A comparative study of protein immobilization techniques for optical immunosensors.

This paper presents the results of a study of a number of antibody immobilization techniques for application to optical immunosensors. In particular, well-known methods such as covalent binding and physical adsorption have been extended to the Langmiur-Blodgett method in an attempt to improve the density and possibly the uniformity of orientation of monoclonal antibodies on an optical surface. The surface density of active immobilized antibodies was determined from enzyme immunoassay and their thickness and refractive index were deduced from ellipsometry. It is shown that, although high surface densities (500 ng/cm2) of antibody can be obtained, the major obstacle to the detection of low concentrations of antigens or haptens is the non-specific binding of foreign molecules to the sensing surface.     

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein–ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol⁻¹) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381–396, 1998. © 1998 Wiley-Liss, Inc.     

Highly sensitive optical chip immunoassays in human serum

Over the past decade the ability of refractometric optical sensors to quantitatively measure a wide range of biomolecules has been demonstrated. These include proteins, nucleic acids, microorganisms, and in competitive formats small molecules such as drugs and pesticides. Furthermore, by using high refractive index nanoparticles to amplify the biomolecular binding signal, sensitivities approaching those of well established diagnostic assays have been achieved. However, to date it has not been possible to show rapid detection of analytes in complex bodily fluids such as serum, in a one-step procedure, due to the interference resulting from non-specific binding (NSB) to the sensor surface. We have carried out preliminary work on the control of interference due to NSB using an optical chip based on the Hartman interferometer. This interferometer configuration employs a reference sensing region that can be functionalized separately from the specific sensing region. Optical chips were stored dry after surface functionalization, and rehydrated in serum. The observed level of background drift in serum was reduced by an order of magnitude when an exposed reference was used, compared to a reference which was blind to the sample. An additional 70% reduction in signal drift in serum was achieved by controlling the surface chemistry of the optical chip using a biotin-poly(ethylene glycol) (PEG) blocking agent. This functionalization procedure was combined with a sandwich assay using gold nanoparticles to develop a one-step assay for human chorionic gonadotropin (hCG) in human serum with a detection limit of 0.1 ng/ml for a 35 min assay.     

Optical chip immunoassay for hCG in human whole blood

We report on the development of an integrated optic chip sensor for performing rapid and sensitive immunoassays with human whole blood using human chorionic gonadotropin (hCG) as the model system. The optical chip is based on the Hartman interferometer, which uses a single planar lightbeam to address multiple interferometers, each comprising a signal/reference pair of sensing regions. The binding of antigen to specific capture antibodies on the signal sensing region causes a change in the refractive index of the surface layer, which is detectable by its effect on the evanescent field of the guided lightbeam. The reference-sensing region is coated with an irrelevant antibody, which optically cancels a large fraction of the non-specific adsorption that occurs on the specific-sensing region when the sensor is tested with clinical specimens. This work extends previous experiments with buffer and human serum to measurements in undiluted whole human blood. Optical chips were stored dry after surface functionalization, and rehydrated with blood. Colloidal gold nanoparticles conjugated to a second anti-hCG monoclonal antibody were used to provide signal amplification, thereby enhancing assay sensitivity, in a one-step procedure with the gold conjugate added to the test sample immediately prior to measurement. Background signals due to non-specific binding (NSB) in blood were found to be higher than those previously reported with human serum. In addition, a high level of background signal was found with the gold conjugate, which had not been observed in experiments with either buffer or serum. Nevertheless, hCG could be detected at 0.5 ng/ml within 10 min of sample application. The sensor response was linear over the concentration range 0.5-5 ng/ml hCG, as compared with the clinically-relevant range 0.3-1.5 ng/ml. Detection at higher concentrations was affected by scattering from large amounts of bound gold nanoparticles. However, initial binding rate measurements could be used to maintain assay quantitation.     

Determination of the refractive index increments of small molecules for correction of surface plasmon resonance data

The refractive index increments (RIIs) of several important low-molecular-weight compounds that bind to DNA or RNA were determined with a differential refractometer for correction of data obtained on surface plasmon resonance (SPR) biosensors. Although the ability to investigate small molecule-macromolecule interactions by SPR is relatively new, the technique is rapidly becoming a primary method to screen focused combinatorial libraries and to quantitatively characterize the interactions between compounds identified as binders and target macromolecules. The most widely used SPR analysis software, BIAevaluation (Biacore, Inc.), assumes that the RIIs of ligand and macromolecule are identical. While the assumption is reasonable for studies involving like molecules such as protein-protein interactions, results presented here demonstrate that RII values for small molecules can be significantly different than those of protein or nucleic acid receptors. The results also show that the RII values can vary greatly depending on the structure of the small molecule. Indeed, the RIIs of the molecules investigated here differ by a factor of 2. Any difference in the RII of interacting molecules must be considered for complete analysis of SPR data. Failure to correct for RII differences can result in serious error in data interpretation, especially for systems involving a ligand:receptor stoichiometry greater than 1. The results serve as the beginning of an SPR correction database for the RIIs of small molecules. Additionally, the results can be used to approximate the RIIs of a variety of other small molecules.     

Manufacturing DNA microarrays of high spot homogeneity and reduced background signal

Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-L-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N:-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.     

Structural parameterization of the binding enthalpy of small ligands

A major goal in ligand and drug design is the optimization of the binding affinity of selected lead molecules. However, the binding affinity is defined by the free energy of binding, which, in turn, is determined by the enthalpy and entropy changes. Because the binding enthalpy is the term that predominantly reflects the strength of the interactions of the ligand with its target relative to those with the solvent, it is desirable to develop ways of predicting enthalpy changes from structural considerations. The application of structure/enthalpy correlations derived from protein stability data has yielded inconsistent results when applied to small ligands of pharmaceutical interest (MW < 800). Here we present a first attempt at an empirical parameterization of the binding enthalpy for small ligands in terms of structural information. We find that at least three terms need to be considered: (1) the intrinsic enthalpy change that reflects the nature of the interactions between ligand, target, and solvent; (2) the enthalpy associated with any possible conformational change in the protein or ligand upon binding; and, (3) the enthalpy associated with protonation/deprotonation events, if present. As in the case of protein stability, the intrinsic binding enthalpy scales with changes in solvent accessible surface areas. However, an accurate estimation of the intrinsic binding enthalpy requires explicit consideration of long-lived water molecules at the binding interface. The best statistical structure/enthalpy correlation is obtained when buried water molecules within 5-7 A of the ligand are included in the calculations. For all seven protein systems considered (HIV-1 protease, dihydrodipicolinate reductase, Rnase T1, streptavidin, pp60c-Src SH2 domain, Hsp90 molecular chaperone, and bovine beta-trypsin) the binding enthalpy of 25 small molecular weight peptide and nonpeptide ligands can be accounted for with a standard error of +/-0.3 kcal x mol(-1).     

An improved coating for the isolation and quantitation of interferon-gamma in spiked plasma using surface plasmon resonance (SPR)

A study was initiated to investigate the use of surface plasmon resonance (SPR) for the detection in plasma of a high pI model protein, recombinant human interferon-gamma (IFN-gamma). Initially a number of self-assembled monolayers (SAMs) and hydrogel-derivatised SAM-coatings were characterised for the adsorptive and desorptive properties of plasma components. Next a monoclonal anti-IFN-gamma antibody, MD-2, was covalently attached to dextran-modified mercaptoundecanoic acid surfaces that performed best. On coatings consisting of carboxyl-modified dextran (CMD) a difference in interaction behaviour was observed when IFN-gamma was injected in either buffer or diluted plasma. During the injection of IFN-gamma in buffer, an acceleration of the interaction process was observed and the signal continued to increase after the injection plug had passed. Upon injection of diluted plasma spiked with IFN-gamma, the response increased without acceleration of the binding process. After the injection was finished, some of the bound material desorbed as expected, resulting in a signal decrease. On non-charged dextrans, the interaction between the antibody-modified surface and IFN-gamma in either plasma or buffer was similar. During sample injection the response increased with a binding rate depending on the concentration of IFN-gamma present in solution. When the injection was finished, some of the bound material was washed away from the surface and only a minor contribution of non-specific adsorbed plasma components was noticeable. From the coatings tested, the non-modified dextran-coated SPR sensor disks prove to be best suited for the detection of IFN-gamma in complex matrices like plasma. The interaction of IFN-gamma in both diluted plasma and buffer is comparable and concentrations of IFN-gamma of 250 ng ml-1 and higher can be detected in both buffer and 100x-diluted plasma. The non-specific adsorption of plasma components is low, whereas the specific IFN-gamma response is relatively high.     

Detection of Staphylococcus aureus enterotoxin B at femtomolar levels with a miniature integrated two-channel surface plasmon resonance (SPR) sensor

Surface plasmon resonance (SPR) biosensors offer the capability for continuous real-time monitoring. The commercial instruments available have been large in size, expensive, and not amenable to field applications. We report here an SPR sensor system based on a prototype two-channel system similar to the single channel Spreeta devices. This system is an ideal candidate for field use. The two-channel design provides a reference channel to compensate for bulk refractive index (RI), non-specific binding and temperature variations. The SPR software includes a calibration function that normalizes the response from both channels, thus enabling accurate referencing. In addition, a temperature-controlled enclosure utilizing a thermo-electric module based on the Peltier effect provides the temperature stability necessary for accurate measurements of RI. The complete SPR sensor system can be powered by a 12V battery. Pre-functionalized, disposable, gold-coated thin glass slides provide easily renewable sensor elements for the system. Staphylococcus aureus enterotoxin B (SEB), a small protein toxin was directly detectable at sub-nanomolar levels and with amplification at femtomolar levels. A regeneration procedure for the sensor surface allowed for over 60 direct detection cycles in a 1-month period.     

The thermodynamics of protein-ligand interaction and solvation: insights for ligand design

Isothermal titration calorimetry is able to provide accurate information on the thermodynamic contributions of enthalpy and entropy changes to free energies of binding. The Structure/Calorimetry of Reported Protein Interactions Online database of published isothermal titration calorimetry studies and structural information on the interactions between proteins and small-molecule ligands is used here to reveal general thermodynamic properties of protein-ligand interactions and to investigate correlations with changes in solvation. The overwhelming majority of interactions are found to be enthalpically favoured. Synthetic inhibitors and biological ligands form two distinct subpopulations in the data, with the former having greater average affinity due to more favourable entropy changes on binding. The greatest correlation is found between the binding free energy and apolar surface burial upon complex formation. However, the free-energy contribution per unit area buried is only 30-50% of that expected from earlier studies of transfer free energies of small molecules. A simple probability-based estimator for the maximal affinity of a binding site in terms of its apolar surface area is proposed. Polar surface area burial also contributes substantially to affinity but is difficult to express in terms of unit area due to the small variation in the amount of polar surface buried and a tendency for cancellation of its enthalpic and entropic contributions. Conventionally, the contribution of apolar desolvation to affinity is attributed to gain of entropy due to solvent release. Although data presented here are supportive of this notion, because the correlation of entropy change with apolar surface burial is relatively weak, it cannot, on present evidence, be confidently considered to be correct. Further, thermodynamic changes arising from small differences between ligands binding to individual proteins are relatively large and, in general, uncorrelated with changes in solvation, suggesting that trends identified across widely differing proteins are of limited use in explaining or predicting the effects of ligand modifications.     

Computational solvent mapping reveals the importance of local conformational changes for broad substrate specificity in mammalian cytochromes P450

Computational solvent mapping moves small organic molecules as probes around a protein surface, finds favorable binding positions, clusters the conformations, and ranks the clusters on the basis of their average free energy. Prior mapping studies of enzymes, crystallized in either substrate-free or substrate-bound form, have shown that the largest number of solvent probe clusters invariably overlaps in the active site. We have applied this method to five cytochromes P450. As expected, the mapping of two bacterial P450s, P450 cam (CYP101) and P450 BM-3 (CYP102), identified the substrate-binding sites in both ligand-bound and ligand-free P450 structures. However, the mapping finds the active site only in the ligand-bound structures of the three mammalian P450s, 2C5, 2C9, and 2B4. Thus, despite the large cavities seen in the unbound structures of these enzymes, the features required for binding small molecules are formed only in the process of substrate binding. The ability of adjusting their binding sites to substrates that differ in size, shape, and polarity is likely to be responsible for the broad substrate specificity of these mammalian P450s. Similar behavior was seen at "hot spots" of protein-protein interfaces that can also bind small molecules in grooves created by induced fit. In addition, the binding of S-warfarin to P450 2C9 creates a high-affinity site for a second ligand, which may help to explain the prevalence of drug-drug interactions involving this and other mammalian P450s.     

Pushing the detection limits: the evanescent field in surface plasmon resonance and analyte-induced folding observation of long human telomeric repeats

Conventional analysis of molecular interactions by surface plasmon resonance is achieved by the observation of optical density changes due to analyte binding to the ligand on the surface. Low molecular weight interaction partners are normally not detected. However, if a macromolecule such as DNA can extend beyond the evanescent field and analyte interaction results in a large-scale contraction, then the refractive index changes due to the increasing amount of macromolecules close to the surface. In our proof-of-principle experiment we could observe the direct folding of long, human telomeric repeats induced by the small analyte potassium using surface plasmon resonance spectroscopy. This work demonstrates the feasibility of new evanescent field-based biosensors that can specifically observe small molecule interactions.     

Long-period gratings in photonic crystal fiber as an optofluidic label-free biosensor

Using long-period gratings (LPG) inscribed in photonic crystal fiber (PCF) and coupling this structure with an optically aligned flow cell, we have developed an optofluidic refractive index transduction platform for label-free biosensing. The LPG-PCF scheme possesses extremely high sensitivity to the change in refractive index induced by localized binding event in different solution media. A model immunoassay experiment was carried out inside the air channels of PCF by a series of surface modification steps in sequence that include adsorption of poly(allylamine hydrochloride) monolayer, immobilization of anti-rat bone sialoprotein monoclonal primary antibody, and binding interactions with non-specific goat anti-rabbit IgG (H+L) and specific secondary goat anti-mouse IgG (H+L) antibodies. These adsorption and binding events were monitored in situ using the LPG-PCF by measuring the shift of the core-to-cladding mode coupling resonance wavelength. Steady and significant resonance changes, about 0.75 nm per nanometer-thick adsorbed/bound bio-molecules, have been observed following the sequence of the surface events with monolayer sensitivity, suggesting the promising potential of LPG-PCF for biological sensing and evaluation.     

Quantitative characterization of binding of small molecules to extracellular matrix

Extracellular matrix (ECM) is a major tissue component that, besides its cell support function, is implicated in cell-cell signaling, wound repair, cell adhesion, and other cell and tissue functions. For small molecules acting in tissues, including chemicals, signaling peptides, effectors, inhibitors, and other man-made and physiological compounds, non-specific binding to ECM is a critical phenomenon affecting their disposition. We describe here a method for a quantitative characterization of the ECM binding, using a solidified ECM layer incubated with medium containing studied small molecules. Working conditions of Matrigel, a commercial basement membrane preparation, were optimized in terms of the protein concentration, surface area, gel layer thickness, solidification time, and mixing speed. The release of proteins from the solidified layer into the buffer was monitored and taken into account. Two major proteins, laminin and collagen IV, dissolve at different rates. The Matrigel stability data, obtained under varying incubation conditions and gentle mixing, can also be useful in other ECM-related research. The experimental binding data, averaged over all binding sites, were analyzed assuming a fast linear binding. The binding constants were determined for 10 small organic molecules for both dissolved proteins and the solidified layer. The binding constants tend to increase with lipophilicity of the compounds, as characterized by the 1-octanol/water partition coefficients.     

Integrated optical surface plasmon resonance immunoprobe for simazine detection.

This paper presents the detailed design and characterisation of a regenerable integrated optical surface plasmon resonance immunoprobe as a detector for the triazine herbicide simazine. A sensor design theoretically optimised for use in the aqueous environment is presented and its fabrication described. Experimental results on the sensitivity to changes in bulk refractive index of the analyte and on non-specific binding of ovalbumin are presented. Binding inhibition immunoassays were conducted for simazine and the lower limit of detection determined to be 0.16 microgram/l using anti-simazine IgG antibodies and 0.11 microgram/l using anti-simazine Fab fragments. A sample test cycle of 20 min was established.     

Liquid phase SPR imaging experiments for biosensors applications

Surface plasmon resonance (SPR) has recently gained attention as a label-free method for the detection of biological molecules binding onto functionalised surfaces. It is one of the most sensitive detection method for monitor variations in the thickness and refractive index in ultra-thin films. Here, the adsorption processes of oligonucleotides onto gold substrates have been investigated in aqueous buffer solution using SPR imaging measurements. The hybridization of a thiol-modified, single stranded oligonucleotide anchored to a gold surface via thiol group, with its complementary sequence has been observed and characterised monitoring the hybridization process by SPR equipment. In situ investigation of smallest changes in SPR imaging measurements dynamically performed in liquid phase in the presence of DNA complementary probes was performed. Infrared spectroscopy and scanning electron microscopy characterisation of the functionalised gold surfaces of the biosensor were compared with the images obtained by SPR experimental apparatus.     

Label-enhanced surface plasmon resonance applied to label-free interaction analysis of small molecules and fragments

Surface plasmon resonance (SPR) is a well-established method for studying interactions between small molecules and biomolecules. In particular, SPR is being increasingly applied within fragment-based drug discovery; however, within this application area, the limited sensitivity of SPR may constitute a problem. This problem can be circumvented by the use of label-enhanced SPR that shows a 100-fold higher sensitivity as compared with conventional SPR. Truly label-free interaction data for small molecules can be obtained by applying label-enhanced SPR in a surface competition assay format. The enhanced sensitivity is accompanied by an increased specificity and inertness toward disturbances (e.g., bulk refractive index disturbances). Label-enhanced SPR can be used for fragment screening in a competitive assay format; the competitive format has the added advantage of confirming the specificity of the molecular interaction. In addition, label-enhanced SPR extends the accessible kinetic regime of SPR to the analysis of very fast fragment binding kinetics. In this article, we demonstrate the working principles and benchmark the performance of label-enhanced SPR in a model system—the interaction between carbonic anhydrase II and a number of small-molecule sulfonamide-based inhibitors.     

AFM and electroanalytical studies of synthetic oligonucleotide hybridization

The first and most important step in the development and manufacture of a sensitive DNA-biosensor for hybridization detection is the immobilization procedure of the nucleic acid probe on the transducer surface, maintaining its mobility and conformational flexibility. MAC Mode AFM images were used to demonstrate that oligonucleotide (ODN) molecules adsorb spontaneously at the electrode surface. After adsorption, the ODN layers were formed by molecules with restricted mobility, as well as by superposed molecules, which can lead to reduced hybridization efficiency. The images also showed the existence of pores in the adsorbed ODN film that revealed large parts of the electrode surface, and enabled non-specific adsorption of other ODNs on the uncovered areas. Electrostatic immobilization onto a clean glassy carbon electrode surface was followed by hybridization with complementary sequences and by control experiments with non-complementary sequences, studied using differential pulse voltammetry. The data obtained showed that non-specific adsorption strongly influenced the results, which depended on the sequence of the ODNs. In order to reduce the contribution of non-specific adsorbed ODNs during hybridization experiments, the carbon electrode surface was modified. After modification, the AFM images showed an electrode completely covered by the ODN probe film, which prevented the undesirable binding of target ODN molecules to the electrode surface. The changes of interfacial capacitance that took place after hybridization or control experiments showed the formation of a mixed multilayer that strongly depended on the local environment of the immobilized ODN.     

Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets: optimization and data analysis

A real-time, label free assay was developed for microbial detection, utilizing double-stranded DNA targets and employing the next generation of an impedimetric sensor array platform designed by Sharp Laboratories of America (SLA). Real-time curves of the impedimetric signal response were obtained at fixed frequency and voltage for target binding to oligonucleotide probes attached to the sensor array surface. Kinetic parameters of these curves were analyzed by the integrated data analysis package for signal quantification. Non-specific binding presented a major challenge for assay development, and required assay optimization. For this, differences were maximized between binding curve kinetic parameters for probes binding to complementary targets versus non-target controls. Variables manipulated for assay optimization included target concentration, hybridization temperature, buffer concentration, and the use of surfactants. Our results showed that (i) different target-probe combinations required optimization of specific sets of variables; (ii) for each assay condition, the optimum range was relatively narrow, and had to be determined empirically; and (iii) outside of the optimum range, the assay could not distinguish between specific and non-specific binding. For each target-probe combination evaluated, conditions resulting in good separation between specific and non-specific binding signals were established, generating high confidence in the SLA impedimetric dsDNA assay results.