Molecular Recognition Agents Research Articles

Structural and thermodynamic insights into antibody light chain tetramer formation through 3D domain swapping

Overexpression of antibody light chains in small plasma cell clones can lead to misfolding and aggregation. On the other hand, the formation of amyloid fibrils from antibody light chains is related to amyloidosis. Although aggregation of antibody light chain is an important issue, atomic-level structural examinations of antibody light chain aggregates are sparse. In this study, we present an antibody light chain that maintains an equilibrium between its monomeric and tetrameric states. According to data from X-ray crystallography, thermodynamic and kinetic measurements, as well as theoretical studies, this antibody light chain engages in 3D domain swapping within its variable region. Here, a pair of domain-swapped dimers creates a tetramer through hydrophobic interactions, facilitating the revelation of the domain-swapped structure. The negative cotton effect linked to the β-sheet structure, observed around 215 nm in the circular dichroism (CD) spectrum of the tetrameric variable region, is more pronounced than that of the monomer. This suggests that the monomer contains less β-sheet structures and exhibits greater flexibility than the tetramer in solution. These findings not only clarify the domain-swapped structure of the antibody light chain but also contribute to controlling antibody quality and advancing the development of future molecular recognition agents and drugs.

Development of On-DNA Thiophene Synthesis for DEL Construction.

Thiophene and its substituted derivatives are a highly important class of heterocyclic compounds, with noteworthy applications in pharmaceutical ingredients. In this study, we leverage the unique reactivity of alkynes to generate thiophenes on-DNA, using a cascade iodination, Cadiot-Chodkiewicz coupling and heterocyclization. This approach, tackling on-DNA thiophene synthesis for the first time, generates diverse, and unprecedented structural and chemical features, which could be significant motifs in DEL screening as molecular recognition agents for drug discovery.

Electrochemical-Based Sensing Platforms for Detection of Glucose and H2O2 by Porous Metal–Organic Frameworks: A Review of Status and Prospects

Establishing enzyme-free sensing assays with great selectivity and sensitivity for glucose and H2O2 detection has been highly required in biological science. In particular, the exploitation of nanomaterials by using noble metals of high conductivity and surface area has been widely investigated to act as selective catalytic agents for molecular recognition in sensing platforms. Several approaches for a straightforward, speedy, selective, and sensitive recognition of glucose and H2O2 were requested. This paper reviews the current progress in electrochemical detection using metal-organic frameworks (MOFs) for H2O2 and glucose recognition. We have reviewed the latest electrochemical sensing assays for in-place detection with priorities including straightforward procedure and manipulation, high sensitivity, varied linear range, and economic prospects. The mentioned sensing assays apply electrochemical systems through a rapid detection time that enables real-time recognition. In profitable fields, the obstacles that have been associated with sample preparation and tool expense can be solved by applying these sensing means. Some parameters, including the impedance, intensity, and potential difference measurement methods have permitted low limit of detections (LODs) and noticeable durations in agricultural, water, and foodstuff samples with high levels of glucose and H2O2.

On-DNA Alkyne Iodination and Acetylenic Coupling as a Useful Tool for DEL Synthesis.

1-Iodoalkynes and 1,3-diynes are versatile chemical intermediates and pharmaceutically valuable ingredients. In this study, copper mediated on-DNA alkyne iodination and Cadiot-Chodkiewicz coupling are developed for the first time. This generates diverse, systematic, and unprecedented topographic structural features, which could be invaluable as molecular recognition agents for drug discovery in DEL screening.

Practical tapered optical fiber system for in-situ label-free sensing of various antigens

A practical tapered optical fiber (TOF) biosensing system was developed for label-free detection using antigen-antibody pairs with repeatable results and a very high degree of sensitivity. This was done by attaching molecular recognition agents to a tapered fiber surface for augmenting sensitivity and specificity of analyte. The entire system included three main parts: a tunable laser, a tapered fiber, and an optical detector. Light from an unpolarized tunable fiber laser was introduced into the tapered fiber from one end, and the transmitted intensity was detected by a photodetector. In the tapered fiber area, the evanescent electromagnetic field, which extends outside the fiber, was able to detect minute changes in the refractive index caused by antigen-antibody pairs. Recorded data was analyzed using an innovative Fourier analysis method to find phase changes, which are directly related to the biomolecular concentration coated on fiber, from which antibody-antigen concentrations are obtained. Two experiments were performed to confirm the concept using two very different agents. The first was the protein Interleukin-8 (IL-8). Repeatable results with a sensitivity of 10 pg/mL were achieved. The second was human coronavirus OC43 (HCoV-OC43), a surrogate viral particle for SARS-CoV-2, with a sensitivity of 50 viruses/mL. Critical sources of error were identified and addressed for the purpose of using the device for real clinical diagnosis in various real-life environments, where viruses can reside in water, phosphate-buffer solution, or saliva, the most popular three environments in real clinical diagnosis. Our device was designed according to the principle that only one specific kind of antibody and antigen can be combined together. The device demonstrated good accuracy to chosen analyte(s) tailored to specific applications and offered the potential to develop a point-of-care device used in clinics, as well as for detecting a variety of viruses and biocontaminants. The reproducibility of TOFs was confirmed through multiple fabrications and consistent results.

Discovery and Characterization of Spike N‐Terminal Domain‐Binding Aptamers for Rapid SARS‐CoV‐2 Detection

The coronavirus disease 2019 (COVID‐19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS‐CoV‐2 spike glycoprotein with high specificity and affinity (<80 nM). Through binding assays and high resolution cryo‐EM, we demonstrate that SNAP1 (SARS‐CoV‐2 spike protein N‐terminal domain‐binding aptamer 1) binds to the S N‐terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV‐inactivated SARS‐CoV‐2 at concentrations as low as 5×105 copies mL−1. SNAP1 is therefore a promising molecular tool for SARS‐CoV‐2 diagnostics.

Discovery and Characterization of Spike N‐Terminal Domain‐Binding Aptamers for Rapid SARS‐CoV‐2 Detection

AbstractThe coronavirus disease 2019 (COVID‐19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS‐CoV‐2 spike glycoprotein with high specificity and affinity (&lt;80 nM). Through binding assays and high resolution cryo‐EM, we demonstrate that SNAP1 (SARS‐CoV‐2 spike protein N‐terminal domain‐binding aptamer 1) binds to the S N‐terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV‐inactivated SARS‐CoV‐2 at concentrations as low as 5×105 copies mL−1. SNAP1 is therefore a promising molecular tool for SARS‐CoV‐2 diagnostics.

Molecular Recognition: Perspective and a New Approach.

This perspective presents an overview of approaches to the preparation of molecular recognition agents for chemical sensing. These approaches include chemical synthesis, using catalysts from biological systems, partitioning, aptamers, antibodies and molecularly imprinted polymers. The latter three approaches are general in that they can be applied with a large number of analytes, both proteins and smaller molecules like drugs and hormones. Aptamers and antibodies bind analytes rapidly while molecularly imprinted polymers bind much more slowly. Most molecularly imprinted polymers, formed by polymerizing in the presence of a template, contain a high level of covalent crosslinker that causes the polymer to form a separate phase. This results in a material that is rigid with low affinity for analyte and slow binding kinetics. Our approach to templating is to use predominantly or exclusively noncovalent crosslinks. This results in soluble templated polymers that bind analyte rapidly with high affinity. The biggest challenge of this approach is that the chains are tangled when the templated polymer is dissolved in water, blocking access to binding sites.

Cyclodextrins as Supramolecular Recognition Systems: Applications in the Fabrication of Electrochemical Sensors.

Supramolecular chemistry, although focused mainly on noncovalent intermolecular and intramolecular interactions, which are considerably weaker than covalent interactions, can be employed to fabricate sensors with a remarkable affinity for a target analyte. In this review the development of cyclodextrin-based electrochemical sensors is described and discussed. Following a short introduction to the general properties of cyclodextrins and their ability to form inclusion complexes, the cyclodextrin-based sensors are introduced. This includes the combination of cyclodextrins with reduced graphene oxide, carbon nanotubes, conducting polymers, enzymes and aptamers, and electropolymerized cyclodextrin films. The applications of these materials as chiral recognition agents and biosensors and in the electrochemical detection of environmental contaminants, biomolecules and amino acids, drugs and flavonoids are reviewed and compared. Based on the papers reviewed, it is clear that cyclodextrins are promising molecular recognition agents in the creation of electrochemical sensors, chiral sensors, and biosensors. Moreover, they have been combined with a host of materials to enhance the detection of the target analytes. Nevertheless, challenges remain, including the development of more robust methods for the integration of cyclodextrins into the sensing unit.

Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events

The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.

Label-Free Detection of Tear Biomarkers Using Hydrogel-Coated Gold Nanoshells in a Localized Surface Plasmon Resonance-Based Biosensor.

The dependence of the localized surface plasmon resonance (LSPR) of noble-metal nanomaterials on refractive index makes LSPR a useful, label-free signal transduction strategy for biosensing. In particular, by decorating gold nanomaterials with molecular recognition agents, analytes of interest can be trapped near the surface, resulting in an increased refractive index surrounding the nanomaterial, and, consequently, a red shift in the LSPR wavelength. Ionic poly( N-isopropylacrylamide- co-methacrylic acid) (PNM) hydrogels were used as protein receptors because PNM nanogels exhibit a large increase in refractive index upon protein binding. Specifically, PNM hydrogels were synthesized on the surface of silica gold nanoshells (AuNSs). This composite material (AuNS@PNM) was used to detect changes in the concentration of two protein biomarkers of chronic dry eye: lysozyme and lactoferrin. Both of these proteins have high isoelectric points, resulting in electrostatic attraction between the negatively charged PNM hydrogels and positively charged proteins. Upon binding lysozyme or lactoferrin, AuNS@PNM exhibits large, concentration-dependent red shifts in LSPR wavelength, which enabled the detection of clinically relevant concentration changes of both biomarkers in human tears. The LSPR-based biosensor described herein has potential utility as an affordable screening tool for chronic dry eye and associated conditions.

Steroid-photosensitizer conjugates: Syntheses and applications

This review focuses on progress in the development of different approaches to the design of steroid ([Formula: see text] estrogens, androgens, cholesterol) conjugates with coordination assemblies of metalloporphyrins, phthalocyanines and related complexes. Porphyrins and phthalocyanines have received considerable attention due to their novel composition, intriguing spectroscopic, photophysical, and redox properties, and potential application in light-harvesting and optoelectronic devices. With the development of more efficient imaging and therapeutic applications, these bio-conjugates are evaluated as multimodality agents (PET, fluorescence imaging) to monitor the mechanism of action of biologically active components in living systems and as agents for molecular recognition, oxygen atom transfer and catalysis. The tetrapyrrole components, which can be coupled via covalent and various non-covalent linkages, may exhibit strong interactions through efficient photo-induced electron and/or energy transfer processes.

Fluorimetric sandwich affinity assay for Staphylococcus aureus based on dual-peptide recognition on magnetic nanoparticles

A novel dual-peptide recognition strategy was designed for sandwich fluorimetric detection of Staphylococcus aureus (S. aureus) utilizing the affinity of bacterial cells for certain peptides. A phage-display peptide derived from a random peptide library was used to functionalize magnetic particles and to specifically capture S. aureus. Magainin I is a broad-spectrum antimicrobial peptide that binds to cell membranes of most bacteria. It was used as a second recognition peptide to form a sandwich complex with S. aureus. By using fluorescein isothiocyanate as the fluorescent label and working at excitation/emission wavelengths of 488/525 nm, S. aureus can be directly detected in the 10 to 10,000 cfu·mL−1 concentration range, with a detection limit as low as 9 cfu·mL−1. The whole detection process can be completed within 90 min. This strategy was successfully applied to the detection of S. aureus in spiked lake water, human urine and apple juice. Respective recovery values ranged from 81% to 110%. The assay is highly sensitive and specific, and fast. The use of synthetic peptides shows many advantages such as lower cost, higher stability and ease of chemical modification compared to other molecular recognition agents such as antibodies, bacteriophages, or aptamers. In our perception, this detection scheme may be extended to many other pathogens if phage-displayed peptides specific for other bacteria are available.

Synthesis of Lipophilic Guanine N-9 Derivatives: Membrane Anchoring of Nucleobases Tailored to Fatty Acid Vesicles.

Covalent or noncovalent surface functionalization of soft-matter structures is an important tool for tailoring their function and stability. Functionalized surfaces and nanoparticles have found numerous applications in drug delivery and diagnostics, and new functionalization chemistry is continuously being developed in the discipline of bottom-up systems chemistry. The association of polar functional molecules, e.g., molecular recognition agents, with soft-matter structures can be achieved by derivatization with alkyl chains, allowing noncovalent anchoring into amphiphilic membranes. We report the synthesis of five new guanine-N9 derivatives bearing alkyl chains with different attachment chemistries, exploiting a synthesis pathway that allows a flexible choice of hydrophobic anchor moiety. In this study, these guanine derivatives were functionalized with C10 chains for insertion into decanoic acid bilayer structures, in which both alkyl chain length and attachment chemistry determined their interaction with the membrane. Incubation of these guanine conjugates, as solids, with a decanoic acid vesicle suspension, showed that ether- and triazole-linked C10 anchors yielded an increased partitioning of the guanine derivative into the membranous phase compared to directly N-9-linked saturated alkyl anchors. Decanoic acid vesicle membranes could be loaded with up to 5.5 mol % guanine derivative, a 6-fold increase over previous limits. Thus, anchor chemistries exhibiting favorable interactions with a bilayer's hydrophilic surface can significantly increase the degree of structure functionalization.

Sensitive Detection of Staphylococcus aureus with Vancomycin-Conjugated Magnetic Beads as Enrichment Carriers Combined with Flow Cytometry.

A novel sandwich strategy was designed to detect Staphylococcus aureus. The strategy is based on an antibacterial agent that captures bacterial cells and a fluorescein-labeled antibody that acts as the signal-output probe. Vancomycin (Van), which exerts a strong antibacterial effect on Gram-positive bacteria, was utilized as a molecular recognition agent to detect pathogenic bacteria. To effectively concentrate S. aureus, we used bovine serum albumin (BSA) as the amplification carrier to modify magnetic beads (MBs), which were then functionalized with Van. To improve the specificity of the method for S. aureus detection, we adopted fluorescein isothiocyanate (FITC)-tagged pig immunoglobulin G (FITC-pig IgG) as the signal probe and the second recognition agent that bound between the Fc fragment of pig IgG and protein A in the surface of S. aureus. To quantify S. aureus, we measured the fluorescence signal by flow cytometry (FCM). The use of multivalent magnetic nanoprobes (Van-BSA-MBs) showed a high concentration efficiency (>98%) at bacterial concentrations of only 33 colony-forming units (CFU)/mL. Furthermore, the sandwich mode (FITC-pig IgG/SA/Van-BSA-MBs) also showed ideal specificity because Van and IgG bound with S. aureus at two distinct sites. The detection limit for S. aureus was 3.3 × 101 CFU/mL and the total detection process could be completed within 120 min. Other Gram-positive bacteria and Gram-negative bacteria, including Listeria monocytogenes, Bacillus cereus, Cronobacter sakazakii, Escherichia coli O157:H7, and Salmonella Enteritidis, negligibly interfered with S. aureus detection. The proposed detection strategy for S. aureus possesses attractive characteristics, such as high sensitivity, simple operation, short testing time, and low cost.

Charged poly(N-isopropylacrylamide) nanogels for use as differential protein receptors in a turbidimetric sensor array.

Due to the high cost and environmental instability of antibodies, there is precedent for developing synthetic molecular recognition agents for use in diagnostic sensors. While these materials typically have lower specificity than antibodies, their cross-reactivity makes them excellent candidates for use in differential sensing routines. In the current work, we design a set of charge-containing poly(N-isopropylacrylamide) (PNIPAM) nanogels for use as differential protein receptors in a turbidimetric sensor array. Specifically, NIPAM was copolymerized with methacrylic acid and modified via carbodiimide coupling to introduce sulfate, guanidinium, secondary amine, or primary amine groups. Modification of the ionizable groups in the network changed the physicochemical and protein binding properties of the nanogels. For high affinity protein-polymer interactions, turbidity of the nanogel solution increased, while for low affinity interactions minimal change in turbidity was observed. Thus, relative turbidity was used as input for multivariate analysis. Turbidimetric assays were performed in two buffers of different pH (i.e., 7.4 and 5.5), but comparable ionic strength, in order to improve differentiation. Using both buffers, it was possible to achieve 100% classification accuracy of eleven model protein biomarkers with as few as two of the nanogel receptors. Additionally, it was possible to detect changes in lysozyme concentration in a simulated tear fluid using the turbidimetric sensor array.

A Synthetic Supramolecular Receptor for the Hydrosulfide Anion.

Hydrogen sulfide (H2 S) has emerged as a crucial biomolecule in physiology and cellular signaling. Key challenges associated with developing new chemical tools for understanding the biological roles of H2 S include developing platforms that enable reversible binding of this important biomolecule. The first synthetic small molecule receptor for the hydrosulfide anion, HS(-) , using only reversible, hydrogen-bonding interactions in a series of bis(ethynylaniline) derivatives, is reported. Binding constants of up to 90 300±8700 m(-1) were obtained in MeCN. The fundamental science of reversible sulfide binding, in this case featuring a key CH⋅⋅⋅S hydrogen bond, will expand the possibility for discovery of sulfide protein targets and molecular recognition agents.

A Synthetic Supramolecular Receptor for the Hydrosulfide Anion

AbstractHydrogen sulfide (H2S) has emerged as a crucial biomolecule in physiology and cellular signaling. Key challenges associated with developing new chemical tools for understanding the biological roles of H2S include developing platforms that enable reversible binding of this important biomolecule. The first synthetic small molecule receptor for the hydrosulfide anion, HS−, using only reversible, hydrogen‐bonding interactions in a series of bis(ethynylaniline) derivatives, is reported. Binding constants of up to 90 300±8700 m−1 were obtained in MeCN. The fundamental science of reversible sulfide binding, in this case featuring a key CH⋅⋅⋅S hydrogen bond, will expand the possibility for discovery of sulfide protein targets and molecular recognition agents.

Rapid and visual detection of Listeria monocytogenes based on nanoparticle cluster catalyzed signal amplification

Foodborne pathogens pose a significant threat to human health worldwide. The identification of foodborne pathogens needs to be rapid, accurate and convenient. Here, we constructed a nanoparticle cluster (NPC) catalyzed signal amplification biosensor for foodborne pathogens visual detection. In this work, vancomycin (Van), a glycopeptide antibiotic for Gram-positive bacteria, was used as the first molecular recognition agent to capture Listeria monocytogenes (L. monocytogenes). Fe3O4 NPC modified aptamer, was used as the signal amplification nanoprobe, specifically recognize to the cell wall of L. monocytogenes. As vancomycin and aptamer recognize L. monocytogenes at different sites, the sandwich recognition showed satisfied specificity. Compared to individual Fe3O4 nanoparticle (NP), NPC exhibit collective effect-enhanced catalytic activity for the color reaction of chromogenic substrate. The change in absorbance or color could represent the concentration of target. Using the Fe3O4 NPC-based signal amplification method, L. monocytogenes whole cells could be directly assayed within a linear range of 5.4×103–108 cfu/mL and a visual limit of detection of 5.4×103 cfu/mL. Fe3O4 NPC-based method was more sensitive than the Fe3O4 NP-based method. All these attractive characteristics of highly sensitivity, visual and labor-saving, make the biosensor possess a potential application for foodborne pathogenic bacteria detection.

Dual-recognition detection of Staphylococcus aureus using vancomycin-functionalized magnetic beads as concentration carriers

Vancomycin, which has a strong antibacterial effect to Gram-positive bacteria, was adopted as one molecular recognition agent for bacterial detection. Magnetic beads (MBs) were functionalized with this antibiotic to effectively concentrate Staphylococcus aureus (S. aureus). In addition, alkaline phosphatase (ALP)-tagged rabbit immunoglobulin G (ALP-IgG) was used as the second recognition agent to improve the specificity based on the binding between the Fc region of rabbit IgG and protein A in the cell wall of S. aureus. MBs-concentrated sandwich complex of vancomycin/S. aureus/ALP-IgG was formed with a one-step incubation protocol. Then ALP chemiluminescent reaction was triggered by injecting substrate solution to quantitate S. aureus. Based on the sandwich molecular recognition mechanism and MBs concentration, an ultrasensitive, specific and rapid method was developed for S. aureus detection. The linear range for S. aureus detection was 12–1.2×106CFUmL−1, with a very low detection limit of 3.3CFUmL−1. The whole detection process could be completed in 75min. Other Gram-positive bacteria and Gram-negative bacteria, including Escherichia coli, Salmonella, Pseudomonas aeruginosa, Micrococcus luteus, Bacillus cereus and Bacillus subtilis, showed negligible interference to S. aureus detection. This method was successfully used to quantitate S. aureus in lake water, milk, human urine and human saliva with acceptable recoveries ranging from 70.0% to 116.7%.