title: Electrochemical detection of leukemia oncogenes using enzyme-loaded carbon nanotube labels.
abstract: We describe an ultrasensitive electrochemical nucleic acid assay amplified by carbon nanotubes (CNTs)-based labels for the detection of human acute lymphocytic leukemia (ALL)-related p185 BCR-ABL fusion transcript. The carboxylated CNTs were functionalized with horseradish peroxidase (HRP) molecules and target-specific detection probes (DP) via diimide-activated amidation and used to label and amplify the target hybridization signal. The activity of captured HRP was monitored by square-wave voltammetry measuring the electroactive enzymatic product in the presence of 2-aminophenol and hydrogen peroxide substrate solution. The signal-amplified assay achieved a detection limit of 83 fM (5 × 10(-18) mol in 60 μL) targets oligonucleotides and has a 4-order-wide dynamic range of target concentration. The resulting assay allowed robust discrimination between the perfect match and a three-base mismatch sequence. When exposed to the full-length (491 bp) DNA oncogene, the approach demonstrated a detection limit of 1 × 10(-16) mol in 60 μL, corresponding to approximately 33 pg of the target gene. The high sensitivity and specificity of the assay enabled a PCR-free detection of target transcripts in as little as 65 ng of mRNA extracted from positive ALL cell lines SUP-B15 in comparison to those obtained from negative cell line HL-60. The approach enables a simple, low-cost and ultrasensitive electrochemical nucleic acid detection in portable devices, point-of-care and early disease diagnostic applications.
authors: Pacific Northwest National Laboratory, Richland, Washington 99352, USA. yuehe.lin@pnnl.gov dbsltm@nus.edu.sg.
authors: Ai-Cheng
authors: AC
authors: Lee
authors: Dan
authors: D
authors: Du
authors: Baowei
authors: B
authors: Chen
authors: Chew-Kiat
authors: CK
authors: Heng
authors: Tit-Meng
authors: TM
authors: Lim
authors: Yuehe
authors: Y
authors: Lin
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D055664:Electrochemical Techniques; D004800:Enzymes, Immobilized / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D037742:Nanotubes, Carbon / Q000737:chemistry*; D009693:Nucleic Acid Hybridization; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics
chemical_list: D004800:Enzymes, Immobilized; D037742:Nanotubes, Carbon; D012333:RNA, Messenger; D006735:Horseradish Peroxidase; D016044:Fusion Proteins, bcr-abl