Hapten immobilization for antibody sensing using a dynamic modification protocol.

Optical fiber (OF) sensors are often limited by the immobilization technique used to associate a specific sensing ligand with the OF surface. This is particularly true when the ligand is biologically active as, for example, in the case of immobilized haptens or antibodies. The dynamic modification protocol is a regenerable and experimentally simple way to immobilize a variety of sensing molecules on an OF surface. Furthermore, the protocol is immune to hydrolysis and not limited by diffusion through a membrane or sol-gel. In this publication the approach is extended by immobilizing the hydrophobic hapten (octadecyl 6-(2,4 dinitrophenyl)aminohexanoic acid) as a means to prepare an OF sensor for antibodies specific for 2,4 dinitrophenyl (DNP). The LOD for anti-DNP is 0.5 nanomolar and the K(apparent) is 1.0+/-0.2x10(6). Nonspecific antibody adsorption is problematic in this sensing approach and was found to limit the quantitative capabilities of the sensor. However, time discrimination can be used to allow the nonspecific antibody to desorb prior to measurement thus minimizing the influence of nonspecific binding on sensor performance.

Electrochemically generated Ru(bpy)(3)(3+)-based chemiluminescence detection in micellar electrokinetic chromatography.

In situ generated Ru(bpy)(3)(3+)-based chemiluminescence (CL) detection will be shown to be compatible with micellar electrokinetic chromatography (MEKC) providing significant advantages over other Ru(3+) generation protocols. The CL reagent, Ru(bpy)(3)(2+) is continuously added post-capillary to avoid precipitating the anionic surfactant used to enhance the separation of neutral analytes. Ru(bpy)(3)(3+) is then electrochemically generated in situ at the interface between the separation capillary and the working electrode, where it can react with specific analytes, for example amines and amino acids to produce chemiluminescent emission. With this scheme, the critical micelle concentration is not exceeded in the detection zone, freeing the analyte to react with the Ru(bpy)(3)(3+) CL reagent. The separation and detection of various underivatized amines will be demonstrated using this methodology. For triethylamine, 70 000 plates per meter are demonstrated with MEKC providing a limit of detection (S/N=2) of 1.5 fmol of injected mass. The experimental approach used to improve the limit of detection while maintaining high separation efficiency will be evaluated and discussed.

Chemiluminescent detection of amines and amino acids using in situ generated Ru(bpy)(3)(3+) following separation by capillary electrophoresis.

A new sensitive chemiluminescent detection method for capillary electrophoresis is described. Underivatized amines and amino acids were detected following capillary electrophoresis separation by their chemiluminescent reaction with Ru(bpy)(3)(3+) generated in situ at 35 mum carbon fibers. Detection limits for triethylamine and proline were 5 and 3 fmol, respectively at a SNR of three. The noise limiting the detectability of separated analytes was determined to exist at the level of the dark noise limit of the PMT used for these studies and additional noise reduction strategies are expected to improve the quantitative aspects of the method. Theoretical plate numbers for proline were approximately 20 000. End column addition of Ru(bpy)(3)(2+) coupled with in situ generation of Ru(bpy)(3)(3+), has been shown to be compatible with the nanoliter elution volumes characteristic of capillary electrophoresis.

Development of a bimodal polarimetric response model for improved quantitation of enantiomeric mixtures under conditions of poor chromatographic resolution.

The combination of a chiral-selective separation mode with polarimetric detection was investigated in terms of its ability to quantitate enantiomeric mixtures of the dansylated derivatives of phenylalanine, threonine, and valine under conditions of poor chromatographic resolution. Using the difference between two Gaussian functions to model the bimodal response obtained using polarimetric detection and incomplete chiral resolution, correlation coefficients as high as 0.999 were obtained when actual enantiomeric fractions were plotted against observed enantiomeric fractions calculated from the fitting procedure. The methodology was evaluated at different levels of chromatographic resolution, and as a function of the sign and magnitude of the specific rotation of the model compounds. Limits of quantitation calculated at different levels of enantiomeric excess demonstrate the potential of the method to quantitate enantiomeric mixtures even under conditions of poor chromatographic resolution. Minimum measurable quantities (MMQ) at, or below the 1.0% enantiomeric excess (ee) level were obtained for the three amino acid derivatives tested under various conditions of chromatographic resolution when the polarimetric data were fit to the bimodal response model. Even under the lowest resolution conditions tested, the MMQ was determined to be at the 5% ee level.

Determination of enantiomeric excess using a chiral selective separation mode and polarimetric detection.

The quantitative capabilities of a system that combines a chiral selective separation mode with polarimetric detection (CSS/PD) were investigated in terms of the ability to quantitate enantiomeric mixtures even under conditions of poor chromatographic resolution. The laser-based polarimetric detection system can provide a rotational sensitivity on the order of 10 mudeg corresponding to a minimum measurable quantity of 10 ng for a compound with a specific rotation of 100 deg (g/ml)(-1) dm(-1). For the chromatographic studies, peak height and peak area were measured as analytical descriptors of the bimodal response function of this polarimetric detector. Analyses of the dansylated enantiomers of the amino acids phenylalanine, threonine, and valine have shown that chromatographic resolution and specific rotation are important factors affecting the shape of the polarimetric response curve. With CSS/PD, enantiomeric materials can be quantitated at both low and high enantiomeric excess (ee) even at resolutions of 0.3-0.4.

Chromatographic analysis of antibiotic materials in food.

The monitoring of food materials for antibiotic residues is an area of increasing concern and importance due to the potential impact on human health. Large-scale screening applications require methods that are rapid, accurate, provide low detection limits and are free from interference. The problem is further complicated by the wide range of chemical functionalities and modes of operation exhibited by the antibiotic materials of physiological significance in use today. As demonstrated, chromatographic methods provide many of the advantages necessary for screening applications. Judicious choice of sample preparation method, separation mode and detection strategy can provide significant immunity from problems associated with the food matrix. Gas chromatography can provide extremely high separation efficiencies, however, only a limited number of antibiotic compounds are inherently volatile enough for direct analysis by gas chromatography. Derivatization to enhance the volatility of the antibiotic is one approach to overcome this limitation. Among the methods available, reversed-phase high-performance liquid chromatography is used extensively for the analysis of many antibiotic systems as it does not require derivatization and it combines relatively high separation efficiencies with low detection limits. The diverse group of properties exhibited by the antibiotic materials in use today suggests that the choice of detection strategy is a key component in the successful development of an analysis technique. Derivatization of the antibiotic material is frequently used to add either a fluorogenic of chromogenic moiety to the antibiotic compound to enhance detection. Derivatization procedures suffer from several limitations which are problematic when making measurements in complicated food matrices. Among the different detection modes utilized for antibiotic analysis, polarimetric detection has the potential to provide extremely selective detection of most antibiotic materials, and this selective response can minimize many of the constraints placed upon the separation system by the sample matrix. Although many of the separation modes used for antibiotic analysis are well developed, separations based on capillary electrophoretic methods have much potential in the field of antibiotic analysis. Future investigations are needed to extend the generality of these techniques and expand their use into the field of food analysis.

Dynamically modified, biospecific optical fiber sensor for riboflavin binding protein based on hydrophobically associated 3-octylriboflavin.

A new approach to the production of optical fiber sensors is described which is based on a dynamic modification procedure. In this approach, the optical fiber surface is rendered hydrophobic through the covalent attachment of a C18 moiety. Specific sensing ligands are then associated with this surface through either their inherent or designed hydrophobicity. To investigate the feasibility of the approach, an optical fiber sensor has been constructed for riboflavin binding protein in which 3-octylriboflavin is associated with the fiber surface. Fluorescence quenching occurs upon binding of the protein to the immobilized riboflavin derivative. The sensor possesses a minimum measurable quantity of 7.3 pmol of binding protein in a probe volume which is less than 10 microL. With this approach, the sensing surface was repetitively regenerated 15 times over a 1-h period with less than a 5% variation in sensor-to-sensor performance. The approach is general, and with minor variations it can be used in a variety of sensing situations.

Effect of pH on the reaction of tris(2,2'-bipyridyl)ruthenium(III) with amino-acids: Implications for their detection.

A new technique for the detection of amino-acids is described, which is based on their chemiluminescence reaction with tris(2,2'-bipyridyl)ruthenium(III). The pH-dependence of this reaction has been investigated and found to be the key experimental parameter in application of this reaction as a detection technique. The chemiluminescence emission obtained is maximal at pH values higher than the N-terminal amino group pK(a) of the amino-acid. The background reaction between the ruthenium reagent and hydroxide ion does not occur with the same efficiency as the amino-acid reaction and the optimum signal to noise ratio is obtained at pH 10. A limit of detection of 30 picomole was found for valine and the response was shown to be linear over two orders of magnitude.

Improvements in the determination of penicillin analogues by HPLC separation and laser-based polarimetric detection.

The combination of HPLC separation and laser-based polarimetric detection is shown to provide unique advantages when applied to the study of penicillin analogues. The mass delectability for penicillin G is 5 ng with this system, which is an order-of-magnitude improvement over other techniques. More importantly, the polarimetric system can provide specific rotation information about eluting species. Individual specific rotations are reported for the (10R)- and (10S)-epimers of both carbenicillin and ticarcillin. These results demonstrate the sensitivity of specific rotation to the arrangement of atoms at or near the chiral centre, suggesting that specific rotation may be used to identify closely related penicillin analogues.

Specific rotation measurements from peak height data, with a Gaussian peak model.

A method is described whereby a sensitive laser-based polarimeter can be used to make very accurate and precise specific rotation measurements on microgram quantities of optically active materials. Flow-injection or liquid chromatography systems provide reproducible introduction of the sample into the polarimetric system. If a Gaussian distribution of the analyte concentration is assumed, the peak height can be used in the determination of specific rotation. This method provides a direct calibration with an absolute standard which yields more accurate and precise results than those obtainable by using peak area.