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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.


Factors affecting the hybridization signal intensity; effect of (A) hybridization time (B) hybridization temperature and (C) target DNA concentration on FRET emission. Excitation was fixed at 488 nm; scan rate: 2 nm/s; slidwidth: 10 nm.
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biosensors-03-00419-f004: Factors affecting the hybridization signal intensity; effect of (A) hybridization time (B) hybridization temperature and (C) target DNA concentration on FRET emission. Excitation was fixed at 488 nm; scan rate: 2 nm/s; slidwidth: 10 nm.

Mentions: Figure A1 (in Appendix) presented the factors affecting the hybridization signal intensity. The FRET intensity increases as the hybridization time increases ranging from 0.5 to 20 min. As can be seen, hybridization time of 10 min is enough to give significant FRET intensity for sensing purposes. Another important parameter is effect of temperature. Hybridization temperature is always discussed relative to the melting temperature of the given oligonucleotide. In this study, the effect of temperature of hybridization was varied from 25 to 65 °C. According to Figure A1(B), the intensity of FRET decreases gradually as the temperature increases. The highest FRET intensity was obtained when hybridization was performed at 25 °C. The hybridization of the DNAs was conducted along with QD that has been conjugated with ssDNA at optimized temperature. High temperature will affect FRET intensity since QD will lose its photoluminescence (PL) properties. Fluorescence decreases as temperature increases. This is due to the increase of molecular motion with increasing temperature, which results in more molecular collisions and subsequent loss of energy [9]. The sensitivity of developed DNA biosensor was studied using different concentration of target DNA. A plot of FRET intensity versus log concentration of target DNA in Figure A1(B) shows that FRET intensities decrease with respect to the logarithm value of the concentration of target DNA. The regression correlation coefficient (R2) is 0.935 and calculated limit of detection (LOD) for the developed system is 3.55 × 10−9 M. The decrease of FRET indicates the increase in negative charge density due to the formation of a DNA duplex which increases the repulsive electrostatic forces between QD and DNA resulting in larger distances and lower efficiency [10]. Besides, a high concentration of analyte will cause difficulty for the light to pass through the sample to instigate excitation. Thus, high concentration of analyte will have low fluorescence, which is known as concentration quenching [11].


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)

Factors affecting the hybridization signal intensity; effect of (A) hybridization time (B) hybridization temperature and (C) target DNA concentration on FRET emission. Excitation was fixed at 488 nm; scan rate: 2 nm/s; slidwidth: 10 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

biosensors-03-00419-f004: Factors affecting the hybridization signal intensity; effect of (A) hybridization time (B) hybridization temperature and (C) target DNA concentration on FRET emission. Excitation was fixed at 488 nm; scan rate: 2 nm/s; slidwidth: 10 nm.
Mentions: Figure A1 (in Appendix) presented the factors affecting the hybridization signal intensity. The FRET intensity increases as the hybridization time increases ranging from 0.5 to 20 min. As can be seen, hybridization time of 10 min is enough to give significant FRET intensity for sensing purposes. Another important parameter is effect of temperature. Hybridization temperature is always discussed relative to the melting temperature of the given oligonucleotide. In this study, the effect of temperature of hybridization was varied from 25 to 65 °C. According to Figure A1(B), the intensity of FRET decreases gradually as the temperature increases. The highest FRET intensity was obtained when hybridization was performed at 25 °C. The hybridization of the DNAs was conducted along with QD that has been conjugated with ssDNA at optimized temperature. High temperature will affect FRET intensity since QD will lose its photoluminescence (PL) properties. Fluorescence decreases as temperature increases. This is due to the increase of molecular motion with increasing temperature, which results in more molecular collisions and subsequent loss of energy [9]. The sensitivity of developed DNA biosensor was studied using different concentration of target DNA. A plot of FRET intensity versus log concentration of target DNA in Figure A1(B) shows that FRET intensities decrease with respect to the logarithm value of the concentration of target DNA. The regression correlation coefficient (R2) is 0.935 and calculated limit of detection (LOD) for the developed system is 3.55 × 10−9 M. The decrease of FRET indicates the increase in negative charge density due to the formation of a DNA duplex which increases the repulsive electrostatic forces between QD and DNA resulting in larger distances and lower efficiency [10]. Besides, a high concentration of analyte will cause difficulty for the light to pass through the sample to instigate excitation. Thus, high concentration of analyte will have low fluorescence, which is known as concentration quenching [11].

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.