A Graphene Oxide–Based Sensing Platform for the Determination of Methicillin-Resistant Staphylococcus aureus Based on Strand-Displacement Polymerization Recycling and Synchronous Fluorescent Signal Amplification

Abstract

To develop new technology for detecting methicillin-resistant Staphylococcus aureus (MRSA), a novel fluorescent biosensor based on Klenow fragment (KF)-assisted target recycling amplification and synchronous fluorescence analysis was created. Carboxy-fluorescein (FAM)-labeled single-stranded DNA (ssDNA) containing a capture probe and a signal probe was adsorbed onto the surface of graphene oxide (GO) via π-stacking interactions, resulting in the fluorescence quenching of the dye. When target and primer were introduced, the fluorescence was restored due to P0 being completely released from the surface of the GO. Meanwhile, by using the KF and exploiting the synergistic effect of FAM and the doublestranded DNA (dsDNA)-SYBR Green I duplex structure, the fluorescence in this detection system was considerably amplified and the sensitivity was improved. The proposed strategy for mecA gene analysis showed a good linear range from 1 to 40 nmol/L, with a lower limit of detection of 0.5 nmol/L. In addition, a bacterial sample harboring the mecA gene was also detected, and its lower detection limit was up to 300 colony-forming units (CFU)/mL. Accordingly, this biosensor exhibits high sensitivity and selectivity and has great potential for early clinical diagnosis and treatment.

Aptamers used for biosensors and targeted therapy

“…GO that can be used to build aptasensors is based on these three main properties. First, GO has a large surface area for binding nucleic acids via π-stacking interactions or chemical modifications [ 122 , 123 ]; second, GO can act as a superb quencher through FRET experiments because of its broad absorption spectrum and high quenching efficiency [ 124 , 125 ]; finally, GO can protect nucleic acids that bind to its surface from being cleaved by nucleases in a biological environment [ 126 , 127 ]. Tan et al designed aptasensor for the analysis of chloramphenicol (CAP), which is based on double signal amplification and GO as an efficient fluorescence quencher.…”

“…Enzyme-assisted target recycling and signal amplification designs have attracted significant attention owing to their high sensitivity [ 134 ]. To date, different types of nucleases, such as polymerases [ 123 , 135 ], exonucleases [ 136 , 137 ], deoxyribonucleases [ 132 , 138 ], and endonucleases [ 139 , 140 ], have been frequently employed to construct aptasensors for many target assays. The different properties of these nucleases can trigger several rounds of target recycling by implementing their endonuclease reaction or polymerization reaction.…”

Application of DNA-Nanosensor for Environmental Monitoring: Recent Advances and Perspectives

“…MRSA containing mecA target ssDNA interacts with carboxy fluorescein (FAM)-ssDNA-GO and removes FAM ssDNA to form double-stranded mecA-ssDNA FAM-SYBR Green I-ssDNA complex in the presence of intercalating fluorescent dye SYBR green I. SYBR green is further incorporated as the polymerase present in the solution extends target DNA using FAM ssDNA as template leading to increase in the fluorescence intensity [ 92 ]. Certain MRSA contains plasmin-sensitive cell wall proteins.…”

Gene Specific DNA Sensors for Diagnosis of Pathogenic Infections

“…On addition of target ssDNA results enhance in fluorescence signal. The limit of detection was found 1-40 nmol L -1 for confirmation of the presence of S. aureus infection [9]. Silica nano particles based platform was also developed using oligonucleotides to detect the presence of S. aureus in whole blood.…”