screening and evaluation of checkpoint inhibitors. We have designed and fabricated a low-density nanostructured plasmonic sensor based on gold nanodisks that enables the direct formation of a functional supported lipid bilayer, which acts as an artificial cell membrane for tumor ligand immobilization. With this biomimetic scaffold, our biosensing approach provides real-time, highly sensitive analysis of immune checkpoint pathways and direct assessment of the blocking effects of monoclonal antibodies in less than 20 min/test. We demonstrate the accuracy of our biomimetic sensor for the study of the programmed cell death protein 1 (PD1) checkpoint pathway, achieving a limit of detection of 6.7 ng/mL for direct PD1/PD-L1 interaction monitoring. Besides, we have performed dose-response inhibition curves for an anti-PD1 monoclonal antibody, obtaining a half maximal inhibitory concentration (IC50) of 0.43 nM, within the same range than those obtained with conventional techniques. Our biomimetic sensor platform combines the potential of plasmonic technologies for rapid label-free analysis with the reliability of cell-based assay in terms of ligand mobility. The biosensor is integrated in a compact user-friendly device for the straightforward implementation in biomedical and pharmaceutical laboratories.

Molecularly imprinted nanogels as synthetic recognition materials for the ultrasensitive detection of periodontal disease biomarkers.

Periodontal disease affects supporting dental structures and ranks among one of the top most expensive conditions to treat in the world. Moreover, in recent years, the disease has also been linked to cardiovascular and Alzheimer's diseases. At present, there is a serious lack of accurate diagnostic tools to identify people at severe risk of periodontal disease progression. Porphyromonas gingivalis is often considered one of the most contributing factors towards disease progression. It produces the Arg- and Lys-specific proteases Rgp and Kgp, respectively. Within this work, a short epitope sequence of these proteases is immobilised onto a magnetic nanoparticle platform. These are then used as a template to produce high-affinity, selective molecularly imprinted nanogels, using the common monomers N-tert-butylacrylamide (TBAM), N-isopropyl acrylamide (NIPAM), and N-(3-aminopropyl) methacrylamide hydrochloride (APMA). N,N-Methylene bis(acrylamide) (BIS) was used as a crosslinking monomer to form the interconnected polymeric network. The produced nanogels were immobilised onto a planar gold surface and characterised using the optical technique of surface plasmon resonance. They showed high selectivity and affinity towards their template, with affinity constants of 79.4 and 89.7 nM for the Rgp and Kgp epitope nanogels, respectively. From their calibration curves, the theoretical limit of detection was determined to be 1.27 nM for the Rgp nanogels and 2.00 nM for the Kgp nanogels. Furthermore, they also showed excellent selectivity against bacterial culture supernatants E8 (Rgp knockout), K1A (Kgp knockout), and W50-d (wild-type) strains in complex medium of brain heart infusion (BHI).

Structural Basis for Recognition of the FLAG-tag by Anti-FLAG M2.

The FLAG-tag/anti-FLAG system is a widely used biochemical tool for protein detection and purification. Anti-FLAG M2 is the most popular antibody against the FLAG-tag, due to its ease of use, versatility, and availability in pure form or as bead conjugate. M2 binds N-terminal, C-terminal and internal FLAG-tags and binding is calcium-independent, but the molecular basis for the FLAG-tag specificity and recognition remains unresolved. Here we present an atomic resolution (1.17 Å) structure of the FLAG peptide in complex with the Fab of anti-FLAG M2, revealing key binding determinants. Five of the eight FLAG peptide residues form direct interactions with paratope residues. The FLAG peptide adopts a 310 helix conformation in complex with the Fab. These structural insights allowed us to rationally introduce point mutations on both the peptide and antibody side. We tested these by surface plasmon resonance, leading us to propose a shorter yet equally binding version of the FLAG-tag for the M2 antibody.

The potential of a novel enzyme-based surface plasmon resonance biosensor for direct detection of dopamine.

Dopamine is one of the significant neurotransmitters and its monitoring in biological fluids is a critical issue in healthcare and modern biomedical technology. Here, we have developed a dopamine biosensor based on surface plasmon resonance (SPR). For this purpose, the carboxymethyl dextran SPR chip was used as a surface to immobilize laccase as a bioaffinity recognition element. Data analysis exhibited that the acidic pH value is the optimal condition for dopamine interaction. Calculated kinetic affinity (KD) (48,545 nM), obtained from a molecular docking study, showed strong association of dopamine with the active site of laccase. The biosensor exhibited a linearity from 0.01 to 189 µg/ml and a lower detection limit of 0.1 ng/ml (signal-to-noise ratio (S/N) = 3) that is significantly higher than the most direct dopamine detecting sensors reported so far. Experiments for specificity in the presence of compounds that can co-exist with dopamine detection such as ascorbic acid, urea and L-dopa showed no significant interference. The current dopamine biosensor with high sensitivity and specificity, represent a novel detection tool that offers a label-free, simple procedure and cost effective monitoring system.