Biosensors and Bioelectronics 28 (2011) 277–283 Contents lists available at ScienceDirect Biosensors and Bioelectronics j our na l ho me page: www.elsevier.com/locate/bios Enzyme-amplified electrochemical biosensor for detection of PML–RAR fusion gene based on hairpin LNA probe Liqing Lin a , Qicai Liu a,c , Liman Wang a , Ailin Liu a,b , Shaohuang Weng a , Yun Lei a , Wei Chen a , Xinhua Lin a,∗ , Yuanzhong Chen a,b,∗∗ a Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350004, China b Fujian Key Lab of Haematology, Fujian Institute of Hematology, the Affiliated Union Hospital of Fujian Medical University, Fuzhou 350000, China c Department of Laboratory Medicine, First Affiliated Hospital of Fujian Medical University, Fuzhou, 350005, China a r t i c l e i n f o Article history: Received 8 May 2011 Received in revised form 14 July 2011 Accepted 14 July 2011 Available online 23 July 2011 Keywords: Enzyme-amplified electrochemical biosensor Hairpin locked nucleic acids Acute promyelocytic leukemia Fusion gene a b s t r a c t In this study, an enzyme-amplified electrochemical biosensor was developed for detection of the promye- locytic leukemia/retinoic acid receptor alpha (PML/RAR) 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 confor- mational 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 enzymati- cally amplified electrochemical current signal. This new biosensor demonstrates its excellent specificity for single-base mismatch and able to detect as little as 83 fM target DNA even in the presence of human serum. We also employed this sensor to directly detect PCR real sample with satisfactory results. © 2011 Elsevier B.V. All rights reserved. 1. Introduction APL is identified as the M 3 subtype of acute myeloid leukemia morphologically. It is characterized by selective expansion of immature myeloid precursors that are blocked at the promyelo- cytic stage (Wang et al., 2011). Cytogenetically, a translocation t(15;17)(q22;q21) is found in more than 95% APL patients, result- ing in the formation of PML–RAR fusion gene (Qi et al., 2010). This gene rearrangement plays an important role in leukemogene- sis through antagonizing retinoic acid signalling and the regulatory pathways mediated by APL. Thus, detection of PML–RAR fusion gene will afford an early diagnosis and monitor of the disease. The reported methods for the detection of PML/RAR fusion gene have included real-time quantitative reverse transcription poly- merase chain reaction (RT-PCR) (Devaraj et al., 1996), fluorescence ∗ Corresponding author at: Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350004, China. Tel.: +86 591 22862016; fax: +86 591 22862016. ∗∗ Corresponding author at: Fujian Institute of Hematology, the Affiliated Union Hospital of Fujian Medical University, Fuzhou 350000, China. Tel: +86 591 83351966; fax: +86 591 83324116. E-mail addresses: xhl1963@sina.com (X. Lin), chenyz@pub3.fz.fj.cn (Y. Chen). in situ hybridization (FISH) (Han et al., 2009), flow cytometry (FCM) (Cullen et al., 2004), chromosome analysis (Yoo et al., 2006), etc. But there were some limitations in these methods, such as time- consuming, poor precision and expensiveness. In recent years, there has been significant progress in developing biosensors for the rapid, cheap and accurate detections of specific gene sequence (Zhang et al., 2008). Various sensing strategies have been developed, aiming at the improvement of sensitivity and selectivity of electrochemical detection (Wang, 1998; Wei et al., 2008; Tosar et al., 2010). Among them, enzyme-based amplifi- cation strategies have been developed to improve the sensitivity of electrochemical DNA sensors (Wei et al., 2008). The selectivity of nucleic acid hybridization assays depends primarily on the selection of the probe and then of the hybridization conditions. Thus, the design of the probe is the most important pre-analytical step. Recently, Koshkin’s group reported a novel oligonucleotide derivative, LNA (Koshkin et al., 1998). LNA nucleotides contain a methylene bridge between the 2 ′ -oxygen and the 4 ′ -carbon of the ribose moiety. The covalent bridge effectively ‘locks’ the ribose in the N-type (3-endo) conformation that is dominant in A-form DNA and RNA. Due to this chemical difference, LNAs differ from DNA molecules in several aspects: The covalent bridge results in a very high affinity for DNA and RNA complementary sequences, with each LNA substitution increasing the melting temperatures 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.07.032