A SERS aptasensor based on Au@Ag bimetallic nanostars-magnetic covalent organic composites for rHuEPO-α detection

The misuse of recombinant human erythropoietin-alpha (rHuEPO-α) for performance enhancement in sports has been a persistent issue. The World Anti-Doping Agency has emphasized the need for reliable detection methods to identify the illicit use of rHuEPO-α, thereby upholding the integrity of sports competitions. Most screening strategies rely on immunological methods for detection. However, these methods often depend on antibodies with limited selectivity, leading to a higher risk of false-positive results. Consequently, there is a pressing need to develop alternative and innovative recognition modules for the precise detection of rHuEPO-α. Aptamers, often termed "chemical antibodies," are well-suited to address this challenge.

The Raman signaling molecule 4-aminothiophene (4-ATP) was initially embedded between gold-silver core-shell nanostars (Au@Ag NSs). The rHuEPO-α aptamer was then conjugated to the Ag shell via an Ag-S covalent bond to assemble the Surface-Enhanced Raman Scattering (SERS) probe. Subsequently, the capture DNA strand was connected to the magnetic covalent organic framework (Fe₃O₄@COF@Au) via "Au-S" bonds. The complementary pairing of the aptamer on the SERS probe with the captured DNA enabled the assembly of the magnetic covalent organic framework and the SERS probe. The specific recognition of rHuEPO-α by the aptamer triggered the separation of the SERS probe from the Fe₃O₄@COF@Au magnetic covalent organic framework, resulting in a decrease in the Raman signal. The detection limit for rHuEPO-α concentration was ultimately determined to be as low as 0.856 pg/mL, exhibiting an inverse linear relationship with the Raman signal.

The aptasensor utilizes its magnetic separation effect to enhance the enrichment and purification of Raman signals, thereby improving detection sensitivity and achieving a lower detection limit. This provides an experimental basis for the construction of SERS aptasensors and the detection of rHuEPO-α. Moreover, the sensor also has the potential to serve as an effective monitoring tool for other trace substances.