Addressing Cu2+ interference for accurate aptamer-based biomarker determinations of Alzheimer’s disease

Aptamers are widely used as a recognition element for Alzheimer's disease (AD)-related biomarker determinations. Due to the existing Cu 2+ in real samples, and its possible coordinations with aptamers and biomarkers, the aptamer-based biomarker determination could be seriously disturbed. In this work, taking Aβ 40 oligomer (Aβ 40 O, a typical biomarker for AD) as a model, the interference of Cu 2+ in the interaction between Aβ 40 O and its aptamer (Apt) was investigated by surface plasmon resonance spectroscopy. The results demonstrated the binding of Cu 2+ to Apt, resulting in significant changes on the original structure of Apt, and the affinity of Apt to Aβ 40 O. The affinity of Apt-Cu 2+ /Aβ 40 O-Cu 2+ (K d : 1.36 µM) was 17 times weaker than that of Apt/Aβ 40 O (K d : 0.08 µM), which induced a poor dynamic range in the Cu 2+ -involved Aβ 40 O determination. The analysis of Aβ 40 O-spiked real samples revealed that the existing Cu 2+ resulted in significant positive errors in CSF analysis, but also complicated errors in serum analysis that depended on the blood collection. Further studies confirmed that EDTA can completely chelate Cu 2+ from Apt-Cu 2+ and Aβ 40 O-Cu 2+ , eliminating the interference of Cu 2+ on the Aβ 40 O/Apt system. Therefore, excess EDTA is highly recommended for Apt-based CSF and blood analysis, even if the samples are from EDTA- or other metal ion chelating agent-involved collection tubes. This work quantitatively revealed the interference of Cu 2+ and provided a simple but effective approach to eliminate the interference, which will improve the existing method to achieve accurate Apt-based biomarker detection in real samples.