Solid-Phase Electrochemical Enzyme Immunoassay with Attomole Detection Limit by Flow Injection Analysis

A sandwich electrochemical enzyme immunoassay with flow injection analysis for the model antigen mouse IgG has been developed with alkaline phosphatase as the enzyme label. The enzyme substrate, 4-aminophenyl phosphate and its enzymatic reaction product, 4-aminophenol have been studied by cyclic and hydrodynamic voltammetry. The determination of 4-aminophenol by flow injection analysis with electrochemical detection (FIAEC) has a linear range of 5.0 × 10−8 to 1.0 × 10−5 M, a detection limit of 2.4 × 10−8 M, and a sample throughput of 72 samples/h. The detection limit is set by a background capacitance response, which depends on the ionic strength difference between the sample and the mobile phase. The sandwich immunoassay has been characterized with respect to substrate concentration for the enzymatic reaction, detection limit, dynamic range and sources of error. Mouse IgG can be determined with a detection limit of 0.81 pg ml−1 by a 30-min substrate incubation time and a six orders of magnitude linear dynamic range

The Effects of Primary Oxy-Salts on Anodizing Magnesium Alloy AZ91D

Anodization is known to be an effective way to slow down the initial corrosion rate of magnesium (Mg) and its alloys. Here, we investigated the specific use of oxy-salts to improve the corrosion resistance of anodizing coatings. Oxy-salts of silicate, phosphate, and carbonate were added separately to a sodium hydroxide alkaline electrolyte used to anodize Mg alloy AZ91D. The process was investigated in terms of anodizing behavior, the surface properties, and the corrosion behavior of AZ91D. Anodizing AZ91D using the silicate- containing electrolyte generated sparks, and produced a thicker and more corrosion-resistant layer than the other oxy-salts. In the process, MgO and SiO2 formed Mg2SiO4 at high temperatures. Coatings from the phosphate- and carbonate- containing electrolyte anodizations did not contain phosphorus or carbon. We also studied the effects of silicate concentration on the corrosion resistance and properties of the surface

Electrochemical Enzyme Immunoassay Using Sequential Saturation Technique in A 20-μl Capillary: Digoxin as A Model Analyte

Capillary enzyme immunoassay with flow-injection analysis for digoxin using the sequential saturation technique has been developed. Glass capillary tubes (10 cm × 0.53 mm i.d.) with immobilized digoxin antibody were used as the immunoassay reactor. The product of enzymatic reaction. 4-aminophenol, was detected amperometrically. The digoxin and the labeled digoxin binding reaction with the immobilized digoxin antibody were completed in 2 and 10 min, respectively. Digoxin was determined in a 20-μl sample with a detection limit of 10 pg ml−1 (200 fg or 260 attomoles) and a 3 orders of magnitude range

ENZYME IMMUNOASSAY WITH ELECTROCHEMICAL DETECTION IN FLOW SYSTEMS

The selectivity of an antigen/antibody reaction combined with the chemical am plif ication feature of an enzyme label enables high specificity and low detection levels to be achieved by enzyme immunoassays. Electrochemical enzyme immunoassay is based on antigen labeled with an enzyme that catalyzes the production of an electroactive product. Hydrodynamic electrochemical techniques such as liquid chromatography and flow injection analysis with electrochemical detection have proved to be very effective for the rapid determination of the enzyme-generated product in enzyme immunoassays. Heterogeneous enzyme imm unoassays in which antibody is adsorbed on the walls of polystyrene cuvettes have been developed. Alkaline phosphatase, which catalyzes the conversion of electroinactive phenylphosphate to phenol, is the enzyme label. The rate of phenol production is measured by either liquid chromatography/electrochemistry or flow injection analysis/electrochemistry with a thin-layer carbon paste electrode at +0.87V vs. the Ag/AgCl reference electrode. An example is an assay for digoxin, a cardiac glycoside used to treat chronic heart disease. The assay has a detection limit of 50 pg/mL and exhibits good correlation with radioimmunoassay on patient serum samples over the therapeutic range of 0.8-2.0 ng/mL. Assays have also been developed for orosomucoid and rabbit imm unoglobulin G. Homogeneous enzyme immunoassays are based on a reduction in catalytic activity of enzyme-labeled drug when bound to antibody. The enzyme label glucose-6-phosphate dehydrogenase, which catalyzes the conversion of NAD+ to NADH, has been used. The drug is quantitated by measuring the rate of NADH production by liquid chromatography/electrochemistry. Good results have been obtained for clinical samples of serum from patients on drug maintenance for both phenytoin, an antiepileptic drug, and digoxin over their therapeutic ranges. The heterogeneous enzyme immunoassay with electrochemical detection has several advantages. The detection limit, as demonstrated for digoxin and IgG, is typically in the low picogram-per-milliliter range and is a function of the antigen-antibody binding constant rather than the ability to detect phenol. Consequently, even lower limits should be attainable. Because the sample is rinsed out of the reagent tubes before adding substrate, problems with interferences from electroactive constituents in the sample and possible fouling of the electrode by the adsorption of protein films are eliminated. The main advantage of a homogeneous immunoassay is the absence of a step for separating the antibody-bound antigen from the free antigen. This usually translates into a simpler, faster assay procedure. However, homogeneous assays are usually restricted to higher detection levels than are heterogeneous assays and are more susceptible to interferences from other sample constituents that would otherwise be removed in a separation step

Heterogeneous Enzyme Immunoassay for Maternal Serum Alpha Fetoprotein by Flow Injection Amperometric Detection of 4-Aminophenol

A sandwich-type heterogeneous enzyme immunoassay with flow-injection analysis for alpha-fetoprotein (AFP) in human serum has been developed. 4-Aminophenol, the product of enzymatic reaction, is detected amperometrically. The interassay CV for this electrochemical enzyme immunoassay was \u3c8.2%, with a minimum detection limit for AFP of 0.163 μg/L. The calibration curve had a linear range of 0.316-100 μg/L. Studies with 48 human maternal serum samples, comparing results by this method with those by a commercial kit, showed a good correlation (r=0.961). This procedure provides an alternative method for determining low concentrations of AFP in human maternal serum

ENZYME IMMUNOASSAYS USING FLOW INJECTION ANALYSIS WITH ELECTROCHEMICAL DETECTION

The utility of FIAEC for enzyme immunoassaysof low detection limit can be constrained bythe existence of a nonfaradaic capacitance current that records with the analytical faradaic signal. Since the magnitudeof this unwanted signal diminishes with smaller potential, conditions can be found where the interference becomes unimportant. This was done here by developing p-aminopheny] phosphate as a substrate for alkaline phosphatase, which yields p-aminophenol as product, oxidizable at between +100 and +300 mV. Additional advantageis gained by matching the compositions of the sample and mobile phase solutions. The useof p-aminopheny] phosphate as a substratefor electrochemical enzyme immunoassay was demonstrated by sensitive assays for theophylline, IgG and alpha-fetoprotein

Blank Response at Glassy Carbon Electrodes in A Flow Injection System

The blank response at glassy carbon electrodes in a flow injection system consists of a transient component and a steady state (residual current) component. The transient response is attributed to nonfaradaic current and faradaic current from the oxidation-reduction of electroactive surface functional groups on glassy carbon. The magnitude of the transient response was dependent on the potential applied to the electrode, temperature, ionic strength and pH of the injected sample solution, and independent of the flow rate. A linear relationship exists between the transient response and the difference in concentration of ionic species or pH between injected sample and the carrier. A differnce of 0.01 M ionic concentration or 0.04 pH unit between carrier and injected sample could change the transient charge by ca. 0.038 μC/cm2. The shape of the transient response due to pH difference at high positive potential indicates that some changes occur on glassy carbon surface in this potential region

Small-Volume Voltammetric Detection of 4-Aminophenol with Interdigitated Array Electrodes and Its Application to Electrochemical Enzyme Immunoassay

A small-volume voltametric detection of 4-aminophenol (PAP) has been developed using an interdigitated array (IDA) microelectrode cell in order to apply the IDA to electrochemical enzyme immunoassay. The signal of PAP at the IDA was steady state, and its magnitude was amplified compared with that of the usual single electrode due to redox cycling of PAP between the two finger sets of the IDA. A linear relationship between PAP concentration and cathodic limiting current was obtained from 1 to 1000 μM, reproducibly. The minimum sample volume in the measurement was reduced to 800 nL. High sample throughout of less than 1-min detection time per sample was achieved on 2-10 μL PAP samples. This IDA cell was applied to the electrochemical enzyme immunoassay of mouse IgG. Alkaline phosphatase was used as the enzyme label. The mouse IgG concentration was evaluated by detecting the concentration of PAP, which is the product of enzymatic reaction of the substrate, 4-aminophenyl phosphate (PAPP). Anti-mouse IgG was covalently immobilized on the glass surface of the small-volume immunowells by carbodiimide coupling. The assay range was 10-1000 ng/mL using 10- μL sample and 20- μL substrate solutions