| Epidermal growth factor receptor(EGFR) is an important signal transducter in cellular pathway. For patients with non-small-cell lung cancer(NSCLC), EGFR gene mutation status is a significant predictor for therapeutic effect of small molecule tyrosine kinase inhibitors(TKIs). Most of the authoritative guidelines both in domestic and abroad recommend detecting EGFR mutation status before targeted medical therapy. However, there is no standard assay for the detection of EGFR so far. DNA electrochemical biosensor attracts more focus due to its high sensitivity, specificity, facility, economics as well as easy performing.Our team has a lots of research groundworks for applying electrochemical DNA biosensor in gene detection. In previous study, we successfully constructed a bovine serum albumin(BSA)-assisted probe carrier platform with excellent reproducibility and sensitivity. In this work, we constructed novel electrochemical DNA biosensors for the detection of EGFR mutation status in NSCLC patients and discrimination of 6 types of hotspot deletions in exon 19 of EGFR. In order to improve the specificity of assay, we designed different DNA sequences according to DNA hybridization characteristics varying in different circumstances. Meanwhile, object to improve sensitivity, we employed nuclease to form hybridization-digestion circle and streptavidin-biotin complex(SABC) assay to amplify detection signal. Based on above technologies, we developed a new detection assay for EGFR gene in NSCLC, and accomplished to detect PCR products of lung cancer tissue. The research finally provided groundworks for wide application of DNA electrochemical biosensor in detection of genes which relevant to diagnose and therapy of disease. The main content of this paper was depicted as follow:(1) In part one, in order to improve the specificity of DNA electrochemical biosensor, we designed 3 pairs of DNA(C, R, C/R) based on DNA hybridization efficiency varies in different reaction phase. The different between 3 pairs of DNA was the deletion position in target DNA, resulting in non-complementary hybridization in electrodes surface, reaction solution and both half, respectively. The experimental results showed that C-type DNA(non-complementary hybridization happened in electrodes surface) exhibited excellent specificity. Meanwhile, we applied the biosensor constructed above to detect synthetic double-stranded DNA(ds DNA). To get target single-stranded DNA(ss DNA), we compared two strategies including denaturization under high temperature and digestion with λ-exo. The result displayed that current signal of ss DNA produced by λ-exo digestion was similar with current signal of synthetic ss DNA, without disadvantage of renaturation and interaction with the sandwich detection system. Consequently, we employed the biosensor to detect PCR products of clinical real samples. Due to the uncertain of the target, we designed 6 different primers and chose the best one. RT-PCR products of real samples were cleaved by λ-Exo to produce target ss DNA for biosensor detection. Through this assay, we can discriminate wild and deletion EGFR according to current signals.(2) The main purpose of the second part was to improve sensitivity of electrochemical biosensor to distinguish 6 types of hotspot deletion in EGFR exon 19. The results of the first part revealed that biosensor with non-complementary hybridization happened in electrode surface showed excellent specificity. In this part, in order to get maximum discrimination, we designed different probes to investigate the relevance of hybridization and the distribution of non-complementary bases in electrode surface, which is a heterogeneous reaction. The result revealed that when non-complementary bases located in the middle of capture probe, the biosensor showed optimization specificity and there was maximum discrimination between single-base mismatched del1 and del2. According to this result and EGFR mutation types, we designed capture probes to detect del1-del6, and divided the deletions into 3 groups according to current signals. This approach accomplished to simplify workload, and discriminated 6 kinds of hotspot deletion types of exon 19 quickly. At the same time, we employed the assay to detect PCR products of real samples, and the result was verified by sequencing, which confirmed the successful application of biosensor for discrimination deletion types in real samples.(3) In the third part, we constructed biosensor by combining BSA-based probe carrier platform with exonuclease-involved hybridization-digestion amplification approach in homogeneous phase medium. Target DNA hybridized with assistant DNA to form ds DNA, which triggered λ-Exo to exert its recognition and digesttion effect on assistant DNA in the formed ds DNA. In hybridization-digestion circles, the current signal amplified via multiple recycling of a small quantity target DNA. In purpose to select the suitable exonuclease, we investigated the effect of Exo-III and λ-Exo, and employed latter for further studies. We performed the experiments to optimize the hybridization-digestion conditions such as reaction time, enzymatic digestion, et al. In addition, in order to amplify the detection signal, we investigated the relevance of current signal and the biotin position in ds DNA on electrode. The result demonstrated that, without space restrain, the shorter distance between biotin and electrode surface resulted in higher current signal. Based on the result, we optimized the position of biotin in sandwich structure. The biosensor constructed in this part showed more sensitively than the previous BSA control immobilization system. The limitation of detection(LOD) was 0.01 n M, and the linear range was 2.0×10-11-4.0×10-10 mol/L. We utilized the biosensor to discriminate wild and deletion EGFR with high specificity.(4) On the ground of the third part results, in the fourth part, we changed the enzymatic digestion object from assistant DNA to reporter probe. We endued reporter probe with two functions including reporting signal and being cleaved by λ-Exo. In this assay, the hybridization-digestion procedure was simplified, and the reaction DNA types was reduced which resulting in the direct reflection of target DNA concentration by reporter detection. Meanwhile, in purpose to increase signal response, we employed SABC system to improve detection sensitivity. We combined horseradish peroxidase(HRP) labeled streptavidin(SA) with HRP labeled biotin to form composites. The composites were used to attach to the biotin modified at the reporter probe via the biotin-SA bridge. Through this way, the quantity of HRP on electrode increased, and the current signal got improved. Via these two signal amplification strategies, the LOD got improved significantly and reached 1 p M. The detection linear range was 2.0×10-12-3.0×10-11 mol/L. The results of the constructed assay for detection of PCR products of real samples were in accordant with those of sequencing, which verified the successful application of biosensor for discrimination wild and deletion EGFR in real samples. |
PDF Full Text Request