Ultra-sensitive detection of protein biomarkers for diagnosis of Alzheimer's disease

Abstract

Beta amyloid peptide, tau, and phosphorylated tau are well recognized as promising biomarkers for the diagnosis of Alzheimer's disease (AD). In this work, we developed a direct, versatile, and ultrasensitive multiplex assay for the quantification of trace amounts of these protein biomarkers for AD in different types of biological fluids including cerebrospinal fluid, serum, saliva, and urine. The detection assay is based on the immunoreaction between the target proteins and their corresponding pair of antibodies followed by fluorescence labelling with a newly developed indolium-based turn-on fluorophore, namely SIM. SIM was tailor-made as a reporter to provide a high signal-to-noise ratio for the detection assay. An exceptionally low limit of detection down to the femto-molar level was achieved in this assay with minute consumption of the sample. This versatile detection assay is capable of reliably quantifying not only the target proteins simultaneously from a CSF sample in an hour but also trace amounts of protein biomarkers in saliva and urine. This assay has a high potential to serve as a practical tool for the diagnosis of AD.

Fig. 1. Molecular structure of SIM.

Fig. 2. Schematic illustration of the detection assay for the direct quantification of target protein biomarkers.

Fig. 3. Selectivity of the detection assay. The Aβ₄₂ nanoprobes were incubated with 0 fM beta amyloid proteins, 500 fM Aβ₄₀, 500 fM Aβ₄₂, and a mixture of 500 fM Aβ₄₀ and 500 fM Aβ₄₂. Error bars show the standard error of mean n = 3 (corrected average net intensity = average net intensity of the sample – average net intensity of the probe) (average net intensity = (1 × 1 square pixel of 100 individual MICs) – (1 × 1 square pixel of 100 individual background areas on the image)/100).

Fig. 4. Quantification of (A) Aβ₄₂, (B) tau₄₄₁, and (C) p-tau₁₈₁ using ELISA and the developed assay using SLAce and SIM as the reporters.

Fig. 5. Quantification of the biomarkers in human (A) CSF and (B) different biological samples by external calibration. Error bars show the standard error of mean n = 3 (average net intensity = (1 × 1 square pixel of 100 individual MICs) – (1 × 1 square pixel of 100 individual background areas on the image)/100).

Fig. 6. The quantification of Aβ₄₂ in human CSF samples as labelled with SIM in the presence of a 10% glycerol solution measured by a spectrofluorimeter. A linear range of 0–2500 fM of Aβ₄₂ was obtained.

Fig. 7. The zero and first order images of the fluorescent labelled magnetic immunocomplexes (top), and the spectra of the magnetic immunocomplexes (bottom).