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

Abstract

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+ (Kd: 1.36µM) was 17 times weaker than that of Apt/Aβ40O (Kd: 0.08µM), 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.