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Anal Chem. 2015 Aug 4;87(15):7746-53. doi: 10.1021/acs.analchem.5b01323. Epub 2015 Jul 14.

DLISA: A DNAzyme-Based ELISA for Protein Enzyme-Free Immunoassay of Multiple Analytes.

Hu R1,2,3, Liu T1, Zhang XB1, Yang Y3, Chen T1,2, Wu C2, Liu Y2, Zhu G1, Huan S1, Fu T1,2, Tan W1,2.

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†Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Molecular Engineering for Theranostics, Hunan University, Changsha 410082, China.
‡Department of Chemistry and Department of Physiology and Functional Genomics, Shands Cancer Center and UF Genetics Institute, Center for Research at the Bio/Nano Interface, University of Florida, Gainesville, Florida 32611-7200, United States.
§College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650092, China.


A DNAzyme-based ELISA, termed DLISA, was developed as a novel protein enzyme-free, triply amplified platform, combining a catalytic and molecular beacon (CAMB) system with a cation exchange reaction for ultrasensitive multiplex fluorescent immunosorbent assay. Classical ELISA, which employs protein enzymes as biocatalysts to afford amplified signals, suffers from poor stability caused by the irreversible denaturation of these enzymes under harsh conditions, such as heat and acidity. Compared with proteins, nucleic acids are more stable and adaptable, and they can be easily produced using a commercial DNA synthesizer. Moreover, the catalytic and cleavage activities of DNAzyme can be achieved in solution; thus, no enzyme immobilization is needed for detection. Taken together, these attributes suggest that a DNAzyme-based ELISA detection approach will be more robust than current ELISA assays. Importantly, the proposed triply amplified DLISA immunoassay method shows ultrasensitive detection of such targets as human IgG with a detection limit of 2 fg/mL (3 × 10(-17) M), which is well within the range of many important disease biomarkers. DLISA can also be used to construct a sensing array for simultaneous multiplexed detection. With these merits, this high-throughput, stable, simple, sensitive, and low-cost multiplex fluorescence immunoassay shows promise for applications in clinical diagnosis.

[Indexed for MEDLINE]

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