A multiple signal amplification electrochemical biosensors based on target DNA recycling for detection of the EGFR mutation status in lung cancer patients

Abstract

In this study, we construct a multiple amplification biosensor combining a hybridization–digestion cycles in a homogeneous reaction in liquid phase, and a modified streptavidin-biotin complex (SABC) method. To reduce the number of DNA types participating in the hybridization–digestion cycles, we design a dual-functional reporter probe with biotin and -PO4 in both distal ends, which can not only hybridize with the target DNA and be recognized by λ-exo but also bind to streptavidin. The target DNA hybridized with the reporter probe, and then the reporter sequence was digested by λ-exo to release the target DNA. After the hybridization–digestion cycles, the remaining reporter probe was captured by the probe mobilized on the gold electrode. Meanwhile, in modified SABC method, we mixed streptavidin-horseradish peroxidase (SA-HRP) and biotin-HRP to form complexes, which bind to biotin on reporter probe to accumulate large amounts of HRP on the electrode, current was further increased on the basis of the HRP-catalyzed reaction. By the combination of this two signal amplification protocols, the detection limit of this biosensor was 1 pM. Wild-type EGFR in clinical samples from lung cancer patients can be successfully discriminated from EGFR mutations by the proposed method. Our research would make the electrochemical biosensor be an excellent candidate for EGFR status detection in clinical diagnosis and prognosis.