Abstract: A noncompetitive heterogeneous enzyme immunoassay for the determination of chicken luteinizing hormone (LH) was equipped with an electrochemical endpoint in order to further enhance its sensitivity. The immunological principle of the original ELISA remained essentially unchanged, except for the fact that the peroxidase label was replaced by alkaline phosphatase, since in the upgraded version of the assay, p-aminophenyl phosphate was to be used as the substrate of alkaline phosphatase. Enzyme-generated p-aminophenol was injected into a flow injection system and detected amperometrically in a thin-layer flow cell with a glassy carbon electrode at 0.325 V vs Ag/AgCl. A classical problem associated with this type of solid-phase immunoassay is the adsorption of proteins other than the capture antibody to the solid phase. The detection sensitivity is therefore often limited by a large background signal observed in the absence of antigen. In the present study, an experiment was designed to examine in each step of the assay the contribution of each of the potential sources of background current. It was shown that the major contribution to the background current was caused by the nonspecific adsorption of biotinylated secondary antibody. Adsorption of the secondary antibody (biotinylated goat anti-rabbit IgG) to the capture antibody (mouse anti-chicken LHbeta) was clearly a case of specific aspecificity, whereas adsorption to the solid phase itself had to be treated as a nonspecific aspecificity. Addition of 0.25% mouse serum to the secondary antibody as a source of mouse immunoglobulin could overcome the cross-reaction and markedly reduced adsorption to capture antibody. The second part of nonspecific adsorption was eliminated by using combinations of Tween 20 and bovine serum albumin as blocking agents. Controlling the adsorption of the biotinylated secondary antibody in this way decreased the detection limit from 39 pg/ml in the original assay to 2.5 pg/ml in the electrochemical version. This way, the plasma volume of samples containing on the order of 1 ng/ml LH was reduced to less than 10 µL. The linear range was 2.5-625 pg/ml. The method allowed us to measure LH in buffer and in adult and juvenile chicken plasma. Copyright 1998 Academic Press.

Abstract: The Tandem-E TSH HS immunoenzymometric assay kit (Hybritech Inc., San Diego, CA, USA) for thyroid stimulating hormone (TSH) was adapted for this assay. Serum containing TSH and mouse monoclonal anti-human TSH antibody conjugated with bovine alkaline phosphatase were placed in a test-tube, then a single monoclonal anti-human TSH IgG coated bead was added. After incubation at 32°C for 2 h, substrate solution, 4-aminophenyl phosphate, was added. The product, 4-aminophenol, was detected by oxidative amperometry in a flow injection system with a vitreous carbon working electrode operated at 325 mV vs. Ag/AgCl, with a stainless-steel auxiliary electrode. The calibration graph was linear from 50 fmol to 100 pmol of 4-aminophenol and the limit of detection was 10.9 fmol. Intra-assay RSD were 8.0% for 0.02-60 miu/l of TSH. The detection limit was 0.01 piu/l of TSH. Results correlated well with those by Bio-Rad immunoradiometric assay (r = 0.992) and an immunochemiluminometric assay (r = 0.986).

Abstract: Electrodes modified by the electrodeposition of poly(3-methylthiophene) were used as chemical sensors for some organic and biological molecules of industrial and medicinal interest. The electrochemical behaviors of ferri/ferrocyanide, catechol, ascorbic acid, hydroquinone, dopamine, epinephrine, acetaminophen, p-aminophenol and NADH were examined by cyclic voltammetry. The results showed that the proposed modified surface catalyzes the oxidation of these compounds. Differential pulse and square wave techniques were used for the analysis of binary mixture of ascorbic acid with catechol, NADH, dopamine and p-aminophenol. Voltammetric peak resolution was also demonstrated for a ternary mixture of ascorbic acid, catechol and p-aminophenol. Polymer coated electrode was also used in an amperometric detector for flow injection analysis of most of the aforementioned compounds. The responses of the polymer electrode were 4-10 times larger as compared to those of platinum. The modified electrode displayed excellent response stability for successive injections and detection limits were 10 ppb for catechol, dopamine, epinephrine, NADH and p-aminophenol, 1 ppb for acetaminophen and 100 ppb for ascorbic acid. Voltammetric peak positions were affected by the nature of the electrolyte and its pH. Also, film thicknesses were shown to be a factor affecting both the current magnitudes and oxidation peak potential of NADH.

Abstract: A low-volume flow-cell electrochemical detector is described for continuous monitoring in liquid chromatography and flow injection analysis. The indicator electrode is the inner surface (0.5 mm2) of a round hole in 0.5 mm thick Pt sheet. Thus, the detection volume is ~0.035 mL. The detector response is linear in the concentration. range 0.4 ng-4 mg, and the detection limit for the test substance 4-aminophenol is 40 pg. (SFS)

Abstract: The combination of liquid chromatography with electrochemical detection (LC-EC) is a powerful analytical tool for determining electroactive compounds in complex matrices. It has found numerous applications especially in the pharmaceutical and clinical areas. This experiment is intended to give students a practical experience with the LC-EC technique. The first part is designed to explore the electrochemistry of p-aminophenol (PAP), the analyte, while the second part deals with separation and identification of PAP in the presence of ascorbic acid and catechol. The improvement in detection limit with electrochemical detection compared to ultraviolet detection is also illustrated.