A universal reusable RGO-loaded cellulose 3D hydrogel electrode via π-π stacking for detection of multiple community-acquired pneumonia pathogens

Community-acquired pneumonia (CAP) is characterized by complex pathogenic agents and similar early clinical manifestations. Existing detection technologies suffer from drawbacks such as complex operation procedures, insufficient sensitivity, and difficulty in distinguishing different pathogens, which fail to meet the demand for rapid and accurate diagnosis at the grassroots units. In this study, a cellulose-sodium alginate-polyaniline-reduced graphene oxide-Ag composite hydrogel (CSPRAG) electrode was successfully fabricated via freeze-drying and in-situ deposition methods, enabling the construction of a label-free electrochemical biosensor with reusability, multi-target detection capability and high sensitivity. Reduced graphene oxide (RGO) on the hydrogel surface forms π-π stacking interactions with DNA probes, and combined with the coordination and electrostatic effects of Ag particles, a stable sensing interface is constructed. Three types of CAP pathogen nucleic acid probes were grafted onto additional RGO carriers to form DNA/RGO composite probes. Through the synergistic dual π-π stacking effect between RGO in the composite probes, DNA bases and RGO on the hydrogel surface, label-free adsorption of the probes is realized. The sensor exhibits a good linear response from 1 fM to 10 μM, with detection limits as low as 0.188 fM, 0.192 fM, and 0.194 fM, respectively. It also demonstrates excellent specificity, stability, and anti-interference capability, maintaining reliable performance after 30 days of storage, 50 cycles of detection, and 15 cycles of probe reuse. Featuring low cost and facile preparation, the CSPRAG electrode provides a novel solution for the rapid screening of CAP pathogens at the grassroots units, holding significant clinical application value.