Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

Journal Article · Sun Aug 01 04:00:00 EDT 2010 · Analytical Chemistry

DOI:https://doi.org/10.1021/ac1006238· OSTI ID:988661

Zhang, Youyu; Tang, Zhiwen; Wang, Jun; Wu, Hong; Maham, Aihui; Lin, Yuehe

A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate due to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 988661
• Report Number(s): PNNL-SA-73376; 24822
• Journal Information: Analytical Chemistry, Journal Name: Analytical Chemistry Journal Issue: 15 Vol. 82; ISSN 0003-2700
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS
AMPLIFICATION
BIOTIN
CONFORMATIONAL CHANGES
DETECTION
DNA
DNA HYBRIDIZATION
DNA hybridization and senquecing
ENZYMES
Environmental Molecular Sciences Laboratory
GOLD
HYBRIDIZATION
PERFORMANCE
PEROXIDASES
PROBES
SHIELDS
SPECIFICITY
SUBSTRATES
TARGETS
THIOLS
enzyme
gold nanoparticles
hairpin DNA
signal amplification