Electrochemical Aptasensor Used To Detect Staphylococcal Enterotoxin B Using GNPS-ZrO₂-Chits Film

Objective:Staphylococcus aureus is an important human pathogen because of its resistance to environmental stress, its increasing antibiotic resistance, and its ability to survive extremely well outside the host. S. aureus produces a number of virulence factors to counter the host’s defense systems. SEB is the most potent toxin in this group and has a remarkable heat stability and resistance to inactivation by proteases in the gastrointestinal tract. They are associated with many other human illnesses, for instance, purpura fulminans, extreme pyrexia, atopic dermatitis, and necrotizing pneumonia. For these reasons, methods that selectively and sensitively detect extremely low levels of SEB are highly desirable.In this work, we developed an electrochemical aptasensor adopting the target-induced aptamer release strategy for SEB detection. Taking advantages of GNPs-ZrO2-Chits, biotin-avidin signaling amplification and horseradish peroxidase,the aptasensor showed a high detection sensitivity.Methods:1. Preparation of GNPs-ZrO2-Chits nanocomposite:Chits was employed to improve ZrO2 dispersion and reduce HAuCl4 in situ, which exhibited good reduction propertie and biocompatibility.2. Preparation of SEB electrochemical aptasensor:The GNPs-ZrO2-Chits nanocomposite was fabricated onto the gold electrode. The capture probe with a thiol group at the 5’-terminal was immobilized on the modifed electrode surface through Au-S bonds, and then hybridized with the aptamer. The aptasensor which based on target-induced aptamer release strategy was used for quantitative detection of SEB.3. Optimization of the experimental variables:The incubation time, the pH of the working solution, the concentration of H2O2 and SA-HRP.4. Evaluation of the aptasensor:The linear range, the detection limit, stability, selectivity and reproducibility.5. SEB detection in real samples:Thirteen serum samples were examined using both the developed aptasensor and a commercially available ELISA method to investigate the possible application of the newly developed technique in clinical analysis.Results:1. TEM was used to characterize the morphologies of ZrO2-Chits, GNPs-Chits, and GNPs-ZrO2-Chits. EDX and UV-vis absorption spectroscopy were also utilized to probe a core-shell structure with GNPs embedded into ZrO2. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to characterize the modification processes.2. The redox reaction on the surface of the aptasensor is a diffusion controllable redox process. When the concentration of SA-HRP was 0.8 μM mL-1, the incubation time was 2h, the pH of the working solution was 5.5, the concentration of H2O2 was 20 mM, the aptasensor exhibited the best behavior. The aptasensor could detect SEB ranging from 2 ng mL-1 to 512 ng/mL, with a detection limit of as low as 0.24 ng/mL (S/N= 3). When the aptasensor was stored in refrigerator at 4℃ for 15 d, the value of current response has fallen by 8.2% compared with the freshly prepared electrode. The aptasensor was incubated in SEA, SEC, and thrombin to replace SEB with the same concentration of 512 ng/mL and to examine the binding selectivity to SEB. Six aptasensors prepared with the same process were tested at 32,64,128,256, and 512 ng/mL, and the relative standard deviation (RSD) of 4.0%,5.6%,4.8%,5.2%, and 3.8% were obtained, respectively. The aptasensor exhibited good stability, selectivity and reproducibility.3. The as-prepared aptasensor was used to analyze SEB in human serum specimens and validated using the ELISA method. Analytical results suggest that the developed assay has a promising alternative approach for detecting SEB in the clinical diagnosis.Conclusions:In summary, a novel electrochemical aptasensor for direct detection of SEB based on GNPs-ZrO2-Chits nanocomposite was successfully developed. The GNPs-ZrO2-Chits film was modified onto the electrode surface to greatly improve the conductivity and electrochemical effective surface area, and through which, the immobilizing amounts of probe were enlarged. Importantly, the target-induced aptamer release strategy could provide a direct sensing platform for detection of SEB and exhibit a broad linear range and low detection limit. The same signal transduction methodology could be applied to construct electrochemical aptasensors for the detection of other toxins like SEA and SEC by employing their corresponding specific aptamers.The developed aptasensor has the potential to become a powerful tool for pathogenic microorganism screening in clinical diagnosis.