This research established a highly sensitive and low-cost electrochemical biosensor for the detection of recombinant human erythropoietin. A sandwich-like gold nanoparticles/Chitosan/gold nanoparticles composite film was prepared on a glassy carbon electrode. The gold nanoparticles/Chitosan/gold nanoparticles film, comprising gold nanoparticles with a large specific surface area, high conductivity, and excellent biocompatibility, was paired with Chitosan, which was naturally high in content, biodegradable, and biocompatible. Immobilization of the erythropoietin receptor on gold nanoparticles/Chitosan/gold nanoparticles films allowed recombinant human erythropoietin to recognize erythropoietin receptor, thereby altering the number of pores. The electrochemical results demonstrated that the gold nanoparticles/Chitosan/gold nanoparticle-modified electrochemical biosensor exhibited a broad detection range (5 × 10 –13 to 5 × 10 –9 g L −1 ) and a low detection limit (5.16 × 10 –14 g L −1 ). The overall preparation process was straightforward, and the detection process was rapid, with an incubation period of only 20 min. Furthermore, the biosensor exhibited high specificity, repeatability, and stability. In conclusion, the electrochemical biosensor was a simple and rapid method for the specific and accurate detection of trace recombinant human erythropoietin, which was suitable for clinical monitoring and the identification of doping. Graphical abstract
In vitro cell experiments have suggested that recombinant human erythropoietin (rhEPO) and peroxisome proliferator activated receptor γ (PPARγ) activation exert protective effects on neurons. This study observed the learning and memory ability, antioxidant capacity and the ratio of apoptotic cells after rhEPO intervention and investigated the relationship among rhEPO, PI3K/Akt and PPARγ in the anti-neural oxidative stress injury process in vivo. The results showed that rhEPO significantly improved the learning and memory abilities of rats subjected to oxidative stress, enhanced the antioxidant capacity of cells, and reduced neuronal apoptosis. Then, the PI3K/Akt and PPARγ pathways were inhibited, and TUNEL staining were used to observe the changes in the effect of rhEPO. After the PI3K/Akt and PPARγ pathways were inhibited, the effect of rhEPO on rats subjected to oxidative stress was significantly weakened, suggesting that both the PI3K/Akt and PPARγ pathways are involved in the process by which rhEPO protects neurons. Finally, Western blotting and immunofluorescence staining were used to observe the changes in PI3K/Akt and PPARγ signalling proteins in the neurons after the rhEPO intervention and to explore the relationship among the three. The results showed that rhEPO significantly increased the levels of the p-Akt and PPARγ proteins and the level of the PPARγ protein in the nucleus, indicating that the PI3K/Akt pathway was located upstream of and regulates PPARγ. In conclusion, this study suggested that rhEPO activates the PI3K/Akt to upregulate PPARγ, enhance the cellular antioxidant capacity, and protect neurons in rats subjected to oxidative stress.
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