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Development of a Fluorescence Resonance Energy Transfer (FRET)-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense.

Mohd Bakhori N, Yusof NA, Abdullah AH, Hussein MZ - Biosensors (Basel) (2013)

Bottom Line: Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system.TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA.This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Department, Faculty Science, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia; E-Mails: noremyliamb@gmail.com (N.M.B.); halim@science.upm.edu.my (A.H.A.); mzobir@science.upm.edu.my (M.Z.H.).

ABSTRACT
An optical DNA biosensor based on fluorescence resonance energy transfer (FRET) utilizing synthesized quantum dot (QD) has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA) via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10(-9) M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

No MeSH data available.


Fluorescence resonance energy transfer between donor QD and acceptor Cy5.
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biosensors-03-00419-f002: Fluorescence resonance energy transfer between donor QD and acceptor Cy5.

Mentions: Hybridization solution contains two probes; reporter probe labeled with Cy5 and ssDNA conjugated with QD. Upon hybridization with target DNA, the reporter probe and CdSe QD-ssDNA will bind with the target forming sandwich hybrid [8]. Hybridization occurs in this system since the sequence of the ssDNA and reporter probe completely match with the sequence of target DNA (ssDNA match one half of the target sequence while reporter probe match the other half of the target). The resulting interaction of the hybrid brings the acceptor, Cy5 and donor QD into close proximity. When excited at 488 nm, QDs emit emission at 640 nm and lead the fluorescence emission from the acceptor Cy5 by means of FRET illumination, as shown in Figure 2.


Development of a Fluorescence Resonance Energy Transfer (FRET)-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense.

Mohd Bakhori N, Yusof NA, Abdullah AH, Hussein MZ - Biosensors (Basel) (2013)

Fluorescence resonance energy transfer between donor QD and acceptor Cy5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263569&req=5

biosensors-03-00419-f002: Fluorescence resonance energy transfer between donor QD and acceptor Cy5.
Mentions: Hybridization solution contains two probes; reporter probe labeled with Cy5 and ssDNA conjugated with QD. Upon hybridization with target DNA, the reporter probe and CdSe QD-ssDNA will bind with the target forming sandwich hybrid [8]. Hybridization occurs in this system since the sequence of the ssDNA and reporter probe completely match with the sequence of target DNA (ssDNA match one half of the target sequence while reporter probe match the other half of the target). The resulting interaction of the hybrid brings the acceptor, Cy5 and donor QD into close proximity. When excited at 488 nm, QDs emit emission at 640 nm and lead the fluorescence emission from the acceptor Cy5 by means of FRET illumination, as shown in Figure 2.

Bottom Line: Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system.TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA.This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Department, Faculty Science, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia; E-Mails: noremyliamb@gmail.com (N.M.B.); halim@science.upm.edu.my (A.H.A.); mzobir@science.upm.edu.my (M.Z.H.).

ABSTRACT
An optical DNA biosensor based on fluorescence resonance energy transfer (FRET) utilizing synthesized quantum dot (QD) has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA) via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10(-9) M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

No MeSH data available.