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 Cu²⁺ in real samples, and its possible coordinations with aptamers and biomarkers, the aptamer-based biomarker determination could be seriously disturbed. In this work, taking Aβ₄₀ oligomer (Aβ₄₀O, a typical biomarker for AD) as a model, the interference of Cu²⁺ in the interaction between Aβ₄₀O and its aptamer (Apt) was investigated by surface plasmon resonance spectroscopy. The results demonstrated the binding of Cu²⁺ to Apt, resulting in significant changes on the original structure of Apt, and the affinity of Apt to Aβ₄₀O. The affinity of Apt-Cu²⁺/Aβ₄₀O-Cu²⁺ (K_d: 1.36 µM) was 17 times weaker than that of Apt/Aβ₄₀O (K_d: 0.08 µM), which induced a poor dynamic range in the Cu²⁺-involved Aβ₄₀O determination. The analysis of Aβ₄₀O-spiked real samples revealed that the existing Cu²⁺ 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 Cu²⁺ from Apt-Cu²⁺ and Aβ₄₀O-Cu²⁺, eliminating the interference of Cu²⁺ on the Aβ₄₀O/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 Cu²⁺ 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.