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Cited by 126 publications
References 8 publications
In this paper, we describe a novel nonlabeled biosensor with high diagnostic potential for rapid and sensitive detection of antigens in complex biological samples. The biosensor comprises a piezoimmunosensor (PZ) displaying a specially constructed recombinant antibody on its surface. The recombinant single-chain fragment variable (scFv) antibody contained a cysteine within the linker amino acid sequence used to join the scFv variable heavy and light chains. The presence of cysteine induced the scFv construct to self-assemble as a densely packed rigid monolayer on the gold surface of a quartz crystal microbalance. scFv molecules in this self-assembled mono-layer (SAM) exhibited a defined orientation and high areal densities, with scFv-modified microbalance surfaces displaying 35 times as many variable antigen-binding sites per square centimeter as surfaces modified with whole antibody. Experimental data show that the scFv SAM PZ is superior to Fab fragment, Fab fragment containing a free sulfhydryl group (i.e., Fab-SH), and whole antibody PZs regarding sensitivity and specificity. Because of their small uniform size (MW ≈ 27000) and the ease with which they can be modified using genetic engineering, scFv's have significant advantages over whole antibodies in microbalance biosensor systems. We demonstrate here that the use of scFv containing a cysteine within the scFv linker sequence (i.e., scFv-cys) for preparation of biosensor surfaces markedly increases the density of available antigen-binding sites, yielding a system that is highly selective, rapid, and capable of detecting low concentrations of antigens in complex samples.Biosensor systems that detect biological and chemical agents have important medical, environmental, public safety, and defense applications. An ideal biosensor would be sensitive, rapid, reliable, robust, and inexpensive. Piezoimmunosensors (PZs) are a type of biosensor utilizing antibodies and a quartz crystal microbalance (QCM) to detect minute changes in mass as antigens bind to the antibodies on the QCM surface. 1,2 Although their diagnostic potential is theoretically quite high, in practice, the usefulness of PZs has been limited by the fact that typical IgG antibodies can trap or nonspecifically bind irrelevant molecules, thus yielding false positive signals in assays. Additionally, there remains some skepticism concerning their applicability as biosensors due to the complexity of the physical properties of biofilms in a liquid that make it difficult to establish an explicit relationship between the added mass and a change in the resonant frequency. The QCM gives a direct response signal that characterizes a binding event between an antibody layer, immobilized on the surface of the QCM or other transducers, and the antigen to be detected. The mass change on the QCM surface is estimated using the Sauerbrey equation, 3 Δf = −2Δmnf 0 2 /[A(µ q ρ q ) 1/2 ], where n is the overtone number, µ q is the shear modulus of the quartz [2.947 × 10 11 g/(cm·s 2 )], ρ q is the density of the ...
In this paper, we describe a novel nonlabeled biosensor with high diagnostic potential for rapid and sensitive detection of antigens in complex biological samples. The biosensor comprises a piezoimmunosensor (PZ) displaying a specially constructed recombinant antibody on its surface. The recombinant single-chain fragment variable (scFv) antibody contained a cysteine within the linker amino acid sequence used to join the scFv variable heavy and light chains. The presence of cysteine induced the scFv construct to self-assemble as a densely packed rigid monolayer on the gold surface of a quartz crystal microbalance. scFv molecules in this self-assembled mono-layer (SAM) exhibited a defined orientation and high areal densities, with scFv-modified microbalance surfaces displaying 35 times as many variable antigen-binding sites per square centimeter as surfaces modified with whole antibody. Experimental data show that the scFv SAM PZ is superior to Fab fragment, Fab fragment containing a free sulfhydryl group (i.e., Fab-SH), and whole antibody PZs regarding sensitivity and specificity. Because of their small uniform size (MW ≈ 27000) and the ease with which they can be modified using genetic engineering, scFv's have significant advantages over whole antibodies in microbalance biosensor systems. We demonstrate here that the use of scFv containing a cysteine within the scFv linker sequence (i.e., scFv-cys) for preparation of biosensor surfaces markedly increases the density of available antigen-binding sites, yielding a system that is highly selective, rapid, and capable of detecting low concentrations of antigens in complex samples.Biosensor systems that detect biological and chemical agents have important medical, environmental, public safety, and defense applications. An ideal biosensor would be sensitive, rapid, reliable, robust, and inexpensive. Piezoimmunosensors (PZs) are a type of biosensor utilizing antibodies and a quartz crystal microbalance (QCM) to detect minute changes in mass as antigens bind to the antibodies on the QCM surface. 1,2 Although their diagnostic potential is theoretically quite high, in practice, the usefulness of PZs has been limited by the fact that typical IgG antibodies can trap or nonspecifically bind irrelevant molecules, thus yielding false positive signals in assays. Additionally, there remains some skepticism concerning their applicability as biosensors due to the complexity of the physical properties of biofilms in a liquid that make it difficult to establish an explicit relationship between the added mass and a change in the resonant frequency. The QCM gives a direct response signal that characterizes a binding event between an antibody layer, immobilized on the surface of the QCM or other transducers, and the antigen to be detected. The mass change on the QCM surface is estimated using the Sauerbrey equation, 3 Δf = −2Δmnf 0 2 /[A(µ q ρ q ) 1/2 ], where n is the overtone number, µ q is the shear modulus of the quartz [2.947 × 10 11 g/(cm·s 2 )], ρ q is the density of the ...
Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for freesolution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R 2 > 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution.backscattering interferometry | assay methodology | molecular interactions | conformation change | hydration change C ontemporary assays enabling single-molecule detection (1,2) have accelerated the sequencing of the human genome (3) and facilitated imaging with extraordinary resolution without labels (4). To most closely approximate the natural state, an interaction assay methodology would interrogate the processes (reaction, molecular interaction, protein folding event, etc.) without perturbation. Label-free chemical and biochemical investigations (5, 6) transduce the desired signal without an exogenous label (fluorescent, radioactive, or otherwise) representing an essential step toward this goal. Many label-free methods require one of the interacting species to be either tethered or immobilized to the sensor surface, introducing a potential perturbation to the natural state of the species (7,8). However, back-scattering interferometry (BSI) is a free-solution label-free technique with the added benefit of sensitivity that rivals fluorescence (9). There are other techniques performed in free solution, such as MS (10, 11) and NMR (12,13) and the widely used isothermal titration calorimetry (ITC) (14, 15). As with NMR, ITC has many advantages, but exhibits modest sensitivity and often requires large sample quantities. Another increasingly popular free-solution approach is microscale thermophoresis (...
The article contains sections titled: 1. Introduction 1.1. Comparison with Other Spectroscopic Methods 1.2. Development and Uses 2. Theoretical Principles 2.1. Electronic States and Orbitals 2.2. Interaction Between Radiation and Matter 2.2.1. Dispersion 2.2.2. Absorption 2.2.3. Scattering 2.2.4. Reflection 2.2.5. Band Intensity 2.3. The Lambert–BeerLaw 2.3.1. Definitions 2.3.2. Deviations from the Lambert ‐ Beer Law 2.4. Photophysics 2.4.1. Energy Level Diagram 2.4.2. Deactivation Processes 2.4.3. Transition Probability and Fine Structure of the Bands 2.5. Chromophores 2.6. Optical Rotatory Dispersion and Circular Dichroism 2.6.1. Generation of Polarized Radiation 2.6.2. Interaction with Polarized Radiation 2.6.3. Optical Rotatory Dispersion 2.6.4. Circular Dichroism and the Cotton Effect 2.6.5. Magnetooptical Effects 3. Optical Components and Spectrometers 3.1. Principles of Spectrometer Construction 3.1.1. Sequential Measurement of Absorption 3.1.2. Multiplex Methods in Absorption Spectroscopy 3.2. Light Sources 3.2.1. Line Sources 3.2.2. Sources of Continuous Radiation 3.2.3. Lasers 3.3. Selection of Wavelengths 3.3.1. Prism Monochromators 3.3.2. Grating Monochromators 3.3.3. Electro‐Acoustic and Opto‐Acoustic Wavelength Generation 3.4. Polarizers and Analyzers 3.5. Sample Compartments and Cells 3.5.1. Closed Compartments 3.5.2. Modular Arrangements 3.5.3. Open Compartments 3.6. Detectors 3.7. Optical Paths for Special Measuring Requirements 3.7.1. Fluorescence Measurement 3.7.2. Measuring Equipment for Polarimetry, ORD, and CD 3.7.3. Reflection Measurement 3.7.4. Ellipsometry 3.8. Effect of Equipment Parameters 3.9. Connection to Electronic Systems and Computers 4. Uses of UV ‐ VIS Spectroscopy in Absorption, Fluorescence, and Reflection 4.1. Identification of Substances and Determination of Structures 4.2. Quantitative Analysis 4.2.1. Determination of Concentration by Calibration Curves 4.2.2. Classical Multicomponent Analysis 4.2.3. Multivariate Data Analysis 4.2.4. Use in Chromatography 4.3. Fluorimetry 4.3.1. Inner Filter Effects 4.3.2. Fluorescene and Scattering 4.3.3. Excitation Spectra 4.3.4. Applications 4.4. Reflectometry 4.4.1. Diffuse Reflection 4.4.2. Color Measurement 4.4.3. Regular Reflection 4.4.4. Determination of Film Thickness 4.4.5. Ellipsometry 4.5. Resonance Methods 4.5.1. SurfacePlasmon Resonance 4.5.2. Grating Couplers 4.5.3. Other Evanescent Methods 4.5.4. Interferometric Methods 4.6. On‐Line Process Control 4.6.1. Process Analysis 4.6.2. Measurement of Film Thicknesses 4.6.3. Optical Sensors 4.7. Measuring Methods Based on Deviations from the Lambert – Beer Law 5. Special Methods 5.1. Derivative Spectroscopy 5.2. Dual‐Wavelength Spectroscopy 5.3. Scattering 5.3.1. Turbidimetry 5.3.2. Nephelometry 5.3.3. Photon Correlation Spectroscopy 5.4. Luminescence, Excitation, and Depolarization Spectroscopy, and Measurement of Lifetimes 5.5. Polarimetry 5.5.1. Sugar Analysis 5.5.2. Cellulose Determination 5.5.3. Stereochemical StructuralAnalysis 5.5.4. Use of Optical Activity Induced by a Magnetic Field 5.6. Photoacoustic Spectroscopy (PAS)
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