Optimal self-referenced sensing using long- and short- range surface plasmons.

Dual-mode surface-plasmon resonance (SPR) sensors use both long- and short- range surface plasmon waves to differentiate surface binding interactions from interfering bulk effects. We have optimized the design of these sensors for minimum surface limit of detection (LOD) using a Cramer-Rao lower bound for spectral shift estimation. Despite trade-offs between resonance width, minimum reflectivity, and sensitivity for the two modes, a range of reasonable design parameters provides nearly optimal performance. Experimental verification using biotin-streptavidin binding as a model system reveals that sensitivity and LOD for dual-mode sensors remains competitive with single-mode sensors while compensating for bulk effects.

Monitoring of bacteria growth using a wireless, remote query resonant-circuit sensor: application to environmental sensing.

A new technique is presented for in-vivo remote query measurement of the complex permittivity spectra of a biological culture solution. A sensor comprised of a printed inductor-capacitor resonant-circuit is placed within the culture solution of interest, with the impedance spectrum of the sensor measured using a remotely located loop antenna; the complex permittivity spectra of the culture is calculated from the measured impedance spectrum. The remote query nature of the sensor platform enables, for example, the in-vivo real-time monitoring of bacteria or yeast growth from within sealed opaque containers. The wireless monitoring technique does not require a specific alignment between sensor and antenna. Results are presented for studies conducted on laboratory strains of Bacillus subtilis, Escherichia coli JM109, Pseudomonas putida and Saccharomyces cerevisiae.

Sensitive and selective liquid chromatographic postcolumn reaction detection system for biotin and biocytin using a homogeneous fluorophore-linked assay.

A homogeneous fluorophore-linked assay was used to develop a postcolumn reaction detection system for high-performance liquid chromatography (HPLC). Biotin and biocytin were chosen as the model analytes. The effluent from the HPLC column was merged with a reagent stream containing avidin that was labeled with fluorescein isothiocyanate (avidin-FITC). The binding of the separated analytes by the labeled avidin was accompanied by an enhancement of the fluorescence intensity at 520 nm. This increase in fluorescence was proportional to the concentration of the analytes and constituted the analytical signal. The procedure was optimized with respect to the reagent concentration and the flow-rate of the reagent solution. Analytical characteristics of the method were determined. The procedure was highly selective for biotin and its derivatives. The detection limits for biotin and biocytin were 89 and 94 pg, respectively, for 20-microliters injections. The developed postcolumn reaction detection system was validated by determining biotin in a liquid vitamin preparation and a horse-feed supplement.

Activity studies of immobilized subtilisin on functionalized pure cellulose-based membranes.

The activity of immobilized subtilisin BPN' on pure cellulose-based membrane support was investigated using site-directed and random immobilization approaches. The catalytic activity of site-directed immobilized subtilisin on pure cellulose fiber-based materials was found to be 81% of that in homogeneous solution, while that of randomly immobilized subtilisin was 27%. Pure cellulose membrane supports provided large surface areas for high enzyme loading without diffusional limitations. The activity of immobilized subtilisin on pure cellulose support was more than twice that on a modified polyether sulfone (MPS) membrane, which was attributed to the higher hydrophilicity of cellulose. Immobilized subtilisin maintained its initial activity for 14 days at 4 degrees C and 7 days at 24 degrees C. The immobilized enzyme could resist higher temperature and operate over a wider range of pH without loss of activity. This study showed that pure cellulose fiber-based membranes are well suited for enzyme immobilization and biocatalysis.

Fiber optic chemical sensor for nitrite based on an electropolymerized cobaltporphyrin film.

Fiber optic sensors for nitrite were prepared by first electrochemically depositing a film of cobalt(II) tetrakis(o-aminophenyl)porphyrin, [Co(o-NH(2))TPP], on the surface of indium(tin) oxide glass slides. Then, the slides with the immobilized porphyrin were positioned at the tip of an optical fiber bundle. The response of the sensors was based on a change in absorbance caused by the interaction between nitrite and the poly[Co(o-NH(2))TPP] film. The sensors had a detection limit of 6 x 10(-9)M nitrite. The selectivity of the sensors was determined under both separate solutions and fixed interference conditions. The sensor had a long lifetime and was reversible.

Enhancement of the emission intensity of fluorophore-labeled avidin by biotin and biotin derivatives. Evaluation of different fluorophores for improved sensitivity.

Fluorescein, Texas Red, Cascade Blue, 7-amino-methylcoumarin-3-acetic acid, and Lucifer Yellow were evaluated as fluorescent labels for homogeneous fluorophore-linked binding assays. Conjugates of avidin with these fluorophores exhibited an enhancement in fluorescence emission in the presence of biotin or biotin derivatives. This property was used in the development of assays for biotin. The biotin-induced fluorescence enhancement of each labeled avidin were compared. Fluorescein led to the most sensitive calibration (dose-response) curve for biotin with a detection limit of 8 x 10(-10)M.

Isoelectric focusing electrophoresis of protein-ligand conjugates: effect of the degree of substitution.

The degree of substitution of protein-ligand conjugates can be determined from the change of the isoelectric point (pI) of the protein when ligand molecules are attached to its surface. Specifically, the pI values of conjugates with known degrees of substitution are obtained by isoelectric focusing electrophoresis and are used to generate a calibration curve that relates these two variables. The shape of the curve is sigmoidal and can be predicted by a theoretical model that takes into account the degree of substitution and the amino acid composition of the protein. By using such a calibration curve, one may estimate the degree of substitution of a given protein-ligand conjugate from its pI value. The applicability of the method is demonstrated with conjugates of pyridoxal 5-phosphate and avidin.

Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves.

Models for predicting the effect of immunological recognition of the bridge group on the dose-response curves obtained with heterogeneous hapten enzyme immunoassays are presented. Appropriate theoretical treatment shows that the greater affinity of antibodies toward the enzyme-labeled species than for the unlabeled hapten analyte results in assays with limited detection capabilities. This problem is compounded when enzyme conjugates possessing multiple haptens are used. In equilibrium type competitive arrangements, the concentrations of binder and labeled hapten may be optimized to some extent to improve assay performance. However, the results presented show that only when assays are performed in a sequential binding mode using carefully controlled timing of reagent incubations can the detection capabilities of the assays be fully maximized for analyte measurements. Unfortunately, it is also shown that such sequential binding approaches render the assays essentially nonselective. The effect of decreasing the affinity of the binder to the enzyme-labeled hapten relative to the unlabeled analyte by using heterologous conjugates in equilibrium arrangements is shown to improve detection capabilities but also at the expense of reduced selectivity. Suggestions for reagent concentrations and conjugate substitution (degree of conjugation), which provide optimized dose-response curves at a given ED50 value, are also presented as are proposals for using different binders which do not exhibit bridging group recognition.

Homogeneous enzyme immunoassay for lipoic acid based on the pyruvate dehydrogenase complex: a model for an assay using a conjugate with one ligand per subunit.

A homogeneous enzyme immunoassay for lipoic acid was developed by using an enzyme-ligand conjugate containing only one ligand per enzyme subunit. Theoretical studies have shown that the traditional use of multisubstituted enzyme-ligand conjugates has limited the detection limits and sensitivity obtainable with these assays. The use of conjugates with a smaller number of ligands should allow for improved assays. The pyruvate dehydrogenase complex was chosen for this study because each polypeptide chain of dihydrolipoyl transacetylase contains one lipoic acid as a covalently attached prosthetic group. Thus, the naturally occurring enzyme can be considered as an enzyme-lipoic acid conjugate. Anti-lipoic acid antibodies were developed in New Zealand White rabbits to be used as the analyte-specific binders. Association and binder dilution curves were prepared in order to optimize the reagent concentrations and the analytical conditions. Unexpected inhibition by free lipoic acid resulted in a biphasic dose-response curve with a detection limit of 5 x 10(-6) M lipoic acid. This technique has several advantages over previous electrochemical and chromatographic techniques for lipoic acid determination.

Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues.

A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 x 10(-8) M for biotin, 1 x 10(-7) M for biocytin, and 2 x 10(-6) M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.

Potentiometric homogeneous enzyme-linked competitive binding assays using adenosine deaminase as the label.

Homogeneous enzyme-linked competitive binding assays for biotin are described that are based on the competition between an enzyme-biotin conjugate and free biotin for a fixed number of binding sites of avidin. Unlike conventional homogeneous enzyme immunoassays, in this system the analyte (biotin) is labeled with adenosine deaminase (ADA), an ammonia-producing enzyme. Consequently, potentiometric rather than photometric methods can be used as means of detection. Several ADA-biotin conjugates were prepared and showed as high as 97% inhibition of the enzymatic activity in the presence of avidin. Addition of free biotin reverses this inhibition in an amount proportional to the concentration of analyte. Relatively steep dose-response curves were observed, leading to a precise and accurate assay for biotin. The detection limits of these curves were as low as 1 x 10(-8) M. Varying the concentration of the reagents in the assay allowed the detection limit and working range to be altered to a desired value. The proposed method was applied in the determination of biotin in a horse-feed supplement.

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.

Defined analyte-enzyme conjugates as signal generators in immunoassays.

We investigated the synthesis of progesterone-horseradish peroxidase (P-HRP) conjugates and products purified by affinity chromatography. The obtained preparations were characterized with an immobilized monoclonal antibody in solid-phase immunoassays. Three homogeneous P-HRP conjugates were isolated. Two preparations were identified to contain a single progesterone ligand on the enzyme molecule. A third preparation contained two progesterone ligands. We postulate that conjugation can occur at two different positions on the enzyme, and that the different microenvironment of the protein structure surrounding the ligand contributes to different binding constants of the conjugates with immunoglobulin. By comparing the effective binding constants derived from affinity chromatography and from Scatchard analysis, we have demonstrated that the divalent conjugate binds to antibody immobilized on planar surfaces only by a single attachment due to steric restriction. Dose-response curves for progesterone using the isolated P-HRP conjugates have been investigated and compared.

Improving the activity of immobilized subtilisin by site-directed attachment through a genetically engineered affinity tag.

An octapeptide affinity tag, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (temied FLAG), was genetically fused to the C-terminus of subtilisin BPN' (SBT) from Bacillus amyloliquefaciens. The fusion protein SBT-FLAG was immobilized to nonporous polystyrene and silica beads both in a site-directed and a random fashion. Site-directed immobilization was achieved by employing the interaction between protein A and a monoclonal antibody specific for the FLAG peptide, while random immobilization was obtained by using glutaraldehyde as a cross-linking reagent. The activity of the immobilized enzymes was compared. It was found that the site-directed subtilisin had higher catalytic efficiency, kcat/KM, which was more than 7-fold of that of the randomly immobilized enzyme. It was also noted that the site-directly immobilized enzyme had superior storage stability over the homogeneous enzyme.

Determination of the extent of protein biotinylation by fluorescence binding assay.

A method was developed to determine the total amount of biotin present in biotinylated protein conjugates. Conjugates of bovine serum albumin, alkaline phosphatase, and horseradish peroxidase were used in this case study. The extent of biotinylation was determined by complete acid hydrolysis or by enzymatic digestion using proteinase K to release biotin from the biotinylated proteins, followed by sensitive detection of biotin using a coupled HPLC-binding assay system. This detection system is based on the enhancement of the fluorescence of streptavidin-FITC by biotin. The extent of biotinylation determined by this method was compared with the values obtained by a conventional colorimetric method that is based on the displacement of the dye 4-hydroxyazobenzene-2-carboxylic acid (HABA) from the binding sites of avidin. It was found that, because the described method determines the amount of liberated biotin after hydrolysis, it does not suffer from steric hindrance problems associated with the ability of biotin on a protein surface to displace HABA from avidin. Therefore, this method can provide a more accurate determination of the extent of biotinylation. It was also determined that the acid hydrolysis of the biotinylated protein was more effective in releasing the conjugated biotin compared to enzymatic digestion by proteinase K.

Pyruvate carboxylase as a model for oligosubstituted enzyme-ligand conjugates in homogeneous enzyme immunoassays.

Theoretical models suggest that the detection capabilities of homogeneous enzyme immunoassays can be improved by the use of oligosubstituted enzyme-ligand conjugates rather than the traditionally used multisubstituted ones. The natural form of pyruvate carboxylase contains four covalently bound biotins (one per subunit) and it can be considered as an oligosubstituted enzyme-biotin conjugate. The enzyme is nearly completely inhibited in the presence of the natural binder for biotin, avidin. When the enzyme is incubated with avidin and free biotin, a competition occurs between the free biotin and the prosthetic group of the enzyme for the avidin. Steep dose-response curves are obtained by relating the observed inhibition to the free biotin concentration. By variation of the amount of avidin or enzyme in the assay, the detection limits of the system can be altered allowing for sensitive determinations over a wide range of biotin concentrations. Such data from real sample analysis of several vitamin supplements are reported.

Improving the activity of immobilized subtilisin by site-specific attachment to surfaces.

Understanding the properties of immobilized proteins is critical to the optimal design of biosensors, bioseparations, and bioreactors. The protease subtilisin BPN' was used as a model protein to study how the orientation of immobilized enzyme molecules on surfaces affects their catalytic properties. To achieve this goal, a single cysteine residue was introduced into the cysteine-free enzyme by site-directed mutagenesis. This cysteine residue was designed to be away from the active site of the enzyme. The enzyme molecules were immobilized through the side-chain sulfhydryl group of the cysteine residue on several supports. This site-specific immobilization method leads to ordered two-dimensional arrays of enzyme molecules on the support surface with the active sites of the enzyme oriented toward the solution phase. Such oriented immobilized subtilisin demonstrated a higher catalytic efficiency compared to subtilisin that was immobilized by a conventional method that leads to random immobilization.

Orientation specific immobilization of organophosphorus hydrolase on magnetic particles through gene fusion.

Recombinant DNA technology has been utilized to fuse an octapeptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG), to the C-terminus of organophosphorus hydrolase (OPH, EC 3.1.8.1), an enzyme capable of hydrolyzing organophosphate compounds, such as insecticides and nerve gas agents. The recombinant OPH-FLAG was immobilized onto magnetic beads coated with protein A in the following ways: (a) site-directly through a monoclonal antibody (MAb) specific for the FLAG peptide; (b) through the MAb that was randomly tethered to the beads using glutaraldehyde; (c) randomly by cross-linking OPH-FLAG to protein-coated beads using glutaraldehyde. Kinetic studies demonstrated that the site-directly immobilized enzyme maintained the highest catalytic efficiency. The orientation specific immobilization strategy described in this article can be applied to other proteins, and therefore, it may find potential applications in the design of biosensors, biocatalysts, and bioreactors having immobilized proteins as their biorecognition elements.

High-performance liquid chromatographic postcolumn reaction detection based on a competitive binding system.

Postcolumn reactions are typically employed to improve detection in high-performance liquid chromatography (HPLC) separation techniques. This study proposes the use of competitive binding principles in designing novel postcolumn reaction schemes. The feasibility of this approach was tested by using the HPLC determination of biotin and biocytin as a model system. The effluent from the HPLC column was merged with a reagent stream containing avidin, whose binding sites were occupied by the dye HABA (2-[4'-hydroxyphenylazo]benzoic acid). HABA was displaced by the analytes from the avidin-HABA complex and the free dye was determined with a UV-vis detector at 345 nm. The procedure was optimized with respect to reactor design, reagent concentrations, and the flow rate of reagent solution. Analytical characteristics of the developed procedure were determined and compared with the direct detection of biotin and biocytin at 220 nm. The postcolumn reaction scheme improved the selectivity and sensitivity of the detection of biotin and biocytin while maintaining similar detection limits.