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The paper illustrates two different concepts for monitoring pesticides and drugs. In the first approach double stranded deoxyribonucleic acid (dsDNA) was used as the sensing material, which intercalated drugs with high affinity. The intercalation of drugs such as Chlorpromazine (CP), Clomipramine (CM) and Imipramine (IP) were investigated. The assay was based on competition between the drug molecule and a fluorescent dye (ToPro-3) for intercalation with dsDNA. The ToPro3 was excited at 642 nm and the emission at 661 nm was detected using a fiber fluorimeter. A linear range of 25–100, 50–200 and 25–100 μM was obtained for CP, CM and IP, respectively. The sensitivity for both CP and CM was 0.14 while it was 0.06 mV/μM for IP. The detection limit was 1.25 μM in all cases. In another approach the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) was investigated based on chemiluminescence detection. The assay was based on competition between the binding of horseradish peroxidase (HRP) labeled versus unlabeled 2,4-D, on immobilized anti-2,4-D monoclonal antibodies (mAbs). Two other peroxidases from tomato (MOP) and tobacco (TOP) were also employed as labels for the 2,4-D assays. The assays were optimized by testing various chemiluminescent substrates for improving the signal intensity and sensitivity of the assays. Based on colorimetry a linear range of 0.35–20 and 0.7–4 μg/ml was obtained for TOP and MOP conjugates, respectively. However, using chemiluminometry both TOP and MOP showed a linear range of 0.5–5000 ng/ml. TOP demonstrated a higher specific activity compared to MOP and HRP. A detection limit of 50 pg/ml for 2,4-D was obtained using TOP conjugate and chemiluminescence detection. Both the fluorimetric and chemiluminometric assays were carried out in glass microcapillaries and detected using a computer coupled photomultiplier tube (PMT) as a photon detector. The responses were also compared with microtitre plate based colorimetric assay.

Based on a molecularly imprinted polymer, a competitive binding assay analogous to competitive enzyme immunoassay has been developed. The assay is specific for the herbicide 2,4-dichlorophenoxyacetic acid and uses, for the first time, an enzyme-labelled conjugate as a tracer. The label tobacco peroxidase allowed for colorimetric and chemiluminescence detection. The molecularly imprinted polymer was synthesised in the form of microspheres by precipitation polymerisation. The polymer efficiently and selectively bound the analyte in aqueous solution. Calibration curves were obtained corresponding to analyte concentrations ranging from 40–600 μg mL−1 for the colorimetric assay, and from 1–200 μg mL−1 for chemiluminescence assay.

Molecular imprinting is a technology that allows for the creation of molecular recognition sites in synthetic polymers via the use of templates. Imprinted materials have been used in various applications, including stationary phases for chromatography, recognition elements in sensors, or antibody binding mimics in immunoassay-type analyses. In the present work, molecularly imprinted enantioselective polymer membranes are described. The amino acid derivative CBZ-tyrosine was chosen as the target compound. A CBZ-l-tyrosine-imprinted polymer was first tested in HPLC mode, and efficient resolution of racemic CBZ-tyrosine was obtained. Imprinted polymer membranes were prepared by casting the polymer in the pores of a polypropylene membrane, using a specifically adapted polymer recipe. In diffusion experiments across the imprinted membranes, enantioselectivity was observed with the diffusion of the l-enantiomer of CBZ-tyrosine (the original template) being faster than that of the d-enantiomer. Chirality 11:465–469, 1999. © 1999 Wiley-Liss, Inc.

Noncovalent molecular imprinting of a synthetic polymer with the herbicide 2,4-dichlorophenoxyacetic acid has been achieved in the presence of the polar solvents methanol and water. Formation of the prearranged complex relied on hydrophobic and ionic interactions between the template and the functional monomer 4-vinylpyridine. The polymer obtained binds the original template with an appreciable selectivity over structurally related compounds. The potential use of micrometer-sized imprinted polymer particles as the recognition element in a radioligand binding assay for 2,4-dichlorophenoxyacetic acid is demonstrated.

A chemiluminescent microformat enzyme linked immuno sorbent assay (ELISA) has been optimized for the simultaneous determination of multiple 2,4-dichlorophenoxyacetic acid (2,4-D) samples. The competitive immunoassay employed a 2,4-DBSA conjugate, anti-2,4-D monoclonal antibodies and alkaline phosphatase (AP) labelled anti-mouse IgG. The bound AP conjugate was determined by quantitating the chemiluminescence emission from the enzymatic decomposition of the luminogenic substrate, CSPD, by AP using a cooled charge coupled device (CCD) camera. The detection limit for the simultaneous determination of multiple samples was 4.3 × 10−10 M corresponding to 96 pg ml−1 or 192 fg well with a coefficient of variation (CV, %) of 12.5%. The linear range of the assay was 4.5 × 10−7–4.5 × 10−10M. The ability of gold coated silicon wafers and glass capillaries to serve as solid phase supports in the imaging ELISA was investigated. The highly reflective gold surfaces improved both the linear range and the sensitivity of the assay, as compared to thick-film patterned surfaces. The capillary supports, on the other hand, lead to a reduction in the linear range and the sensitivity of the assay, as compared to the thick-film patterned surfaces. Initial studies indicate that the capillaries guide the light and may provide a built-in mechanism for collecting the emitted light. Strategies for further development of support materials for imaging-based detectors will be discussed.

A flat-well microformat competitive enzyme-linked immunosorbent chemiluminescent assay for the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) is described. Thick-film technology was used to pattern a hydrophobic layer 100 μm thick onto glass microscope slides to form an array of 2 × 2 mm(2) squares. These flat wells were able to hold 2 μL of reagents, corresponding to a height of ∼500 μm, with minimal contamination risk. The hydrophobic ink used to pattern the surfaces allowed significantly larger volumes of samples to be applied when compared with surfaces patterned with nonhydrophobic inks. This reduced evaporation effects and permitted greater pipetting accuracy, thereby improving assay reproducibility. A competitive immunoassay was developed based on the ability of free 2,4-D hapten to inhibit binding of anti-2,4-D monoclonal antibodies to 2,4-D-bovine serum albumin conjugate adsorbed onto the glass support. The support was subsequently incubated with alkaline phosphatase (AP) labeled anti-mouse IgG. The amount of AP conjugate bound was determined by quantitating the chemiluminescent emission produced from the enzymatic breakdown of CSPD substrate by AP using a cooled CCD camera. The detection limit of the single-sample microformat assay was 2.7 × 10(-)(11) M, or 6 pg of 2,4-D. The linear ranges of the single-sample and multisample assays were 4.5 × 10(-)(8)-4.5 × 10(-)(11) and 4.5 × 10(-)(7)-1.66 × 10(-)(10) M, respectively. In comparison, the detection limits of a tube-based chemiluminescent assay using standard luminometer and of a colorimetric ELISA were 45 × 10(-)(11) and 9.9 × 10(-)(8) M, respectively. The ability to scale the thick-film-based microformat assay makes it an ideal candidate for the development of affinity arrays and high-throughput assay formats. Prospects for further improvements of this imaging ELISA strategy will be discussed.

In this paper, a novel strategy for multicomponent analysis of two classes of pesticides such as triazines (atrazine and simazine) and phenoxyalkanoic acids (2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-chlorophenoxyacetic acid (CPOAc), phenoxyacetic acid (POAc)) employing immuno-arrays is demonstrated. The approach is based on cross-reactive arrays of specific antibody pairs coupled to chemometric pattern recognition. The monoclonal antibody pairs employed in this work (atrazine–simazine and 2,4-D) are specific towards a set of analytes and preclude a particular set of others present in the sample matrix. Antibody pairs of atrazine, simazine, and 2,4-D are used to discriminate and quantify analyte of interest. Atrazine was quantified in presence of trace concentration of simazine and that of 2,4-D. The combinatorial cross-reactivity of antibody pairs towards simazine, atrazine and 2,4-D is used to distinguish among different classes of analytes and their influence on the signal suppression in immuno-techniques. These sensors exclude recognition by carbamates such as carbaryl and carbofuran.

The application of gold-coated glass capillaries for the design of a sensitive chemiluminescent immunoassay for 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. The gold coating on the glass capillaries has been partially characterized and its effect on enhancing the signal intensity has been measured. A simple photo-multiplier tube-based photon detector is used for this purpose. At least three times improvement in the signal intensity is observed compared to uncoated glass capillaries, with a consequent improvement in the sensitivity of detection. Using such gold-coated glass capillaries, 2,4-D in the range 10(-9) to 10(-13) mol/l is detectable at a precision of +/-15% (CV%) and a limit of detection of 10(-15) mol/l is achievable. The possibility of using such gold-coated capillaries with a portable multianalytical set-up for field studies is also demonstrated.

A microformat ELISA has been developed for the simultaneous determination of multiple 2,4-dichlorophenoxyacetic acid (2,4-D) samples using a CCD-camera and highly effficient chemiluminescent enzyme substrates. The linear range was 4.5×10−10 – 4.5×10−7 M with a lower detection limit of 4.3×10−10 M, corresponding to 96 pg/ml or 192 pg/well with a CV of 12.5%. The use of gold coated silicon wafers and glass capillaries as solid phase supports in the imaging ELISA is also described. The highly reflective gold surfaces improved the linear range and the sensitivity as compared to thick-film patterned surfaces. Sampling in capillary tubes reduces the sensitivity and the linear range, but there are indications that the capillaries can act as light guides and thereby improve the light collection efficiency.

The application of a newly isolated transgenic tobacco peroxidase (TOP) as a chemiluminescent label for immunoassay purposes is described for the first time. The enzyme has been oxidized with m-periodate and subsequently coupled to the model compound 2,4-dichlorophenoxyacetic acid (2,4-D) using a carbodiimide method. As compared to the native horseradish peroxidase used in control experiments, the TOP enzyme showed significantly higher efficiency of coupling to the antigen and no loss of the specific activity was observed. The obtained 2,4-D-TOP conjugate demonstrated unique properties in chemiluminescent detection. The latter allowed the minimization of the conjugate concentration due to the superior chemiluminescent activity of the enzyme. A highly sensitive capillary chemiluminescent immunoassay using the 2,4-D-TOP conjugate as labeled competitor is reported. Direct competitive ELISA has been performed using a specific monoclonal antibody immobilized onto the sol-gel treated glass capillary surface. A modified photomultiplier tube with a special holder for a capillary was used for the resulting chemiluminescent signal detection. The typical standard calibration curve for the 2,4-D pesticide detection is linear between 30 pg and 500 ng/mL.