Measurement of alkaline phosphatase isoenzymes in individual mouse bone marrow fibroblast cells based on capillary electrophoresis with on-capillary enzyme-catalyzed reaction and electrochemical detection.
A novel method for the determination of alkaline phosphatase (ALP) isoenzymes in individual fibroblast cells of mouse bone marrow was developed by combining capillary electrophoresis with an on-capillary enzyme-catalyzed reaction and electrochemical detection. In this method, a single an cell, followed by 5.0 x 10(-2) mol/L Na2B4O7- 3.0 x 10(-2) mol/L NaCl (pH 9.8) as cell lysis solution, was injected into the inlet of the separation capillary by electromigration. The cell was lysed by applying a voltage of 2 kV. The ALP isoenzymes in the cell were preseparated at 20 kV for 1 min, and then allowed to react for 30 min with disodium phenyl phosphate as enzyme substrate in the running buffer. ALP converted disodium phenyl phosphate into its product, phenol, at a relatively high reaction rate without consumption, with resultant amplification of the signal on prolonged reaction time, producing an adequate amount of product for final detection. A mass detection limit as low as 3.5 x 10(-21) mol/L (corresponding to 1.5 nU) was achieved. Finally, the two zones of products generated by ALP isoenzymes were detected at the outlet of the capillary by using the end-capillary amperometric detection at a carbon fiber microdisk bundle electrode with a constant potential.
Sun
Xuemei
X
School of Chemistry and Chemical Engineering, Shandong University, Jinan, China.
Jin
Wenrui
W
Li
Dong
D
Bai
Zengliang
Z
D000469:Alkaline Phosphatase / Q000032:analysis*; D000818:Animals; D001853:Bone Marrow / Q000201:enzymology*; D002478:Cells, Cultured; D019075:Electrophoresis, Capillary; D005347:Fibroblasts / Q000201:enzymology*; D007527:Isoenzymes / Q000032:analysis*; D051379:Mice
D007527:Isoenzymes; D000469:Alkaline Phosphatase