FLOW-INJECTION CHEMILUMINESCENT METHOD FOR AN ENZYME-LABELED DNA PROBE

A flow-injection chemiluminescent (CL) method was investigated for the determination of hydrogen peroxide generated from an enzyme-labelled DNA probe. Biotinylated DNA probes were immobilized onto a membrane and labelled with biotinylated glucose oxidase by a streptavidin bridge and placed in a microtitre well. The glucose oxidase-labelled sample was incubated in a solution of glucose and the enzymatically produced hydrogen peroxide was determined using now injection with a peroxyoxalate chemiluminescence reaction. The peroxide generated is related to the amount of the labelled DNA probe. The important factors for increasing the sensitivity of the CL measurement are to maximize the production of hydrogen peroxide and to reduce the non-specific binding of the enzyme label on the membrane. This CL-flow-injection measurement using biotinylated glucose oxidase-labelled DNA probes was applied to a DNA dot-blot hybridization assay and compared with a visual colorimetric method using 5-bromo-4-chloroindolyl phosphate and nitro blue tetrazolium as a chromogenic substrate for detection of an alkaline phosphatase label. The CL method was quantitative over the range 100 pg-100 ng of target DNA with a precision (r.s.d.) of 4-11%. In comparison, the colorimetric method was useful for the range 10 pg-l ng.