Label-Free Photonic Crystal Biosensor Integrated Microfluidic Chip for Determination of Kinetic Reaction Rate Constants

We demonstrate a photonic crystal integrated microfluidic chip that is compatible with a 384-well microplate format for measuring kinetic reaction rate constants in high-throughput biomolecular interaction screening applications. The device enables low volume kinetic analysis of protein-protein interactions with low flow latency, and control of five analyte flow channels with a single control point. The structure is fabricated with a replica molding process that produces the submicron photonic crystal structure simultaneously with the micrometer-scale fluid channel structure. The device significantly reduces the kinetic assay time required compared with a conventional biosensor microplate in which reagents reach the active detection surface by diffusion. Using the photonic crystal sensor fluid network system, we demonstrate determination of the kinetic association/dissociation rate constants between immobilized ligands and analytes in the flow stream, using the heparin/lactoferrin system as an example.     

A computational reaction-diffusion model for the analysis of transport-limited kinetics

Optical, evanescent wave biosensors have become popular tools for quantitatively characterizing the kinetic properties of biomolecular interactions. Analyzing data from biosensor experiments, however, is often complicated when mass-transfer influences the detection kinetics. We present a computational, transport-kinetic model that can be used to analyze transport-limited biosensor data. This model describes a typical biosensor experiment in which a soluble analyte diffuses through a flow chamber and binds to a receptor immobilized on the transducer surface. Analyte transport in the flow chamber is described by the diffusion equation while the kinetics of analyte-surface association and dissociation are captured by a reactive boundary condition at the sensor surface. Numerical integration of the model equations and nonlinear least-squares fitting are used to compare model kinetic data to experimental results and generate estimates for the rate constants that describe analyte detection. To demonstrate the feasibility of this model, we use it to analyze data collected for the binding of fluorescently labeled trinitrobenzene to immobilized monoclonal anti-TNT antibodies. A successful analysis of this antigen-antibody interaction is presented for data collected with a fluorescence-based fiber-optic immunoassay. The results of this analysis are compared with the results obtained with existing methods for analyzing diffusion-limited kinetic data.     

A quantitative assessment of heterogeneity for surface-immobilized proteins

Many biotechnological applications use protein receptors immobilized on solid supports. Although, in solution, these receptors display homogeneous binding affinities and association/dissociation kinetics for their complementary ligand, they often display heterogeneous binding characteristics after immobilization. In this study, a fluorescence-based fiber-optic biosensor was used to quantify the heterogeneity associated with the binding of a soluble analyte, fluorescently labeled trinitrobenzene, to surface-immobilized monoclonal anti-TNT antibodies. The antibodies were immobilized on silica fiber-optic probes via five different immobilization strategies. We used the Sips isotherm to assesses and compare the heterogeneity in the antibody binding affinity and kinetic rate parameters for these different immobilization schemes. In addition, we globally analyzed kinetic data with a two-compartment transport-kinetic model to analyze the heterogeneity in the analyte-antibody kinetics. These analyses provide a quantitative tool by which to evaluate the relative homogeneity of different antibody preparations. Our results demonstrate that the more homogeneous protein preparations exhibit more uniform affinities and kinetic constants.     

A kinetic study of analyte-receptor binding and dissociation for surface plasmon resonance biosensors applications

A fractal analysis, which takes into account the effect of surface heterogeneity brought about by ligand immobilization on the reaction kinetics in surface plasmon resonance (SPR) biosensors, is presented. The binding and dissociation of estrogen receptors (ERs), ERa and ER/spl alpha/ and ER/spl beta/, in solution to different ligands immobilized on the SPR biosensor is analyzed within the fractal framework. The heterogeneity on the biosensor surface is made quantitative by using a single number, the fractal dimension D/sub f/. The analysis provides physical insights into the binding of these receptors to different ligands and compounds, particularly the endocrine disrupting compounds (EDCs). These EDCs have deleterious effects on humans and on wildlife. Single- and dual-fractal models were employed to fit the ER-binding data obtained from the literature. Values of the binding and dissociation rate coefficient and fractal dimensions were obtained from a regression analysis provided by Corel Quattro Pro, 8.0. Values for the affinity K/sub D/(=k/sub d//k/sub a/) were also calculated. This provides us with some extra flexibility in designing biomolecular assays. The analysis should provide further information on the mode of action and interaction of EDCs with the ERs. This would help in the design of agents and modulators against these EDCs.     

Quartz crystal microbalance detection of glutathione-protected nanoclusters using antibody recognition

A quartz crystal microbalance (QCM) immunosensor was developed for the quantitative detection of glutathione-protected nanoclusters. Advantages intrinsic to QCM were employed to make it an attractive alternative to other immunosensing techniques. We have addressed challenges in the area of QCM mass sensing through experimental correlation between damping resistance and frequency change for a reliable mass measurement. Electrode functionalization was optimized with the use of protein A to immobilize and present polyclonal IgG for antigen binding. This method was developed for the detection of glutathione (antigen)-protected clusters of nanometer size with high surface area and thiolate valency. Quantitation of glutathione-nanocluster binding to immobilized polyclonal antibody provides equilibrium constants (K(a) = (3.6 +/- 0.2) x 10(5) M(-1)) and kinetic rate constants (k(f) = (5.4 +/- 0.7) x 10(1) M(-1) s(-1) and k(r) = (1.5 +/- 0.4) x10(-4) s(-1)) comparable to literature reports. These observations further imply that immunoreactive nanoparticles have potential in medical diagnostics and materials assembly.     

Nonregeneration protocol for surface plasmon resonance: study of high-affinity interaction with high-density biosensors

Surface plasmon resonance (SPR) has been used in determining kinetics and thermodynamics of biological interaction in the past decades. One difficulty encountered in this technology is the need for a proper regeneration, which means the removal of analytes from the bound complexes to regenerate the activity of the ligands. Regeneration is not always practical since the harsh regeneration reagents may destroy the bioactivity of the ligands. It is even more difficult for complexes with high affinity constants. In this paper, we report a nonregeneration protocol for SPR techniques in which subsequent ligand/analyte interactions can be measured without regeneration; thus ligand biological activity could be retained. Kinetics, binding models, and mathematics of this protocol are discussed in detail using rabbit IgG as the analyte and engineered recombinant antibody A10B single-chain fragment variables (scFv) as the ligand. The affinity constant of rabbit IgG binding with A10B scFv measured by using a nonregeneration protocol was (2.5 +/- 0.2) x 10(7) M(-1), which was comparable with the value determined with a conventional regeneration SPR method ((2.2 +/- 1.5) x 10(7) M(-1)) and quartz crystal microbalance (1.9 x 10(7) M(-1)). A paradigm of streptavidin-biotin binding was analyzed to validate this protocol. The affinity constant for each binding subunit of streptavidin to the immobilized biotin was determined to be (7.3 +/- 0.2) x 10(6) M(-1), which was comparable with the solution-based value of 2 x 10(7) M(-1). The nonregeneration protocol requires a relatively high ligand density on the biosensor surface so that more data points can be obtained before surface saturation. The small size of scFv enables them to be constructed in the biosensors for such purpose.     

Biosensor for asparagine using a thermostable recombinant asparaginase from Archaeoglobus fulgidus

Asparaginase from the hyperthermophilic microorganism Archaeoglobus fulgidus was cloned and expressed in Escherichia coli as a fusion protein with a polyhistidine tail. After heat treatment to denature most of the native E. coli proteins, the enzyme was purified by an immobilized metal ion affinity chromatography method. The activity of the enzyme was determined by monitoring the change in ammonium concentration in solution. It was found that the enzyme is thermostable at temperatures as high as 85 degrees C. The KM for L-asparagine was 8 x 10(-5) and 5 x 10(-6) M at 37 and 70 degrees C, respectively. The catalytic activity for L-asparagine was 5-fold higher than for D-asparagine. The enzyme was immobilized in front of an ammonium-selective electrode and used to develop a biosensor for asparagine. The biosensor had a detection limit of 6 x 10(-5) M for L-asparagine. Unlike a sensor based on asparaginase from E. coli, the biosensor based on recombinant asparaginase from A. fulgidus demonstrated higher stability.     

Characterization by potentiometric procedures of the Acid-base and metal binding properties of two new classes of immobilized metal ion affinity adsorbents developed for protein purification

The acid-base protonation constants of two recently introduced chelating ligands for protein purification, O-phosphoserine and 8-hydroxyquinoline immobilized onto Sepharose CL-4B, and the stability constants of their derived immobilized metal ion chelate complexes have been determined by potentiometric methods. The data confirm that immobilization thermodynamically constrains the ligands, with the electron-withdrawing characteristics of the group linking the ligand to the support material affecting the magnitude of the stability constant of the immobilized metal ion complex vis-à-vis the free ligand-metal ion complex in solution. The influence of buffer composition, ionic strength, and pH on the stability constant of the immobilized hard metal ion chelate complexes has also been examined. Collectively, the results have confirmed that coordination complexes with stoichiometries other than the simply 1:1 ML-type exist with these systems, with hard metal ions exhibiting a preference for hydrolytic M(OH)(m)L(n) complexes where m or n > 1. These findings on the participation of coordination complexes of different stoichiometry depending on the characteristics of the chelating ligand and metal ion have fundamental implications for the interpretation of immobilized metal ion affinity chromatographic separation of proteins.     

Ligand binding to nicotinic acetylcholine receptor investigated by surface plasmon resonance

Ligand binding to the nicotinic acetylcholine receptor is studied by surface plasmon resonance. Biotinylated bungarotoxin, immobilized on a streptavidin-coated gold film, binds nicotinic acetylcholine receptor both in detergent-solubilized and in lipid vesicle-reconstituted form with high specificity. In the latter case, nonspecific binding to the sensor surface is significantly reduced by reconstituting the receptor into poly(ethylene glycol)-lipid-containing sterically stabilized vesicles. By preincubation of a bulk nicotinic acetylcholine receptor sample with the competing ligands carbamoylcholine and decamethonium bromide, the subsequent specific binding of the receptor to the surface-immobilized bungarotoxin is reduced, depending on the concentration of competing ligand. This competition assay allows the determination of the dissociation constants of the acetylcholine receptor-carbamoylcholine complex. A K(D) = 3.5 × 10(-)(6) M for the detergent-solubilized receptor and a K(D) = 1.4 × 10(-)(5) M for the lipid vesicle-reconstituted receptor are obtained. For decamethonium bromide, a K(D) = 4.5 × 10(-)(5) M is determined for the detergent-solubilized receptor. This approach is of general importance for investigating ligand-receptor interactions in case of small ligand molecules by mass-sensitive techniques.     

Nonlinear analyte concentration gradients for one-step kinetic analysis employing optical microring resonators

Conventional methods to probe the binding kinetics of macromolecules at biosensor surfaces employ a stepwise titration of analyte concentrations and measure the association and dissociation to the immobilized ligand at each concentration level. It has previously been shown that kinetic rates can be measured in a single step by monitoring binding as the analyte concentration increases over time in a linear gradient. We report here the application of nonlinear analyte concentration gradients for determining kinetic rates and equilibrium binding affinities in a single experiment. A versatile nonlinear gradient maker is presented, which is easily applied to microfluidic systems. Simulations validate that accurate kinetic rates can be extracted for a wide range of association and dissociation rates, gradient slopes, and curvatures, and with models for mass transport. The nonlinear analyte gradient method is demonstrated with a silicon photonic microring resonator platform to measure prostate specific antigen-antibody binding kinetics.     

Revealing equilibrium and rate constants of weak and fast noncovalent interactions

Rate and equilibrium constants of weak noncovalent molecular interactions are extremely difficult to measure. Here, we introduced a homogeneous approach called equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM) to determine k(on), k(off), and K(d) of weak (K(d) > 1 μM) and fast kinetics (relaxation time, τ < 0.1 s) in quasi-equilibrium for multiple unlabeled ligands simultaneously in one microreactor. Conceptually, an equilibrium mixture (EM) of a ligand (L), target (T), and a complex (C) is prepared. The mixture is introduced into the beginning of a capillary reactor with aspect ratio >1000 filled with T. Afterward, differential mobility of L, T, and C along the reactor is induced by an electric field. The combination of differential mobility of reactants and their interactions leads to a change of the EM peak shape. This change is a function of rate constants, so the rate and equilibrium constants can be directly determined from the analysis of the EM peak shape (width and symmetry) and propagation pattern along the reactor. We proved experimentally the use of ECEEM for multiplex determination of kinetic parameters describing weak (3 mM > K(d) > 80 μM) and fast (0.25 s ≥ τ ≥ 0.9 ms) noncovalent interactions between four small molecule drugs (ibuprofen, S-flurbiprofen, salicylic acid and phenylbutazone) and α- and β-cyclodextrins. The affinity of the drugs was significantly higher for β-cyclodextrin than α-cyclodextrin and mostly determined by the rate constant of complex formation.     

Electrochemical investigation of Pb2+ binding and transport through a polymerized crystalline colloidal array hydrogel containing benzo-18-crown-6

The transport of Pb2+ through a sensory gel, a polymerized crystalline colloidal array hydrogel with immobilized benzo-18-crown-6, is important for understanding and optimizing the sensor. Square wave voltammetry at a Hg/Au electrode reveals many parameters. The partition coefficient for Pb2+ into a control gel (no crown ether), K(p), is 1.00 +/- 0.018 (errors reported are SEM). The porosity, epsilon, of the gel is 0.90 +/- 0.01. Log K(c) for complexation in the gel is 2.75 +/- 0.014. Log K(c) in aqueous solution for Pb2+ with the ligand 4-acryloylamidobenzo-18-crown-6 is 3.01 +/- 0.010 with dissociation rate k(d) = (8.34 +/- 0.45) x 10(2) s(-1) and association rate k(f) = (8.79 +/- 0.025) x 10(7) M(-1) s(-1). The partition coefficient of the ligand 4-acryloylamidobenzo-18-crown-6 into the control gel, K(p,L) is 2.07 +/- 0.15. The diffusion coefficient of Pb2+ in the control gel is 6.72 x 10(-6) +/- 0.12 cm(2)/s. For the sensor gel, but not control gel, diffusion coefficients are location dependent. The range of diffusion coefficients for Pb2+ in the probed locations was found to be (6.11-12.60) x 10(-7) cm(2)/s for 0.91 mM Pb2+ and (2.84-9.39) x 10(-7) cm(2)/s for 0.35 mM Pb2+. Lead binding in the sensor gel is slightly less avid than in solution. This is attributed, in part, to the demonstrated affinity of the ligand 4-acryloylamidobenzo-18-crown-6 to the gel. Diffusion coefficients determined for the sensor gel were found to be location dependent. This is attributed to heterogeneities in the crown concentration in the gel. Analysis of diffusion coefficients and rate constants show that diffusion and not chemical relaxation will limit the time response of the material.     

Binding kinetics of biomolecule interaction at ultralow concentrations based on gold nanoparticle enhancement

Measuring the kinetic constants of protein-protein interactions at ultralow concentrations becomes critical in characterizing biospecific affinity, and exploring the feasibility of clinical diagnosis with respect to detection sensitivity, efficiency and accuracy. In this study, we propose a method that can calculate the binding constants of protein-protein interactions in sandwich assays at ultralow concentrations at the pg/mL level, using a localized surface plasmon coupled fluorescence fiber-optic biosensor (LSPCF-FOB). We discuss a two-compartment model to achieve reaction-limited kinetics under the stagnant conditions of the reaction chamber. The association rate constant, dissociation rate constant, and the equilibrium dissociation constant, that is, k(a), k(d), K(D), respectively, of the kinetics of binding between total prostate-specific antigen (t-PSA) and anti-t-PSA at concentrations from 0.1 pg/mL to 1 ng/mL, were measured either in PBS or in human serum. This is the first time that k(a), k(d), and K(D) have been measured at such a low concentration range in a complex sample such as human serum.     

氧化锌纳米线尿酸氧化酶固定性能

本文利用石英晶体微天平研究了氧化锌纳米线对尿酸氧化酶的固定性能和作为尿酸传感器的应用.本文在PBS溶液中对尿酸氧化酶进行固定,并且利用固定的传感器作为尿酸传感器,能检测浓度范围从5.0 × 10-6到8×10-5 molL-1的尿酸含量,所有结果都说明氧化锌纳米线是一种很好的生物传感器的材料.     

Determination of drug-plasma protein binding kinetics and equilibria by chromatographic profiling: exemplification of the method using L-tryptophan and albumin.

Drug-plasma protein binding may greatly influence the bioavailability and metabolism of a plasma-borne drug, the bound form being partially protected from the metabolic fate of the unbound drug. Traditionally, equilibrium values (e.g., percentage binding) for drug-protein binding have been measured to rationalize in vivo phenomena. However, such studies overlook the influence of kinetics. A rapid method of simultaneously determining kinetic rate constants and equilibrium constants from chromatographic profiles has been developed, based on the use of immobilized protein columns and HPLC. By measuring the chromatographic profiles (the position and width) of a retained and an unretained compound one can directly determine both the rate and equilibrium constants. Results are presented for the binding of L-tryptophan to human serum albumin to exemplify the method. The association equilibrium constant (Ka) and the association and dissociation rate constants (k(a) and k(d), respectively) were thereby measured in an aqueous pH 7.4 environment at 37 degrees C as 0.84 10(4) M(-1), 5.8 10(4) M(-1) s(-1), and 6.9 s(-1), respectively. These compare favorably with previously published results. The described method may be used in quantitative structure-property relationship-based rational drug discovery or for the rationalization of drug pharmacokinetics.     

Biosensors based on membrane-bound enzymes immobilized in a 5-(octyldithio)-2-nitrobenzoic acid layer on gold electrodes

Gold electrodes were modified through chemisorption of 5-(octyldithio)-2-nitrobenzoic acid (ODTNB). ODTNB includes a long chain in a short-length thio acid, providing a heterogeneous-like alkanethiol layer after adsorption on gold electrodes. Membrane-bound enzymes, in particular D-fructose dehydrogenase (FDH), D-gluconate dehydrogenase (GADH), and L-lactic dehydrogenase (cytochrome b2) (Cyb2), were immobilized onto ODTNB-modified gold electrodes simply by adsorption. The short-length thio acid may provide electrostatic interactions with enzyme surface charges, while the alkanethiolate enables hydrophobic interaction with the largely lipophilic, membrane-bound enzymes. The immobilization of FDH, GADH, and Cyb2 onto ODTNB-modified gold surfaces has been studied with the quartz crystal microbalance (QCM). Spectrophotometric and electrochemical assays indicate that the immobilized enzyme retains its enzymatic activity after immobilization onto the ODTNB-modified gold surface. The amount of immobilized (and active) enzyme was estimated from QCM to be of the order of 2.5 x 10(-12)-5.3 x 10(-12) mol x cm(-2). A fructose biosensor was developed, making use of a gold surface modified with ODTNB and fructose dehydrogenase, employing hydroxymethylferrocene as a mediator in solution. Calibration curves exhibited a linear relation between the biosensor response and the substrate concentration up to 0.7 mM. Statistical analysis gave an excellent linear correlation (r = 0.9993) and a sensitivity of 6.1 mM(-1) fructose. The biosensor shows a significant stable catalytic current for at least 25 days.     

Understanding Ligand Interaction with Different Structures of G-Quadruplex DNA: Evidence of Kinetically Controlled Ligand Binding and Binding-Mode Assisted Quadruplex Structure Alteration

The study of ligand interaction with G-quadruplex DNA is an active research area, because many ligands are shown to bind G-quadruplex structures, showing anticancer effects. Here, we show, for the first time, how fluorescence correlation spectroscopy (FCS) can be used to study binding kinetics of ligands with G-quadruplex DNA at the single molecule level. As an example, we study interaction of a benzo-phenoxazine ligand (Cresyl Violet, CV) with antiparallel and (3 + 1) hybrid G-quadruplex structures formed by human telomeric sequence. By using simple modifications in FCS setup, we describe how one can extract the reaction kinetics from diffusion-coupled correlation curves. It is found that the ligand (CV) binds stronger, by an order of magnitude, to a (3 + 1) hybrid structure, compared to an antiparallel one. Ensemble-averaged time-resolved fluorescence experiments are also carried out to obtain the binding equilibrium constants (K) of ligand-quadruplex interactions in bulk solution for the first time, which are found to match very well with FCS results. Global analysis of FCS data provides association (k(+)) and dissociation (k(-)) rates of the ligand in the two structures. Results indicate that stronger ligand binding to the (3 + 1) hybrid structure is controlled by the dissociation rate, rather than the association rate of ligand in the quadruplexes. Circular dichroism (CD) and induced-CD spectra show that the ligand not only binds at different conformations in the quadruplexes, but also induces antiparallel structure to form a mixed-type hybrid structure in Na(+) solution. However, in K(+) solution, the ligand stabilizes the (3 + 1) hybrid structure. Molecular docking studies predict the possible differences in binding sites of the ligand inside two quadruplexes, which strongly support the experimental observations. Results suggest that different binding modes of the ligand to the quadruplex structures actually assist the alteration of structures differently.     

An aptamer-based quartz crystal protein biosensor

We developed a quartz crystal biosensor designed to detect concentrations and ligand affinity parameters of free unlabeled proteins in real time. Using a model system with human IgE as the analyte and single-stranded DNA aptamers or an anti-IgE antibody as immobilized ligands, we could demonstrate that aptamers were equivalent to antibodies in terms of specificity and sensitivity. Both receptor types selectively detected 0.5 nmol/L of IgE. In addition, the aptamer receptors tolerated repeated affine layer regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity. Because of the small size and nonprotein nature of the aptamers, they were immobilized in a dense, well-oriented manner, thus extending the linear detection range to 10-fold higher concentrations of IgE. In addition to demonstrating for the first time that an aptamer-based biosensor can specifically and quantitatively detect an analyte in various complex protein mixes, the aptamer-ligand proved to be relatively heat resistant and stable over several weeks. Since aptamers consist of nucleic acids, well-established chemistry can be applied to produce optimized affine layers on biosensors that may be developed to specifically detect proteins in solution for analysis of proteomes.     

Selection of smart aptamers by methods of kinetic capillary electrophoresis

We coin the term "smart aptamers" -- aptamers with predefined binding parameters (k(on), k(off), Kd) of aptamer-target interaction. Aptamers, in general, are oligonucleotides, which are capable of binding target molecules with high affinity and selectivity. They are considered as potential therapeutic targets and also thought to rival antibodies in immunoassay-like analyses. Aptamers are selected from combinatorial libraries of oligonucleotides by affinity methods. Until now, technological limitations have precluded the development of smart aptamers. Here, we report on two kinetic capillary electrophoresis techniques applicable to the selection of smart aptamers. Equilibrium capillary electrophoresis of equilibrium mixtures was used to develop aptamers with predefined equilibrium dissociation constants (Kd), while nonequilibrium capillary electrophoresis of equilibrium mixtures facilitated selection of aptamers with different dissociation rate constants (k(off)). Selections were made for MutS protein, for which aptamers have never been previously developed. Both theoretical and practical aspects of smart aptamer development are presented, and the advantages of this new type of affinity probes are described.     

Optical resonance-enhanced absorption-based near-field immunochip biosensor for allergen detection

An optical immunochip biosensor has been developed as a rapid method for allergen detection in complex food matrixes, and its application evaluated for the detection of the egg white allergens, ovalbumin and ovomucoid. The optical near-field phenomenon underlying the basic principle of the sensor design is called resonance-enhanced absorption (REA), which utilizes gold nanoparticles (Au NPs) as signal transducers in a highly sensitive interferometric setup. Using this approach, a novel, simple, and rapid colorimetric solid-phase immunoassay on a planar chip substrate was realized in direct and sandwich assay formats, with a detection system that does not require any instrumentation for readout. Semiquantitative immunochemical responses are directly visible to the naked eye of the analyst. The biosensor shows concentration-dependent color development by capturing antibody-functionalized Au NPs on allergen-coated chips and has a detection limit of 1 ng/mL. To establish a rapid method, we took advantage of the physicochemical microenvironment of the Au NP-antibody bioconjugate to be bound directly over an interacting poly(styrene-methyl methacrylate) interlayer by an immobilized antigen. In the direct assay format, a coating time with allergen of only 5 min under "soft" nondenaturing conditions was sufficient for accurate reproducibility and sensitivity. In conclusion, the REA-based immunochip sensor is easy to fabricate, is reproducible and selective in its performance, has minimal technical requirements, and will enable high-throughput screening of affinity binding interactions in technological and medical applications.