Biomolecular Interaction Analysis Research Articles

A Raman spectral marker for the iso-octyl chain structure of cholesterol.

Raman spectroscopy provides label-free, specific analysis of biomolecular structure and interactions. It could have a greater impact with improved characterization of complex fingerprint vibrations. Many Raman peaks have been assigned to cholesterol, for example, but the molecular vibrations associated with those peaks are not known. In this report, time-dependent density functional theory calculations of the Raman spectrum of cholesterol are compared to measurements on microcrystalline powder to identify 23 peaks in the Raman spectrum. Among them, a band of six peaks is found to be sensitive to the conformational structure of cholesterol's iso-octyl chain. Calculations on 10 conformers in this spectral band are fit to experimental spectra to probe the cholesterol chain structure in purified powder and in phospholipid vesicles. In vesicles, the chain is found to bend perpendicular to the steroid rings, supporting the case that the chain is a dynamic structure that contributes to lipid condensation and other effects of cholesterol in biomembranes. Statement of Significance: Here we use density functional theory to identify a band of six peaks in cholesterol's Raman spectrum that is sensitive to the conformational structure of cholesterol's chain. Raman spectra were analyzed to show that in fluid-phase lipid membranes, about half of the cholesterol chains point perpendicular to the steroid rings. This new method of label-free structural analysis could make significant contributions to our understanding of cholesterol's critical role in biomembrane structure and function. More broadly, the results show that computational quantum chemistry Raman spectroscopy can make significant new contributions to molecular structure when spectra are interpreted with computational quantum chemistry.

Distributed interferometric fiber tip biosensors for a multi-channel and label-free biomolecular interaction analysis.

This paper describes fabrication and implementation of distributed optical fiber tip biosensor probes for simultaneously measuring label-free biomolecular interactions at multiple locations. Biosensor probes at the tip of a single-mode fiber are Fabry-Perot etalons that are functionalized with a capture layer for a specific biomolecule. A coherence multiplexing method is implemented to separate data collected from distributed biosensors in a single data stream. Multiplexing is achieved by using fiber tip biosensors of varying etalon lengths with the same or different capture layers for each biosensing channel. Experiments demonstrating simultaneous multi-channel recording of protein-to-protein interaction sensorgrams with fiber tip biosensor probes are presented.

Xanthohumol inhibits non-small cell lung cancer via directly targeting T-lymphokine-activated killer cell-originated protein kinase.

Xanthohumol is a principal prenylated chalcone isolated from hops. Previous studies have shown that xanthohumol was effective against various types of cancer, but the mechanisms, especially the direct targets for xanthohumol to exert an anticancer effect, remain elusive. Overexpression of T-lymphokine-activated killer cell-originated protein kinase (TOPK) promotes tumorigenesis, invasion and metastasis, implying the likely potential for targeting TOPK in cancer prevention and treatment. In the present study, we found that xanthohumol significantly inhibited the cell proliferation, migration and invasion of non-small cell lung cancer (NSCLC) in vitro and suppressed tumor growth in vivo, which is well correlated with inactivating TOPK, evidenced by reduced phosphorylation of TOPK and its downstream signaling histone H3 and Akt, and decreased its kinase activity. Moreover, molecular docking and biomolecular interaction analysis showed that xanthohumol was able to directly bind to the TOPK protein, suggesting that TOPK inactivation by xanthohumol is attributed to its ability to directly interact with TOPK. The findings of the present study identified TOPK as a direct target for xanthohumol to exert its anticancer activity, revealing novel insight into the mechanisms underlying the anticancer activity of xanthohumol.

Optimizing Upconversion Nanoparticles for FRET Biosensing.

Upconversion nanoparticles (UCNPs) are some of the most promising nanomaterials for bioanalytical and biomedical applications. One important challenge to be still solved is how UCNPs can be optimally implemented into Förster resonance energy transfer (FRET) biosensing and bioimaging for highly sensitive, wash-free, multiplexed, accurate, and precise quantitative analysis of biomolecules and biomolecular interactions. The many possible UCNP architectures composed of a core and multiple shells doped with different lanthanoid ions at different ratios, the interaction with FRET acceptors at different possible distances and orientations via biomolecular interaction, and the many and long-lasting energy transfer pathways from the initial UCNP excitation to the final FRET process and acceptor emission make the experimental determination of the ideal UCNP-FRET configuration for optimal analytical performance a real challenge. To overcome this issue, we have developed a fully analytical model that requires only a few experimental configurations to determine the ideal UCNP-FRET system within a few minutes. We verified our model via experiments using nine different Nd-, Yb-, and Er-doped core-shell-shell UCNP architectures within a prototypical DNA hybridization assay using Cy3.5 as an acceptor dye. Using the selected experimental input, the model determined the optimal UCNP out of all theoretically possible combinatorial configurations. An extreme economy of time, effort, and material was accompanied by a significant sensitivity increase, which demonstrated the powerful feat of combining a few selected experiments with sophisticated but rapid modeling to accomplish an ideal FRET biosensor.

Label-Free Multiplexed Microfluidic Analysis of Protein Interactions Based on Photonic Crystal Surface Mode Imaging.

High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.

Affinity Exploration of SARS-CoV-2 RBD Variants to mAb-Functionalized Plasmonic Metasurfaces for Label-Free Immunoassay Boosting.

Plasmonic metasurfaces (PMs) functionalized with the monoclonal antibody (mAb) are promising biophotonic sensors for biomolecular interaction analysis and convenient immunoassay of various biomarkers, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Previous PM biosensing suffers from the slow affinity detection rate and lack of sufficient immunoassay studies on various SARS-CoV-2 variants. Here, we develop a high-efficiency affinity testing method based on label-free PM sensors with mAbs and demonstrate their binding characteristics to 12 spike receptor binding domain (RBD) variants of SARS-CoV-2. In addition to the research of plasmonic near-field influence on surface biomolecule sensing, we provide a comprehensive report about the Langmuir binding equilibrium of molecular kinetics between 12 SARS-CoV-2 RBD variants and mAb-functionalized PMs, which plays a crucial role in label-free immunosensing. A high-affinity mAb can be combined with the highly sensitive propagating plasmonic mode to boost the detection of SARS-CoV-2 variants. Owing to a better understanding of molecular dynamics on PMs, we develop an ultrasensitive biosensor of the SARS-CoV-2 Omicron variant. The experiments show great distinguishment of P < 0.0001 from respiratory diseases induced by other viruses, and the limit of detection is 2 orders smaller than the commercial colloidal gold immunoassay. Our study shows the label-free biosensing by low-cost wafer-scale PMs, which will provide essential information on biomolecular interaction and facilitate high-precision point-of-care testing for emerging SARS-CoV-2 variants in the future.

Pocketable Biosensor Based on Quartz-Crystal Microbalance and Its Application to DNA Detection

Quartz-crystal microbalance (QCM) is a technique that can measure nanogram-order masses. When a receptor is immobilized on the sensor surface of a QCM device, the device can detect chemical molecules captured by the mass change. Although QCM devices have been applied to biosensors that detect biomolecules without labels for biomolecular interaction analysis, most highly sensitive QCM devices are benchtop devices. We considered the fabrication of an IC card-sized QCM device that is both portable and battery-powered. Its miniaturization was achieved by repurposing electronic components and film batteries from smartphones and wearable devices. To demonstrate the applicability of the card-sized QCM device as a biosensor, DNA-detection experiments were performed. The card-sized QCM device could detect specific 10-mer DNA chains while discerning single-base differences with a sensitivity similar to that of a conventional benchtop device. The card-sized QCM device can be used in laboratories and in various other fields as a mass sensor.

Application of Nanopore Sensors for Biomolecular Interactions and Drug Discovery.

Biomolecular interactions, such as protein-protein, protein-nucleic acid, and protein/nucleic acid-ligand interactions, play crucial roles in various cellular signaling and biological processes, and offer attractive therapeutic targets in numerous human diseases. Currently, drug discovery is limited by the low efficiency and high cost of conventional ensemble-averaging-based techniques for biomolecular interaction analysis and high-throughput drug screening. Nanopores are an emerging technology for single-molecule sensing of biomolecules. Owing to the notable merits of single-molecule sensing, nanopore sensors have contributed tremendously to nucleic acid sequencing and disease diagnostics. In this minireview, we summarize the recent developments and outlooks in single-molecule sensing of various biomolecular interactions for drug discovery applications using biological and solid-state nanopore sensors.

Accurate Correction of the "Bulk Response" in Surface Plasmon Resonance Sensing Provides New Insights on Interactions Involving Lysozyme and Poly(ethylene glycol).

Surface plasmon resonance is a very well-established surface sensitive technique for label-free analysis of biomolecular interactions, generating thousands of publications each year. An inconvenient effect that complicates interpretation of SPR results is the “bulk response” from molecules in solution, which generate signals without really binding to the surface. Here we present a physical model for determining the bulk response contribution and verify its accuracy. Our method does not require a reference channel or a separate surface region. We show that proper subtraction of the bulk response reveals an interaction between poly(ethylene glycol) brushes and the protein lysozyme at physiological conditions. Importantly, we also show that the bulk response correction method implemented in commercial instruments is not generally accurate. Using our method, the equilibrium affinity between polymer and protein is determined to be KD = 200 μM. One reason for the weak affinity is that the interaction is relatively short-lived (1/koff < 30 s). Furthermore, we show that the bulk response correction also reveals the dynamics of self-interactions between lysozyme molecules on surfaces. Besides providing new insights on important biomolecular interactions, our method can be widely applied to improve the accuracy of SPR data generated by instruments worldwide.

Microscopies Enabled by Photonic Metamaterials.

In recent years, the biosensor research community has made rapid progress in the development of nanostructured materials capable of amplifying the interaction between light and biological matter. A common objective is to concentrate the electromagnetic energy associated with light into nanometer-scale volumes that, in many cases, can extend below the conventional Abbé diffraction limit. Dating back to the first application of surface plasmon resonance (SPR) for label-free detection of biomolecular interactions, resonant optical structures, including waveguides, ring resonators, and photonic crystals, have proven to be effective conduits for a wide range of optical enhancement effects that include enhanced excitation of photon emitters (such as quantum dots, organic dyes, and fluorescent proteins), enhanced extraction from photon emitters, enhanced optical absorption, and enhanced optical scattering (such as from Raman-scatterers and nanoparticles). The application of photonic metamaterials as a means for enhancing contrast in microscopy is a recent technological development. Through their ability to generate surface-localized and resonantly enhanced electromagnetic fields, photonic metamaterials are an effective surface for magnifying absorption, photon emission, and scattering associated with biological materials while an imaging system records spatial and temporal patterns. By replacing the conventional glass microscope slide with a photonic metamaterial, new forms of contrast and enhanced signal-to-noise are obtained for applications that include cancer diagnostics, infectious disease diagnostics, cell membrane imaging, biomolecular interaction analysis, and drug discovery. This paper will review the current state of the art in which photonic metamaterial surfaces are utilized in the context of microscopy.

Multiplexed Biosensing and Bioimaging Using Lanthanide-Based Time-Gated Förster Resonance Energy Transfer.

The necessity to scrutinize more and more biological molecules and interactions both in solution and on the cellular level has led to an increasing demand for sensitive and specific multiplexed diagnostic analysis. Photoluminescence (PL) detection is ideally suited for multiplexed biosensing and bioimaging because it is rapid and sensitive and there is an almost unlimited choice of fluorophores that provide a large versatility of photophysical properties, including PL intensities, spectra, and lifetimes.The most frequently used technique to detect multiple parameters from a single sample is spectral (or color) multiplexing with different fluorophores, such as organic dyes, fluorescent proteins, quantum dots, or lanthanide nanoparticles and complexes. In conventional PL biosensing approaches, each fluorophore requires a distinct detection channel and excitation wavelength. This drawback can be overcome by Förster resonance energy transfer (FRET) from lanthanide donors to other fluorophore acceptors. The lanthanides' multiple and spectrally narrow emission bands over a broad spectral range can overlap with several different acceptors at once, thereby allowing FRET from one donor to multiple acceptors. The lanthanides' extremely long PL lifetimes provide two important features. First, time-gated (TG) detection allows for efficient suppression of background fluorescence from the biological environment or directly excited acceptors. Second, temporal multiplexing, for which the PL lifetimes are adjusted by the interaction with the FRET acceptor, can be used to determine specific biomolecules and/or their conformation via distinct PL decays. The high signal-to-background ratios, reproducible and precise ratiometric and homogeneous (washing-free) sensing formats, and higher-order multiplexing capabilities of lanthanide-based TG-FRET have resulted in significant advances in the analysis of biomolecular recognition. Applications range from fundamental analysis of biomolecular interactions and conformations to high-throughput and point-of-care in vitro diagnostics and DNA sequencing to advanced optical encoding, using both liquid and solid samples and in situ, in vitro, and in vivo detection with high sensitivity and selectivity.In this Account, we discuss recent advances in lanthanide-based TG-FRET for the development and application of advanced immunoassays, nucleic acid sensing, and fluorescence imaging. In addition to the different spectral and temporal multiplexing approaches, we highlight the importance of the careful design and combination of different biological, organic, and inorganic molecules and nanomaterials for an adjustable FRET donor-acceptor distance that determines the ultimate performance of the diagnostic assays and conformational sensors in their physiological environment. We conclude by sharing our vision on how progress in the development of new sensing concepts, material combinations, and instrumentation can further advance TG-FRET multiplexing and accelerate its translation into routine clinical practice and the investigation of challenging biological systems.

A Review: Surface Plasmon Resonance-Based Biosensor for Early Screening of SARS-CoV2 Infection

Rapid viral diagnosis is essential to contain the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) in the community and ensure effective patient management. However, the current gold standard for diagnosis of viral disease depends on the time-consuming molecular technique. Surface plasmon resonance (SPR) phenomenon has been widely applied in the analysis of biomolecular interactions and analytes detection, which is currently being investigated as a quick diagnostic method of numerous viral infections. In this regard, researchers have performed experimental studies dedicated to enhancing the sensitivity and specificity of the SPR sensor performance for utilization in the medical diagnostic field. In this review, we summarized various SPR application including the recent advancements and advantages of different types of SPR-based biosensor such prism-based, localized surface plasmon resonance (LSPR), fiber optic, optical grating, and optical waveguide for the detection of SARS-CoV2. We proposed various technical recommendations to improve the sensitivity, accuracy, time response, and cost-saving means of the SPR sensor. A special focus was set for sensitivity enhancement aspects in terms of the type of prism, nanomaterials, support layer with type and shape of optical fibers for early, swift, and ultra-sensitive detection of viral infection. With the highlighted impacts of SPR application, this review frames the current potential and challenges of SPR in comparison to the current gold standard of viral infection diagnostic strategy.

Microchip Free-Flow Electrophoresis for Bioanalysis, Sensing, and Purification.

The separation of complex mixtures is ubiquitous throughout molecular biology, and techniques such as gel-based electrophoresis are common laboratory practice. Such methods are not without their drawbacks, however, which include non-specific interactions between analyte and the separation matrix, poor yields in purification and non-continuous analyte throughput. Microfluidic techniques, which exploit physical phenomena unique to the microscale, promise to improve many aspects of traditional laboratory procedures. These methods offer a quantitative, solution-based alternative to traditional gel electrophoresis, with rapid measurement times enabling the analysis of transient or weak biomolecular interactions that would be challenging to observe with traditional methods. Here, we present a protocol for the lithographic fabrication and operation of microfluidic chips capable of free-flow electrophoretic (FFE) fractionation and analysis of biological analytes. We demonstrate the efficacy of our approach through a protein-sensing methodology based on FFE fractionation of DNA-protein mixtures. In addition, the FFE technique described here can be readily adapted to suit a variety of preparative and analytical applications, providing information on the charge, zeta-potential, and interactions of analytes.

A Versatile One-Step Competitive Fiber Optic Surface Plasmon Resonance Bioassay Enabled by DNA Nanotechnology.

Fiber optic surface plasmon resonance (FO-SPR)-based biosensors have emerged as powerful tools for biomarker detection due to their ability for real-time analysis of biomolecular interactions, cost-effectiveness, and user-friendliness. However, as (FO-)SPR signals are determined by the mass of the target molecules, the detection of low-molecular-weight targets remains challenging and currently requires tedious labeling and preparation steps. Therefore, in this work, we established a new concept for low-molecular-weight target detection by implementing duplexed aptamers on an FO-SPR sensor. In this manner, we enabled one-step competitive detection and could achieve significant signals, independent of the weight of the target molecules, without requiring labeling or preprocessing steps. This was demonstrated for the detection of a small molecule (ATP), protein (thrombin), and ssDNA target, thereby reaching detection limits of 72 μM, 36 nM, and 30 nM respectively and proving the generalizability of the proposed bioassay. Furthermore, target detection was successfully achieved in 10-fold diluted plasma, which demonstrated the applicability of the assay in biologically relevant matrices. Altogether, the developed one-step competitive FO-SPR bioassay opens up possibilities for the detection of low-molecular-weight targets in a fast and straightforward manner.

Comparison of methods for quantitative biomolecular interaction analysis

In order to perform good kinetic experiments, not only the experimental conditions have to be optimized, but the evaluation procedure as well. The focus of this work is the in-depth comparison of different approaches and algorithms to determine kinetic rate constants for biomolecular interaction analysis (BIA). The different algorithms are applied not only to flawless simulated data, but also to real-world measurements. We compare five mathematical approaches for the evaluation of binding curves following pseudo-first-order kinetics with different noise levels. In addition, reflectometric interference spectroscopy (RIfS) measurements of two antibodies are evaluated to determine their binding kinetics. The advantages and disadvantages of the individual approach will be investigated and discussed in detail. In summary, we will raise awareness on how to evaluate and judge results from BIA by using different approaches rather than having to rely on “black box” closed (commercial) software packages.

Biochemical Sensing with Nanoplasmonic Architectures: We Know How but Do We Know Why?

Here, the research field of nanoplasmonic sensors is placed under scrutiny, with focus on affinity-based detection using refractive index changes. This review describes how nanostructured plasmonic sensors can deliver unique advantages compared to the established surface plasmon resonance technique, where a planar metal surface is used. At the same time, it shows that these features are actually only useful in quite specific situations. Recent trends in the field are also discussed and some devices that claim extraordinary performance are questioned. It is argued that the most important challenges are related to limited receptor affinity and nonspecific binding rather than instrumental performance. Although some nanoplasmonic sensors may be useful in certain situations, it seems likely that conventional surface plasmon resonance will continue to dominate biomolecular interaction analysis. For detection of analytes in complex samples, plasmonics may be an important tool, but probably not in the form of direct refractometric detection.

Plasmonic mode coupling and thin film sensing in metal\u2013insulator\u2013metal structures

Optical sensors based on surface plasmon resonance (SPR) in the attenuated total reflection (ATR) configuration in layered media have attracted considerable attention over the past decades owing to their ability of label free sensing in biomolecular interaction analysis, and highly sensitive detection of changes in refractive index and thickness, i.e. the optical thickness, of thin film adsorbates (thin film sensing). Furthermore, SPR is highly sensitive to the refractive index of the medium adjacent to the bare metal, and it allows for bulk sensing as well. When deposited at the metal/air interface, an adsorbed layer disturbs the highly localized, i.e. bound, wave at this interface and changes the plasmon resonance to allow for sensing in angular or wavelength interrogation and intensity measurement modes. A high degree of sensitivity is required for precise and efficient sensing, especially for biomolecular interaction analysis for early stage diagnostics; and besides conventional SPR (CSPR), several other configurations have been developed in recent years targeting sensitivity, including long-range SPR (LRSPR) and waveguide-coupled SPR (WGSPR) observed in MIM structures, referred here to by MIM modes, resulting from the coupling of SPRs at I/M interfaces, and Fano-type resonances occurring from broad and sharp modes coupling in layered structures. In our previous research, we demonstrated that MIM is better than CSPR for bulk sensing, and in this paper, we show that CSPR is better than MIM for thin film sensing for thicknesses of the sensing layer (SL) larger than 10 nm. We discuss and compare the sensitivity of CSPR and MIM for thin film sensing by using both experiments and theoretical calculations based on rigorous electromagnetic (EM) theory. We discuss in detail MIM modes coupling and anti-crossing, and we show that when a thin film adsorbate, i.e. a SL), is deposited on top of the outermost-layer of an optimized MIM structure, it modifies the characteristics of the coupled modes of the structure, and it reduces the electric field, both inside the SL and at the SL/air interface, and as a result, it decreases the sensitivity of the MIM versus the CSPR sensor. Our work is of critical importance to plasmonic mode coupling using MIM configurations, as well as to optical bio- and chemical-sensing.

Erratum: Principles for Sensitive and Robust Biomolecular Interaction Analysis: The Limits of Detection and Resolution of Diffraction-Limited Focal Molography [Phys. Rev. Applied 11, 014056 (2019)

Erratum: Principles for Sensitive and Robust Biomolecular Interaction Analysis: The Limits of Detection and Resolution of Diffraction-Limited Focal Molography [Phys. Rev. Applied 11, 014056 (2019)

Engineering the penetration depth of nearly guided wave surface plasmon resonance towards application in bacterial cells monitoring

Photonic techniques based on evanescent waves sensing (such as the surface plasmon resonance (SPR) method) using plasmonic and nanostructured metallic/semiconductor materials hold huge potential in biosensing and associated analysis of biomolecular interactions. However, conventional SPR suffers from low penetration depths (<300 nm), limiting the applications for the surface interactions and analysis of larger biomolecules, such as for bacteria cells with a typical size of ∼1 μm. These cases result in the measured signal being non-monotonic with concentration, making the technique unreliable for high concentrations. Infrared wavelengths can be used, but then signal contrast suffers, and the instruments required for mid-infrared or longer wavelengths are prohibitively expensive. With this in mind, we developed a “nearly” guided SPR (NGWSPR) structure to enhance the performance of these sensors by increasing penetration depth and figure of merit using wavelengths in the optical telecommunication window where off-the-shelf instruments are available at low cost. The use of this technique for monotonic detection of cultured live Escherichia coli bacterial cells is demonstrated, thus opening a pathway to utilize and promote the approach for biosensing, biomedical research and industrial applications.

In situ analysis of biomolecular interactions on nano/microparticles in formulations

Nano/microparticle-based formulations are attracting more and more attention in the drug and vaccine formulation field. Analytical methodologies are needed for analyzing the function of the protein molecules before and after their adsorption onto particles. Here, we report a fluorescence imaging-based high content analysis (HCA) for analyzing bimolecular interactions on the particle surface. Subtle conformational alteration of antigen epitope overlapping were shown to be feasible using this microplate-based HCA method. This HCA is highly amenable for automation, making it a method of choice for early formulation development as well as for stability test of products in late stage development.