Ferrocenylethyl Phosphate:  An Improved Substrate for the Detection of Alkaline Phosphatase by Cathodic Stripping Ion-Exchange Voltammetry. Application to the Electrochemical Enzyme Affinity Assay of Avidin

Abstract

The ferrocenylethyl phosphate disodium salt was synthesized and used as a new substrate for alkaline phosphatase (AP). The enzyme-generated ferroceneethanol was selectively and sensitively detected at a Nafion film-coated electrode by anodic preconcentration of the ferricinium salt, followed by cyclic voltammetry. The accumulated ferricinium units could be expelled from the polymer film in their neutral form by cathodic stripping, and so the Nafion-modified electrode could be reused for more than 10 measurements with a standard deviation less than 3%. Values of 0.75 mM for the Michaelis constant and 1.42 μmol s(-)(1) (mg of protein)(-)(1) for the maximal velocity were found. The regenerable Nafion-coated electrode was employed for the indirect detection of AP down to 2 × 10(-)(12) M and for the noncompetitive heterogeneous enzyme assay of avidin, whose detection limit was 5 × 10(-)(12) M.

Synthesis, properties and crystal structures of ferrocene derivatives containing pyrazinium and quinoxalinium units

“…In each case, the CH 2 group gives rise to a resonance signal close to 6 ppm in the 1 H NMR spectrum; the corresponding 13 C NMR signal lies close to 60 ppm. These values compare well with those of closely related species such as, for example, [FcCH 2 py] + [6] and [FcCH 2 bipy] + [7]. Owing to the quadrupole moment of 14 N (I = 1), the 13 C NMR signal of the tertiary carbon atom adjacent to the formally cationic alkylated N atom of 2 shows characteristic broadening; in the case of 1 14 N, 13 C coupling is resolved (t, 1 J = 9 Hz) for the magnetically equivalent tertiary carbon atoms bonded to the alkylated N atom.…”

Electrochemical Detection of Secreted Alkaline Phosphatase: Implications to Cell Based Assays

“…In all cases reported, the detection time represents a compromise between assay speed and magnitude of response. Although more sensitive detection methods for alkaline phosphatase have been reported in the literature, e.g., [6,7], the detection levels reported in this work were deemed suf®cient.…”

3-Indoxyl Phosphate: an Alkaline Phosphatase Substrate for Enzyme Immunoassays with Voltammetric Detection

“…A variety of alternative substrates have been synthezized with the aim of overcoming these disadvantages. Ferrocene-based compounds [4][5][6], p-hydroxyphenyl phosphate [7] or p-aminophenyl phosphate [8][9][10][11][12][13][14] are described in the literature. The last one appears to be very useful for the quantitation of mouse IgG [8], theophylline [9], digoxin [10] and some other antigens [11][12][13], or simply for the measurement of the AP activity [14].…”