Pneumolysin ELISA with Adsorptive Voltammetric Detection of Indigo in a Flow System

Bobes, Cecilia Fernández; Fernández‐Abedul, M. Teresa; Costa-Garcı́a, Agustı́n

doi:10.1002/1521-4109(200105)13:7<559::aid-elan559>3.0.co;2-6

Cited by 16 publications

References 33 publications

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3‐Indoxyl Phosphate as an Electrochemical Substrate for Horseradish Peroxidase

2004

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In this work 3-indoxyl phosphate (3-IP), an alkaline phosphatase substrate, is demonstrated to be a suitable substrate for horseradish peroxidase (HRP). HRP catalyzes the oxidation of 3-IP in presence of hydrogen peroxide (H 2 O 2 ) generating the product indigo blue, which is an aromatic heterocycle compound insoluble in aqueous solutions. This product was easily converted into its soluble parent compound indigo carmine (IC) (by addition of fuming sulfuric acid to the reaction media) which has a reversible voltammetric peak at the formal potential of À 0.15 V (vs. Ag pseudo-reference electrode) when a screen-printed carbon electrode (SPCE) is used. Parameters that influence the enzymatic reaction, such as pH, temperature, substrate concentration and reaction time have been optimized. Moreover, the enzyme apparent kinetic constants (V max , K M ) for both substrates (3-IP and H 2 O 2 ) have been calculated. Indirect measurements of HRP activity in solution were carried out not only by cyclic voltammetry but also using amperometric detection in a flow system. The detection limits were 6.86 Â 10 À12 and 5.68 Â 10 À12 M, respectively. Thus, 3-IP is the first substrate that could be used for alkaline phosphatase (AP) and HRP, the most common enzymatic labels in affinity assays.

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3‐Indoxyl Phosphate as an Electrochemical Substrate for Horseradish Peroxidase

2004

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Gold Electrodes for Detection of Enzyme Assays with 3‐Indoxylphosphate as Substrate

2004

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The advantageous characteristics of gold not only for its electrochemical behavior but also for the unique adsorptive protein characteristics are the basis of this paper. 3-Indoxyl phosphate (3-IP) has previously been demonstrated to be a very convenient substrate for the electrochemical detection, using carbon-based electrodes, of enzymeimmunoassays (EIAs) that employ not only alkaline phosphatase (AP) but also horseradish peroxidase (HRP) as label. Combination of both: gold electrodes and 3-IP has not been studied and it produces a very suitable detection for EIAs. The electrochemical behavior of indigo, product of the enzymatic hydrolisis of 3-IP, has been thoroughly studied for the first time on disk and band gold electrodes. The possibility of electrodepositing gold on the bands and automation has also been considered. The detection has been applied for the determination of osteocalcin with an HRP-based ELISA (enzyme-linked immunosorbent assay).

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Voltammetric Determination of Leucoindigo Adsorbed on Pretreated Carbon Paste Electrodes: Its Application in a Flow System

2004

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The determination of leucoindigo adsorbed on the surface of pretreated carbon paste electrodes has been performed by cyclic (CV) and alternating current voltammetry (ACV). The water-soluble compound called leucoindigo is obtained through the reduction of water insoluble indigo in an alkaline media and in presence of a dithionite salt (Na 2 S 2 O 4 ) as a reducing agent. Cyclic voltammograms of leucoindigo show two reversible electrodic processes, in the aqueous 0.1 M Tris-HCl pH 7.2, at the formal potential of À 0.4 V and þ 0.3 V (vs. Ag/AgCl/sat. KCl). In a batch protocol, leucoindigo was quantified by CV with a change of medium between accumulation and detection steps. Reversible voltammetric processes of leucoindigo were greatly enhanced by other voltammetric technique like alternating current voltammetry. Limit of detection in the nanomolar range was achieved for a 2 min accumulation time by ACV in a batch procedure. A flow system was also employed, with adsorptive voltammetric detection of leucoindigo, since this automates the methodology and decreases analysis time. Parameters related to the electrochemical technique are optimized and calibration plots obtained are reported. These data provide useful information about the suitability of using leucoindigo in the detection system of alkaline phosphatase (AP) and horseradish peroxidase (HRP) based affinity devices, in which indigo is generated by the enzymatic hydrolysis of the 3-indoxyl phosphate substrate.

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Pneumolysin ELISA with Adsorptive Voltammetric Detection of Indigo in a Flow System

Cited by 16 publications

References 33 publications

3‐Indoxyl Phosphate as an Electrochemical Substrate for Horseradish Peroxidase

3‐Indoxyl Phosphate as an Electrochemical Substrate for Horseradish Peroxidase

Gold Electrodes for Detection of Enzyme Assays with 3‐Indoxylphosphate as Substrate

Voltammetric Determination of Leucoindigo Adsorbed on Pretreated Carbon Paste Electrodes: Its Application in a Flow System

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