Development of An Impedimetric Aptasensor for the Detection of Staphylococcus aureus

Abstract

In combination with electrochemical impedance spectroscopy, aptamer-based biosensors are a powerful tool for fast analytical devices. Herein, we present an impedimetric aptasensor for the detection of the human pathogen Staphylococcus aureus. The used aptamer targets protein A, a surface bound virulence factor of S. aureus. The thiol-modified protein A-binding aptamer was co-immobilized with 6-mercapto-1-hexanol onto gold electrodes by self-assembly. Optimization of the ratio of aptamer to 6-mercapto-1-hexanol resulted in an average density of 1.01 ± 0.44 × 1013 aptamer molecules per cm2. As shown with quartz crystal microbalance experiments, the immobilized aptamer retained its functionality to bind recombinant protein A. Our impedimetric biosensor is based on the principle that binding of target molecules to the immobilized aptamer decreases the electron transfer between electrode and ferri-/ferrocyanide in solution, which is measured as an increase of impedance. Microscale thermophoresis measurements showed that addition of the redox probe ferri-/ferrocyanide has no influence on the binding of aptamer and its target. We demonstrated that upon incubation with various concentrations of S. aureus, the charge-transfer resistance increased proportionally. The developed biosensor showed a limit of detection of 10 CFU·mL−1 and results were available within 10 minutes. The biosensor is highly selective, distinguishing non-target bacteria such as Escherichia coli and Staphylococcus epidermidis. This work highlights the immense potential of impedimetric aptasensors for future biosensing applications.

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

“…Two of the aptamers (PA#2/8 and PA-C8) are very suitable as capture molecules and in particular PA#2/8 as reporter molecule. We have previously shown that PA#2/8 is functional in different assay designs and is able to recognize intact cells of S. aureus [ 32 ]. Our next steps will focus on the integration of the aptamers in detection assays, e.g., sandwich-type assays like ELONA or lateral flow assays (LFA), which are suitable for the practicable pathogen detection.…”

“…We have previously described the selection of DNA aptamers for the specific binding to Protein A as a cell surface component of Staphylococcus aureus [ 30 ]. We found one promising aptamer that was extensively characterized and evaluated for its ability to recognize and bind to Protein A in a whole cell context of the bacterial pathogen [ 31 , 32 ]. In this work, we applied NGS to the same selected aptamer pool to compare the outcome with that of Sanger sequencing and perhaps to expand our portfolio of Protein A-binding aptamers.…”

Antimicrobial Peptides: Powerful Biorecognition Elements to Detect Bacteria in Biosensing Technologies

“…A large number of impedimetric biosensors for whole bacteria detection have been described in recent years ( Table 1 ). The most common bioreceptors employed in this case are enzymes coated on nanoparticles [ 45 ], antibodies [ 14 , 57 , 58 , 59 , 60 ], aptamers [ 71 ], lectins [ 75 , 76 , 77 ] and phages [ 88 , 89 ]. The use of AMPs represents the main focus of this review and will be covered comprehensively in the following sections.…”

“…© 2010 Elsevier B.V. ELECTROCHEMICAL BIOSENSORS: ( A ) Aptamer functionalized impedimetric biosensor. Figure reproduced from [ 71 ]; ( B ) Potentiometric biosensor. Reprinted from [ 70 ], with permission from Elsevier.…”

Application of Aptamer-Based Biosensor for Rapid Detection of Pathogenic Escherichia coli

“…Compared with optical sensors, electrochemical sensors [ 59 , 60 , 61 ] are easy to achieve miniaturization and portability, and can be used for turbid medium detection. Therefore, aptamer-based electrochemical sensors for pathogenic E. coli detection have also attracted much attention [ 62 , 63 , 64 ].…”