Monoclonal Antibody-Based Flow-Through Immunosensor for Analysis of Carbaryl Miguel A. Gonza ´lez-Martı´nez, ² Sergi Morais, ² Rosa Puchades, ² Angel Maquieira,* ,² Antonio Abad, ‡ and Angel Montoya ‡ Departamento de Quı ´ mica, ETSIA, and Laboratorio Integrado de Bioingenierı ´ a, Universidad Polite ´ cnica de Valencia, 46071 Valencia, Spain Immunosensor systems have been developed for the rapid and sensitive determination of pesticides, using the insecticide carbaryl as a model analyte. The systems are based on the principle of heterogeneous competitive enzyme immunoassay and used mouse monoclonal anti- carbaryl antibodies either in solution (indirect format) or immobilized (direct format). In both formats, enzyme label (horseradish peroxidase, HRP) and fluorometric detection were employed. In the direct format, antibodies were immobilized on a hydrazide-derivatized agarose gel, and 6 -[((1 -naphthyloxy)carbonyl)amino]hexanoic acid (CNH hapten) conjugated to HRP was used as the enzyme tracer. The limit of detection was 26 ng L -1 (11 min/ assay), and the useful life of the sensor was 6 0 -7 0 cycles. In the indirect format, CNH conjugated to bovine serum albumin was immobilized on an N-hydroxysuccinimide- derivatized agarose gel, and a rabbit anti-mouse antibody labeled with HRP was used as a secondary immu- noreagent. The limit of detection was 284 ng L -1 (17 min/ assay), and the useful life of the sensor was 160 - 2 0 0 cycles. The developed methods were applied to the analysis of commercial drinking water and apple juice spiked with carbaryl. Interassay RSD ranged from 9 to 3 9 % in the direct format and from 4 to 2 8 % in the indirect format. Recoveries were between 62 and 109% in the direct format and between 78 and 124% in the indirect format. The results were compared with those obtained by enzyme-linked immunosorbent assay as reference method and indicated the suitability of the immunosensor for quality control in water and food analysis. The extensive use of pesticide formulations in agriculture, together with the increasingly rigorous regulations of the presence of contaminants in the environment and food products, has brought about the need to develop sensitive and accurate methods for the detection and quantitation of pesticide residues. Pesticide analysis methods have been widely reviewed by Sherma. 1 As an alternative to the commonly used chromatographic methods, immunoassay techniques for the detection of pollutants have grown steadily in recent years. 2 Immunoassays can be performed with different formats, most of them being enzyme immunoassays. To facilitate sample handling and analysis automation, on-line methods based on the use of immunosensors are being devel- oped. 3 Immunosensors are devices that use immunochemical principles to carry out analysis in a rapid and automated way. Flow- based heterogeneous immunosensors (flow-through sensors) combine the sensitivity and selectivity of immunoassays with the precision and ability to be automated of flow techniques. 4 Stability and reusability are key factors in the performance of sensors based on heterogeneous immunoassays. The ideal situation would be to integrate a reversible antibody-antigen reaction in a sensor that is able to maintain the same activity through a high number of assays. However, most immunosensors can be used only for a limited number of assays. In flow immunosensors, antibodies or antigens are immobilized on a suitable support, which must be changed or regenerated by disrupting the antigen-antibody complex. The sensitivity of enzyme immunoassays is primarily determined by the affinity of the antibody used. However, high-affinity antibodies need harsh chemical conditions to accomplish an effective immunosurface regeneration, with the subsequent loss of activity. Hence, a compromise between sensitivity and sensor regeneration is necessary. In this sense, the use of specific monoclonal antibodies (MAbs) is advisable, since it allows the selection of the immu- noreagent with the most suitable properties. In the development of flow-based immunosensors, two different alternatives should be considered, i.e., antibody or antigen/ hapten immobilization, the first one being the most common approach found in the literature. 4 The main advantage of this option is the economy of expensive antibodies and the reduction of assay steps. The technique of immobilization of the antigen or hapten, although less employed, has the advantage that the regeneration process can be performed without loss of activity of the immobilized reagent. Another important issue to be addressed in immunosensor development is the choice of immobilization support. 5 To enhance sensitivity, packed bed reactors offer good performance since they minimize sample dispersion. 6 The most used particulate supports for immobilization are glass, silica, and agarose-based gels, and for all of them several methods have been proposed to solve the critical problem of protein immobilization. 7 † Departamento de Quı ´mica, ETSIA. ‡ Laboratorio Integrado de Bioingenierı ´a. (1) Sherma, J. Anal. Chem. 1993 , 65, 40R-50R. (2) Sherry, J. P. Crit. Rev. Anal. Chem. 1992 , 23 (4), 217-300. (3) Marco, M. P.; Gee, S.; Hammock, B. D. Trends Anal. Chem. 1995 , 14 (7), 341-349. (4) Puchades, R.; Maquieira, A. Crit. Rev. Anal. Chem. 1996 , 26 (4), 195-218. (5) Thompson, R. Q.; Kim, H.; Miller, C. E. Anal. Chim. Acta 1987 , 198, 165- 172. (6) Ho, M. H. Methods Enzymol. 1988 , 137, 271-287. (7) Scouten, W. H. Methods Enzymol. 1987 , 135, 30-65. Anal. Chem. 1997, 69, 2812-2818 2812 Analytical Chemistry, Vol. 69, No. 14, July 15, 1997 S0003-2700(96)01068-2 CCC: $14.00 © 1997 American Chemical Society