提出了以固体辣根过氧化物酶(HRP)对过氧化氢氧化邻苯二胺的催化作用为基础的测定HRP及其标记物的电化学方法.测定中以Au-Pt/PAN/GCE为工作电极,并详细叙述其制备过程.将一定浓度的HRP按规定方法固定在上述修饰电极上制得HRP/Au-Pt/PAN/GCE修饰电极,将此电极浸入含5.0×10-3mol·L-1邻苯二胺及2.5×10-3mol·L-1过氧化氢的磷酸盐缓冲溶液(pH 5.0)中,反应10 min后将电极取出,记录溶液中酶催化反应产物的方波伏安峰及峰电流.结果表明:酶催化反应前,底物在工作电极上于-0.488 V(vs.SCE)处有明显的还原峰,在酶催化反应后,在-0.584 V处出现一个更大的还原峰,电位负移160 mV,且峰电流明显增大.峰电流值(Ip)与修饰在Au-Pt/PAN/GCE电极上的HRP的含量在1.0×10-2~2.0×102μg·L-1之间呈线性关系,方法的检出限(3S/N)为3.0 ng·L-1.

Based on the catalytic action of solid horseradish peroxidase (HRP) on oxidation of o-phenylenediamine by hydrogen peroxide,an electrochemical method for determination of HRP and its labelled enzyme HRP-IgG was proposed.The Au-Pt/PAN/GCE was used as working electrode,and its preparation was described in detail.A definite amount of HRP was immobilized on the modified electrode to give a modified electrode of HRP/Au-Pt/PAN/GCE,which was placed into a substrate solution of pH 5.0 PBS containing definite amounts of o-phenylenediamine and reacted for 10 min.The HRP/Au-Pt/PAN/GCE was taken out,and the square wave voltammetric peak of the product of catalytic reaction in the substrate solution was recorded by an electrochemical analyzer.As shown by the experimental results,a well-defined reduction peak at the potential of -0.488 V (vs.SCE) was obtained at the working electrode in the base solution before the enzyme catalytic reaction,and a much larger reduction peak at the potantial of -0.584 V (vs.SCE) was observed after the enzyme catalytic reaction,with remarkable enhancement of the peak current (Ip).It was found that linear relationship between the values of Ip and mass concentration of HRP on the modified GCE was kept in the range of 1.0×10-2~2.0×102μg·L-1.Detection limit (3S/N) of HRP by the method was found to be 3.0 ng·L-1.