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Abstract: In this study, an enzyme-amplified electrochemical biosensor was developed for detection of the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia (APL). This new sensor employs a hairpin locked nucleic acids (LNAs) probe dually labeled with biotin and carboxyfluorescein molecule (FAM). The probe is immobilized at a streptavidin-modified electrode surface via the biotin–streptavidin bridge, and FAM serves as an affinity tag for the peroxidase conjugate binding. Initially, the immobilized hairpin probe was in the “closed” state in the absence of the target, which shielded FAM from being approached by the bulky anti-FAM-HRP conjugate due to the steric effect. Target binding opens the hairpin structure of the probe, the probe undergoes a significant conformational change, forcing FAM away from the electrode. As a result, the FAM label becomes accessible by the anti-FAM-HRP, and the target hybridization event can be sensitively transduced via the enzymatically amplified electrochemical current signal. This new biosensor demonstrates its excellent specificity for single-base mismatch and able to detect as little as 83fM target DNA even in the presence of human serum. We also employed this sensor to directly detect PCR real sample with satisfactory results. [Copyright &y& Elsevier]

Abstract: An amplified chronocoulometric Locked nucleic acid (LNA) sensor (CLS) for selective electrochemical detection of K-ras mutation was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. Thiolated-hairpin LNA probe with palindrome structure of stem was immobilized on the gold nanoparticles modified gold electrode (NG/AuE). It can be cleaved by EcoRI in the absence of K-ras mutation-type DNA (complementary with the loop part of hairpin probe), but cannot be cleaved in the presence of mutation-type DNA. The difference before and after enzymatic cleavage was then monitored by chronocoulometric biosensor. Electrochemical signals are generated by chronocoulometric interrogation of Hexaammineruthenium (III) chloride (RuHex) that quantitatively binds to surface-confined hairpin LNA probe via electrostatic interactions. The results suggested this CLS had a good specificity to distinguish the K-ras mutation-type, wild-type and non-complementary sequence. There was a good linear relationship between the charge and the logarithmic function of K-ras mutation-type DNA concentration. The detection limit had been estimated as 0.5fM. It is possible to qualitatively and quantitatively detect K-ras point mutation in pancreatic cancer. [Copyright &y& Elsevier]

Abstract: In this study, a kind of nanoporous gold electrode (NPG) prepared with repetitive square-wave oxidation reduction cycle (SWORC) was reported. The active surface area of the proposed NPG electrode was 9.9 times larger than that of a bare flat one characterized by cyclic voltammetry (CV). An electrochemical DNA biosensor based on NPG electrode was fabricated for detection of promyelocytic leukemia/retinoic acid receptor α (PML/RARα) fusion gene in acute promyelocytic leukemia (APL) by using Methylene Blue (MB) as an electroactive indicator. Differential pulse voltammetry (DPV) was employed to monitor the hybridization reaction on the probe modified electrode. The decrease of the peak current of MB was observed upon hybridization of the probe with target DNA. The results indicated that the peak current was linear with the concentration of complementary strand in the range of 60pM to 220pM with a detection limit of 6.7pM. This new biosensor exhibited excellent sensitivity and selectivity and had been used for an assay of PCR real sample with a satisfactory result. [Copyright &y& Elsevier]