A novel electrochemical DNA biosensor for transgenic soybean detection based on triple signal amplification

A novel electrochemical DNA biosensor was developed and MON89788 of soybean transgenic gene sequence was detected based on a strategy of rolling circle amplification (RCA) and gold nanoparticle cube (AuNPC)-labeled multiple probes. First, the mercapto-modified capture DNA was immobilized on the surface of the Fe 3 O 4 @Au magnetic nanoparticles via an Au–S bond, and the capture DNA was opened and complementarily hybridized with the target DNA to form a double-stranded DNA. In the 10 × reaction buffer, Exonuclease III (ExoIII) specifically recognized and sheared the double-stranded DNA to release the target DNA, which led to the next round of reaction. Afterward, AuNP cube-loaded ssDNA (AuNPC/DNA) was added with the rolling circle reaction with the help of Phi29 DNA polymerase and T4 ligase . Finally, [Ru(NH 3 ) 6 ] 3+ was attracted directly by the anionic phosphate of ssDNA via electrostatic interaction . The determination was carried out by using chronocoulometry (CC), and the CC signal was recorded. The mass amount of DNA strands extended infinitely on the AuNPs cube and numerous [Ru(NH 3 ) 6 ] 3+ were absorbed, thus the detected signal was highly amplified. The corresponding CC signal showed a good linear relationship with the logarithm of the target DNA concentration in the range of 1 × 10 −16 to 1 × 10 −7  mol L −1 , with a detection limit of 4.5 × 10 −17  mol L −1 . Specific gene sequence of MON89788 in soybean samples was determined, and the recoveries ranged from 97.3% to 102.0%. This sensor is one of the most sensitive sensors for genetic sequence assessment at present. Moreover, it demonstrates good selectivity , stability, and reproducibility.