Detection Of Biomolecular Interactions Research Articles

Real-time, label-free detection of biomolecular interactions in sandwich assays by the oblique-incidence reflectivity difference technique.

One of the most important goals in proteomics is to detect the real-time kinetics of diverse biomolecular interactions. Fluorescence, which requires extrinsic tags, is a commonly and widely used method because of its high convenience and sensitivity. However, in order to maintain the conformational and functional integrality of biomolecules, label-free detection methods are highly under demand. We have developed the oblique-incidence reflectivity difference (OI-RD) technique for label-free, kinetic measurements of protein-biomolecule interactions. Incorporating the total internal refection geometry into the OI-RD technique, we are able to detect as low as 0.1% of a protein monolayer, and this sensitivity is comparable with other label-free techniques such as surface plasmon resonance (SPR). The unique advantage of OI-RD over SPR is no need for dielectric layers. Moreover, using a photodiode array as the detector enables multi-channel detection and also eliminates the over-time signal drift. In this paper, we demonstrate the applicability and feasibility of the OI-RD technique by measuring the kinetics of protein-protein and protein-small molecule interactions in sandwich assays.

Research on Protein Thermal Condensation Detection Based on Phase Modulation SPR Imaging

The existing of labeling methods can not completely meet the requirements of protein detection. It is currently in urgent need of sensitive, labeless, real-time and high throughput protein detection methods. A novel SPR imaging biomolecular interatction detection method based on time domain phase modulation is presented in this thesis. Experimental apparatus of SPR imaging biomolecular interaction detection based on TDPM is established. Biomolecular interaction is detected. 2×2 lysozyme array chip is prepared and lysozyme thermal condensation is detected by the experimental apparatus. SPR curves of the interaction are obtained and kinetic parameters are calculated. It can sensitively acquire real-time phase change caused by biomolecular interaction based on interference imaging, and resolve related bioinformation, which is a potential tool for proteomics research.

Publisher’s Note: Focal Molography: Coherent Microscopic Detection of Biomolecular Interaction [Phys. Rev. X4, 031024 (2014)

Received 17 October 2014DOI:https://doi.org/10.1103/PhysRevX.4.049901This article is available under the terms of the Creative Commons Attribution 3.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.© 2014 American Physical Society

Visualization of the interaction between tris and lysozyme with a biosensor based on total internal reflection imaging ellipsometry

Weak affinity interactions among biomolecules have attracted more and more attentions for they play a significant role in organisms. However, it is difficult to characterize these interactions due to the limitation of the detection approaches. The biosensor based on total internal reflection imaging ellipsometry (TIRIE) is a real-time analysis method for biomolecular interaction detection with the advantages of high throughput and sensitivity. The interaction between tris and lysozyme (LZM), a typical example of weak affinity interactions, has been detected with TIRIE as a trial. Tris is immobilized on the gold thin film substrate to form the biosensing surface and then LZM as well as its negative controls is delivered to the tris-immobilized surface. The interaction process between tris and LZM is recorded by TIRIE biosensor to form a real-time curve and its dissociation constant is deduced as 7.6×10−5M, which is in agreement with the results in the reported work by other investigators. The results indicate that TIRIE biosensor is competent for weak affinity interaction analysis.

Molecular Rotors As Conditionally Fluorescent Labels for Rapid Detection of Biomolecular Interactions

We demonstrate the use of fluorescent molecular rotors as probes for detecting biomolecular interactions, specifically peptide-protein interactions. Molecular rotors undergo twisted intramolecular charge transfer upon irradiation, relax via the nonradiative torsional relaxation pathway, and have been typically used as viscosity probes. Their utility as a tool for detecting specific biomolecular interactions has not been explored. Using the well characterized p53-Mdm2 interaction as a model system, we designed a 9-(2-carboxy-2-cyanovinyl) julolidine-based p53 peptide reporter, JP1-R, which fluoresces conditionally only upon Mdm2 binding. The reporter was used in a rapid, homogeneous assay to screen a fragment library for antagonists of the p53-Mdm2 interaction, and several inhibitors were identified. Subsequent validation of these hits using established secondary assays suggests increased sensitivity afforded by JP1-R. The fluorescence of molecular rotors contingent upon target binding makes them a versatile tool for detecting specific biomolecular interactions.

Regio- and chemoselective immobilization of proteins on gold surfaces.

Protein chips are powerful tools as analytical and diagnostic devices for detection of biomolecular interactions, where the proteins are covalently or noncovalently attached to biosensing surfaces to capture and detect target molecules or biomarkers. Thus, fabrication of biosensing surfaces for regio- and chemoselective immobilization of biomolecules is a crucial step for better biosensor performance. In our previous studies, a regio- and chemoselective immobilization strategy was demonstrated on glass surfaces. This strategy is now used to regioselectively attach proteins to self-assembled monolayers (SAMs) on gold surfaces. Recombinant green fluorescent protein (GFP), glutathione S-transferase (GST), and antibody-binding protein G, bearing a C-terminal CVIA motif, were prepared and a farnesyl analogue with an ω-alkyne moiety was attached to the sulfhydryl moiety in the cysteine side chain by protein farnesyltransferase. The proteins, modified with the bioorthogonal alkyne functional group, were covalently and regioselectively immobilized on thiol or dithiocarbamate (DTC) SAMs on a gold surface by a Huigsen [3 + 2] cycloaddition reaction with minimal nonspecific binding. A concentration-dependent increase of fluorescence intensity was observed in wells treated with GFP on both thiol- and DTC-SAMs. The highly ordered, densely packed layer allowed for a high loading of immobilized protein, with a concomitant increase in substrate binding capacity. The DTC-SAMs were substantially more resistant to displacement of the immobilized proteins from the gold surface by β-mercaptoethanol than alkane-thiol SAMs.

A photonic crystal biosensor assay for ferritin utilizing iron-oxide nanoparticles

Iron deficiency anemia afflicts 1 in 3 individuals, mostly women and children worldwide. A novel application using iron-oxide nanoparticles (IONPs) and a photonic crystal (PC) optical biosensor as an immunodiagnostic platform for detection of serum ferritin, a biomarker for iron deficiency, is presented. Human liver ferritin (450kDa), clinical serum controls, and three commercially available ferritin ELISA tests were used to evaluate the PC biosensor assay in terms of inter- and intra-assay variability, spike-recovery (%), limit of detection (LOD), and matrix effects on binding. For the PC biosensor, signal response from label-free, sandwich with secondary antibody (pAb), and pAb functionalized with iron-oxide nanoparticles (FpAb) assays were detected using the Biomolecular Interaction Detection (BIND) system. Bland–Altman analysis was used to evaluate agreement between expected values for ferritin in control sera and each of the detection platforms. Inter- and intra-assay variability of the PC biosensor were both <10%. Percent mean recovery (±%RSD) of ferritin from two control sera samples were 94.3% (13.1%) and 96.9% (7.6%). Use of FpAb in PC biosensor resulted in two orders of magnitude increase in sensitivity compared to label-free assay; capable of measuring serum ferritin as low as 26ng/mL. In comparison to ELISA tests, the PC biosensor assay had the lowest bias (−1.26; 95% CI [−3.0–5.5]) and narrower limit of agreement (−11.6–9.1ng/mL) when determining ferritin concentrations from control sera. These proof-of-concept studies support the use of IONPs to enhance detection sensitivity of PC biosensors for determination of biomarkers of nutritional status.

Simultaneous Detection of Bio-Molecular Interactions and Surface Topography using Photonic Force Microscopy

We developed a photonic force microscope that can map multiple parameters simultaneously, including the surface topography and biomolecular interactions. To track the position of the probe bead and to determine contact position with the sample surface, we adopted a video analysis method using the diffraction pattern of monochromatic light passing through the probe bead. To demonstrate the capability of the microscope, we report the simultaneous measurement of the molecule distribution of DNA oligonucleotides on the surface, the binding strength of DNA hybridization between the bead and surface, and the topography of the smooth molded surface.

Simultaneous detection of biomolecular interactions and surface topography using photonic forcemicroscopy

We developed a photonic force microscope that can map multiple parameters simultaneously, including the surface topography and biomolecular interactions. To track the position of the probe bead and to determine contact position with the sample surface, we adopted a video analysis method using the diffraction pattern of monochromatic light passing through the probe bead. To demonstrate the capability of the microscope, we report the simultaneous measurement of the molecule distribution of DNA oligonucleotides on the surface, the binding strength of DNA hybridization between the bead and surface, and the topography of the smooth molded surface.

Dynamic imaging of enzymatic events at polyelectrolyte-disrupted phospholipid membranes using liquid crystals

In this study, we employed a simple liquid crystal (LC)-based system to dynamically image enzymatic events at the aqueous/LC interface decorated with polyelectrolyte-disrupted phospholipid membranes. Since polyelectrolytes were shown to disrupt the arrangement of the self-assembled phospholipid monolayer and induced a dark-to-bright shift in the optical response of LCs that support the phospholipid membrane, we observed that the transfer of an aqueous solution of protease onto the polyelectrolyte-disrupted phospholipid membrane resulted in a gradual recovery of the optical response of LCs from bright to dark appearance. Due to the enzymatic event that occurs at the aqueous/LC interface, the generated polyelectrolyte fragments desorbed from the interface to the bulk solution. This led to the restoration of the disrupted phospholipid monolayer, which resulted in recovery of the optical response. These results suggest that the polyelectrolyte-decorated membrane-supported LCs could be potentially used to examine a range of biological interactions that involve polyelectrolytes. Furthermore, the LC-based system holds great promise for label-free and real-time investigation and detection of biomolecular interactions coupled to membrane disruption and restoration, which might have potential utility in the clinical diagnosis and treatment of membrane-associated disease.

Carbon nanomaterials field-effect-transistor-based biosensors

Carbon nanomaterials field-effect transistor (FET)-based electrical biosensors provide significant advantages over the current gold standards, holding great potential for realizing direct, label-free, real-time electrical detection of biomolecules in a multiplexed manner with ultrahigh sensitivity and excellent selectivity. The feasibility of integrating them with current complementary metal oxide semiconductor platform and a fluid handling module using standard microfabrication technology opens up new opportunities for the development of low-cost, low-noise, portable electrical biosensors for use in practical future devices. In this article, we review recent progress in the rapidly developing area of biomolecular interaction detection using FET-based biosensors based on the carbon nanomaterials single-walled carbon nanotubes (SWNTs) and graphene. Detection scenarios include DNA–DNA hybridization, DNA–protein interaction, protein function and cellular activity. In particular, we will highlight an amazing property of SWNT- or graphene-FETs in biosensing: their ability to detect biomolecules at the single-molecule level or at the single-cell level. This is due to the size comparability and the surface compatibility of the carbon nanomaterials with biological molecules. We also summarize some current challenges the scientific community is facing, including device-to-device heterogeneity and the lack of system integration for uniform device array mass production. The detection of biological events is crucial in many life science applications such as disease diagnosis and the analysis of biological systems. Song Liu and Xuefeng Guo from the Beijing National Laboratory for Molecular Sciences and Peking University now review the use of field-effect transistors (FET) based on carbon nanomaterials for biosensing applications. Biosensors made from graphene or carbon nanotubes-based FETs have several advantages. They are very sensitive to environmental parameters such as surface charges or pH values. Furthermore, these nanostructures are very good charge conductors, allowing for a fast response to external changes. In particular sensors based on transistor geometries are of benefit, as biological reactions taking place at their surface directly influence the charge transport through the device. Although reliability remains an issue, such carbon-derived biosensors have already shown protein reactions and detection down to the single molecule level, clearly establishing their potential for practical applications. In this article, we review recent progress in the rapidly developing area of biomolecular interaction detection with excellent selectivity and ultrahigh sensitivity, such as DNA-DNA hybridization, DNA-protein interaction, protein function, and cellular activity, using FET-based biosensors based on the carbon nanomaterials single-walled carbon nanotubes (SWNTs) and graphenes. We also summarize some current challenges the scientific community is facing, including device-to-device heterogeneity and the lack of system integration for uniform device array mass-production.

Nanobiosensors Based on Localized Surface Plasmon Resonance for Biomarker Detection

Localized surface plasmon resonance (LSPR) is induced by incident light when it interacts with noble metal nanoparticles that have smaller sizes than the wavelength of the incident light. Recently, LSPR‐based nanobiosensors were developed as tools for highly sensitive, label‐free, and flexible sensing techniques for the detection of biomolecular interactions. In this paper, we describe the basic principles of LSPR‐based nanobiosensing techniques and LSPR sensor system for biomolecule sensing. We also discuss the challenges using LSPR nanobiosensors for detection of biomolecules as a biomarker.

“Peak tracking chip” for label-free optical detection of bio-molecular interaction and bulk sensing

A novel imaging method for bulk refractive index sensing or label-free bio-molecular interaction sensing is presented. This method is based on specially designed "Peak tracking chip" (PTC) involving "tracks" of adjacent resonant waveguide gratings (RWG) "micropads" with slowly evolving resonance position. Using a simple camera the spatial information robustly retrieves the diffraction efficiency, which in turn transduces either the refractive index of the liquids on the tracks or the effective thickness of an immobilized biological layer. Our intrinsically multiplex chip combines tunability and versatility advantages of dielectric guided wave biochips without the need of costly hyperspectral instrumentation. The current success of surface plasmon imaging techniques suggests that our chip proposal could leverage an untapped potential to routinely extend such techniques in a convenient and sturdy optical configuration toward, for instance for large analytes detection. PTC design and fabrication are discussed with challenging process to control micropads properties by varying their period (step of 2 nm) or their duty cycle through the groove width (steps of 4 nm). Through monochromatic imaging of our PTC, we present experimental demonstration of bulk index sensing on the range [1.33-1.47] and of surface biomolecule detection of molecular weight 30 kDa in aqueous solution using different surface densities. A sensitivity of the order of 10(-5) RIU for bulk detection and a sensitivity of the order of ∼10 pg mm(-2) for label-free surface detection are expected, therefore opening a large range of application of our chip based imaging technique. Exploiting and chip design, we expect as well our chip to open new direction for multispectral studies through imaging.

Synthesis of ultrathin silver shell on gold core for reducing substrate effect of LSPR sensor

The refractive index sensitivity of gold@silver core-shell nanoparticles on indium tin oxide glass was investigated. The thin silver shell was electrodeposited on gold surface by controlling the applied potential and electrodeposition cycles. The refractive index sensitivity at gold@silver core-shell nanoparticles with ultrathin silver shell (~ 0.7 nm) to bulk refractive index was enhanced 76% than that of the Au nanoparticles (Au core) deposited on ITO substrate. This core-shell platform was also applied to detection of biomolecular interactions using the streptavidin-biotin assay. The strategy would reduce greatly the substrate effect of plasmonic sensors.

Characteristics of Protein G-modified BioFET

Label-free detection of biomolecular interactions was performed using BioFET(Biologically sensitive Field-Effect Transistor) and SPR(Surface Plasmon Resonance). Qualitative information on the immobilization of an anti-IgG and antibody-antigen interaction was gained using the SPR analysis system. The BioFET was used to explore the pI value of the protein and to monitor biomolecular interactions which caused an effective charge change at the gate surface resulting in a drain current change. The results show that the BioFET can be a useful monitoring tool for biomolecular interactions and is complimentary to the SPR system.

Biomolecular and electrochemical charge detection by a micromechanical electrometer

A micromechanical electrometer is applied to the label-free detection of biomolecular interactions and electrochemical charge sensing. The primary element of the electrometer is a micromechanical variable capacitor to modulate and convert a dc charge to an ac voltage output, thereby limiting the effects of low frequency noise on charge detection. At room temperature and ambient pressure the noise-limited charge resolution of a micromechanical electrometer based on this principle is found to be 3 e/ Hz , enabling the potential detection of charged single molecule binding on electrode surfaces. The detection principle is validated by several experiments. Biomolecular binding experiments are conducted on an external gold electrode situated within a custom-designed flow cell and electrically connected to the micromachined electrometer. The concepts are validated by demonstrating the detection of biotin–streptavidin binding and DNA hybridization. Furthermore, it is shown that the electrometer can be applied for the detection of the redox system ferrocyanide/ferricyanide to describe Nernstian behaviour due to well defined charge transfer on the electrode surface at different concentration ratios as expected.

Fabrication and bio-functionalization of tetrahedral amorphous carbon thin films for bio sensor applications

Tetrahedral amorphous carbon (ta-C) thin films are a promising material for use as biocompatible interfaces in applications such as in-vivo biosensors. However, the functionalization of ta-C film surface, which is a pre-requisite for biosensors, remains a big challenge due to its chemical inertness. We have investigated the bio-functionalization of ta-C films fabricated under specific physical conditions through the covalent attachment of functional biomolecular probes of peptide nucleic acid (PNA) to ta-C films, and the effect of fabrication conditions on the bio-functionalization. The study showed that the functional bimolecular probes such as protected long-chain ω-unsaturated amine (TFAAD) can be covalently attached to the ta-C surface through a well-defined structure. With the given fabrication process, electrochemical methods can be applied to the detection of biomolecular interaction, which establishes the basis for the development of stable, label-free biosensors.

AlGaN/GaN FET for DNA hybridization detection

AbstractWe present the application of AlGaN/GaN field effect transistor (FET) for detection of biomolecular interactions, demonstrated by a DNA hybridization process. We immobilized a monolayer of single strand probe DNAs on the active gate surface of an AlGaN/GaN FET device and monitored the drain current during the affinity binding process with the presence of both matched and mismatched DNA molecules. The immobilization quality was first studied by X‐ray photoelectron spectroscopy (XPS) and fluorescence images. We then observed from the time‐resolved electrical measurement that significant and specific signals were solely upon the hybridization between the probe DNAs and the matched DNAs. The IDS–VDS characteristics of devices before and after hybridization were compared with a notable shift indicating the binding of DNA molecules.

Real-time and label-free detection of biomolecular interactions by oblique-incidence reflectivity difference method

We successfully conduct the label-free and real-time detection of the interactions between epoxy groups and rabbit IgG and 5′ CTT CAG GTC ATG AGC CTG AT 3′ oligonucleotide, and between the hybridization of 5′ CTT CAG GTC ATG AGC CTG AT 3′ and its complementary 3′ GAA GTC CAG TAC TCG GAC TA 5′ oligonucleotide, by the oblique-incidence reflectivity difference (OI-RD) method. The dynamic curves of OI-RD signals, corresponding to the kinetic processes of biomolecular combination or hybridization, are acquired. In our case, the combination of epoxy groups with rabbit IgG and 5′ CTT CAG GTC ATG AGC CTG AT 3′ oligonucleotide need almost one and a half hours and about two hundred seconds, respectively; and the hybridization of the two oligonucleotides needs about five hundred seconds. The experimental results show that the OI-RD is a promising method for the real-time and label-free detection of biomolecular interactions.

Photopotential Imaging on Functionalized Surfaces Dedicated to Label-Free Detection of Biomolecular Interactions

We present a label-free biosensor capable to map local potential by photogenerating charge carriers in a field effect device. It consists in an amplitude-modulated laser beam scanning through an electrolyte/insulator/semiconductor structure at very low illumination around the equilibrium state of semiconductor. A proof of principle is demonstrated by depositing a self-assembled monolayer on the dielectric and by showing a clear photopotential change between naked and covered areas.