Oligonucleotide labelled lipase as a new sensitive hybridization probe and its use in bio‐assays and biosensors

Abstract

Radiolabelled polynucleotide probes have been employed extensively for the detection of complementary nucleic acids by specific hybridization. Within the last few years, various methods have been developed using enzyme‐labelled probes to avoid unstable and hazardous isotopes. These assays, based on photometry, fluorescenec, and chemiluminescence, have helped to overcome the use of radioactive probes.To increase the performance of a non‐radioactive DNA detection system, the labelling enzyme should remain stable under hybridization conditions which allow the formation of a 15–25 bp long DNA–DNA duplex (T_m = 50–70°C). Therefore, the use of unstable phosphatase and peroxidase conjugates must be avoided due to the composition of the hybridization mixture and the high temperature. By screening various hydrolytic enzymes to fit the special demands, fungal lipases turned out to be the most practical. They offer high sensitivity due to an extremely high turnover number, stability at room temperature for several years, thermostability under working conditions and an easy design of various chromogenic, fluorescent and electrochemical active substrates.Several types of silanized, oxidized and unmodified metal sensors and also standard microtitre plates modified with amino groups were used for the immobilization of oligonuleotides. A sandwich hybridization using the lipase‐labelled oligonucleotide probe and a terminal immobilized capture DNA on a microtitre plate or sensor surface combined with a rapid hybridization in solution simplifies and improves the performance of the DNA detection system.