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Piezoelectric Sensor for Determination of Genetically Modified Soybean Roundup Ready ® in Samples not Amplified by PCR

View Article: PubMed Central

ABSTRACT

The chemically modified piezoelectrodes were utilized to develop relatively cheap and easy to use biosensor for determination of genetically modified Roundup Ready soybean (RR soybean). The biosensor relies on the immobilization onto gold piezoelectrodes of the 21-mer single stranded oligonucleotide (probes) related to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, which is an active component of an insert integrated into RR soybean genome. The hybridization reaction between the probe and the target complementary sequence in solution was monitored. The system was optimized using synthetic oligonucleotides, which were applied for EPSPS gene detection in DNA samples extracted from animal feed containing 30% RR soybean amplified by the PCR and nonamplified by PCR. The detection limit for genomic DNA was in the range of 4.7·105 numbers of genom copies contained EPSPS gene in the QCM cell. The properties such as sensitivity and selectivity of piezoelectric senor presented here indicated that it could be applied for the direct determination of genetically modified RR soybean in the samples non-amplified by PCR.

No MeSH data available.


Steps of modification of the gold piezoelectrode.
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f1-sensors-07-01462: Steps of modification of the gold piezoelectrode.

Mentions: The Au electrode surface was cleaned with piranha solution consisting of H2O2 (30%) and concentrated H2SO4 (98%) in a 7:3 ratio. The crystal was dipped in this solution for 5 min, then thoroughly washed with deionized water and dried under nitrogen. This process was repeated twice. After washing, the gold surfaces of QCM electrodes were immediately immersed for 2 hours into 5 mM solution of 3,3′-dithiodipropionic acid di-(N-succinimidyl ester) in chloroform. Subsequently, the free surface of gold was blocked by immersion for 1 h into 1 mM solution of 6-mercapto-1-hexanol in ethanol. Then, the electrodes were modified by avidin (aqueous solution, 0.2 mg/ml) for 1 h. Next, they were exposed for 1 h to a 1 mM aqueous solution of 2-aminoethanol (pH 8.00, adjusted with HCl). Piezoelectrodes modified according to the above procedure were dept in immobilization buffer in the QCM cell. After frequency stabilization (± 1 Hz), the solution of biotinylated DNA probe was injected. The concentration of the DNA probe in the QCM cell was 300 nM (Figure 1).


Piezoelectric Sensor for Determination of Genetically Modified Soybean Roundup Ready ® in Samples not Amplified by PCR
Steps of modification of the gold piezoelectrode.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814863&req=5

f1-sensors-07-01462: Steps of modification of the gold piezoelectrode.
Mentions: The Au electrode surface was cleaned with piranha solution consisting of H2O2 (30%) and concentrated H2SO4 (98%) in a 7:3 ratio. The crystal was dipped in this solution for 5 min, then thoroughly washed with deionized water and dried under nitrogen. This process was repeated twice. After washing, the gold surfaces of QCM electrodes were immediately immersed for 2 hours into 5 mM solution of 3,3′-dithiodipropionic acid di-(N-succinimidyl ester) in chloroform. Subsequently, the free surface of gold was blocked by immersion for 1 h into 1 mM solution of 6-mercapto-1-hexanol in ethanol. Then, the electrodes were modified by avidin (aqueous solution, 0.2 mg/ml) for 1 h. Next, they were exposed for 1 h to a 1 mM aqueous solution of 2-aminoethanol (pH 8.00, adjusted with HCl). Piezoelectrodes modified according to the above procedure were dept in immobilization buffer in the QCM cell. After frequency stabilization (± 1 Hz), the solution of biotinylated DNA probe was injected. The concentration of the DNA probe in the QCM cell was 300 nM (Figure 1).

View Article: PubMed Central

ABSTRACT

The chemically modified piezoelectrodes were utilized to develop relatively cheap and easy to use biosensor for determination of genetically modified Roundup Ready soybean (RR soybean). The biosensor relies on the immobilization onto gold piezoelectrodes of the 21-mer single stranded oligonucleotide (probes) related to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, which is an active component of an insert integrated into RR soybean genome. The hybridization reaction between the probe and the target complementary sequence in solution was monitored. The system was optimized using synthetic oligonucleotides, which were applied for EPSPS gene detection in DNA samples extracted from animal feed containing 30% RR soybean amplified by the PCR and nonamplified by PCR. The detection limit for genomic DNA was in the range of 4.7·105 numbers of genom copies contained EPSPS gene in the QCM cell. The properties such as sensitivity and selectivity of piezoelectric senor presented here indicated that it could be applied for the direct determination of genetically modified RR soybean in the samples non-amplified by PCR.

No MeSH data available.