Characterization Of Biomolecular Interactions Research Articles

Integrated Signal Amplification on a Fiber Optic SPR Sensor Using Duplexed Aptamers.

Throughout the past decades, fiber optic surface plasmon resonance (FO-SPR)-based biosensors have proven to be powerful tools for both the characterization of biomolecular interactions and target detection. However, as FO-SPR signals are generally related to the mass that binds to the sensor surface, multistep processes and external reagents are often required to obtain significant signals for low molecular weight targets. This increases the time, cost, and complexity of the respective bioassays and hinders continuous measurements. To overcome these requirements, in this work, cis-duplexed aptamers (DAs) were implemented on FO-SPR sensors, which underwent a conformational change upon target binding. This induced a spatial redistribution of gold nanoparticles (AuNPs) upon specific target binding and resulted in an amplified and concentration-dependent signal. Importantly, the AuNPs were covalently conjugated to the sensor, so the principle does not rely on multistep processes or external reagents. To implement this concept, first, the thickness of the gold fiber coating was adapted to match the resonance conditions of the surface plasmons present on the FO-SPR sensors with those on the AuNPs. As a result, the signal obtained due to the spatial redistribution of the AuNPs was amplified by a factor of 3 compared to the most commonly used thickness. Subsequently, the cis-DAs were successfully implemented on the FO-SPR sensors, and it was demonstrated that the DA-based FO-SPR sensors could specifically and quantitatively detect an ssDNA target with a detection limit of 230 nM. Furthermore, the redistribution of the AuNPs was proven to be reversible, which is an important prerequisite for continuous measurements. Altogether, the established DA-based FO-SPR bioassay holds much promise for the detection of low molecular weight targets in the future and opens up possibilities for FO-SPR-based continuous biosensing.

Liquid crystal-amplified optofluidic biosensor for ultra-highly sensitive and stable protein assay

Protein assays show great importance in medical research and disease diagnoses. Liquid crystals (LCs), as a branch of sensitive materials, offer promising applicability in the field of biosensing. Herein, we developed an ultrasensitive biosensor for the detection of low-concentration protein molecules, employing LC-amplified optofluidic resonators. In this design, the orientation of LCs was disturbed by immobilized protein molecules through the reduction of the vertical anchoring force from the alignment layer. A biosensing platform based on the whispering-gallery mode (WGM) from the LC-amplified optofluidic resonator was developed and explored, in which the spectral wavelength shift was monitored as the sensing parameter. The microbubble structure provided a stable and reliable WGM resonator with a high Q factor for LCs. It is demonstrated that the wall thickness of the microbubble played a key role in enhancing the sensitivity of the LC-amplified WGM microcavity. It is also found that protein molecules coated on the internal surface of microbubble led to their interactions with laser beams and the orientation transition of LCs. Both effects amplified the target information and triggered a sensitive wavelength shift in WGM spectra. A detection limit of 1 fM for bovine serum albumin (BSA) was achieved to demonstrate the high-sensitivity of our sensing platform in protein assays. Compared to the detection using a conventional polarized optical microscope (POM), the sensitivity was improved by seven orders of magnitude. Furthermore, multiple types of proteins and specific biosensing were also investigated to verify the potential of LC-amplified optofluidic resonators in the biomolecular detection. Our studies indicate that LC-amplified optofluidic resonators offer a new solution for the ultrasensitive real-time biosensing and the characterization of biomolecular interactions.

Probing Biomolecular Interactions by a Pattern-Forming Peptide-Conjugate Sensor.

As a key mechanism underpinning many biological processes, protein self-organization has been extensively studied. However, the potential to apply the distinctive, nonlinear biochemical properties of such self-organizing systems to biotechnological problems such as the facile detection and characterization of biomolecular interactions has not yet been explored. Here, we describe an in vitro assay in a 96-well plate format that harnesses the emergent behavior of the Escherichia coli Min system to provide a readout of biomolecular interactions. Crucial for the development of our approach is a minimal MinE-derived peptide that stimulates MinD ATPase activity only when dimerized. We found that this behavior could be induced via any pair of foreign, mutually binding molecular entities fused to the minimal MinE peptide. The resulting MinD ATPase activity and the spatiotemporal nature of the produced protein patterns quantitatively correlate with the affinity of the fused binding partners, thereby enabling a highly sensitive assay for biomolecular interactions. Our assay thus provides a unique means of quantitatively visualizing biomolecular interactions and may prove useful for the assessment of domain interactions within protein libraries and for the facile investigation of potential inhibitors of protein–protein interactions.

Some like it Hot: Quantification of Biomolecular Interactions using MicroScale Thermophoresis

Characterization of biomolecular interactions, such as protein-protein, protein-nucleic acid or protein-small molecule, provides critical insights into cellular processes, and is essential for the development of drug diagnostics and therapeutics.Here we present a novel, label-free and tether-free technology to analyze picomolar to millimolar affinities of biomolecular interactions by MicroScale Thermophoresis (MST). The entropy of the hydration shell surrounding molecules determines thermophoretic movement. MST exploits this principle by measuring interactions using optically generated temperature gradients. MST detects changes in the size, charge and hydration shell of molecules and measures biomolecule interactions under close-to-native conditions: immobilization-free and in bioliquids of choice, including cell lysates, blood, and serum. Thus, MST measures interactions under close-to-native conditions, and without laborious sample purification. Here, we demonstrate how MST determines the picomolar to millimolar affinities of various protein:protein and protein:small molecule interactions. MST assays are highly adaptable to fit to the diverse requirements of different and complex biomolecules. NanoTemper´s unique technology is ideal for studies requiring flexibility and sensitivity at the experimental scale, making MST suitable for basic research investigations and pharmaceutical applications.

Ara h 1 protein–antibody dissociation study: evidence for binding inhomogeneities on a molecular scale

The characterization of biomolecular interactions is essential when designing novel biosensors, since the interaction between the bioreceptor and the ligand determines important biosensing parameters such as sensitivity and selectivity. In this paper we study the interaction of the trimeric Ara h 1 protein with a monoclonal anti-Ara h 1 antibody by means of magnetic force-induced dissociation. The proteins were bound to magnetic particles and polystyrene surfaces by EDC/NHS reaction chemistry and by physisorption, respectively. Two different molecular configurations have been investigated, with either the Ara h 1 protein on the particles or the Ara h 1 protein on the polystyrene surface. A model with a Gaussian distribution of energy barriers for dissociation gives an adequate description for the measured multi-exponential decays. We hypothesize that distributions of molecular orientations as well as experimentally induced variations may underlay the observed distributions. The two molecular configurations show a different peak value of the energy distribution. Similarly, SPR experiments for two distinct configurations (either Ara h 1 protein on the surface, or anti-Ara h 1 antibody on the surface) also show clear differences in dissociation behavior. We hypothesize that the multivalency of the involved molecules leads to different modes of binding. The results of this work highlight the importance of molecular inhomogeneities when studying the interaction processes of biomolecular complexes.

Phase‐sensitive surface plasmon resonance biosensors: methodology, instrumentation and applications

AbstractSurface Plasmon Resonance (SPR) has become a central tool for label‐free characterization of biomolecular interactions. Based on monitoring of amplitude characteristics, conventional SPR sensors have been extensively explored, commercialized and applied for studies of many important interactions (antigen‐antibody, protein‐ligand etc), but this technology still lacks of sensitivity for the detection of relatively small and low copy number compounds. Phase‐sensitive SPR has recently emerged as an upgrade of this technology to resolve the sensitivity issue. Profiting from a sharp phase jump under SPR and ultra‐sensitive tools of its control, this technology offers up to 100‐time improvement of the detection limit, giving access to the detection of trace amounts of small molecular weight analytes (drugs etc). This paper intends to provide a tutorial on basic concepts of phase detection in SPR sensing, compare the performance of phase‐ and amplitude‐sensitive sensors, review recent progress in the development and applications of phase‐sensitive SPR sensors, and outline future prospects and trends of this technology.

A Novel Approach to Label-Free Sensing: Diffractive Optics Technology (dot®)

Label-free detection methods have played a very significant role in drug design and refinement. They have been used primarily during secondary screening and for in-depth characterization of biomolecular interactions. Misconceptions about the accessibility of these platforms, since they often require specialized training, throughput and robustness in complex media have hampered their adoption in the earliest phases of discovery not to mention their significant and unrealized potential in qualifying reagents for high throughput screening or during novel assay development. A new wave of more cost effective, robust and accessible platforms has made significant inroads, demonstrating that significant information can be derived from these methods all along the drug discovery research continuum. One of these recent entrants, the dotLab System uses diffractive optics technology (dot) to detect biomolecular interactions and can be used for a wide variety of applications in the study of a broad spectrum of biological analytes including proteins, DNA and even microorganisms.

Residual dipolar couplings: synergy between NMR and structural genomics.

Structural genomics is on a quest for the structure and function of a significant fraction of gene products. Current efforts are focusing on structure determination of single-domain proteins, which can readily be targeted by X-ray crystallography, NMR spectroscopy and computational homology modeling. However, comprehensive association of gene products with functions also requires systematic determination of more complex protein structures and other biomolecules participating in cellular processes such as nucleic acids, and characterization of biomolecular interactions and dynamics relevant to function. Such NMR investigations are becoming more feasible, not only due to recent advances in NMR methodology, but also because structural genomics is providing valuable structural information and new experimental and computational tools. The measurement of residual dipolar couplings in partially oriented systems and other new NMR methods will play an important role in this synergistic relationship between NMR and structural genomics. Both an expansion in the domain of NMR application, and important contributions to future structural genomics efforts can be anticipated.