In Situ Antigen Immobilization for Stable Organic-Phase Immunoelectrodes

Abstract

A new method based on enzymatic single-step in situ synthesis of hapten-carrier conjugates on electrodes is described yielding stable, reproducible, and reusable organic-phase immunoelectrodes (OPIEs). The electrodes developed were tailored for analyte detection in organic solvents and allow for the analysis of soil extracts without further sample processing and cleanup. Catalyzed by transglutaminase from a variant of Streptoverticillium mobaraense, the reaction proceeds in aqueous solution with and without addition of organic media in only 1.5 hours. In this study, the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was chosen as model compound and chemically amino-functionalized prior to its enzymatic immobilization. The high reproducibility of the immobilization procedure allowed for batch calibration of the immunoelectrodes. Moreover, pure methanol or treatment with diluted sulfuric acid used for regeneration studies did not disturb the hapten layer. The OPIE consists of screen-printed carbon electrodes, monoclonal anti-2,4-D antibodies, and the immunochemical recognition reaction and was optimized with regard to a high stability in organic media. For electrochemical detection, horseradish peroxidase was used as enzyme label together with H2O2 as substrate and hexacyanoferrate (II)/(III) as mediator. The OPIE showed high stability upon storage over 93 days. Response times of 17 s (t95) were found to be advantageous compared to those of other biosensors. Including the immunochemical reactions, the complete assay takes 30 min. A calibration curve for 2,4-D in 30% methanol/buffer obtained with 70 electrodes within 4 weeks revealed a detection limit of 9 mg/L, a sensitivity of 1.3 nA L mg-1 cm-2, and a repeatability of 6.8%. Although we calculated a lowered repeatability for reused electrodes of 13.4% and a slightly decreased sensitivity of 0.9 nA L mg-1 cm-2, multiple-used OPIEs could also be applied for calibration.

Gold Electrode Modification with Thiolated Hapten for the Design of Amperometric and Piezoelectric Immunosensors

“…Recycling of immunosensors involves dissociation of the antibody-antigen complex. It can be performed with chaotropic or acidic buffer or with organic [12]. In this study, the solution of 2.5 M MgCl 2 was used and demonstrated the desorption of the adsorbed antibody (mAb10c10).…”

“…However, chemical synthesis of hapten-protein conjugates is often laborious and hard to reproduce. In certain cases, a hapten itself was attached directly to an activated surface [2,12]. The conjugation and immobilization procedure offers new and advantageous features for the immunosensor development such as high reproducibility, easy calibration and extraordinary stability in organic-phases.…”

Organic phase immunosensors

“…The main research in this area has been provided by Kröger et al [41][42][43] and Jülicher et al [44], working in collaboration to develop a field-based tool for the quantification of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in aqueous/ organic solvent soil extracts. The assay was centred on a disposable 3-electrode assembly prepared using screen-printing technology, an approach amenable to the mass-manufacture of devices with fabrication materials being selected for compatibility with the organic-phase [45].…”

“…Jülicher et al [44] utilised a novel, highly reproducible in situ antigen immobilisation procedure, capable of immobilising 2,4-D directly onto graphite carbon working electrode surfaces. A transglutaminase enzyme was used to catalyse an acyl-transfer reaction between casein glutaminyl residues and the primary amine group of a 2,4-D amide derivative [49].…”

“…The costs involved in monitoring an environment can be substantially reduced with an effective on-site screening programme. To date, organic-phase immunosensor technology has mainly focused on pesticides analysis (atrazine [37][38][39], diphenylurea derivates [40], 2,4-dichlorophenoxyacetic acid [41][42][43][44]), although the measurement of polychlorinated biphenyls has also been reported [39].…”