Graphene Oxide-Based Fluorometric Determination of the eta Gene in Pseudomonas aeruginosa Using Nicking Enzyme-Mediated Cyclic Signal Amplification

Abstract

A convenient and highly sensitive bioassay employing graphene oxide (GO) as a nanocarrier and nicking enzyme (Nb.BbvC I)-assisted signal amplification was developed to detect Pseudomonas aeruginosa. Furthermore, the assay employed a DNA probe with a capture probe (CP) and a help probe (HP), which were designed to bind the target and signal probe (SP), respectively. In the absence of the target, the SP did not bind to the HP due to steric effects. Consequently, the fluorescence of carboxyfluorescein (FAM) was quenched by the black hole quenching group (BHQ). The CP specifically recognized the introduced target, which opened the HP that was bound to the SP. Nb.BbvC I cleaved the formed nicking site, which restored the fluorescence and simultaneously released the HP for the next round of reaction. The target densities, which ranged from 10 to 10⁷ colony-forming units (CFU) mL⁻¹, and the fluorescence intensity had a linear relationship. The limit of detection (LOD) under the optimal conditions was 5 CFU mL⁻¹. Moreover, this bioassay performed well in the analysis of water samples. These results demonstrate that this method is highly specific and sensitive for determining P. aeruginosa and is promising for the clinical diagnosis of infectious diseases.

Keywords:

• Enzyme-assisted recycling amplification
• fluorescent assay
• graphene oxide
• Pseudomonas aeruginosa detection
• quenching

Disclosure statement

The authors declare that they have no competing interests.

Additional information

Funding

We would like to thank National Natural Science Foundation (81803964), Natural Science Foundation of Hunan Province (2019JJ50693, 2020JJ4465, 2018JJ3573), Scientific Research Project Funded by Education Department of Hunan Province (18A493, 18C1156), Hunan Key Laboratory Cultivation Base of Research and Development of Novel Pharmaceutical Preparations (2016TP1029), Excellent Young Innovators of Changsha (kq2009092) and Foundation of Education Bureau of Hunan Province (18A493) for the financial support. We would like to thank Editage for English language editing.