Label-free Detection Of Biomolecular Interactions Research Articles

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.

Enhancing the sensitivity of plasmonic optical fiber sensors by analyzing the distribution of the optical modes intensity.

Improving the sensitivity of plasmonic optical fiber sensors constitutes a major challenge as it could significantly enhance their sensing capabilities for the label-free detection of biomolecular interactions or chemical compounds. While many efforts focus on developing more sensitive structures, we present here how the sensitivity of a sensor can be significantly enhanced by improving the light analysis. Contrary to the common approach where the global intensity of the light coming from the core is averaged, our approach is based on the full analysis of the retro-reflected intensity distribution that evolves with the refractive index of the medium being analyzed. Thanks to this original and simple approach, the refractive index sensitivity of a plasmonic optical fiber sensor used in reflection mode was enhanced by a factor of 25 compared to the standard method. The reported approach opens exciting perspectives for improving the remote detection as well as for developing new sensing strategies.

Self-assembly of robust gold nanoparticle monolayer architectures for quantitative protein interaction analysis by LSPR spectroscopy

Localized surface plasmon resonance (LSPR) detection offers highly sensitive label-free detection of biomolecular interactions. Simple and robust surface architectures compatible with real-time detection in a flow-through system are required for broad application in quantitative interaction analysis. Here, we established self-assembly of a functionalized gold nanoparticle (AuNP) monolayer on a glass substrate for stable, yet reversible immobilization of Histidine-tagged proteins. To this end, one-step coating of glass substrates with poly-L-lysine graft poly(ethylene glycol) functionalized with ortho-pyridyl disulfide (PLL-PEG-OPSS) was employed as a reactive, yet biocompatible monolayer to self-assemble AuNP into a LSPR active monolayer. Site-specific, reversible immobilization of His-tagged proteins was accomplished by coating the AuNP monolayer with tris-nitrilotriacetic acid (trisNTA) PEG disulfide. LSPR spectroscopy detection of protein binding on these biocompatible functionalized AuNP monolayers confirms high stability under various harsh analytical conditions. These features were successfully employed to demonstrate unbiased kinetic analysis of cytokine-receptor interactions.Graphical abstract

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.

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.

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.

Nano and micro structures for label-free detection of biomolecules

Nano and micro structure-based biosensors are promising tool for label-free detection of biomolecular interactions with great accuracy. This review gives a brief survey on nano and micro platforms to sense a variety of analytes such as DNA, proteins and viruses. Among incredible nano and micro structure for bio-analytical applications, the scope of this paper will be limited to micro and nano resonators and nanowire field-effect transistors. Nanomechanical motion of the resonators transducers biological information to readable signals. They are commonly combined with an optical, capacitive or piezo-resistive detection systems. Binding of target molecule to the modified surface of nanowire modulates the current of the nanowire through electrical field-effect. Both detection methods have advantages of label-free, real-time and high sensitive detection. These structures can be extended to fabricate array-type sensors for multiplexed detection and high-throughput analysis. The biosensors based on these structures will be applied to lab-on-a-chip platforms and point-of-care diagnostics. Basic concepts including detection mechanisms and trends in their fields will be covered in this review.

Low Noise Detection of Biomolecular Interactions with Signal-Locking Surface Plasmon Resonance

Surface plasmon resonance (SPR) is a popular technique for label-free detection of biomolecular interactions at a surface. SPR yields quantitative kinetic association and dissociation constants of surface interactions such as the binding of two molecular species, one present in the liquid phase and the other immobilized at the surface. Current state-of-the-art SPR systems extract kinetic constants from measurements of the step response of the interaction versus time. The step response measurement is subject to the influence of noise and drift disturbances that limit its minimum-detectable mass changes. This paper presents a new SPR technique that measures the biomolecular interaction not in time but over a very narrow frequency range under periodic excitation. The measured response is, thus, locked to a very specific narrow band signal. This narrow band spectral sensing scheme has a very high degree of rejection to uncorrelated spurious signals. The signal-locked SPR technique was implemented using a chemical modulator chip connected to a set of functionalized Au sensing sites downstream. Binding experiments for a model system of carbonic anhydrase-II (CA-II) analyte and immobilized 4-(2-aminoethyl)benzenesulfonamide (ABS) ligand display a 100-fold (20 dB) improvement in the measured signal-to-noise ratio (SNR) when using the new technique compared to the SNR achieved using the conventional step response method.

Specific and reversible immobilization of histidine-tagged proteins on functionalized siliconnanowires

Silicon nanowire (Si NW)-based field effect transistors (FETs) have shown great potential asbiosensors (bioFETs) for ultra-sensitive and label-free detection of biomolecularinteractions. Their sensitivity depends not only on the device properties, but also on thefunction of the biological recognition motif attached to the Si NWs. In this study, we showthat SiNWs can be chemically functionalized with Ni:NTA motifs, suitable for thespecific immobilization of proteins via a short polyhistidine tag (His-tag) at closeproximity to the SiNW surface. We demonstrate that the proteins preserve theirfunction upon immobilization onto SiNWs. Importantly, the protein immobilizationon the Si NWs is shown to be reversible after addition of EDTA or imidazole,thus allowing the regeneration of the bioFET when needed, such as in the caseof proteins having a limited lifetime. We anticipate that our methodology mayfind a generic use for the development of bioFETs exploiting functional proteinassays because of its high compatibility to various types of NWs and proteins.

Investigation of sensing mechanism and signal amplification in carbon nanotube based microfluidic liquid-gated transistors via pulsating gate bias

The advent of a carbon nanotube liquid-gated transistor (LGFET) for biosensing applications allows the possibility of real-time and label-free detection of biomolecular interactions. The use of an aqueous solution as dielectric, however, has traditionally restricted the operating gate bias (VG) within |VG| < 1 V, due to the electrolysis of water. Here, we propose pulsed-gating as a facile method to extend the operation window of LGFETs to |VG| > 1 V. A comparison between simulation and experimental results reveals that at voltages in excess of 1 V, the LGFET sensing mechanism has a contribution from two factors: electrostatic gating as well as capacitance modulation. Furthermore, the large IDS drop observed in the |VG| > 1 V region indicates that pulsed-gating may be readily employed as a simple method to amplify the signal in the LGFET and pushes the detection limit down to attomolar concentration levels, an order of magnitude improvement over conventionally employed DC VG biasing.

Nanogap biosensors for electrical and label-free detection of biomolecular interactions

We demonstrate nanogap biosensors for electrical and label-free detection of biomolecularinteractions. Parallel fabrication of nanometer distance gaps has been achieved using asilicon anisotropic wet etching technique on a silicon-on-insulator (SOI) wafer with a finelycontrollable silicon device layer. Since silicon anisotropic wet etching resulted in atrapezoid-shaped structure whose end became narrower during the etching, the nanogapstructure was simply fabricated on the device layer of a SOI wafer. The nanogap deviceswere individually addressable and a gap size of less than 60 nm was obtained. Wedemonstrate that the nanogap biosensors can electrically detect biomolecular interactionssuch as biotin/streptavidin and antigen/antibody pairs. The nanogap devices show acurrent increase when the proteins are bound to the surface. The current increasesproportionally depending upon the concentrations of the molecules in the range of 100 fg ml−1–100 ng ml−1 at 1 V bias. It is expected that the nanogap developed here could be a highly sensitivebiosensor platform for label-free detection of biomolecular interactions.

Label-Free Detection of Biomolecular Interactions Using BioLayer Interferometry for Kinetic Characterization

The analysis of biomolecular interactions is key in the drug development process. Label-free biosensor methods provide information on binding, kinetics, concentration, and the affinity of an interaction. These techniques provide real-time monitoring of interactions between an immobilized ligand (such as a receptor) to an analyte in solution without the use of labels. Advances in biosensor design and detection using BioLayer Interferometry (BLI) provide a simple platform that enables label-free monitoring of biomolecular interactions without the use of flow cells. We review the applications of BLI in a wide variety of research and development environments for quantifying antibodies and proteins and measuring kinetics parameters.

Spectral-domain optical coherence phase microscopy for label-free multiplexed protein microarray assay

Quantitative measurement of affinities and kinetics of various biomolecular interactions such as protein–protein, protein–DNA and receptor–ligand is central to our understanding of basic molecular and cellular functions and is useful for therapeutic evaluation. Here, we describe a laser-scanning quantitative imaging method, referred to as spectral-domain optical coherence phase microscopy, as an optical platform for label-free detection of biomolecular interactions. The instrument is based on a confocal interferometric microscope that enables depth-resolved quantitative phase measurements on sensor surface with high spatial resolution and phase stability. We demonstrate picogram per square millimeter surface mass sensitivity, and show its sensing capability by presenting static and dynamic detection of multiplexed protein microarray as immobilized antigens capture their corresponding antibodies.

Label-free detection of biomolecular interactions in real time with a nano-porous silicon-based detection method

BackgroundWe describe a biosensor platform for monitoring molecular interactions that is based on the combination of a defined nano-porous silicon surface, coupled to light interferometry. This platform allows the label-free detection of protein-protein and protein-DNA interactions in defined, as well as complex protein mixtures. The silicon surface can be functionalized to be compatible with traditional carboxyl immobilization chemistries, as well as with aldehyde-hydrazine bioconjugation chemistries.ResultsWe demonstrate the utility of the new platform in measuring protein-protein interactions of purified products in buffer, in complex mixtures, and in the presence of different organic solvent spikes, such as DMSO and DMF, as these are commonly used in screening chemical compound libraries.ConclusionNano-porous silicon, when combined with white light interferometry, is a powerful technique for the measurement of protein-protein interactions. In addition to studying the binary interactions of biomolecules in clean buffer systems, the newly developed surfaces are also suited for studying interactions in complex samples, such as plasma.

Mechanism and sensitivity of the intrinsic charge detection of biomolecular interactions by field effect devices

For many biotechnological applications a quantitative and label-free detection of biomolecular interactions is becoming of outstanding importance. The here presented analytical description of the direct charge detection by field effect devices introduces a straightforward tool for quantitative analysis. By combined measurements of the pH and electrolyte concentration response the absolute amount of adsorbed surface charge can now be determined.

A novel optical method providing for high-sensitivity and high-throughput biomolecular interaction analysis

We present a novel method for label-free detection of biomolecular interactions based on the surface plasmon resonance (SPR) phenomenon. The method enables simultaneous and extremely sensitive analysis of a large number of samples arranged in a 2D array format on the sensor surface. To realize the approach, a prototype of the optical sensor was constructed. With this sensor, interaction kinetics are sampled at a 10 Hz rate, and the dynamic range of the system extends from 1.32 to 1.38 refractive index units (RIU). While the fluidics for a highly multiplexed system are not yet developed, we optimized the prototype for simultaneous sensing of up to 110 interaction sites. In this configuration, extremely low root-mean-squared (RMS) noise, 2.6×10−8RIU/Hz, is obtained. This low noise level corresponds to sensitivity exceeding that of any commercial SPR instrument—even those with little or no multiplexing capability. Simple software changes allow the user to trade off dynamic range and multiplexing in exchange for additional sensitivity enhancement. This capability is demonstrated in experiments we perform characterizing the interaction of carbonic anhydrase II with a small molecule, carboxybenzenesulfonamide.

Microplate-based, label-free detection of biomolecular interactions: applications in proteomics

This review describes a new type of label-free optical biosensor that is inexpensively manufactured from continuous sheets of plastic film and incorporated into standard format microplates to enable highly sensitive, high-throughput detection of small molecules, proteins and cells. The biosensor and associated detection instrumentation are applied to review two fundamental limiting issues for assays in proteomics research and drug discovery: requirement for quantitative measurement of protein concentration and specific activity, and measurements made with complex systems in highly parallel measurements. SRU BIosystems, Inc.’s BIND™ label-free detection will address these issues using data examples for hybridoma screening, epitope binning and mapping, small-molecule screening, and cell-based functional assays. The review describes several additional applications that are under development for the system, and the key issues that will drive adoption of the technology over the next 5 years.

Field Effect of Screened Charges: Electrical Detection of Peptides and Proteins by a Thin‐Film Resistor

For many biotechnological applications the label-free detection of biomolecular interactions is becoming of outstanding importance. In this Article we report the direct electrical detection of small peptides and proteins by their intrinsic charges using a biofunctionalized thin-film resistor. The label-free selective and quantitative detection of small peptides and proteins is achieved using hydrophobized silicon-on-insulator (SOI) substrates functionalized with lipid membranes that incorporate metal-chelating lipids. The response of the nanometer-thin conducting silicon film to electrolyte screening effects is taken into account to determine quantitatively the charges of peptides. It is even possible to detect peptides with a single charge and to distinguish single charge variations of the analytes even in physiological electrolyte solutions. As the device is based on standard semiconductor technologies, parallelization and miniaturization of the SOI-based biosensor is achievable by standard CMOS technologies and thus a promising basis for high-throughput screening or biotechnological applications.