Catalysis‐Electrochemical Detection of Horseradish Peroxidase at Zeptomole Level in Capillary Zone Electrophoresis

Capillary zone electrophoresis with catalysis‐electrochemical detection has been developed and applied to determining horseradish peroxidase (HRP) at zeptomole levels. In this method, an on‐line enzyme catalysis reactor with a reaction capillary was designed. Isoenzymes of HRP were separated by capillary zone electrophoresis, and then they catalyzed the enzyme substrate 3,3′,5,5′‐tetramethylbenzide (TMB(Red)) and H 2 O 2 in the reaction capillary. The reaction product, TMB(Ox), could be determined using amperometric detection on a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Because of enzyme amplification, a significant amount of TMB(Ox) could be produced for detection. Therefore, the limit of detection (LOD) of HRP is very low. The optimum conditions of the method are 1.5×10 −2  mol/L borate (pH 7.4) for the run buffer, 2×10 −3  mol/L for the concentration of H 2 O 2 , 2×10 −4  mol/L TMB(Red)+2.0×10 −2  mol/L citrate‐phosphate (pH 5.0) for the substrate solution, 40 cm for the liquid pressure height, 20 kV for the separation voltage, 100 mV for the detection potential. HRP could be measured with a detection limit of 4.8×10 −12  mol/L or 47.5 zmol (S/N=3). The linear range is from 2.40×10 −11 to 2.40×10 −8  mol/L. Using this method, commercial HRP was measured at zeptomole within ten minutes.