Study Of Biomolecular Interactions Research Articles

Three-Dimensional (3D) Surface-Enhanced Raman Spectroscopy (SERS) Substrates for Sensing Low-Concentration Molecules in Solution

The use of surface-enhanced Raman spectroscopy (SERS) in liquid solutions has always been challenging due to signal fluctuations, inconsistent data, and difficulties in obtaining reliable results, especially at very low analyte concentrations. In our study, we introduce a new method using a three-dimensional (3D) SERS substrate made of silica microparticles (SMPs) with attached plasmonic nanoparticles (NPs). These SMPs were placed in low-concentration analyte solutions for SERS analysis. In the first approach to perform SERS in a 3D environment, glycerin was used to immobilize the particles, which enabled high-resolution SERS imaging. Additionally, we conducted time-dependent SERS measurements in an aqueous solution, where freely suspended SMPs passed through the laser focus. In both scenarios, EFs larger than 200 were achieved, which enabled the detection of low-abundance analytes. Our study demonstrates a reliable and reproducible method for performing SERS in liquid environments, offering significant advantages for the real-time analysis of dynamic processes, sensitive detection of low-concentration molecules, and potential applications in biomolecular interaction studies, environmental monitoring, and biomedical diagnostics.

Covalent modification of protein by chemical probe in living cells for structural and interaction studies.

Cellular activities are predominantly carried out by proteins that can dynamically adopt different structural conformations and differentially interact with other biomolecules according to cellular needs. Chemical probes are small molecules used to selectively interact and modulate the activities of specific proteins to study their functions such as the validation of potential drug targets. The remarkable performance of AlphaFold algorithms in the prediction of protein structures has pivoted interest toward elucidating the intracellular dynamics of protein structural conformation where covalent modification of proteins by chemical probes could be used to shed light upon. However, due to the barrier to entry by cell membrane and the general unfavorable reactive conditions of the intracellular environment, most studies using reactive chemical probes are still conducted on purified proteins and cell lysates. Nevertheless, recent progresses have been made in designing chemical probes with improved membrane permeability, stability and reactivity. This paper surveys the literature on recent advancements in membrane-permeable chemical probes and their applications with protein mass spectrometry for the intracellular studies of protein structural conformations and biomolecular interactions.

Development of a carboxymethyl chitosan functionalized slide for small molecule detection using oblique-incidence reflectivity difference technology.

Label-free optical biosensors have become powerful tools in the study of biomolecular interactions without the need for labels. High throughput and low detection limit are desirable for rapid and accurate biomolecule detection. The oblique-incidence reflectivity difference (OI-RD) technique is capable of detecting thousands of biomolecular interactions in a high-throughput mode, specifically for biomolecules larger than 1000 Da. In order to enhance the detection capability of OI-RD for small molecules (typically < 500 Da), we have developed a three-dimensional biochip that utilized carboxymethyl chitosan (CMCS) functionalized slides. By investigating various factors such as sonication time, protein immobilization time, CMCS molecular weight, and glutaraldehyde (GA) functionalization time, we have achieved a detection limit of 6.8 pM for avidin (68 kDa). Furthermore, accurate detection of D-biotin with a molecular weight of 244 Da has also been achieved. This paper presents an effective solution for achieving both high throughput and low detection limits using the OI-RD technique in the field of biomolecular interaction detection.

Evaluation of Co(II) Schiff base complexes: Catalysis, theoretical and biomolecular interaction studies

Two Schiff base ligands, namely H2L1 [3-(2‑hydroxy-phenylimino)-1,3-dihydro-indole-2-one] and H2L2 [3-(2-mercapto-phenylimino)-1,3-dihydro-indole-2–1], were successfully synthesized. Furthermore, the ligands were subsequently complexed with [CoCl2(PPh3)3] to form CoL1 and CoL2, respectively. The synthesized metal complexes were investigated using FTIR, UV–Visible, and NMR spectroscopic techniques. Catalytic efficiency was evaluated for the oxidation of alcohol and C6H5-H5C6Coupling reactions. DFT study verified the structural parameters of the prepared Co2+ complexes were made reliable. The interaction of Co2+ Schiff base complexes with BSA and DNA were studied using an in-silico docking approach.

Sustainable and effective reconditioning of SPR gold chips functionalized with molecularly imprinted polynorepinephrine

Surface Plasmon Resonance (SPR) technology has revolutionized the study of affinity-based biomolecular interactions, offering label-free and real-time analysis capabilities. However, traditional SPR gold chips have been viewed as disposable due to challenges in post-use reconditioning, leading to significant resource wastage and increased costs. To address this issue, we propose a novel approach utilizing polynorepinephrine-based (PNE) Molecularly Imprinted Bio-Polymers (MIBPs) as alternative receptors to conventional antibodies. Self-adhesive MIBPs do not require covalent immobilization. This enables efficient and rapid chip functionalization and post-use removal, facilitating multiple reuses of the plasmon source without compromising analytical performance. We conducted a thorough characterization and data analysis, confirming the robustness and repeatability of a single MIBP-functionalized chip for human IgG detection. 10 cycles of reconditioning and reuse, assayed by 60 kinetic calibrations, were performed. Our findings demonstrate the potential indefinite reuse of SPR chips facilitated by PNE MIBPs, with implications for streamlining test development and routine implementation in SPR biosensing applications. Therefore, they represent a sustainable solution to the longstanding challenge of disposable SPR gold chips also by reducing the reliance on animal-derived Abs for bioanalytic testing. Being also extremely low-cost and green, PNE-based MIBPs minimize the ecological footprint associated with traditional SPR assays. Overall, our work represents a significant advancement towards the development of reusable SPR biosensors. It promises a more sustainable and cost-effective future for biomedical research and diagnostic applications, with application on other transducers and bioanalytical platforms.

Periplasmic electron transfer network in Geobacter sulfurreducens revealed by biomolecular interaction studies

AbstractMultiheme cytochromes located in different compartments are crucial for extracellular electron transfer in the bacterium Geobacter sulfurreducens to drive important environmental processes and biotechnological applications. Recent studies have unveiled that for particular sets of electron terminal acceptors, discrete respiratory pathways selectively recruit specific cytochromes from both the inner and outer membranes. However, such specificity was not observed for the abundant periplasmic cytochromes, namely the triheme cytochrome family PpcA‐E. In this work, the distinctive NMR spectroscopic signatures of these proteins in different redox states were explored to monitor pairwise interactions and electron transfer reactions between each pair of cytochromes. The results showed that the five proteins interact transiently and can exchange electrons between each other revealing intra‐promiscuity within the members of this family. This discovery is discussed in the light of the establishment of an effective electron transfer network by this pool of cytochromes. This network is advantageous to the bacteria as it enables the maintenance of the functional working potential redox range within the cells.

Novel Multifunctional Meta-Surface Plasmon Resonance Chip Microplate for High-Throughput Molecular Screening.

The utilization of surface plasmon resonance (SPR) sensors for real-time label-free molecular interaction analysis is already being employed in the fields of in vitro diagnostics and biomedicine. However, the widespread application of SPR technology is hindered by its limited detection throughput and high cost. To address this issue, this study introduces a novel multifunctional MetaSPR high-throughput microplate biosensor featuring 3D nanocupsarray structure, aiming to achieve high-throughput screeningwith a reduced cost and enhanced speed. Different types of MetaSPR sensors and analytical detection methods have been developed for accurateantibody subtype identification, epitope binding, affinity determination, antibody collocation, and quantitative detection， greatly promoting the screening and analysis ofearly-stageantibody drugs. The MetaSPR platform combined with nano-enhanced particles amplifies the detection signal and improves the detection sensitivity, making it more convenient, sensitive, and efficient than traditional ELISA. The findings demonstrate that the MetaSPR biosensor is a new practical technology detection platform that can improve the efficiency of biomolecular interaction studies with unlimited potential for new drug development.

Trends in surface plasmon resonance biosensing: materials, methods, and machine learning.

Surface plasmon resonance (SPR) proves to be one of the most effective methods of label-free detection and has been integral for the study of biomolecular interactions and the development of biosensors. This trend delves into the latest SPR research and progress built upon the Kretschmann configuration, a pivotal platform, and highlights three key developments that have enhanced the capabilities of the technique. We will first cover a range of explorations of novel plasmonic materials that have shaped SPR performance. Innovative signal transduction and collection, which leverages traditional materials and emerging alternatives, will then be discussed. Finally, the evolving landscape of data analysis, including the integration of machine learning algorithms to navigate complex SPR datasets, will be reviewed. We will also discuss the implementation of these improvements that have enabled new biosensing functions. These advancements not only pave the way for enhanced biosensing in general but also open new avenues for the technique to play a more significant role in research concerning human health.

Fluorescence-Based Methods for the Study of Biomolecular Interactions.

Fluorescence-Based Methods for the Study of Biomolecular Interactions.

MARTin-an open-source platform for microarray analysis.

Background: Microarray technology has brought significant advancements to high-throughput analysis, particularly in the comprehensive study of biomolecular interactions involving proteins, peptides, and antibodies, as well as in the fields of gene expression and genotyping. With the ever-increasing volume and intricacy of microarray data, an accurate, reliable and reproducible analysis is essential. Furthermore, there is a high level of variation in the format of microarrays. This not only holds true between different sample types but is also due to differences in the hardware used during the production of the arrays, as well as the personal preferences of the individual users. Therefore, there is a need for transparent, broadly applicable and user-friendly image quantification techniques to extract meaningful information from these complex datasets, while also addressing the challenges posed by specific microarray and imager formats, which can flaw analysis and interpretation. Results: Here we introduce MicroArray Rastering Tool (MARTin), as a versatile tool developed primarily for the analysis of protein and peptide microarrays. Our software provides state-of-the-art methodologies, offering researchers a comprehensive tool for microarray image quantification. MARTin is independent of the microarray platform used and supports various configurations including high-density formats and printed arrays with significant x and y offsets. This is made possible by granting the user the ability to freely customize parts of the application to their specific microarray format. Thanks to built-in features like adaptive filtering and autofit, measurements can be done very efficiently and are highly reproducible. Furthermore, our tool integrates metadata management and integrity check features, providing a straightforward quality control method, along with a ready-to-use interface for in-depth data analysis. This not only promotes good scientific practice in the field of microarray analysis but also enhances the ability to explore and examine the generated data. Conclusion: MARTin has been developed to empower its users with a reliable, efficient, and intuitive tool for peptidomic and proteomic array analysis, thereby facilitating data-driven discovery across disciplines. Our software is an open-source project freely available via the GNU Affero General Public License licence on GitHub.

Trauma diagnostic-related target proteins and their detection techniques.

Trauma is a significant health issue that not only leads to immediate death in many cases but also causes severe complications, such as sepsis, thrombosis, haemorrhage, acute respiratory distress syndrome and traumatic brain injury, among trauma patients. Target protein identification technology is a vital technique in the field of biomedical research, enabling the study of biomolecular interactions, drug discovery and disease treatment. It plays a crucial role in identifying key protein targets associated with specific diseases or biological processes, facilitating further research, drug design and the development of treatment strategies. The application of target protein technology in biomarker detection enables the timely identification of newly emerging infections and complications in trauma patients, facilitating expeditious medical interventions and leading to reduced post-trauma mortality rates and improved patient prognoses. This review provides an overview of the current applications of target protein identification technology in trauma-related complications and provides a brief overview of the current target protein identification technology, with the aim of reducing post-trauma mortality, improving diagnostic efficiency and prognostic outcomes for patients.

Microscale Thermophoresis Analysis of Chromatin Interactions.

Architectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and regulation through the interaction with a diverse array of other protein factors. Detailed knowledge on the binding modes involved is crucial for our understanding of how these protein-protein and protein-DNA interactions shape the functional landscape of chromatin in all kingdoms of life: bacteria, archaea, and eukarya.Microscale thermophoresis (MST) is a biophysical technique for the study of biomolecular interactions. It has seen increasing application in recent years thanks to its solution-based nature, rapid application, modest sample demand, and the sensitivity of the thermophoresis effect to binding events.Here, we describe the use of MST in the study of chromatin interactions. The emphasis lies on the wide range of ways in which these experiments are set up and the diverse types of information they reveal. These aspects are illustrated with four very different systems: the sequence-dependent DNA compaction by architectural protein HMfB, the sequential binding of core histone complexes to histone chaperone APLF, the impact of the nucleosomal context on the recognition of histone modifications, and the binding of a viral peptide to the nucleosome. Special emphasis is given to the key steps in the design, execution, and analysis of MST experiments in the context of the provided examples.

Determining Macromolecular Structures Using Cryo-Electron Microscopy.

Structural insights into macromolecular and protein complexes provide key clues about the molecular basis of the function. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful structural biology method for studying protein and macromolecular structures at high resolution in both native andnear-native states. Despite the ability to get detailed structural insights into the processes underlying protein function using cryo-EM, there has been hesitancy amongst plant biologists to apply the method for biomolecular interaction studies. This is largely evident from the relatively fewer structural depositions of proteins and protein complexes from plant origin in electron microscopy databank. Even though the progress has been slow, cryo-EM has significantly contributed to our understanding of the molecular biology processes underlying photosynthesis, energy transfer in plants, besides viruses infecting plants. This chapter introduces sample preparation for both negative-staining electron microscopy (NSEM) and cryo-EM for plant proteins and macromolecular complexes and data analysis using single particle analysis for beginners.

Synthesis, characterization, biomolecular interaction, cytotoxicity, and computational studies of quinoxaline-based platinum(II) complexes

The combination of ninhydrin and o-phenylenediamine, followed by their reaction with a phenylhydrazine derivative produces quinoxaline-based ligands (L1 – L6). These ligands react with K2PtCl4 to form Pt(II) complexes (I – VI). The ligands and Pt(II) chelate were characterized through various spectroscopic and analytical techniques, like 1H NMR, 13C NMR, C, H, N-elemental analysis, IR spectroscopy, and mass spectrometry. DFT calculations were used to optimizethe structures of metal complexes. To investigate the binding mode of complexes to CT-DNA/BSA, absorption titration, viscosity measurements, and molecular docking analysis were employed. The results indicated that the DNA binding mechanism involved intercalation. The antibacterial efficacy of the compounds was evaluated against five strains of bacteria. The lower minimum inhibitory concentration (MIC) of complexes suggests that they are more potent than quinoxaline-based ligands. The synthesized compounds were evaluated for cytotoxicity using brine shrimp. The LC50 values of ligands and complexes ranged from 7.64 to 11.45 μg/mL and 5.27 to 7.02 μg/mL, respectively. The capacity of the molecule to suppress cell proliferation using the MCF-7 cancer cell was tested, and the IC50 value was discovered to be comparable to that of astandard drug. Thus in this work, complexes demonstrated intercalation as the binding mode with CT-DNA/BSA and exhibited enhanced antibacterial efficacy compared to the ligands. The compounds also showed promising cytotoxicity against brine shrimp and MCF-7 cancer cells, with comparable IC50 values to a standard drug, indicating their potential as therapeutic agents.

Using transition density models to interpret experimental optical spectra of exciton-coupled cyanine (iCy3)2 dimer probes of local DNA conformations at or near functional protein binding sites.

Exciton-coupled chromophore dimers are an emerging class of optical probes for studies of site-specific biomolecular interactions. Applying accurate theoretical models for the electrostatic coupling of a molecular dimer probe is a key step for simulating its optical properties and analyzing spectroscopic data. In this work, we compare experimental absorbance and circular dichroism (CD) spectra of 'internally-labeled' (iCy3)2 dimer probes inserted site-specifically into DNA fork constructs to theoretical calculations of the structure and geometry of these exciton-coupled dimers. We compare transition density models of varying levels of approximation to determine conformational parameters of the (iCy3)2 dimer-labeled DNA fork constructs. By applying an atomistically detailed transition charge (TQ) model, we can distinguish between dimer conformations in which the stacking and tilt angles between planar iCy3 monomers are varied. A major strength of this approach is that the local conformations of the (iCy3)2 dimer probes that we determined can be used to infer information about the structures of the DNA framework immediately surrounding the probes at various positions within the constructs, both deep in the duplex DNA sequences and at sites at or near the DNA fork junctions where protein complexes bind to discharge their biological functions.

Quercetin-phenylalanine 3d-transition metal-based {Co(II), Ni(II) & Cu(II)} intercalative therapeutic agents: DNA & BSA interaction studies in vitro and cleavage activity

New Quercetin-phenylalanine metal-based therapeutic agents of the formulation [Qu(Phe)M(II).(H2O)2].NO3 where M(II) = Co(II) and Ni(II) and [Qu(Phe)Cu(II).(H2O)2] were synthesized and their structure was predicted by IR, UV–vis, EPR and ESI-MS spectroscopic techniques. The bio-molecular interaction studies of the Quercetin-phenylalanine complexes, 1–3 with ct-DNA and BSA were performed using a battery of complimentary biophysical techniques. The corroborative results of these experiments revealed strong binding propensity via electrostatic interactions probably through minor grove binding towards ct-DNA, therapeutic target. The binding affinity of Quercetin-phenylalanine complexes 1–3 was quantified by determining binding constants values, Kb, Ksv, and the magnitude of binding propensity followed the order 3 > 1 > 2, implicating the preferential binding of Cu(II) complex 3 with ct-DNA. The cleavage studies were performed with complexes using gel electrophoretic mobility assay. The complexes 1–3 demonstrated efficient cleaving ability by the hydrolytic cleavage pathway involving hydroxyl (OH) radicals. BSA binding profile of Quercetin-phenylalanine metal therapeutics 1–3 was studied in order to understand the drug carrier potential of these compounds and found that complex 3 was capable of binding preferentially with BSA as compared to other complexes.

Formation, biomolecular interaction and cytotoxicity studies of new organoruthenium Schiff base compounds

Cytotoxic effects of arene ruthenium(II) compounds have been fine-tuned by the inherent lipophilic character of their arene moiety and the inclusion of different biomarkers within their coordination spheres. Herein, we demonstrate the formation and characterization of novel diamagnetic p-cymene ruthenium compounds: (µ-S)2[Ru(η6-p-cymene)(tmc)]2(PF6)2 (1) (Htmc = 1-((thiophen-2-yl)methylene)thiosemicarbazide), [Ru(η6-p-cymene)(ap)Cl](PF6) (2) (ap = 4-aminoantipyrene), [Ru(η6-p-cymene)(cinap)Cl](PF6) (3) (cinap = 1,5-dimethyl-2-phenyl-4-10-1,2-dihydro-3H-pyrazol-3-one), and [Ru(η6-p-cymene)(cumbh)Cl] (4) (cumbh = N-(4-isopropylbenzylidene)benzohydrazide)). The compounds were less toxic than the cisplatin and NAMI-A controls in all cell lines tested and, except for 3, were generally more cytotoxic to the breast cancer cells than the HeLa cells, displaying half maximal inhibitory concentration (IC50) values in the micromolar range. The effect of compounds 1–4 on long-term survival using a clonogenic assay was assessed in the TNBC HCC1806 cell, where all the compounds reduced colony formation thus adversely affected the long-term survival of the HCC1806 cells. Inhibitory effects of the compounds 1–4 on Topoisomerase I (Topo I) function were assessed. Biomolecular interaction investigations between human genomic DNA and the individual metal complexes indicated that they are likely groove-binders or partial intercalators with intrinsic binding constants in the order of 104 M−1.

Tetrameric Antibody Complexes and Affinity Tag Peptides for the Selective Immobilization and Imaging of Single Quantum Dots.

Colloidal semiconductor quantum dots (QDs) are of widespread interest as fluorescent labels for bioanalysis and imaging applications. Single-particle measurements have proven to be a very powerful tool for better understanding the fundamental properties and behaviors of QDs and their bioconjugates; however, a recurring challenge is the immobilization of QDs in a solution-like environment that minimizes interactions with a bulk surface. Immobilization strategies for QD-peptide conjugates are particularly underdeveloped within this context. Here, we present a novel strategy for the selective immobilization of single QD-peptide conjugates using a combination of tetrameric antibody complexes (TACs) and affinity tag peptides. A glass substrate is modified with an adsorbed layer of concanavalin A (ConA) that binds a subsequent layer of dextran that minimizes nonspecific binding. A TAC with anti-dextran and anti-affinity tag antibodies binds to the dextran-coated glass surface and to the affinity tag sequence of QD-peptide conjugates. The result is spontaneous and sequence-selective immobilization of single QDs without any chemical activation or cross-linking. Controlled immobilization of multiple colors of QDs is possible using multiple affinity tag sequences. Experiments confirmed that this approach positions the QD away from the bulk surface. The method supports real-time imaging of binding and dissociation, measurements of Förster resonance energy transfer (FRET), tracking of dye photobleaching, and detection of proteolytic activity. We anticipate that this immobilization strategy will be useful for studies of QD-associated photophysics, biomolecular interactions and processes, and digital assays.

Sequential Two-Photon Delayed Fluorescence Anisotropy for Macromolecular Size Determination

Time-resolved fluorescence anisotropy (FA) uses the fluorophore depolarization rate to report on rotational diffusion, conformation changes, and intermolecular interactions in solution. Although FA is a rapid, sensitive, and nondestructive tool for biomolecular interaction studies, the short (∼ns) fluorescence lifetime of typical dyes largely prevents the application of FA on larger macromolecular species and complexes. By using triplet shelving and recovery of optical excitation, we introduce optically activated delayed fluorescence anisotropy (OADFA) measurements using sequential two-photon excitation, effectively stretching fluorescence anisotropy measurement times from the nanosecond scale to hundreds of microseconds. We demonstrate this scheme for measuring slow depolarization processes of large macromolecular complexes, derive a quantitative rate model, and perform Monte Carlo simulations to describe the depolarization process of OADFA at the molecular level. This setup has great potential to enable future biomacromolecular and colloidal studies.

Digital Magnetic Detection of Biomolecular Interactions with Single Nanoparticles.

Biomolecular interactions compose a fundamental element of all life forms and are the biological basis of many biomedical assays. However, current methods for detecting biomolecular interactions have limitations in sensitivity and specificity. Here, using nitrogen-vacancy centers in diamond as quantum sensors, we demonstrate digital magnetic detection of biomolecular interactions with single magnetic nanoparticles (MNPs). We first developed a single-particle magnetic imaging (SiPMI) method on 100 nm-sized MNPs with negligible magnetic background, high signal stability, and accurate quantification. The single-particle method was performed on biotin-streptavidin interactions and DNA-DNA interactions in which a single-base mismatch was specifically differentiated. Subsequently, SARS-CoV-2-related antibodies and nucleic acids were examined by a digital immunomagnetic assay derived from SiPMI. In addition, a magnetic separation process improved the detection sensitivity and dynamic range by more than 3 orders of magnitude and also the specificity. This digital magnetic platform is applicable to extensive biomolecular interaction studies and ultrasensitive biomedical assays.