Electrochemiluminescence Biosensor Based on an Efficient Antifouling Cell Membrane Hydrogel for the Direct Detection of Alkaline Phosphatase in Human Serum and Cell Lysate

Electrochemiluminescence (ECL) biosensors are an ideal choice for the real-time and sensitive detection of biomarkers. However, there are many interfering biomolecules in human serum or cell lysate that can affect the ECL signal through nonspecific interface adsorption. To solve this issue, an efficient ECL antifouling biosensor based on a red blood cell membrane (RBCM) hydrogel was developed for the direct detection of alkaline phosphatase (ALP) in human serum and cell lysate. Among them, the RBCM hydrogel was prepared by embedding the purified RBCM vesicles into [1,2-distearoyl-sn-glycerol-3-phosphoethanolamine]-[polyethylene]-[acrylamide] (DSPE-PEG-AM), which endowed the antifouling interface with high stability and biocompatibility. Meanwhile, compared to RBCM, the constructed RBCM hydrogel improved the electron transfer rate of the antifouling interface. Furthermore, luminescent copper nanosheets (Cu NSs) and quencher MnO₂ NSs were encapsulated in the RBCM hydrogel, which greatly reduced the distance between Cu NSs and MnO₂ NSs, thereby effectively improving the signal quenching efficiency. In the presence of ALP, l-ascorbic acid 2-phosphate trisodium salt was hydrolyzed to ascorbic acid, thus disrupting the structure of MnO₂ NSs to restore the ECL signal. Based on the above sensing strategies, the constructed ECL biosensor achieved an ultrasensitive detection of ALP with a wide linear range (10^-5 to 10⁴ U·L^-1) and a detection limit as low as 3.4 × 10^-6 U·L^-1 (3δ/k), demonstrating potential application value in clinical diagnosis.